Patent Grant
12031164
A sequence listing contained in the file named “GeneSequenceListing.TXT” which is 621,774 bytes (measured in MS-Windows®) and created on Mar. 17, 2020, and comprises 388 sequences, is incorporated herein by reference in its entirety.
The present invention relates to fusion proteins containing a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion proteins further comprise an enzyme having ACC deaminase activity, a phospholipase, a lipase, a xylanase, a xylosidase, a lactonase, a chitosanase, a protease, a glucanase, a phytase, an acid phosphatase, a pectinase, a mannanase, and/or an expansin protein. The invention further relates to recombinant Bacillus cereus family members that express the fusion proteins, exosporium fragments derived from the recombinant Bacillus cereus family members, and formulations containing the recombinant Bacillus cereus family members or exosporium fragments. Plant seeds treated with the recombinant Bacillus cereus family members, exosporium fragments, or formulations are also provided. Animal feed additives and animal feed compositions comprising the recombinant Bacillus cereus family members or exosporium fragments derived from the recombinant Bacillus cereus family members are also provided. The invention further relates to methods for stimulating plant growth and/or promoting plant health using the recombinant Bacillus cereus family members, exosporium fragments, or formulations. Method for delivering enzymes to an animal using the recombinant Bacillus cereus family members or exosporium fragments derived from the recombinant Bacillus cereus family members are also provided.
Within the zone surrounding a plant's roots is a region called the rhizosphere. In the rhizosphere, bacteria, fungi, and other organisms compete for nutrients and for binding to the root structures of the plant. Both detrimental and beneficial bacteria and fungi can occupy the rhizosphere. The bacteria, fungi, and the root system of the plant can all be influenced by the actions of peptides, enzymes, and other proteins in the rhizosphere. Augmentation of soil or treatment of plants with certain of these peptides, enzymes, or other proteins would have beneficial effects on the overall populations of beneficial soil bacteria and fungi, create a healthier overall soil environment for plant growth, improve plant growth, and provide for the protection of plants against certain bacterial and fungal pathogens. However, previous attempts to introduce peptides, enzymes, and other proteins into soil to induce such beneficial effects on plants have been hampered by the low survival of enzymes, proteins, and peptides in soil. Additionally, the prevalence of proteases naturally present in the soil can lead to degradation of the proteins in the soil. The environment around the roots of a plant (the rhizosphere) is a unique mixture of bacteria, fungi, nutrients, and roots that has different qualities than that of native soil. The symbiotic relationship between these organisms is unique, and could be altered for the better with inclusion of exogenous proteins. The high concentration of fungi and bacteria in the rhizosphere causes even greater degradation of proteins due to abnormally high levels of proteases and other elements detrimental to proteins in the soil. In addition, enzymes and other proteins introduced into soil can dissipate away from plant roots quickly.
Thus, there exists a need in the art for a method for effectively delivering peptides, enzymes, and other proteins to plants (e.g., to plant root systems) and for extending the period of time during which such molecules remain active. Furthermore, there exists a need in the art for a method of selectively targeting such peptides, enzymes, and proteins to the rhizosphere and to plant leaves and plant roots in particular.
A fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises an enzyme having 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity. The enzyme having ACC deaminase activity comprises:
Another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises a phospholipase. The phospholipase:
Yet another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a lipase. The lipase comprises:
Another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises a xylanase. The xylanase comprises:
A further fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises a xylosidase. The xylosidase comprises:
Another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a lactonase. The lactonase comprises:
Yet another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises a chitosanase. The chitosanase:
A further fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises a protease. The protease comprises:
Another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a glucanase. The glucanase:
Yet another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises an expansin protein. The expansin protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 316.
A further fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises a phytase. The phytase comprises:
Another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises an acid phosphatase. The acid phosphatase comprises:
Yet another fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a pectinase. The pectinase comprises a pectolyase.
A further fusion protein is provided. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises a mannanase.
A recombinant Bacillus cereus family member is provided. The recombinant Bacillus cereus family member expresses a fusion protein. The fusion protein can be any of the fusion proteins described herein.
Exosporium fragments are provided. The exosporium fragments are derived from a recombinant Bacillus cereus family member. The recombinant Bacillus cereus family member can be any of the recombinant Bacillus cereus family members described herein. The exosporium fragments can comprise any of the fusion proteins described herein.
A formulation is provided. The formulation comprises any of the recombinant Bacillus cereus family members described herein. The formulation further comprises an agriculturally acceptable carrier.
Another formulation is provided. The formulation comprises exosporium fragments derived from any of the recombinant Bacillus cereus family members described herein. The formulation further comprises an agriculturally acceptable carrier.
Yet another formulation is provided. The formulation comprises a recombinant Bacillus cereus family member that expresses a fusion protein. Alternatively, or in addition, the formulation comprises exosporium fragments derived from a Bacillus cereus family member that expresses a fusion protein. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises an acid phosphatase. The formulation further comprises a second enzyme.
Yet another formulation is provided. The formulation comprises a recombinant Bacillus cereus family member that expresses a fusion protein. Alternatively, or in addition, the formulation comprises exosporium fragments derived from a Bacillus cereus family member that expresses a fusion protein. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein also comprises a phospholipase C. The formulation further comprises a second enzyme.
A treated plant seed is provided. The plant seed can be treated with any of the recombinant Bacillus cereus family members described herein. The recombinant Bacillus cereus family member can express any of the fusion proteins described herein.
Another treated plant seed is provided. The plant seed can be treated with any of the exosporium fragments described herein. The exosporium fragments can be derived from any of the Bacillus cereus family members described herein. The exosporium fragments can comprise any of the fusion proteins described herein.
Yet another treated plant seed is provided. The plant seed can be treated with any of the formulations described herein.
An animal feed additive is provided. The animal feed additive can comprise any of the recombinant Bacillus cereus family members that expresses any of the fusion proteins described herein. Alternatively, or in addition, the animal feed additive can comprise exosporium fragments derived from any of the Bacillus cereus family members that expresses any of the fusion protein described herein.
An animal feed composition is also provided. The animal feed composition comprises animal feed and any of the recombinant Bacillus cereus family members that expresses a fusion protein described herein. Alternatively, or in addition, the animal feed composition can comprise animal feed and exosporium fragments derived from any of the recombinant Bacillus cereus family members described herein.
A method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a recombinant Bacillus cereus family member to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The recombinant Bacillus cereus family member can comprise any of the recombinant Bacillus cereus family members described herein. The recombinant Bacillus cereus family member can express any of the fusion proteins described herein.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying exosporium fragments to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The exosporium fragments can comprise exosporium fragments derived from any of the recombinant Bacillus cereus family members described herein. The exosporium fragments can comprise any of the fusion proteins described herein.
Yet another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a formulation to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The formulation can comprise any of the formulations described herein.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a recombinant Bacillus cereus family member expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a xylosidase, a histidine protease, an acid phosphatase, or a combination of any thereof.
Yet another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying exosporium fragments derived from spores of a recombinant Bacillus cereus family member expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a xylosidase, a histidine protease, an acid phosphatase, or a combination of any thereof.
A further method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a recombinant Bacillus cereus family member expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises an acid phosphatase. The method further comprises applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying exosporium fragments derived from spores of a recombinant Bacillus cereus expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises an acid phosphatase. The method further comprises applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a recombinant Bacillus cereus family member expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a phospholipase C. The method further comprises applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying exosporium fragments derived from spores of a recombinant Bacillus cereus expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a phospholipase C. The method further comprises applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
A method for delivering an enzyme to an animal is provided. The method comprises feeding to the animal any of the recombinant Bacillus cereus family members that express a fusion protein described herein. Alternatively, or in addition, the method comprises feeding to the animal exosporium fragments derived from spores of any of the recombinant Bacillus cereus family members that express a fusion protein described herein.
Other objects and features will be in part apparent and in part pointed out hereinafter.
When the articles “a,” “an,” “one,” “the,” and “said” are used herein, they mean “at least one” or “one or more” unless otherwise indicated.
The term “Bacillus cereus family member” as used herein refers to any Bacillus species that is capable of producing an exosporium. Thus, the Bacillus cereus family of bacteria includes the species Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, and Bacillus toyoiensis. Bacillus cereus family members are also referred to in the art as “Bacillus cereus senso lato.”
The terms “comprising,” “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.
The term “free enzyme” as used herein refers to an enzyme preparation that is substantially free of intact cells. The term “free enzyme” includes, but is not limited to, crude cell extracts containing an enzyme, partially purified, substantially purified, or purified enzyme. Free enzymes can optionally be immobilized on a chemical matrix or support to allow for controlled release of the enzyme. The term “immobilizing” as used herein in reference to immobilizing an enzyme on a matrix or support refers to the binding of the enzyme to the matrix or support such that the enzyme is maintained on the matrix or support or released from the support over a controlled period of time, instead of dissipating into the environment in an uncontrolled manner. Illustrative matrices and supports include, but are not limited to, charcoal, biochar, nanocarbon, agarose, an alginate, cellulose, a cellulose derivative, silica, plastic, stainless steel, glass, polystyrene, a ceramic, dolomite, a clay, diatomaceous earth, talc, a polymer, a gum, a water-dispersable material, and combinations of any thereof.
The term “foliar” used herein with respect to the application of enzymes or recombinant microorganisms to plants means that the enzyme or recombinant microorganism is applied to one or more aerial portions of the plant, including stems, leaves, fruits, flowers, or other exposed aerial portions of the plant.
The term “fusion protein” as used herein refers to a protein having a polypeptide sequence that comprises sequences derived from two or more separate proteins. A fusion protein can be generated by joining together a nucleic acid molecule that encodes all or part of a first polypeptide with a nucleic acid molecule that encodes all or part of a second polypeptide to create a nucleic acid sequence which, when expressed, yields a single polypeptide having functional properties derived from each of the original proteins.
The term “germination rate” as used herein refers to the number of seeds that germinate during a particular time period. For example, a germination rate of 85% indicates that 85 out of 100 seeds germinate during a given time period.
The term “inactivate” or “inactivation” as used herein in reference to the inactivation of spores of a recombinant Bacillus cereus family member means that the spores are unable to germinate, or that the spores can germinate, but are damaged such that germination does not result in a living bacterium. The terms “partially inactivate” or “partial inactivation” mean that a percentage of the spores are inactivated, but that some spores retain the ability to germinate and return to a live, replicating state. The term “genetic inactivation” refers to inactivation of spores a recombinant Bacillus cereus family member by a mutation of the spore's DNA that results in complete or partial inactivation of the spore. The terms “physical inactivation” and “chemical inactivation” refer to inactivation of spores using any physical or chemical means, e.g., by heat treatment, gamma irradiation, x-ray irradiation, UV-A irradiation, UV-B irradiation, or treatment with a solvent such as glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, chloroform, phenol, or any combination thereof.
The terms “native sequence,” “native amino acid sequence,” “wild-type sequence,” and “wild-type amino acid sequence” are used interchangeably herein to refer to an amino acid sequence as it exists in a naturally occurring protein.
A “plant growth medium” includes any material that is capable of supporting the growth of a plant.
The terms “promoting plant growth” and “stimulating plant growth” are used interchangeably herein, and refer to the ability to enhance or increase at least one of the plant's height, weight, leaf size, root size, fruit size, or stem size, and/or the ability to increase protein yield from the plant and/or to increase crop yield.
The term “recombinant” as used in reference to the bacteria described herein encompasses bacteria having any genetic modification as compared to wild-type bacteria of the same type, including bacteria that have been modified to delete of a gene or a portion of a gene (e.g., bacteria that have a “knock-out” of a gene), as well as bacteria that have been modified to express an exogenous peptide or protein.
The term “rhizosphere” is used interchangeably with “root zone” to denote that segment of the soil that surrounds the roots of a plant and is influenced by them.
The term “synergistically effective amount” as used herein refers an amount of a first substance (e.g., a first enzyme) that when used in combination with a second substance (e.g., a second enzyme) produces a biological effect that is greater than the sum of the biological effects of each of the respective first and second substances when used alone.
The term “targeting sequence” as used herein refers to a polypeptide sequence that, when present as part of a longer polypeptide or a protein, results in the localization of the longer polypeptide or the protein to a specific subcellular location. The targeting sequences described herein result in localization of proteins to the exosporium of a Bacillus cereus family member.
I. Fusion Proteins for Expression in Bacillus Cereus Family Members
The present invention relates to fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion proteins further comprise an ACC deaminase, a phospholipase, a lipase, a xylanase, a xylosidase, a lactonase, a chitosanase, a protease, a glucanase, a phytase, an acid phosphatase, a pectinase, a mannanase, and/or an expansin protein. When expressed in Bacillus cereus family member bacteria, these fusion proteins are targeted to the exosporium layer of the spore and are physically oriented such that the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the expansin protein, the phytase, the acid phosphatase, the pectinase, or the mannanase is displayed on the outside of the spore.
This Bacillus exosporium display (BEMD) system can be used to deliver the ACC deaminases, the phospholipases, the lipases, the xylanases, the xylosidases, the lactonases, the chitosanases, the proteases, the glucanases, the phytases, the acid phosphatases, the pectinases, the mannanases, and/or the expansin proteins to plants (e.g., to plant foliage, fruits, flowers, stems, or roots) or to a plant growth medium such as soil. Enzymes and proteins delivered to the soil or another plant growth medium in this manner persist and exhibit activity in the soil for extended periods of time. Introduction of recombinant Bacillus cereus family member bacteria expressing the fusion proteins described herein into soil or the rhizosphere of a plant leads to a beneficial enhancement of plant growth in many different soil conditions. The use of the BEMD to create these enzymes allows them to continue to exert their beneficial results to the plant and the rhizosphere over the first months of a plants life.
In addition, as is described further hereinbelow, the BEMD system can be modified such that the exosporium of the recombinant Bacillus cereus family member can be removed from the spore, generating exosporium fragments containing the fusion proteins. The exosporium fragments can also be used to deliver the ACC deaminases, the phospholipases, the lipases, the xylanases, the xylosidases, the lactonases, the chitosanases, the proteases, the glucanases, the phytases, the acid phosphatases, the pectinases, the mannanases, and/or the expansin proteins to plants in a cell-free preparation.
A. Targeting Sequences, Exosporium Proteins, and Exosporium Protein Fragments for Targeting Enzymes Having ACC Deaminase Activity, Phospholipases, Lipases, Xylanases, Xylosidases, Lactonases, Chitosanases, Proteases, Glucanases, Phytases, Acid Phosphatases, Pectinases, Mannanases, and Expansin Proteins to the Exosporium of a Bacillus cereus Family Member
For ease of reference, descriptions of the amino acid sequences for the targeting sequences, exosporium proteins, and exosporium protein fragments that can be used for targeting of enzymes or proteins (e.g., enzymes having ACC deaminase activity, phospholipases, lipases, xylanases, xylosidases, lactonases, chitosanases, proteases, glucanases, phytases, acid phosphatases, pectinases, mannanases, and/or expansin proteins) to the exosporium of a Bacillus cereus family members, are provided in Table 1 together with their SEQ ID NOs.
Bacillus is a genus of rod-shaped bacteria. The Bacillus cereus family of bacteria includes any Bacillus species that is capable of producing an exosporium. Thus, the Bacillus cereus family of bacteria includes the species Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, and Bacillus toyoiensis. Under stressful environmental conditions, Bacillus cereus family bacteria undergo sporulation and form oval endospores that can stay dormant for extended periods of time. The outermost layer of the endospores is known as the exosporium and comprises a basal layer surrounded by an external nap of hair-like projections. Filaments on the hair-like nap are predominantly formed by the collagen-like glycoprotein BclA, while the basal layer is comprised of a number of different proteins. Another collagen-related protein, BclB, is also present in the exosporium and exposed on endospores of Bacillus cereus family members. BclA, the major constituent of the surface nap, has been shown to be attached to the exosporium with its amino-terminus (N-terminus) positioned at the basal layer and its carboxy-terminus (C-terminus) extending outward from the spore.
It was previously discovered that certain sequences from the N-terminal regions of BclA and BclB could be used to target a peptide or protein to the exosporium of a Bacillus cereus family member endospore (see U.S. Patent Application Publication Nos. 2010/0233124 and 2011/0281316, and Thompson et al., Targeting of the BclA and BclB proteins to the Bacillus anthracis spore surface, Molecular Microbiology 70(2):421-34 (2008)). It was also found that the BetA/BAS3290 protein of Bacillus anthracis localized to the exosporium. Further targeting sequences, as well as exosporium proteins and fragments of exosporium proteins, that can be incorporated into a fusion protein and used to target a peptide or protein of interest to the exosporium of a recombinant Bacillus cereus family member are described in U.S. Patent Application Publication Nos. 2016/0031948 and 2016/0108096, which are incorporated by reference herein in their entirety.
In particular, amino acids 20-35 of BclA from Bacillus anthracis Sterne strain have been found to be sufficient for targeting to the exosporium. A sequence alignment of amino acids 1-41 of BclA (SEQ ID NO: 1) with the corresponding N-terminal regions of several other Bacillus cereus family exosporium proteins and Bacillus cereus family proteins having related sequences is shown in
Additional proteins from Bacillus cereus family members also contain the conserved targeting region. In particular, in
Amino acids 1-41 of BclA from B. thuringiensis (SEQ ID NO: 382) and amino acids 1-41 of BclA from B. anthracis (SEQ ID NO: 384) are identical to SEQ ID NO: 2 and are thus not depicted in
Any portion of BclA which includes amino acids 20-35 can be used as to target a fusion protein to the exosporium. In addition, full-length exosporium proteins or exosporium protein fragments can be used for targeting the fusion proteins to the exosporium. Thus, full-length BclA or a fragment of BclA that includes amino acids 20-35 can be used for targeting to the exosporium. For example, full length BclA (SEQ ID NO: 2, 382, or _384) or a midsized fragment of BclA that lacks the carboxy-terminus such as SEQ ID NO: 95 or 385 (amino acids 1-196 of BclA) or 383 (amino acids 1-166 of BclA) can be used to target the fusion proteins to the exosporium. Midsized fragments such as the fragments of SEQ ID NO: 95, 383, and 385 have less secondary structure than full length BclA and have been found to be suitable for use as a targeting sequence. The targeting sequence can also comprise much shorter portions of BclA which include amino acids 20-35, such as SEQ ID NO: 1 (amino acids 1-41 of BclA), amino acids 1-35 of SEQ ID NO: 1, amino acids 20-35 of SEQ ID NO: 1, or SEQ ID NO: 96 (a methionine residue linked to amino acids 20-35 of BclA). Even shorter fragments of BclA which include only some of amino acids 20-35 also exhibit the ability to target fusion proteins to the exosporium. For example, the targeting sequence can comprise amino acids 22-31 of SEQ ID NO: 1, amino acids 22-33 of SEQ ID NO: 1, or amino acids 20-31 of SEQ ID NO: 1.
Alternatively, any portion of BetA/BAS3290, BAS4623, BclB, BAS1882, the KBAB4 2280 gene product, the KBAB4 3572 gene product, B. cereus VD200 exosporium leader peptide, B. cereus VD166 exosporium leader peptide, B. cereus VD200 hypothetical protein IKG_04663, B. weihenstephensis KBAB4 YVTN β-propeller protein, B. weihenstephensis KBAB4 hypothetical protein bcerkbab4_2363, B. weihenstephensis KBAB4 hypothetical protein bcerkbab4_2131, B. weihenstephensis KBAB4 triple helix repeat containing collagen, B. mycoides 2048 hypothetical protein bmyco0001_21660, B. mycoides 2048 hypothetical protein bmyc0001_22540, B. mycoides 2048 hypothetical protein bmyc0001_21510, B. thuringiensis 35646 collagen triple helix repeat protein, B. cereus hypothetical protein WP_69652, B. cereus exosporium leader WP016117717, B. cereus exosporium peptide WP002105192, B. cereus hypothetical protein WP87353, B. cereus exosporium peptide 02112369, B. cereus exosporium protein WP016099770, B. thuringiensis hypothetical protein YP006612525, B. mycoides hypothetical protein TIGR03720, B. cereus ATCC 10987 collagen triple helix repeat domain protein, B. cereus E33L collagen-like protein, B. weihenstephanensis KBAB4 triple helix repeat-containing collagen, B. thuringiensis str. Al Hakam hypothetical protein BALH_2230, B. cereus ATCC 14579 triple helix repeat-containing collagen, B. cereus collagen triple helix repeat, B. cereus ATCC 14579 triple helix repeat-containing collagen, B. cereus E33L hypothetical protein BCZK1835, B. weihenstephanensis KBAB4 triple helix repeat-containing collagen, B. cereus ATCC 14579 triple helix repeat-containing collagen, B. cereus ATCC 14579 hypothetical protein BC4725, B. cereus E33L hypothetical protein BCZK4476, B. anthracis str. ‘Ames Ancestor’ triple helix repeat-containing collagen, B. thuringiensis serovar konkukian str. 97-27 BclA protein, B. cereus ATCC 10987 conserved hypothetical protein, B. cereus ATCC 14579 triple helix repeat-containing collagen, B. cereus exosporium leader peptide partial sequence, or B. weihenstephanensis hypothetical protein ER45_27600 which includes the amino acids corresponding to amino acids 20-35 of BclA can serve as the targeting sequence.
As can be seen from
Furthermore, any amino acid sequence comprising amino acids 20-35 of BclA, or any of the above-listed corresponding amino acids, can serve as the targeting sequence.
The targeting sequence can comprise amino acids 1-35 of SEQ ID NO: 1, amino acids 20-35 of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 96, amino acids 22-31 of SEQ ID NO: 1, amino acids 22-33 of SEQ ID NO: 1, or amino acids 20-31 of SEQ ID NO: 1. Alternatively, the targeting sequence can consist of amino acids 1-35 of SEQ ID NO: 1, amino acids 20-35 of SEQ ID NO: 1, SEQ ID NO: 1, or SEQ ID NO: 96. Alternatively, the targeting sequence can consist of amino acids 22-31 of SEQ ID NO: 1, amino acids 22-33 of SEQ ID NO: 1, or amino acids 20-31 of SEQ ID NO: 1. Alternatively, the exosporium protein can comprise full length BclA (SEQ ID NO: 2), or the exosporium protein fragment can comprise a midsized fragment of BclA that lacks the carboxy-terminus, such as SEQ ID NO: 95 (amino acids 1-196 of BclA). Alternatively, the exosporium protein fragment can consist of SEQ ID NO: 95.
The targeting sequence can comprise amino acids 2-35 of SEQ ID NO: 1; amino acids 5-35 of SEQ ID NO: 1; amino acids 8-35 of SEQ ID NO: 1; amino acids 10-35 of SEQ ID NO: 1; or amino acids 15-35 of SEQ ID NO: 1.
The targeting sequence can comprise amino acids 1-27 of SEQ ID NO: 3, amino acids 12-27 of SEQ ID NO: 3, or SEQ ID NO: 3, or the exosporium protein can comprise full length BetA/BAS3290 (SEQ ID NO: 4). It has also been found that a methionine residue linked to amino acids 12-27 of BetA/BAS3290 can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 97. Alternatively, the targeting sequence can comprise amino acids 14-23 of SEQ ID NO: 3, amino acids 14-25 of SEQ ID NO: 3, or amino acids 12-23 of SEQ ID NO: 3.
The targeting sequence can comprise amino acids 2-27 of SEQ ID NO: 3; amino acids 5-27 of SEQ ID NO: 3; amino acids 8-27 of SEQ ID NO: 3; or amino acids 10-27 of SEQ ID NO: 3.
The targeting sequence can comprise amino acids 1-38 of SEQ ID NO: 5, amino acids 23-38 of SEQ ID NO: 5, SEQ ID NO: 5, or SEQ ID NO: 371 (a methionine residue linked to amino acids 23-38 of BAS4623) or the exosporium protein can comprise full length BAS4623 (SEQ ID NO: 6).
The targeting sequence can comprise amino acids 2-38 of SEQ ID NO: 5; amino acids 5-38 of SEQ ID NO: 5; amino acids 8-38 of SEQ ID NO: 5; amino acids 10-38 of SEQ ID NO: 5; amino acids 15-38 of SEQ ID NO: 5; or amino acids 20-38 of SEQ ID NO: 5.
Alternatively, the targeting sequence can comprise amino acids 1-28 of SEQ ID NO: 7, amino acids 13-28 of SEQ ID NO: 7, SEQ ID NO: 7, or SEQ ID NO: 372 (a methionine residue linked to amino acids 13-28 of BclB) or the exosporium protein can comprise full length BclB (SEQ ID NO:8).
The targeting sequence can comprise amino acids 2-28 of SEQ ID NO: 7; amino acids 5-28 of SEQ ID NO: 7; amino acids 8-28 of SEQ ID NO: 7; or amino acids 10-28 of SEQ ID NO: 7.
The targeting sequence can comprise amino acids 1-24 of SEQ ID NO: 9, amino acids 9-24 of SEQ ID NO: 9, or SEQ ID NO: 9, or the exosporium protein can comprise full length BAS1882 (SEQ ID NO: 10). A methionine residue linked to amino acids 9-24 of BAS1882 can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 105.
The targeting sequence can comprise amino acids 2-24 of SEQ ID NO: 9; amino acids 5-24 of SEQ ID NO: 9; or amino acids 8-24 of SEQ ID NO: 9.
The targeting sequence can comprise amino acids 1-33 of SEQ ID NO:11, amino acids 18-33 of SEQ ID NO: 11, or SEQ ID NO: 11, or the exosporium protein can comprise the full length B. weihenstephensis KBAB4 2280 gene product (SEQ ID NO: 12). A methionine residue linked to amino acids 18-33 of the B. weihenstephensis KBAB4 2280 gene product can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 98.
The targeting sequence can comprise amino acids 2-33 of SEQ ID NO: 11; amino acids 5-33 of SEQ ID NO: 11; amino acids 8-33 of SEQ ID NO: 11; amino acids 10-33 of SEQ ID NO: 11; or amino acids 15-33 of SEQ ID NO: 11.
The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO: 13, amino acids 18-33 of SEQ ID NO: 13, or SEQ ID NO:13, or the exosporium protein can comprise the full length B. weihenstephensis KBAB4 3572 gene product (SEQ ID NO:14). A methionine residue linked to amino acids 18-33 of the B. weihenstephensis KBAB4 3572 gene product can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 99.
The targeting sequence can comprise amino acids 2-33 of SEQ ID NO: 13; amino acids 5-33 of SEQ ID NO: 13; amino acids 8-33 of SEQ ID NO: 13; amino acids 10-33 of SEQ ID NO: 13; or amino acids 15-33 of SEQ ID NO: 13.
Alternatively, the targeting sequence can comprise amino acids 1-43 of SEQ ID NO: 15, amino acids 28-43 of SEQ ID NO: 15, or SEQ ID NO:15, or the exosporium protein can comprise full length B. cereus VD200 exosporium leader peptide (SEQ ID NO:16).
The targeting sequence can comprise amino acids 2-43 of SEQ ID NO: 15; amino acids 5-43 of SEQ ID NO: 15; amino acids 8-43 of SEQ ID NO: 15; amino acids 10-43 of SEQ ID NO: 15; amino acids 15-43 of SEQ ID NO: 15; amino acids 20-43 of SEQ ID NO: 15; or amino acids 25-43 of SEQ ID NO: 15.
The targeting sequence can also comprise amino acids 1-27 of SEQ ID NO: 17, amino acids 12-27 of SEQ ID NO: 17, or SEQ ID NO: 17, or the exosporium protein can comprise full-length B. cereus VD166 exosporium leader peptide (SEQ ID NO:18). A methionine residue linked to amino acids 12-27 of the B. cereus VD166 exosporium leader peptide can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 100.
The targeting sequence can comprise amino acids 2-27 of SEQ ID NO: 17; amino acids 5-27 of SEQ ID NO: 17; amino acids 8-27 of SEQ ID NO: 17; or amino acids 10-27 of SEQ ID NO: 17.
The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO: 19, amino acids 18-33 of SEQ ID NO: 19, or SEQ ID NO:19, or the exosporium protein can comprise full length B. cereus VD200 hypothetical protein IKG_04663 (SEQ ID NO:20).
The targeting sequence can comprise amino acids 2-33 of SEQ ID NO: 19; amino acids 5-33 of SEQ ID NO: 19; amino acids 8-33 of SEQ ID NO: 19; amino acids 10-33 of SEQ ID NO: 19; or amino acids 15-33 of SEQ ID NO: 19.
Alternatively, the targeting sequence comprises amino acids 1-33 of SEQ ID NO: 21, amino acids 18-33 of SEQ ID NO: 21, or SEQ ID NO:21, or the exosporium protein can comprise full length B. weihenstephensis KBAB4 YVTN β-propeller protein (SEQ ID NO: 22). A methionine residue linked to amino acids 18-33 of the B. weihenstephensis KBAB4 YVTN β-propeller protein can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 101.
The targeting sequence can comprise amino acids 2-33 of SEQ ID NO: 21; amino acids 5-33 of SEQ ID NO: 21; amino acids 8-33 of SEQ ID NO: 21; amino acids 10-33 of SEQ ID NO: 21; or amino acids 15-33 of SEQ ID NO: 21.
The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO: 23, amino acids 9-24 of SEQ ID NO: 23, or SEQ ID NO:23, or the exosporium protein can comprise full length B. weihenstephensis KBAB4 hypothetical protein bcerkbab4_2363 (SEQ ID NO:24). A methionine residue linked to amino acids 9-24 of B. weihenstephensis KBAB4 hypothetical protein bcerkbab4_2363 can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 102.
The targeting sequence can comprise amino acids 2-24 of SEQ ID NO:23; amino acids 5-24 of SEQ ID NO: 23; or amino acids 8-24 of SEQ ID NO: 23.
The targeting sequence comprise amino acids 1-24 of SEQ ID NO: 25, amino acids 9-24 of SEQ ID NO: 25, or SEQ ID NO: 25, or the exosporium protein can comprise full length B. weihenstephensis KBAB4 hypothetical protein bcerkbab4_2131 (SEQ ID NO:26). A methionine residue linked to amino acids 9-24 of B. weihenstephensis KBAB4 hypothetical protein bcerkbab4_2131 can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 103.
The targeting sequence can comprise amino acids 2-24 of SEQ ID NO: 25; amino acids 5-24 of SEQ ID NO: 25; or amino acids 8-24 of SEQ ID NO: 25.
Alternatively, the targeting sequence comprises amino acids 1-30 of SEQ ID NO: 27, amino acids 15-30 of SEQ ID NO: 27, or SEQ ID NO:27, or the exosporium protein can comprise full length B. weihenstephensis KBAB4 triple helix repeat containing collagen (SEQ ID NO:28).
The targeting sequence can comprise amino acids 2-30 of SEQ ID NO: 27; amino acids 5-30 of SEQ ID NO: 27; amino acids 8-30 of SEQ ID NO: 27; or amino acids 10-30 of SEQ ID NO: 27.
The targeting sequence can also comprise amino acids 1-33 of SEQ ID NO: 29, amino acids 18-33 of SEQ ID NO: 29, or SEQ ID NO:29, or the exosporium protein can comprise full length B. mycoides 2048 hypothetical protein bmyco0001_21660 (SEQ ID NO:30).
The targeting sequence can comprise amino acids 2-33 of SEQ ID NO: 29; amino acids 5-33 of SEQ ID NO: 29; amino acids 8-33 of SEQ ID NO: 29; amino acids 10-33 of SEQ ID NO: 29; or amino acids 15-33 of SEQ ID NO: 29.
The targeting sequence can also comprise amino acids 1-24 of SEQ ID NO: 31, amino acids 9-24 of SEQ ID NO: 31, or SEQ ID NO:31, or the exosporium protein can comprise full length B. mycoides 2048 hypothetical protein bmyc0001_22540 (SEQ ID NO:32). A methionine residue linked to amino acids 9-24 of B. mycoides 2048 hypothetical protein bmyc0001_22540 can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 104.
The targeting sequence can comprise amino acids 2-24 of SEQ ID NO: 31; amino acids 5-24 of SEQ ID NO: 31; or amino acids 8-24 of SEQ ID NO: 31.
Alternatively, the targeting sequence comprises amino acids 1-15 of SEQ ID NO: 33, SEQ ID NO:33, or the exosporium protein comprises full length B. mycoides 2048 hypothetical protein bmyc0001_21510 (SEQ ID NO:34).
The targeting sequence can also comprise amino acids 1-16 of SEQ ID NO: 35, SEQ ID NO:35, or the exosporium protein can comprise full length B. thuringiensis 35646 collagen triple helix repeat protein (SEQ ID NO:36).
The targeting sequence can comprise amino acids 1-29 of SEQ ID NO:43, amino acids 14-29 of SEQ ID NO: 43, or SEQ ID NO: 43, or the exosporium protein can comprise full length B. cereus hypothetical protein WP_69652 (SEQ ID NO: 44).
The targeting sequence can comprise amino acids 2-29 of SEQ ID NO: 43; amino acids 5-29 of SEQ ID NO: 43; amino acids 8-29 of SEQ ID NO: 43; or amino acids 10-29 of SEQ ID NO: 43.
Alternatively, the targeting sequence can comprise amino acids 1-35 of SEQ ID NO: 45, amino acids 20-35 of SEQ ID NO: 45, or SEQ ID NO: 45, or the exosporium protein can comprise full length B. cereus exosporium leader WP016117717 (SEQ ID NO: 46). A methionine residue linked to amino acids 20-35 of B. cereus exosporium leader WP016117717 can be used as a targeting sequence. Thus, the targeting sequence can comprise SEQ ID NO: 106.
The targeting sequence can comprise amino acids 2-35 of SEQ ID NO: 45; amino acids 5-35 of SEQ ID NO: 45; amino acids 8-35 of SEQ ID NO: 45; amino acids 10-35 of SEQ ID NO: 45; or amino acids 15-35 of SEQ ID NO: 45.
The targeting sequence can comprise amino acids 1-43 of SEQ ID NO: 47, amino acids 28-43 of SEQ ID NO: 47, or SEQ ID NO: 47, or the exosporium protein can comprise full length B. cereus exosporium peptide WP002105192 (SEQ ID NO: 48).
The targeting sequence can comprise amino acids 2-43 of SEQ ID NO: 47; amino acids 5-43 of SEQ ID NO: 47; amino acids 8-43 of SEQ ID NO: 47; amino acids 10-43 of SEQ ID NO: 47; amino acids 15-43 of SEQ ID NO: 47; amino acids 20-43 of SEQ ID NO: 47; or amino acids 25-43 of SEQ ID NO: 47.
The targeting sequence can comprise amino acids 1-32 of SEQ ID NO: 49, amino acids 17-32 of SEQ ID NO: 49, or SEQ ID NO: 49, or the exosporium protein can comprise full length B. cereus hypothetical protein WP87353 (SEQ ID NO: 50).
The targeting sequence can comprise amino acids 2-32 of SEQ ID NO: 49; amino acids 5-32 of SEQ ID NO: 49; amino acids 8-32 of SEQ ID NO: 49; amino acids 10-32 of SEQ ID NO: 49; or amino acids 15-32 of SEQ ID NO: 49.
Alternatively, the targeting sequence can comprise amino acids 1-33 of SEQ ID NO: 51, amino acids 18-33 of SEQ ID NO: 51, or SEQ ID NO: 51, or the exosporium protein can comprise full length B. cereus exosporium peptide 02112369 (SEQ ID NO: 52).
The targeting sequence can comprise amino acids 2-33 of SEQ ID NO: 51; amino acids 5-33 of SEQ ID NO: 51; amino acids 8-33 of SEQ ID NO: 51; amino acids 10-33 of SEQ ID NO: 51; or amino acids 15-33 of SEQ ID NO: 51.
The targeting sequence can comprise amino acids 1-33 of SEQ ID NO: 53, amino acids 18-33 of SEQ ID NO: 53, or SEQ ID NO: 53, or the exosporium protein can comprise full length B. cereus exosporium protein WP016099770 (SEQ ID NO: 54).
The targeting sequence can comprise amino acids 2-33 of SEQ ID NO: 53; amino acids 5-33 of SEQ ID NO: 53; amino acids 8-33 of SEQ ID NO: 53; amino acids 10-33 of SEQ ID NO: 53; or amino acids 15-33 of SEQ ID NO: 53.
Alternatively, the targeting sequence can comprise acids 1-30 of SEQ ID NO: 55, amino acids 15-30 of SEQ ID NO: 55, or SEQ ID NO: 55, or the exosporium protein can comprise full length B. thuringiensis hypothetical protein YP006612525 (SEQ ID NO: 56).
The targeting sequence can comprise amino acids 2-30 of SEQ ID NO: 55; amino acids 5-30 of SEQ ID NO: 55; amino acids 8-30 of SEQ ID NO: 55; or amino acids 10-30 of SEQ ID NO: 55.
The targeting sequence can comprise amino acids 1-130 of SEQ ID NO: 57, amino acids 115-130 of SEQ ID NO: 57, or SEQ ID NO: 57, or the exosporium protein can comprise full length B. mycoides hypothetical protein TIGR03720 (SEQ ID NO: 58).
The targeting sequence can comprise amino acids 2-130 of SEQ ID NO: 57; amino acids 5-130 of SEQ ID NO: 57; amino acids 10-130 of SEQ ID NO: 57; amino acids 20-130 of SEQ ID NO: 57; amino acids 30-130 of SEQ ID NO: 57; amino acids 40-130 of SEQ ID NO: 57; amino acids 50-130 of SEQ ID NO: 57; amino acids 60-130 of SEQ ID NO: 57; amino acids 70-130 of SEQ ID NO: 57; amino acids 80-130 of SEQ ID NO: 57; amino acids 90-130 of SEQ ID NO: 57; amino acids 100-130 of SEQ ID NO: 57; or amino acids 110-130 of SEQ ID NO: 57.
The targeting sequence can comprise amino acids 1-30 of SEQ ID NO: 59; or SEQ ID NO: 59; or the exosporium protein can comprise full length B. cereus ATCC 10987 collagen triple helix repeat domain protein (SEQ ID NO: 60).
The targeting sequence can comprise amino acids 2-30 of SEQ ID NO: 59; amino acids 4-30 of SEQ ID NO: 59; or amino acids 6-30 of SEQ ID NO: 59.
The targeting sequence can comprise amino acids 1-33 of SEQ ID NO: 61; amino acids 18-33 of SEQ ID NO: 61; or SEQ ID NO: 61; or the exosporium protein can comprise full length B. cereus E33L collagen-like protein (SEQ ID NO: 62).
The targeting sequence can comprise amino acids 2-33 of SEQ ID NO: 61; amino acids 5-33 of SEQ ID NO: 61; amino acids 10-33 of SEQ ID NO: 61; or amino acids 15-33 of SEQ ID NO: 61.
The targeting sequence can comprise amino acids 1-35 of SEQ ID NO: 63; or SEQ ID NO: 63; or the exosporium protein can comprise full length B. weihenstephanensis KBAB4 triple helix repeat-containing collagen (SEQ ID NO: 64).
The targeting sequence can comprise amino acids 2-35 of SEQ ID NO: 63; amino acids 5-35 of SEQ ID NO: 63; amino acids 8-35 of SEQ ID NO: 63; amino acids 10-35 of SEQ ID NO: 63; or amino acids 15-35 of SEQ ID NO: 63.
The targeting sequence can comprise amino acids 1-24 of SEQ ID NO: 65; acids 9-24 of SEQ ID NO: 65; SEQ ID NO: 65; or SEQ ID NO: 107; or the exosporium protein can comprise full length B. thuringiensis str. Al Hakam hypothetical protein BALH_2230 (SEQ ID NO: 66).
The targeting sequence can comprise amino acids 2-24 of SEQ ID NO: 65; or amino acids 5-24 of SEQ ID NO: 65.
The targeting sequence can comprise acids 1-27 of SEQ ID NO: 67; amino acids 12-27 of SEQ ID NO: 67; or SEQ ID NO: 67; or the exosporium protein can comprise full length B. cereus ATCC 14579 triple helix repeat-containing collagen (SEQ ID NO: 68).
The targeting sequence can comprise amino acids 2-27 of SEQ ID NO: 67; amino acids 5-27 of SEQ ID NO: 67; or amino acids 10-27 of SEQ ID NO: 67.
The targeting sequence can comprise amino acids 1-38 of SEQ ID NO: 69; amino acids 23-38 of SEQ ID NO: 69; or SEQ ID NO: 69; or the exosporium protein can comprise full length B. cereus collagen triple helix repeat (SEQ ID NO: 70).
The targeting sequence can comprise amino acids 2-38 of SEQ ID NO: 69; amino acids 5-38 of SEQ ID NO: 69; amino acids 10-38 of SEQ ID NO: 69; or amino acids 15-38 of SEQ ID NO: 69.
The exosporium protein can comprise full length B. cereus ATCC 14579 triple helix repeat-containing collagen (SEQ ID NO: 72).
The targeting sequence can comprise SEQ ID NO: 73, or the exosporium protein can comprise full length B. cereus E33L hypothetical protein BCZK1835 (SEQ ID NO: 74).
The targeting sequence can comprise amino acids 1-42 of SEQ ID NO: 75; amino acids 27-42 of SEQ ID NO: 75; or SEQ ID NO: 75; or the exosporium protein can comprise full length B. weihenstephanensis KBAB4 triple helix repeat-containing collagen (SEQ ID NO: 76).
The targeting sequence can comprise amino acids 2-42 of SEQ ID NO: 75; amino acids 5-42 of SEQ ID NO: 75; amino acids 10-42 of SEQ ID NO: 75; amino acids 15-42 of SEQ ID NO: 75; amino acids 20-42 of SEQ ID NO: 75; or amino acids 25-42 of SEQ ID NO: 75.
The targeting sequence can comprise amino acids 1-24 of SEQ ID NO: 77; amino acids 9-24 of SEQ ID NO: 77; or SEQ ID NO: 77; or the exosporium protein can comprise full length B. cereus ATCC 14579 triple helix repeat-containing collagen (SEQ ID NO: 78).
The targeting sequence can comprise amino acids 2-24 of SEQ ID NO: 77; or amino acids 5-24 of SEQ ID NO: 77;
The exosporium protein can comprise full length B. cereus ATCC 14579 hypothetical protein BC4725 (SEQ ID NO: 80).
The targeting sequence can comprise amino acids 1-38 of SEQ ID NO: 81; amino acids 23-38 of SEQ ID NO: 81; or SEQ ID NO: 81; or the exosporium protein can comprise full length B. cereus E33L hypothetical protein BCZK4476 (SEQ ID NO: 82).
The targeting sequence can comprise amino acids 2-38 of SEQ ID NO: 81; acids 5-38 of SEQ ID NO: 81; amino acids 10-38 of SEQ ID NO: 81; amino acids 15-38 of SEQ ID NO: 81; or amino acids 20-38 of SEQ ID NO: 81.
The targeting sequence can comprise amino acids 1-34 of SEQ ID NO: 83; or SEQ ID NO: 83; or the exosporium protein can comprise full length B. anthracis str. ‘Ames Ancestor’ triple helix repeat-containing collagen (SEQ ID NO: 84).
The exosporium protein can comprise full length B. thuringiensis serovar konkukian str. 97-27 BclA protein (SEQ ID NO: 86).
The targeting sequence can comprise amino acids 1-28 of SEQ ID NO: 87; amino acids 13-28 of SEQ ID NO: 87; or SEQ ID NO: 87; or the exosporium protein can comprise full length B. cereus ATCC 10987 conserved hypothetical protein (SEQ ID NO: 88).
The targeting sequence can comprise amino acids 2-28 of SEQ ID NO: 87; amino acids 5-28 of SEQ ID NO: 87; or amino acids 10-28 of SEQ ID NO: 87.
The targeting sequence can comprise amino acids 1-28 of SEQ ID NO: 89; or SEQ ID NO: 89; or the exosporium protein can comprise full length B. cereus ATCC 14579 triple helix repeat-containing collagen (SEQ ID NO: 90).
The targeting sequence can comprise amino acids 2-28 of SEQ ID NO: 89; amino acids 5-28 of SEQ ID NO: 89; or amino acids 10-28 of SEQ ID NO: 89
The targeting sequence can comprise amino acids 1-93 of SEQ ID NO: 91; or SEQ ID NO: 91; or the exosporium protein can comprise B. cereus exosporium leader peptide partial sequence (SEQ ID NO: 92).
The targeting sequence can comprise amino acids 2-93 of SEQ ID NO: 91; amino acids 10-93 of SEQ ID NO: 91; amino acids 20-93 of SEQ ID NO: 91; amino acids 30-93 of SEQ ID NO: 91; amino acids 40-93 of SEQ ID NO: 91; amino acids 50-93 of SEQ ID NO: 91; or amino acids 60-93 of SEQ ID NO: 91.
The targeting sequence can comprise amino acids 1-130 of SEQ ID NO: 93; or SEQ ID NO: 93; or the exosporium protein can comprise B. weihenstephanensis) hypothetical protein ER45_27600, partial sequence (SEQ ID NO: 94).
The targeting sequence can comprise amino acids 2-130 of SEQ ID NO: 93; amino acids 10-130 of SEQ ID NO: 93; amino acids 20-130 of SEQ ID NO: 93; or amino acids 30-130 of SEQ ID NO: 93.
The targeting sequence can comprise amino acids 1-35 of SEQ ID NO:382, amino acids 20-35 of SEQ ID NO: 382, SEQ ID NO: 382, or SEQ ID NO: 383.
The targeting sequence can comprise amino acids 1-35 of SEQ ID NO:384, amino acids 20-35 of SEQ ID NO: 384, SEQ ID NO: 384, or SEQ ID NO: 385.
Furthermore, as illustrated in the Examples provided hereinbelow, it has been found that sequences shorter than amino acids 20-35 of BclA can be used to target a fusion protein to the exosporium of a recombinant Bacillus cereus family member. In particular, amino acids 20-33 of BclA, amino acids 20-31 of BclA, amino acids 21-33 of BclA, or amino acids 23-31 of BclA can be used to target a fusion protein to the exosporium of a recombinant Bacillus cereus family member. Thus, the targeting sequence can consist of amino acids 20-33 of SEQ ID NO: 1, amino acids 20-31 of SEQ ID NO: 1, amino acids 21-33 of SEQ ID NO: 1, or amino acids 23-31 of SEQ ID NO: 1. The corresponding regions of any of the SEQ ID NOs. shown in
Even shorter regions within amino acids 20-35 of BclA can also be used for targeting a fusion protein to the exosporium of a recombinant Bacillus cereus family member. In particular, any amino acid sequence that includes amino acids 25-30 of SEQ ID NO: 1 or the corresponding amino acids from any of the sequences shown in
In addition, it can readily be seen from the sequence alignment in
With respect to amino acids 25-35 of BclA, the corresponding amino acids of BetA/BAS3290 are about 90.9% identical, the corresponding amino acids of BAS4623 are about 72.7% identical, the corresponding amino acids of BclB are about 54.5% identical, the corresponding amino acids of BAS1882 are about 72.7% identical, the corresponding amino acids of the KBAB4 2280 gene product are about 90.9% identical, and the corresponding amino acids of the KBAB4 3572 gene product are about 81.8% identical. The sequence identities over this region for the remaining sequences are listed in
Thus, the targeting sequence can comprise an amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 54%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 54%.
The targeting sequence can also comprise an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%.
The targeting sequence can also comprise an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%.
The targeting sequence can also comprise an amino acid sequence having at least about 56% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 56% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%.
The targeting sequence can comprise an amino sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%. Alternatively, the targeting sequence can consist of an amino acid sequence consisting of 16 amino acids and having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 of SEQ ID NO:1 is at least about 72%.
The targeting sequence can comprise an amino acid sequence having at least 68% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 81%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least 68% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 81%.
The targeting sequence can also comprises an amino sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 of SEQ ID NO:1 is at least about 72%.
The targeting sequence can also comprise an amino sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 81%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 of SEQ ID NO:1 is at least about 81%.
The targeting sequence can also comprise an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 81%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 81% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 81%.
The targeting sequence can comprise an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 90%. Alternatively, the targeting sequence consists of an amino acid sequence consisting of 16 amino acids and having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 90%.
The skilled person will recognize that variants of the above sequences can also be used as targeting sequences, so long as the targeting sequence comprises amino acids 20-35 of BclA, the corresponding amino acids of BetA/BAS3290, BAS4263, BclB, BAS1882, the KBAB4 2280 gene product, or the KBAB 3572 gene product, or a sequence comprising any of the above noted sequence identities to amino acids 20-35 and 25-35 of BclA is present.
Certain Bacillus cereus family exosporium proteins which lack regions having homology to amino acids 25-35 of BclA can also be used to target a peptide or protein to the exosporium of a Bacillus cereus family member. In particular, the fusion proteins can comprise an exosporium protein comprising SEQ ID NO: 108 (B. mycoides InhA), an exosporium protein comprising SEQ ID NO: 109 (B. anthracis Sterne BAS1141 (ExsY)), an exosporium protein comprising SEQ ID NO: 110 (B. anthracis Sterne BAS1144 (BxpB/ExsFA)), an exosporium protein comprising SEQ ID NO: 111 (B. anthracis Sterne BAS1145 (CotY)), an exosporium protein comprising SEQ ID NO: 112 (B. anthracis Sterne BAS1140), an exosporium protein comprising SEQ ID NO: 113 (B. anthracis H9401 ExsFB), an exosporium protein comprising SEQ ID NO: 114 (B. thuringiensis HD74 InhA1), an exosporium protein comprising SEQ ID NO: 115 (B. cereus ATCC 10876 ExsJ), an exosporium protein comprising SEQ ID NO: 116 (B. cereus ExsH), an exosporium protein comprising SEQ ID NO: 117 (B. anthracis Ames YjcA), an exosporium protein comprising SEQ ID NO: 118 (B. anthracis YjcB), an exosporium protein comprising SEQ ID NO: 119 (B. anthracis Sterne BclC), an exosporium protein comprising SEQ ID NO: 120 (Bacillus thuringiensis serovar konkukian str. 97-27 acid phosphatase), an exosporium protein comprising SEQ ID NO: 121 (B. thuringiensis HD74 InhA2), an exosporium protein comprising SEQ ID NO: 122 (B. mycoides InhA3), or an exosporium protein comprising SEQ ID NO: 381 (B. anthracis CotY variant). Inclusion of an exosporium protein comprising any of SEQ ID NOs: 108-122 or 381 in the fusion proteins described herein will result in targeting to the exosporium of a B. cereus family member.
Moreover, exosporium proteins having a high degree of sequence identity with any of the full-length exosporium proteins or the exosporium protein fragments described above can also be used to target a peptide or protein to the exosporium of a Bacillus cereus family member. Thus, the fusion protein can comprise an exosporium protein or exosporium protein fragment comprising an amino acid sequence having at least 85% identity with any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 381.
Alternatively, the fusion protein can comprise an exosporium protein having at least 90% identity with any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 381.
The fusion protein can comprise an exosporium protein having at least 95% identity with any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 381.
The fusion protein can comprise an exosporium protein having at least 98% identity with any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 381.
The fusion protein can comprise an exosporium protein having at least 99% identity with any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 381.
The fusion protein can comprise an exosporium protein having 100% identity with any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, and 381.
Any of the targeting sequences, exosporuim proteins, or exosporium protein fragments can be used to target any protein or peptide of interest, including, but not limited to, the enzymes and expansin proteins described herein, to the exosporium of a recombinant Bacillus cereus family member.
For example, any of the targeting sequences, exosporium proteins, or exosporium protein fragments (e.g., any of SEQ ID NOs. 381-385) can be used to target a protein or peptide of interest (e.g., an endoglucanase such as the endoglucanase of SEQ ID NO: 293; a phospholipase such as the phospholipase of SEQ ID NO: 250; a xyloglucanase such as the xyloglucanase of SEQ ID NO: 300; a protease such as the protease of SEQ ID NO: 303 or 304; or an ACC deaminase such as the ACC deaminase of SEQ ID NO: 249) to the exosporium of a recombinant Bacillus cereus family member.
During sporulation of a recombinant Bacillus cereus family member expressing any of the fusion proteins described herein, the targeting motif, exosporium protein, or exosporium protein fragment is recognized by the spore exosporium assembly machinery and directed to the exosporium, resulting in display of the protein or peptide of interest portion of the fusion protein (e.g., the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the phytase, the acid phosphatase, the pectinase, the mannanase, or the expansin protein) on the outside of the spore.
As illustrated further by the Examples provided hereinbelow, the use of different targeting sequences allows for control of the expression level of the fusion protein on the surface of the Bacillus cereus family member spore. Use of certain of the targeting sequences described herein will result in a higher level of expression of the fusion protein, whereas use of others of the targeting sequences will result in lower levels of expression of the fusion protein on the surface of the spore.
In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid.
In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment, can comprise an alanine residue at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.
In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment can further comprise a methionine, serine, or threonine residue at the amino acid position immediately preceding the first amino acid of the targeting sequence, exosporium protein, or exosporium protein fragment or at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.
B. ACC Deaminases, Phospholipases, Lipases, Xylanases, Xylosidases, Lactonases, Chitosanases, Proteases, Glucanases, Phytases, Acid Phosphatases, Pectinases, Mannanases, and Expansin Proteins
The fusion proteins can comprise an enzyme having ACC deaminase activity, a phospholipase, a lipase, a xylanase, a xylosidase, a lactonase, a chitosanase, a protease, a glucanase, a phytase, an acid phosphatase, a pectinase, a mannanase, and/or an expansin protein. For ease of reference, illustrative sequences for wild-type and modified ACC deaminase enzymes, as well as sequences for the other enzymes and the expansin proteins that can be used in connection with the fusion proteins, recombinant Bacillus cereus family members, exosporium fragments, formulations, seeds, and methods described herein, are provided below.
1.
The fusion proteins can comprise an enzyme having ACC deaminase activity. ACC deaminases and
Thus, an enzyme having increased ACC deaminase activity would be beneficial for use in agriculture in order to reduce ACC levels and thereby reduce ethylene levels. Application of ACC deaminase to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed can stimulate plant growth, promote plant health (e.g., by increasing nutrient uptake), and slow fruit ripening. These effects in turn lead to increased yields, early season vigor, and resistance of plants to early season stresses. ACC deaminases can also protect plants from pathogens as well as abiotic stresses.
As explained in greater detail below, mutations can be made in enzymes that exhibit
Naturally occurring ACC deaminase is not a secreted protein. ACC deaminases are found in many types of microorganisms, including bacteria of the Phyla Bacteriodetes, Firmicutes, and Actinobacteria, and bacteria of the genera Pseudomonas, Bacillus, Rhizobium, Bradyrhizobium, as well as many others. However, the ACC deaminases found in these bacteria are intracellular, and have limited exposure to the substrate ACC from the host plants that they colonize. Display of an enzyme having ACC deaminase activity on the outside of Bacillus cereus family member spores using the BEMD system described herein allows for increased contact between the enzyme and its substrate upon application of the spores to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed.
For ease of reference, illustrative
In Table 2, SEQ ID NOs. 234-237 are nucleotide sequences for wild-type enzymes that exhibit both ACC deaminase and
In Table 3, SEQ ID NOs. 242-245 are amino acid sequences for wild-type enzymes that exhibit both ACC deaminase and
Bacillus thuringiensis
Bacillus pseudomycoides
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus pseudomycoides
Bacillus thuringiensis
Bacillus thuringiensis,
2. Phospholipases
Phospholipases are enzymes that cleave phospholipids. Different types of phospholipases cleave phospholipids at different positions. Phospholipases A1, A2 and B cleave the SN-1 and/or SN-2 acyl chains, resulting in the release of fatty acids and lysophospholipids. Phospholipase C (PLC) and phospholipase D (PLD) act on the head group of the phospholipid instead. PLCs cleave phospholipids into diacylglycerides and head group phosphates. PLDs cleave phospholipids into phosphatidic acid and the head group. Many phospholipases are membrane-associated. However, some phospholipases are secreted and others are localized intracellularly. Phospholipases in general play important roles in signal transduction pathways and can be found in a wide range of organisms, including bacteria, yeast, plants, animals and viruses.
Phospholipase C (EC 3.1.4.3) cleaves phospholipids just prior to the phosphate group into diacylglycerols and the corresponding head group phosphates. Phospholipase C is characterized by the site of cleavage on the phospholipid (the phosphorous-oxygen bond on the carbon chain side of the phosphate head), which distinguishes it from other phospholipases. General activators of the PLC class of enzymes typically include heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, calcium (Ca2+), and phospholipids.
Phospholipase D (EC 3.1.4.4) is another enzyme of the phospholipase superfamily. The principal substrate of phospholipase D (PLD) is phosphatidylcholine, which is hydrolyzed to produce a signal molecule phosphatidic acid and soluble choline. Phospholipase D is involved in membrane trafficking, cytoskeletal reorganization, receptor-mediated endocytosis, exocytosis and cell migration.
In soil, phospholipases can break down phospholipids naturally the soil (e.g., phospholipids from plant debris and/or lysed microbes), and release fatty acids and phospholipid head groups. The phospholipid head groups act as natural reservoirs of excess phosphate, which is a valuable extractable nutrient for both plants and microbes, and the conversion of phospholipids to phosphatidic acid by phospholipases is the first step before the phosphate group can be released from the molecule.
In addition, plants secrete phospholipids from the root cap during development of roots into the rhizosphere. These phospholipids, together with complex polysaccharides and monosaccharides, recruit beneficial microbes and fungi to the plant roots/rhizosphere and also provide a nutrient source for the microbes. Plants also use the secreted phospholipids as natural secreted surfactants, which allows the plant access to additional nutrients that are bound to the soil, such as phosphate. Conversion of phospholipids to phosphatidic acid maintains the surfactant properties while simultaneously allowing for release of nutrients into the soil.
Phospholipases can be used for any of the plant growth stimulating or plant health promoting purposes described herein, but are especially suitable for stimulating plant growth, increasing nutrient uptake, and/or increasing root development and nodulation. Increasing root nodulation enhances the ability of the plant to form symbiotic relationships with nitrogen fixing microorganisms in the soil, resulting in increased nitrogen uptake and enhanced growth rates. These effects also lead to decreased susceptibility to environmental stresses such as drought.
For ease of reference, illustrative phospholipase amino acid sequences are provided in Table 4 below, together with their SEQ ID NOs.
HENDGGQRFGVIPRWSAEDKHKEGVNSHLWIVNRAIDIMSRNTT
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus
thuringiensis serovar
Bacillus cereus ATCC
Clostridium perfringens
Streptomyces chromofuscus
Bacillus cereus
Bacillus cereus
Acidovorax avenae
Bacillus licheniformis
CCDEYLQTPAAPHDIDSKLPHKLSWSADNPTNTDVNTHYWLFK
Listeria monocytogenes
Listeria monocytogenes
Bacillus thuringiensis
The native amino acid sequences of the phospholipases of SEQ ID NOs. 250 and 252 include the signal peptide sequence MKKKVLALAAAITLVAPLQSVAFA (SEQ ID NO: 317) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NOs. 250 and 252. This signal peptide is not included in SEQ ID NOs. 250 and 252. However, the signal peptide of SEQ ID NO: 317, or another signal peptide, can optionally be included at the amino-terminus of the phospholipases of SEQ ID NO: 250 or 252, or at the amino-terminus of any of the other enzymes or expansin proteins described herein. Where the signal peptide of SEQ ID NO: 317 is included at the amino terminus of the phospholipase of SEQ ID NO: 250, this enzyme is referred to herein by ID Code E95, E62, or E80.
The native amino acid sequence of the phospholipase of SEQ ID NO: 250 also includes a propeptide (HENDGGQRFGVIPRW, SEQ ID NO: 261). This propeptide is underlined in the sequence of SEQ ID NO: 250 shown in Table 4 above. This propeptide can be present, as shown in SEQ ID NO: 250 in Table 4 above, or can optionally be removed as shown in SEQ ID NO: 251 in Table 4 above.
The native amino acid sequence of the phospholipase of SEQ ID NO: 373 includes a propeptide (CCDEYLQTPAAPHDIDSKLPHKLS, SEQ ID NO: 388). This propeptide is underlined in the sequence of SEQ ID NO: 373 shown in Table 4 above. This propeptide can be present, as shown in SEQ ID NO: 373 in Table 4 above, or can optionally be removed as shown in SEQ ID NO: 374 in Table 4 above.
The native amino acid sequence of the phospholipase of SEQ ID NO: 253 includes the signal peptide MKGKLLKGVLSLGVGLGALYSGTSAQAE (SEQ ID NO: 318) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 253. This signal peptide is not included in SEQ ID NO: 253. However, the signal peptide of SEQ ID NO: 318, or another signal peptide, can optionally be included at the amino terminus of the phospholipase of SEQ ID NO: 253, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the phospholipase of SEQ ID NO: 254 includes the signal peptide MKKKVLALAAAITVVAPLQSVAFA (SEQ ID NO: 319) at the amino terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 254. This signal peptide is not included in SEQ ID NO: 254. However, the signal peptide of SEQ ID NO: 319, or another signal peptide, can optionally be included at the amino terminus of the phospholipase of SEQ ID NO: 254, or at the amino terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the phospholipase of SEQ ID NO: 255 includes the signal peptide MKRKICKALICATLATSLWAGASTKVYAW (SEQ ID NO: 320) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 255. This signal peptide is not included in SEQ ID NO: 255. However, the signal peptide of SEQ ID NO: 320, or another signal peptide, can optionally be included at the amino terminus of the phospholipase of SEQ ID NO: 255, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the phospholipase of SEQ ID NO: 256 includes the signal peptide MLAGPLAAALPARA (SEQ ID NO: 321) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 256. This signal peptide is not included in SEQ ID NO: 256. However, the signal peptide of SEQ ID NO: 321, or another signal peptide, can optionally be included at the amino terminus of the phospholipase of SEQ ID NO: 256, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
Alternatively, the signal peptide
can optionally be added to SEQ ID NO: 256, at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 256. The signal peptide of SEQ ID NO: 322 can also optionally be included at the amino terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the phospholipase of SEQ ID NO: 257 includes the signal peptide MKKKVLALAAAITLVAPLQNVAFA (SEQ ID NO: 323) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 257. This signal peptide is not included in SEQ ID NO: 257. However, the signal peptide of SEQ ID NO: 323, or another signal peptide, can optionally be included at the amino-terminus of the phospholipase of SEQ ID NO: 257, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the phospholipase of SEQ ID NO: 373 includes the signal peptide MKFKNVVLGMCLTASVLVFPVTIKANA (SEQ ID NO: 376) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 373. This signal peptide is not included in SEQ ID NO: 373. However the signal peptide of SEQ ID NO: 376, or another signal peptide, can optionally be included at the amino-terminus of the phospholipase of SEQ ID NO: 373, or at the amino-terminus of any of the other enzymes or expansin proteins described herein. Where the signal peptide of SEQ ID NO: 376 is included at the amino terminus of the phospholipase of SEQ ID NO: 373, this enzyme is referred to herein by ID Code D473.
The native amino acid sequence of the phospholipase of SEQ ID NO: 375 includes the signal peptide MSNKKLILKLFICSTIFITFVFALHDKQVVA (SEQ ID NO: 377) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 375. This signal peptide is not included in SEQ ID NO: 375. However the signal peptide of SEQ ID NO: 377 or another signal peptide, can optionally be included at the amino terminus of the phospholipase of SEQ ID NO: 375, or at the amino terminus of any of the other enzymes or expansin proteins described herein. Where the signal peptide of SEQ ID NO: 377 is included at the amino terminus of the phospholipase of SEQ ID NO: 375, this enzyme is referred to herein by ID Code E258.
3. Lipases
Lipases are enzymes that exert their enzymatic activity on lipids, cleaving fatty acid chains off of larger lipid molecules such as triglycerides. Lipases are a class of enzymes which have the ability to act at the interface between aqueous and organic phases. Lipases primarily catalyze the hydrolysis of ester bonds in water-insoluble lipid substrates (e.g., longer acyl chain length derivatives).
Lipases can be used for any of the plant growth stimulating or plant health-promoting purposes described herein, but are particularly well-suited for stimulating plant growth and enhancing nutrient uptake. These effects in turn lead to increased crop yields, improved early season vigor, and decreased susceptibility of plants to early season stresses.
For ease of reference, illustrative lipase amino acid sequences are provided in Table 5 below, together with their SEQ ID NOs.
subtilis str. 168
The native amino acid sequence of the lipase of SEQ ID NO: 263 includes the signal peptide MKFVKRRIIALVTILMLSVTSLFALQPSAKA (SEQ ID NO: 324) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 263. This signal peptide is not included in SEQ ID NO: 263. However, the signal peptide of SEQ ID NO: 324, or another signal peptide, can optionally be included at the amino terminus of the lipase of SEQ ID NO: 263, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the lipase of SEQ ID NO: 264 includes the signal peptide MARTMRSRVVAGAVACAMSIAPFAGTTAVMTLATTHAAMAATAP (SEQ ID NO: 325) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 264. This signal peptide is not included in SEQ ID NO: 264. However, the signal peptide of SEQ ID NO: 325, or another signal peptide, can optionally be included at the amino-terminus of the lipase of SEQ ID NO: 264, or at the amino-terminus of any of the other enzymes of expansin proteins described herein.
The native amino acid sequence of the lipase of SEQ ID NO: 265 includes the signal peptide MGIFDYKNLGTEGSKTLFADAMA (SEQ ID NO: 326) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 265. This signal peptide is not included in SEQ ID NO: 265. However, the signal peptide of SEQ ID NO: 326, or another signal peptide, can optionally be included at the amino-terminus of SEQ ID NO: 265, or at the amino terminus of any of the other enzymes or expansin proteins described herein.
4. Xylanases
Xylanases (EC 3.2.1.8) are a class of enzymes that act on the polysaccharide xylan, a common sugar found in plants and in the soil. Xylans consist of a β-1,4-linked xylose main chain in which individual xylose molecules often are decorated with methyl glucuronic acid and arabinose. Arabinoxylans are the primary hemicelluloses in mature monocot tissues. Xylanases catalyze the cleavage of internal β-1,4-bonds between two xylose molecules within polysaccharide main chains.
Xylanases can be used as a seed treatment, delivered to the plant growth medium (e.g., via in furrow application or as a soil amendment), or applied as a foliar treatment onto plants to generate smaller sugar chains that can be taken up by the plant or used to feed the surrounding microbiome.
For ease of reference, illustrative xylanase amino acid sequences are provided in Table 6 below, together with their SEQ ID NOs.
stearothermophilus (Bacillus stearothermophilus)
The native amino acid sequence of the xylanase of SEQ ID NO: 267 includes the signal peptide MCENLEMLNLSLAKTYKDYFKIGAAVTA (SEQ ID NO: 327) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 267. This signal peptide is not included in SEQ ID NO: 267. However, the signal peptide of SEQ ID NO: 327, or another signal peptide, can optionally be included at the amino terminus of the xylanase of SEQ ID NO: 267, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the xylanase of SEQ ID NO: 268 includes the signal peptide MFKFKKNFLVGLSAALMSISLFSATASA (SEQ ID NO: 328) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 268. This signal peptide is not included in SEQ ID NO: 268. However, the signal peptide of SEQ ID NO: 238, or another signal peptide, can optionally be included at the amino terminus of the xylanase of SEQ ID NO: 268, or at the amino-terminus of any of the other enzymes or expansin proteins described herein. Where the xylanase of SEQ ID NO: 268 includes the signal peptide of SEQ ID NO: 328 at its amino terminus, this enzyme is referred to herein by ID Code E100.
The native amino acid sequence of the xylanase of SEQ ID NO: 269 includes the signal peptide MRKKCSVCLWILVLLLSCLSGKSAYA (SEQ ID NO: 329) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 269. This signal peptide is not included in SEQ ID NO: 269. However, the signal peptide of SEQ ID NO: 329, or another signal peptide, can optionally be included at the amino terminus of the xylanase of SEQ ID NO: 269, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the xylanase of SEQ ID NO: 270 includes the signal peptide MKLKKKMLTLLLTASMSFGLFGATSSA (SEQ ID NO: 330) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 270. This signal peptide is not included in SEQ ID NO: 270. However, the signal peptide of SEQ ID NO: 330, or another signal peptide, can optionally be included at the amino terminus of the xylanase of SEQ ID NO: 270, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the xylanase of SEQ ID NO: 273 includes the signal peptide MIPRIKKTICVLLVCFTMLSVMLGPGATEVLA (SEQ ID NO: 331) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 273. This signal peptide is not included in SEQ ID NO: 273. However, the signal peptide of SEQ ID NO: 331, or another signal peptide, can optionally be included at the amino terminus of the xylanase of SEQ ID NO: 273, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
5. Xylosidases
Xylosidases (EC 3.2.1.37) are a class of enzymes that cleave single xylose molecules off of shorter fragments of xylan, a common polysaccharide found in plants and in the soil. Xylans consist of a β-1,4-linked xylose main chain in which individual xylose molecules often are decorated with methyl glucuronic acid and arabinose. Arabinoxylans are the primary hemicelluloses in mature monocot tissues. Xylosidases catalyze the cleavage of terminal β-1,4-bonds in the xylose main chain of xylans.
Xylosidases can be used as a seed treatment, delivered to the plant growth medium (e.g., via in furrow application or as a soil amendment), or applied as a foliar treatment onto plants to generate smaller sugar chains that can be taken up by the plant or used to feed the surrounding microbiome.
For ease of reference, illustrative xylosidase amino acid sequences are provided in Table 7 below, together with their SEQ ID NOs.
6. Lactonases
Lactonases can be used for any of the plant growth stimulating or plant health promoting purposes described herein, but are especially suitable for decreasing the susceptibility of plants to pathogens. Lactonases are also described as acyl-homoserine lactonases and are metalloenzymes produced by certain species of bacteria. For example, lactonases can be found in bacteria of the Phyla Bacteriodetes, Firmicutes, Actinobacteria, and in bacteria of the genera of Pseudomonas and Bacillus, as well as others. Lactonases target and inactivate acylated homoserine lactones. Lactonases hydrolyze the ester bonds of small hormone-like molecules commonly known as homoserine lactones. In the hydrolysis of these lactone bonds, lactonase acts to prevent these homoserine lactones from binding to their transcriptionally-regulated targets and thereby interfere with quorum sensing. However, lactonase secretion from naturally occurring bacteria that colonize soil or plants is limited and inducible, and thus it would be desirable to providing higher levels of lactonase to the environment of a plant.
Lactonases can be applied to plants (e.g., foliarly or as a seed treatment) or a plant growth medium in order to reduce the levels of lactones in the environment. Without being bound to any particular theory, it is believed that this reduction in the level of lactones can in turn lead to reduction in plant disease, as well as a secondary increase in plant growth and development.
For ease of reference, illustrative lactonase amino acid sequences are provided in Table 8 below, together with their SEQ ID NOs.
7. Chitosanases
Chitosanases can be used for any of the plant growth stimulating or plant health promoting purposes described herein, but are especially suitable for increasing nutrient uptake and increasing plant growth. This in turn leads to increased crop yield, improved early season vigor, and decreased susceptibility to early season stresses. Chitosanases are also useful for protecting plants from pathogens.
For ease of reference, illustrative chitosanase amino acid sequences are provided in Table 9 below, together with their SEQ ID NOs.
subtilis str. 168)
The native amino acid sequence of the chitosanase of SEQ ID NO: 279 includes the signal peptide MKISMQKADFWKKAAISLLVFTMFFTLMMSETVFA (SEQ ID NO: 332) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 279. This signal peptide is not included in SEQ ID NO: 279. However, the signal peptide of SEQ ID NO: 332, or another signal peptide, can optionally be included at the amino terminus of the chitosanase of SEQ ID NO: 279, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the chitosanase of SEQ ID NO: 280 includes the signal peptide MHSQHRTARIALAVVLTAIPASLATAGVGYASTQASTAVK (SEQ ID NO: 333) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 280. This signal peptide is not included in SEQ ID NO: 280. However, the signal peptide of SEQ ID NO: 333, or another signal peptide, can optionally be included at the amino-terminus of the chitosanase of SEQ ID NO: 280, or at the amino terminus of any of the other enzymes or expansin proteins described herein.
8. Glucanases
Glucanases use water to break chemical bonds between individual glucose molecules in glucans, which are long chain polysaccharides. Glucans can be broken down into two types, alpha glucan, consisting of primarily alpha chains of glucose molecules, and beta glucans, consisting of primarily beta chains of glucose molecules. Common alpha glucans include dextrans, glycogens, pullalans, and starch. Alpha glucans generally include combinations of alpha 1,4; alpha 1,6, and/or alpha 1,3 glucans and branches. Glucanases that are specific for cleaving alpha linkages are called alpha-glucanases. Beta glucanases are specific to beta linkages between glucans. Common beta glucans include cellulose, laminarin, lichenin, zymosan. Beta glucans are commonly found with b1,3; b1,4, and/or b1,6 linkages between glucose molecules. Glucanases can be either “exo” or “endo” depending on the location of the cleavage of the polysaccharide. Alpha-, beta-, exo- and endo-glucanases are all effective for stimulating plant growth.
β-1,4-endoglucanases comprise a class of enzymes (EC 3.2.1.4) that function in the endohydrolysis of the (1→4)-β-
Xyloglucanases (EC 3.2.1.151) comprise a class of enzymes that catalyze the cleavage of xyloglucan oligosaccharides. Xyloglucans consist of β-1,4-linked glucose main chain primarily decorated with α-1,6-linked xylose. Xyloglucans are the primary hemicelluloses in dicot plants. Xyloglucanases catalyze the endo-hydrolysis of the internal β-1,4 bonds in the glucose main chain of xyloglucans. This results in specific oligosaccharide mixtures arising from cleavage at specific sites, usually unbranched, glucosyl residues.
Lichenases (EC 3.2.1.73) are a class of enzymes also referred to as β-1,3-1,4-endoglucanase that specifically catalyze the cleavage of β-1,4 bonds preceded by a β-1,3 linkage in a glucan backbone. Mixed-linked glucans consist solely of glucose monomers, which are linked by β-1,4 and β-1,3 bonds. Mixed linked-glucans occur in monocot seeds and in the cell walls of developing monocot tissues such as corn and wheat.
Glucanases can be used for any of the plant growth stimulating or plant health promoting purposes described herein, but are particularly useful for increasing nutrient uptake and stimulating plant growth. This in turn leads to increased crop yield, improved early season vigor, and decreased susceptibility to early season stresses. Glucanases can also be used for protecting plants from pathogens and for reducing susceptibility to an environmental stress in a plant.
For ease of reference, illustrative glucanase amino acid sequences are provided in Table 10 below, together with their SEQ ID NOs.
subtilis str. 168) (ID CODE: E112)
subtilis str. 168, without carbohydrate-binding domain)
The native amino acid sequence of the glucanase of SEQ ID NO: 293 includes the signal peptide MKRSISIFITCLLITLLTMGGMIASPASA (SEQ ID NO: 334) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 293. This signal peptide is not included in SEQ ID NO: 293. However, the signal peptide of SEQ ID NO: 334, or another signal peptide, can optionally be included at the amino terminus of the glucanase of SEQ ID NO: 293, or at the amino-terminus of any of the other enzymes or expansin proteins described herein. Where the signal peptide of SEQ ID NO: 334 is included at the amino terminus of SEQ ID NO: 293, this enzyme is referred to herein by ID Code E94.
The carbohydrate-binding domain can optionally be removed from the glucanase of SEQ ID NO: 293 by truncating the last 167 amino acids of the protein. SEQ ID NO: 294 provides the sequence for this glucanase wherein the carbohydrate-binding domain has been removed. The native signal peptide (SEQ ID NO: 334) is not included in SEQ ID NO: 294. However, the signal peptide of SEQ ID NO: 334, or another signal peptide, can optionally be included at the amino terminus of the glucanase of SEQ ID NO: 294. Where the signal peptide of SEQ ID NO: 334 is included at the amino terminus of SEQ ID NO: 294, this enzyme is referred to herein by ID Code EE113.
The native amino acid sequence of the glucanase of SEQ ID NO: 295 includes the signal peptide MPYLKRVLLLLVTGLFMSLFAVTATASA (SEQ ID NO: 335) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 295. This signal peptide is not included in SEQ ID NO: 295. However, the signal peptide of SEQ ID NO: 335, or another signal peptide, can optionally be included at the amino terminus of the glucanase of SEQ ID NO: 295, or at the amino-terminus of any of the other enzymes or expansin proteins described herein. Where the signal peptide of SEQ ID NO: 335 is included at the amino terminus of SEQ ID NO: 295, this enzyme is referred to herein by ID Code E111.
The native amino acid sequence of the glucanase of SEQ ID NO: 296 includes the signal peptide MKNRVISLLMASLLLVLSVIVAPFYKA (SEQ ID NO: 336) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 296. This signal peptide is not included in SEQ ID NO: 296. However, the signal peptide of SEQ ID NO: 336, or another signal peptide, can optionally be included at the amino terminus of the glucanase of SEQ ID NO: 296, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the glucanase of SEQ ID NO: 297 includes the signal peptide MKRSQTSEKRYRQRVLSLFLAVVMLASIGLLPTSKVQA (SEQ ID NO: 337) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 297. This signal peptide is not included in SEQ ID NO: 297. However, the signal peptide of SEQ ID NO: 337, or another signal peptide, can optionally be included at the amino terminus of the glucanase of SEQ ID NO: 297, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the glucanase of SEQ ID NO: 298 includes the signal peptide MKPSHFTEKRFMKKVLGLFLVVVMLASVGVLPTSKVQA (SEQ ID NO: 338) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 298. This signal peptide is not included in SEQ ID NO: 298. However, the signal peptide of SEQ ID NO: 338, or another signal peptide, can optionally be included at the amino terminus of the glucanase of SEQ ID NO: 298, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the glucanase of SEQ ID NO: 299 includes the signal peptide MFKKWKKFGISSLALVLVAAVAFTGWSAKASA (SEQ ID NO: 339) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 299. This signal peptide is not included in SEQ ID NO: 299. However, the signal peptide of SEQ ID NO: 339, or another signal peptide, can optionally be included at the amino-terminus of the glucanase of SEQ ID NO: 299, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the glucanase of SEQ ID NO: 300 includes the signal peptide MKNNHLLKSILLWGAVCIIVLAGPLSAFA (SEQ ID NO: 340) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 300. This signal peptide is not included in SEQ ID NO: 300. However, the signal peptide of SEQ ID NO: 340, or another signal peptide, can optionally be included at the amino-terminus of the glucanase of SEQ ID NO: 300, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the glucanase of SEQ ID NO: 302 includes the signal peptide MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA (SEQ ID NO: 341) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 302. This signal peptide is not included in SEQ ID NO: 302. However, the signal peptide of SEQ ID NO: 341, or another signal peptide, can optionally be included at the amino-terminus of the glucanase of SEQ ID NO: 302, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
9. Proteases
Proteases can be used for any of the plant growth stimulating or plant health promoting purposes described herein, but are particularly useful for increasing nutrient uptake and stimulating plant growth. This in turn leads to increased crop yield, improved early season vigor, and decreased susceptibility to early season stresses. Proteases are also useful for protecting plants from pathogens.
For ease of reference, illustrative protease amino acid sequences are provided in Table 11 below, together with their SEQ ID NOs.
The native amino acid sequence of the protease of SEQ ID NO: 304 includes the signal peptide MKKGIIRFLLVSFVLFFALSTGITGVQA (SEQ ID NO: 342) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 304. This signal peptide is not included in SEQ ID NO: 304. However, the signal peptide of SEQ ID NO: 342, or another signal peptide, can optionally be included at the amino terminus of the protease of SEQ ID NO: 304, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the protease of SEQ ID NO: 306 includes the signal peptide MVVFSKTAALVLGLSTAVSA (SEQ ID NO: 343) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 306. This signal peptide is not included in SEQ ID NO: 306. However, the signal peptide of SEQ ID NO: 343, or another signal peptide, can optionally be included at the amino-terminus of the protease of SEQ ID NO: 306, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
10. Mannanases
Mannanases (EC 3.2.1.78) are a class of enzymes that catalyze the cleavage of internal β-1,4-bonds between monosaccharide units in mannose/glucose main chain polysaccharides such as mannans. Mannans are minor constituents of cell walls in higher plants and consist of a β-1,4-linked mannose main chain, which may contain β-1,4-linked glucose monomers. Individual mannose monomers can further be decorated with galactose monomers. The small sugars that are released from polysaccharides through the action of mannanases can be taken up by plants as carbon sources and can also feed the inherent microbes that surround the plant.
For ease of reference, illustrative mannanase amino acid sequences are provided in Table 12 below, together with their SEQ ID NOs.
The native amino acid sequence of the mannanase of SEQ ID NO: 307 includes the signal peptide MAKLQKGTILTVIAALMFVILGSAAPKAAA (SEQ ID NO: 344) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 307. This signal peptide is not included in SEQ ID NO: 307. However, the signal peptide of SEQ ID NO: 344, or another signal peptide, can optionally be included at the amino-terminus of the mannanase of SEQ ID NO: 307, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the mannanase of SEQ ID NO: 308 includes the signal peptide MFKKHTISLLIIFLLASAVLAKPIEA (SEQ ID NO: 345) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 308. This signal peptide is not included in SEQ ID NO: 308. However, the signal peptide of SEQ ID NO: 345, or another signal peptide, can optionally be included at the amino-terminus of the mannanase of SEQ ID NO: 308, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
11. Pectinases
Pectinases act on pectin and related polysaccharides to release small sugars. Specifically, pectin lyases (EC 4.2.2.10), also referred to as pectolyases, and endopolygalacturonases (EC 3.2.1.15) are both enzyme classes that catalyze the cleavage of internal β-1,4-bonds in galacturonan main chains. Galacturonans are found within the plant cell walls. However, they are primarily known as pectin, which is the main constituent of the middle lamella. Galacturonans mainly consist of α-1,4-linked galacturonic acid monomers, which can be esterified and extensively decorated. The small sugars that are released from polysaccharides through the action of pectinases can be taken up by a plant as a carbon source and can also feed the inherent microbes that surround the plant.
For ease of reference, illustrative pectinase amino acid sequences are provided in Table 13 below, together with their SEQ ID NOs.
The native amino acid sequence of the pectolyase of SEQ ID NO: 309 includes the signal peptide MPSAKPLFCLATLAGAALA (SEQ ID NO: 346) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 309. This signal peptide is not included in SEQ ID NO: 309. However, the signal peptide of SEQ ID NO: 346, or another signal peptide, can optionally be included at the amino-terminus of the pectolyase of SEQ ID NO: 309, or at the amino-terminus of any of the other enzymes or expansin proteins described herein. Where the pectolyase of SEQ ID NO: 309 includes the signal peptide of SEQ ID NO: 346 at its amino terminus, this enzyme is referred to herein by ID Code D405.
The native amino acid sequence of the pectolyase of SEQ ID NO: 310 includes the signal peptide MKRLCLWFTVFSLFLVLLPGKALG (SEQ ID NO: 347) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 310. This signal peptide is not included in SEQ ID NO: 310. However, the signal peptide of SEQ ID NO: 347, or another signal peptide, can optionally be included at the amino-terminus of the pectolyase of SEQ ID NO: 310, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
12. Acid Phosphatases
Acid phosphatases act on insoluble and less soluble forms of phosphates in the soil, and release them from for uptake by plants. Acid phosphatase are a family of enzymes expressed widely across kingdoms. These enzymes non-specifically catalyze the hydrolysis of monoesters and anhydrides of phosphoric acid to produce inorganic phosphate and are distinct from other phosphatases due to their activity in acidic conditions. Plant acid phosphatases have been localized in the cytosol, vacuoles, and cell walls and are important for mobilizing organic phosphates in the soil. Acid phosphatases hydrolyze phytins, ATP, protein phosphates, nucleotide phosphates, and have a role in general metabolic reactions.
For ease of reference, descriptions of illustrative acid phosphatase amino acid sequences are provided in Table 14 below, together with their SEQ ID NOs.
The native amino acid sequence of the acid phosphatase of SEQ ID NO: 311 includes the signal peptide MARGSMAAVLAVLAVAALRCAPAAA (SEQ ID NO: 348) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 311. This signal peptide is not included in SEQ ID NO: 311. However, the signal peptide of SEQ ID NO: 348, or another signal peptide, can optionally be included at the amino-terminus of the acid phosphatase of SEQ ID NO: 311, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the acid phosphatase of SEQ ID NO: 312 includes the signal peptide MRGLGFAALSLHVLLCLANGVSSRRTSSYV (SEQ ID NO: 349) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 312. This signal peptide is not included in SEQ ID NO: 312. However, the signal peptide of SEQ ID NO: 349, or another signal peptide, can optionally be included at the amino-terminus of the acid phosphatase of SEQ ID NO: 312, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the acid phosphatase of SEQ ID NO: 378 includes the signal peptide MKMKRGITTLLSVAVLSTSLVACSGTPEKTVA (SEQ ID NO: 379) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 378. This signal peptide is not included in SEQ ID NO: 378. However, the signal peptide of SEQ ID NO: 379, or another signal peptide, can optionally be included at the amino terminus of the acid phosphatase of SEQ ID NO: 378, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
13. Phytases
Phytases act on phytic acids in soil, a source of free phosphate for plant growth. Phytases remove select phosphates off of the phytic acids, and the freed phosphates can be taken up by nearby plants.
For ease of reference, descriptions of illustrative phytase amino acid sequences are provided in Table 15 below, together with their SEQ ID NOs.
The native amino acid sequence of the phytase of SEQ ID NO: 313 includes the signal peptide MWWGSLRLLLLLAAAVAA (SEQ ID NO: 350) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 313. This signal peptide is not included in SEQ ID NO: 313. However, the signal peptide of SEQ ID NO: 350, or another signal peptide, can optionally be included at the amino-terminus of the phytase of SEQ ID NO: 313, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the phytase of SEQ ID NO: 314 includes the signal peptide MWWGSLRLLLLLAAAVAA (SEQ ID NO: 350) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 314. This signal peptide is not included in SEQ ID NO: 314. However, the signal peptide of SEQ ID NO: 350, or another signal peptide, can optionally be included at the amino-terminus of the phytase of SEQ ID NO: 314, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the phytase of SEQ ID NO: 315 includes the signal peptide MGIWRGSLPLLLLAA (SEQ ID NO: 351) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 315. This signal peptide is not included in SEQ ID NO: 315. However, the signal peptide of SEQ ID NO: 351, or another signal peptide, can optionally be included at the amino-terminus of the phytase of SEQ ID NO: 315, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
The native amino acid sequence of the phytase of SEQ ID NO: 380 includes the signal peptide MKVPKTMLLSTAAGLLLSLTATSVSA (SEQ ID NO: 354) at the amino-terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 380. This signal peptide is not included in SEQ ID NO: 380. However, the signal peptide of SEQ ID NO: 354, or another signal peptide can optionally be included at the amino-terminus of the phytase of SEQ ID NO: 380, or at the amino-terminus of any of the other enzymes or expansin proteins described herein.
14. Expansin Proteins
Expansin proteins aid plant walls in expanding during growth of the plant. Expansins are thus particularly useful in any of the methods for stimulating plant growth described herein.
For ease of reference, an illustrative expansin amino acid sequences is provided in Table 16 below, together with its SEQ ID NOs.
The native amino acid sequence of the expansin protein of SEQ ID NO: 316 includes the signal peptide MKKIMSAFVGMVLLTIFCFSPQASA (SEQ ID NO: 352) at the amino terminus of the sequence, immediately preceding the first amino acid of SEQ ID NO: 316. This signal peptide is not included in SEQ ID NO: 316. However, the signal peptide of SEQ ID NO: 352, or another signal peptide, can optionally be included at the amino terminus of the protease of SEQ ID NO: 316, at the amino terminus of any of the enzymes described herein, or at the amino terminus of another expansin protein.
15. Signal Peptides
As described above, many of the native amino acid sequences of the enzymes and expansin proteins include signal peptides. When a fusion protein comprises an enzyme or expansin protein whose native sequence includes a signal peptide, the enzyme or expansin protein can be used without the signal peptide. Alternatively, the native signal peptide (or another signal peptide) can optionally be included at the amino terminus of the enzyme or expansin protein sequence, immediately preceding the first amino acid of the enzyme or expansin sequence.
In addition, a signal peptide can optionally be included at the amino terminus of the enzymes or expansin proteins whose native sequences do not include a signal peptide.
For ease of reference, amino acid sequences for illustrative signal peptides that can be added to any of the enzymes or expansin proteins described herein are provided below in Table 17. Any of the signal peptides listed in Table 17 below can be added at the amino terminus of any of the enzymes or expansin proteins described herein.
Bacillus thuringiensis
Bacillus thuringiensis serovar israelensis 4Q7
Bacillus cereus ATCC 10987
Clostridium perfingens
Streptomyces chromofuscus
Streptomyces chromofuscus
Bacillus cereus
Bacillus subtilis subsp. subtilis str. 168
Burkholderia cepacia
Pseudomonas fluorescens
Caldicellulosiruptor saccharolyticus
Bacillus subtilis subsp. subtilis str. 168
Bacillus subtilis subsp. subtilis str. 168
Geobacillus stearothermophilus
Bacillus subtilis strain 168
Bacillus subtilis subsp. subtilis str. 168
Streptomyces species N174
Bacillus subtilis subsp. subtilis str. 168
Bacillus subtilis subsp. subtilis str. 168
Clostridium thermocellum ATCC 27405
Bacillus circulans
Bacillus circulans
Paenibacillus pabuli
Bacillus licheniformis
Bacillus subtilis 168
Bacillus subtilis subsp. subtilis str. 168
Aspergillus saitoi
Bacillus circulans
Bacillus subtilis strain 168
Aspergillus niger
Bacillus subtilis strain 168
Triticum aestivum
Triticum aestivum
Triticum aestivum
Triticum aestivum
Bacillus subtilis subsp. subtilis str. 168
Bacillus subtilis subsp. subtilis str. 168
Bacillus subtilis subsp. subtilis str. 168
Bacillus subtilis subsp. subtilis str. 168
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus pseudomycoides
Bacillus thuringiensis serovar israelensis 4Q7
Listeria monocytogenes
Bacillus thuringiensis
Bacillus thuringiensis
C. Fusion Proteins for Expression in Recombinant Bacillus cereus Family Members
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member are provided. The fusion proteins further comprise an enzyme having ACC deaminase activity, a phospholipase, a lipase, a xylanase, a xylosidase, a lactonase, a chitosanase, a protease, a glucanase, a phytase, an acid phosphatase, a pectinase, a mannanase, and/or an expansin protein.
In any of the fusion proteins described herein, the fusion protein can comprise: (1) a targeting sequence comprising an amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 54%; (2) a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 1; (3) a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1; (4) a targeting sequence comprising SEQ ID NO: 1; (5) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 2; (6) a targeting sequence comprising amino acids 2-35 of SEQ ID NO: 1; (7) a targeting sequence comprising amino acids 5-35 of SEQ ID NO: 1; (8) a targeting sequence comprising amino acids 8-35 of SEQ ID NO: 1; (9) a targeting sequence comprising amino acids 10-35 of SEQ ID NO: 1; (10) a targeting sequence comprising amino acids 15-35 of SEQ ID NO: 1; (11) a targeting sequence comprising amino acids 1-27 of SEQ ID NO: 3; (12) a targeting sequence comprising amino acids 12-27 of SEQ ID NO: 3; (13) a targeting sequence comprising SEQ ID NO: 3; (14) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 4; (15) a targeting sequence comprising amino acids 2-27 of SEQ ID NO: 3; (16) a targeting sequence comprising amino acids 5-27 of SEQ ID NO: 3; (17) a targeting sequence comprising amino acids 8-27 of SEQ ID NO: 3; (18) a targeting sequence comprising amino acids 10-27 of SEQ ID NO: 3; (19) a targeting sequence comprising amino acids 1-38 of SEQ ID NO: 5; (20) a targeting sequence comprising amino acids 23-38 of SEQ ID NO: 5; (21) a targeting sequence comprising SEQ ID NO: 5; (22) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 6; (23) a targeting sequence comprising amino acids 2-38 of SEQ ID NO: 5; (24) a targeting sequence comprising amino acids 5-38 of SEQ ID NO: 5; (25) a targeting sequence comprising amino acids 8-38 of SEQ ID NO: 5; (26) a targeting sequence comprising amino acids 10-38 of SEQ ID NO: 5; (27) a targeting sequence comprising amino acids 15-38 of SEQ ID NO: 5; (28) a targeting sequence comprising amino acids 20-38 of SEQ ID NO: 5; (29) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 7; (30) a targeting sequence comprising amino acids 13-28 of SEQ ID NO: 7; (31) a targeting sequence comprising SEQ ID NO: 7; (32) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 8; (33) a targeting sequence comprising amino acids 2-28 of SEQ ID NO: 7; (34) a targeting sequence comprising amino acids 5-28 of SEQ ID NO: 7; (35) a targeting sequence comprising amino acids 8-28 of SEQ ID NO: 7; (36) a targeting sequence comprising amino acids 10-28 of SEQ ID NO: 7; (37) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 9; (38) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 9; (39) a targeting sequence comprising SEQ ID NO: 9; (40) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 10; (41) a targeting sequence comprising amino acids 2-24 of SEQ ID NO: 9; (42) a targeting sequence comprising amino acids 5-24 of SEQ ID NO: 9; (43) a targeting sequence comprising amino acids 8-24 of SEQ ID NO: 9; (44) a targeting sequence comprising amino acids 1-33 of SEQ ID NO:11; (45) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 11; (46) a targeting sequence comprising SEQ ID NO: 11; (47) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 12; (48) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 11; (49) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 11; (50) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 11; (51) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 11; (52) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 11; (53) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 13; (54) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 13; (55) a targeting sequence comprising SEQ ID NO:13; (56) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:14; (57) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 13; (58) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 13; (59) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 13; (60) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 13; (61) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 13; (62) a targeting sequence comprising amino acids 1-43 of SEQ ID NO: 15; (63) a targeting sequence comprising amino acids 28-43 of SEQ ID NO: 15; (64) a targeting sequence comprising SEQ ID NO:15; (65) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:16; (66) a targeting sequence comprising amino acids 2-43 of SEQ ID NO: 15; (67) a targeting sequence comprising amino acids 5-43 of SEQ ID NO: 15; (68) a targeting sequence comprising amino acids 8-43 of SEQ ID NO: 15; (69) a targeting sequence comprising amino acids 10-43 of SEQ ID NO: 15; (70) a targeting sequence comprising amino acids 15-43 of SEQ ID NO: 15; (71) a targeting sequence comprising amino acids 20-43 of SEQ ID NO: 15; (72) a targeting sequence comprising amino acids 25-43 of SEQ ID NO: 15; (73) a targeting sequence comprising amino acids 1-27 of SEQ ID NO: 17; (74) a targeting sequence comprising amino acids 12-27 of SEQ ID NO: 17; (75) a targeting sequence comprising SEQ ID NO:17; (76) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:18; (77) a targeting sequence comprising amino acids 2-27 of SEQ ID NO: 17; (78) a targeting sequence comprising amino acids 5-27 of SEQ ID NO: 17; (79) a targeting sequence comprising amino acids 8-27 of SEQ ID NO: 17; (80) a targeting sequence comprising amino acids 10-27 of SEQ ID NO: 17; (81) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 19; (82) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 19; (83) a targeting sequence comprising SEQ ID NO:19; (84) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:20; (85) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 19; (86) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 19; (87) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 19; (88) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 19; (89) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 19; (90) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 21; (91) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 21; (92) a targeting sequence comprising SEQ ID NO:21; (93) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:22; (94) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 21; (95) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 21; (96) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 21; (97) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 21; (98) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 21; (99) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 23; (100) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 23; (101) a targeting sequence comprising SEQ ID NO:23; (102) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:24; (103) a targeting sequence comprising amino acids 2-24 of SEQ ID NO:23; (104) a targeting sequence comprising amino acids 5-24 of SEQ ID NO: 23; (105) a targeting sequence comprising amino acids 8-24 of SEQ ID NO: 23; (106) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 25; (107) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 25; (108) a targeting sequence comprising SEQ ID NO:25; (109) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:26; (110) a targeting sequence comprising amino acids 2-24 of SEQ ID NO: 25; (111) a targeting sequence comprising amino acids 5-24 of SEQ ID NO: 25; (112) a targeting sequence comprising amino acids 8-24 of SEQ ID NO: 25; (113) a targeting sequence comprising amino acids 1-30 of SEQ ID NO: 27; (114) a targeting sequence comprising amino acids 15-30 of SEQ ID NO: 27; (115) a targeting sequence comprising SEQ ID NO:27; (116) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:28; (117) a targeting sequence comprising amino acids 2-30 of SEQ ID NO: 27; (118) a targeting sequence comprising amino acids 5-30 of SEQ ID NO: 27; (119) a targeting sequence comprising amino acids 8-30 of SEQ ID NO: 27; (120) a targeting sequence comprising amino acids 10-30 of SEQ ID NO: 27; (121) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 29; (122) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 29; (123) a targeting sequence comprising SEQ ID NO:29; (124) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:30; (125) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 29; (126) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 29; (127) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 29; (128) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 29; (129) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 29; (130) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 31; (131) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 31; (132) a targeting sequence comprising SEQ ID NO:31; (133) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:32; (134) a targeting sequence comprising amino acids 2-24 of SEQ ID NO: 31; (135) a targeting sequence comprising amino acids 5-24 of SEQ ID NO: 31; (136) a targeting sequence comprising amino acids 8-24 of SEQ ID NO: 31; (137) a targeting sequence comprising amino acids 1-15 of SEQ ID NO: 33; (138) a targeting sequence comprising SEQ ID NO:33; (139) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:34; (140) a targeting sequence comprising amino acids 1-16 of SEQ ID NO: 35; (141) a targeting sequence comprising SEQ ID NO:35; (142) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO:36; (143) a targeting sequence comprising amino acids 1-29 of SEQ ID NO:43; (144) a targeting sequence comprising amino acids 14-29 of SEQ ID NO: 43; (145) a targeting sequence comprising SEQ ID NO: 43; (146) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 44; (147) a targeting sequence comprising amino acids 2-29 of SEQ ID NO: 43; (148) a targeting sequence comprising amino acids 5-29 of SEQ ID NO: 43; (149) a targeting sequence comprising amino acids 8-29 of SEQ ID NO: 43; (150) a targeting sequence comprising amino acids 10-29 of SEQ ID NO: 43; (151) a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 45; (152) a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 45; (153) a targeting sequence comprising SEQ ID NO: 45; (154) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 46; (155) a targeting sequence comprising amino acids 2-35 of SEQ ID NO: 45; (156) a targeting sequence comprising amino acids 5-35 of SEQ ID NO: 45; (157) a targeting sequence comprising amino acids 8-35 of SEQ ID NO: 45; (158) a targeting sequence comprising amino acids 10-35 of SEQ ID NO: 45; (159) a targeting sequence comprising amino acids 15-35 of SEQ ID NO: 45; (160) a targeting sequence comprising amino acids 1-43 of SEQ ID NO: 47; (161) a targeting sequence comprising amino acids 28-43 of SEQ ID NO: 47; (162) a targeting sequence comprising SEQ ID NO: 47; (163) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 48; (164) a targeting sequence comprising amino acids 2-43 of SEQ ID NO: 47; (165) a targeting sequence comprising amino acids 5-43 of SEQ ID NO: 47; (166) a targeting sequence comprising amino acids 8-43 of SEQ ID NO: 47; (167) a targeting sequence comprising amino acids 10-43 of SEQ ID NO: 47; (168) a targeting sequence comprising amino acids 15-43 of SEQ ID NO: 47; (169) a targeting sequence comprising amino acids 20-43 of SEQ ID NO: 47; (170) a targeting sequence comprising amino acids 25-43 of SEQ ID NO: 47; (171) a targeting sequence comprising amino acids 1-32 of SEQ ID NO: 49; (172) a targeting sequence comprising amino acids 17-32 of SEQ ID NO: 49; (173) a targeting sequence comprising SEQ ID NO: 49; (174) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 50; (175) a targeting sequence comprising amino acids 2-32 of SEQ ID NO: 49; (176) a targeting sequence comprising amino acids 5-32 of SEQ ID NO: 49; (177) a targeting sequence comprising amino acids 8-32 of SEQ ID NO: 49; (178) a targeting sequence comprising amino acids 10-32 of SEQ ID NO: 49; (179) a targeting sequence comprising amino acids 15-32 of SEQ ID NO: 49; (180) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 51; (181) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 51; (182) a targeting sequence comprising SEQ ID NO: 51; (183) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 52; (184) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 51; (185) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 51; (186) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 51; (187) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 51; (188) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 51; (189) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 53; (190) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 53; (191) a targeting sequence comprising SEQ ID NO: 53; (192) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 54; (193) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 53; (194) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 53; (195) a targeting sequence comprising amino acids 8-33 of SEQ ID NO: 53; (196) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 53; (197) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 53; (198) a targeting sequence comprising amino acids 1-30 of SEQ ID NO: 55; (199) a targeting sequence comprising amino acids 15-30 of SEQ ID NO: 55; (200) a targeting sequence comprising SEQ ID NO: 55; (201) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 56; (202) a targeting sequence comprising amino acids 2-30 of SEQ ID NO: 55; (203) a targeting sequence comprising amino acids 5-30 of SEQ ID NO: 55; (204) a targeting sequence comprising amino acids 8-30 of SEQ ID NO: 55; (205) a targeting sequence comprising amino acids 10-30 of SEQ ID NO: 55; (206) a targeting sequence comprising amino acids 1-130 of SEQ ID NO: 57; (207) a targeting sequence comprising amino acids 115-130 of SEQ ID NO: 57; (208) a targeting sequence comprising SEQ ID NO: 57; (209) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 58; (210) a targeting sequence comprising amino acids 2-130 of SEQ ID NO: 57; (211) a targeting sequence comprising amino acids 5-130 of SEQ ID NO: 57; (212) a targeting sequence comprising amino acids 10-130 of SEQ ID NO: 57; (213) a targeting sequence comprising amino acids 20-130 of SEQ ID NO: 57; (214) a targeting sequence comprising amino acids 30-130 of SEQ ID NO: 57; (215) a targeting sequence comprising amino acids 40-130 of SEQ ID NO: 57; (216) a targeting sequence comprising amino acids 50-130 of SEQ ID NO: 57; (217) a targeting sequence comprising amino acids 60-130 of SEQ ID NO: 57; (218) a targeting sequence comprising amino acids 70-130 of SEQ ID NO: 57; (219) a targeting sequence comprising amino acids 80-130 of SEQ ID NO: 57; (220) a targeting sequence comprising amino acids 90-130 of SEQ ID NO: 57; (221) a targeting sequence comprising amino acids 100-130 of SEQ ID NO: 57; (222) a targeting sequence comprising amino acids 110-130 of SEQ ID NO: 57; (223) an exosporium protein fragment comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 95; (224) a targeting sequence comprising SEQ ID NO: 96; (225) a targeting sequence comprising SEQ ID NO: 97; (226) a targeting sequence comprising SEQ ID NO: 98; (227) a targeting sequence comprising SEQ ID NO: 99; (228) a targeting sequence comprising SEQ ID NO: 100; (229) a targeting sequence comprising SEQ ID NO: 101; (230) a targeting sequence comprising SEQ ID NO: 102; (231) a targeting sequence comprising SEQ ID NO: 103; (232) a targeting sequence comprising SEQ ID NO: 104; (233) a targeting sequence comprising SEQ ID NO: 105; (234) a targeting sequence comprising SEQ ID NO: 106; (235) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 108; (236) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 109; (237) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 110; (238) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 111; (239) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 112; (240) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 113; (241) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 114; (242) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 115; (243) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 116; (244) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 117; (245) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 118; (246) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 119; (247) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 120; (248) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 121; (249) a targeting sequence comprising amino acids 22-31 of SEQ ID NO: 1; (250) a targeting sequence comprising amino acids 22-33 of SEQ ID NO: 1; (251) a targeting sequence comprising amino acids 20-31 of SEQ ID NO: 1; (252) a targeting sequence comprising amino acids 14-23 of SEQ ID NO: 3; (253) a targeting sequence comprising amino acids 14-25 of SEQ ID NO: 3; (254) a targeting sequence comprising amino acids 12-23 of SEQ ID NO: 3; (255) a targeting sequence comprising amino acids 1-30 of SEQ ID NO: 59; (256) a targeting sequence comprising SEQ ID NO: 59; (257) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 60; (258) a targeting sequence comprising amino acids 2-30 of SEQ ID NO: 59; (259) a targeting sequence comprising amino acids 4-30 of SEQ ID NO: 59; (260) a targeting sequence comprising amino acids 6-30 of SEQ ID NO: 59; (261) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 61; (262) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 61; (263) a targeting sequence comprising SEQ ID NO: 61; (264) an exosporium protein comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 62; (265) a targeting sequence comprising amino acids 2-33 of SEQ ID NO: 61; (266) a targeting sequence comprising amino acids 5-33 of SEQ ID NO: 61; (267) a targeting sequence comprising amino acids 10-33 of SEQ ID NO: 61; (268) a targeting sequence comprising amino acids 15-33 of SEQ ID NO: 61; (269) a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 63; (270) a targeting sequence comprising SEQ ID NO: 63; (271) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 64; (272) a targeting sequence comprising amino acids 2-35 of SEQ ID NO: 63; (273) a targeting sequence comprising amino acids 5-35 of SEQ ID NO: 63; (274) a targeting sequence comprising amino acids 8-35 of SEQ ID NO: 63; (275) a targeting sequence comprising amino acids 10-35 of SEQ ID NO: 63; (276) a targeting sequence comprising amino acids 15-35 of SEQ ID NO: 63; (277) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 65; (278) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 65; (279) a targeting sequence comprising SEQ ID NO: 65; (280) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 66; (281) a targeting sequence comprising SEQ ID NO: 107; (282) a targeting sequence comprising amino acids 2-24 of SEQ ID NO: 65; (283) a targeting sequence comprising amino acids 5-24 of SEQ ID NO: 65; (284) a targeting sequence comprising amino acids 1-27 of SEQ ID NO: 67; (285) a targeting sequence comprising amino acids 12-27 of SEQ ID NO: 67; (286) a targeting sequence comprising SEQ ID NO: 67; (287) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 68; (288) an targeting sequence comprising amino acids 2-27 of SEQ ID NO: 67; (289) a targeting sequence comprising amino acids 5-27 of SEQ ID NO: 67; (290) a targeting sequence comprising amino acids 10-27 of SEQ ID NO: 67; (291) a targeting sequence comprising amino acids 1-38 of SEQ ID NO: 69; (292) a targeting sequence comprising amino acids 23-38 of SEQ ID NO: 69; (293) a targeting sequence comprising SEQ ID NO: 69; (294) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 70; (295) a targeting sequence comprising amino acids 2-38 of SEQ ID NO: 69; (296) a targeting sequence comprising amino acids 5-38 of SEQ ID NO: 69; (297) a targeting sequence comprising amino acids 10-38 of SEQ ID NO: 69; (298) a targeting sequence comprising amino acids 15-38 of SEQ ID NO: 69; (299) an exosporium protein comprising SEQ ID NO: 72; (300) a targeting sequence comprising SEQ ID NO: 73; (301) an exosporium protein comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 74; (302) a targeting sequence comprising amino acids 1-42 of SEQ ID NO: 75; (303) a targeting sequence comprising amino acids 27-42 of SEQ ID NO: 75; (304) a targeting sequence comprising SEQ ID NO: 75; (305) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 76; (306) a targeting sequence comprising amino acids 2-42 of SEQ ID NO: 75; (307) a targeting sequence comprising amino acids 5-42 of SEQ ID NO: 75; (308) a targeting sequence comprising amino acids 10-42 of SEQ ID NO: 75; (309) a targeting sequence comprising amino acids 15-42 of SEQ ID NO: 75; (310) a targeting sequence comprising amino acids 20-42 of SEQ ID NO: 75; (311) a targeting sequence comprising amino acids 25-42 of SEQ ID NO: 75; (312) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 77; (313) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 77; (314) a targeting sequence comprising SEQ ID NO: 77; (315) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 78; (316) a targeting sequence comprising amino acids 2-24 of SEQ ID NO: 77; (317) a targeting sequence comprising amino acids 5-24 of SEQ ID NO: 77; (318) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 80; (319) a targeting sequence comprising amino acids 1-38 of SEQ ID NO: 81; (320) a targeting sequence comprising amino acids 23-38 of SEQ ID NO: 81; (321) a targeting sequence comprising SEQ ID NO: 81; (322) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 82; (323) a targeting sequence comprising amino acids 2-38 of SEQ ID NO: 81; (324) a targeting sequence comprising amino acids 5-38 of SEQ ID NO: 81; (325) a targeting sequence comprising amino acids 10-38 of SEQ ID NO: 81; (326) a targeting sequence comprising amino acids 15-38 of SEQ ID NO: 81; (327) a targeting sequence comprising amino acids 20-38 of SEQ ID NO: 81; (328) a targeting sequence comprising amino acids 1-34 of SEQ ID NO: 83; (329) a targeting sequence comprising SEQ ID NO: 83; (330) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 84; (331) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 86; (332) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 87; (333) a targeting sequence comprising amino acids 13-28 of SEQ ID NO: 87; (334) a targeting sequence comprising SEQ ID NO: 87; (335) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 88; (336) a targeting sequence comprising amino acids 2-28 of SEQ ID NO: 87; (337) a targeting sequence comprising amino acids 5-28 of SEQ ID NO: 87; (338) a targeting sequence comprising amino acids 10-28 of SEQ ID NO: 87; (339) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 89; (340) a targeting sequence comprising SEQ ID NO: 89; (341) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 90; (342) a targeting sequence comprising amino acids 2-28 of SEQ ID NO: 89; (343) a targeting sequence comprising amino acids 5-28 of SEQ ID NO: 89; (344) a targeting sequence comprising amino acids 10-28 of SEQ ID NO: 89; (345) a targeting sequence comprising amino acids 1-93 of SEQ ID NO: 91; (346) a targeting sequence comprising SEQ ID NO: 91; (347) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 92; (348) a targeting sequence comprising amino acids 2-93 of SEQ ID NO: 91; (349) a targeting sequence comprising amino acids 10-93 of SEQ ID NO: 91; (350) a targeting sequence comprising amino acids 20-93 of SEQ ID NO: 91; (351) a targeting sequence comprising amino acids 30-93 of SEQ ID NO: 91; (352) a targeting sequence comprising amino acids 40-93 of SEQ ID NO: 91; (353) a targeting sequence comprising amino acids 50-93 of SEQ ID NO: 91; (354) a targeting sequence comprising amino acids 60-93 of SEQ ID NO: 91; (355) a targeting sequence comprising amino acids 1-130 of SEQ ID NO: 93; (356) a targeting sequence comprising SEQ ID NO: 93; (357) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 94; (358) a targeting sequence comprising amino acids 2-130 of SEQ ID NO: 93; (359) a targeting sequence comprising amino acids 10-130 of SEQ ID NO: 93; (360) a targeting sequence comprising amino acids 20-130 of SEQ ID NO: 93; (361) a targeting sequence comprising amino acids 30-130 of SEQ ID NO: 93; (362) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 122; (363) a targeting sequence consisting of amino acids 20-33 of SEQ ID NO: 1; (364) a targeting sequence consisting of amino acids 21-33 of SEQ ID NO: 1; (365) a targeting sequence consisting of amino acids 23-31 of SEQ ID NO: 1; (366) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 96; (367) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 96; (368) a targeting sequence consisting of amino acids 12-25 of SEQ ID NO: 3; (369) a targeting sequence consisting of amino acids 13-25 of SEQ ID NO: 3; (370) a targeting sequence consisting of amino acids 15-23 of SEQ ID NO: 3; (371) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 97; (372) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 98; (373) a targeting sequence consisting of amino acids 23-36 of SEQ ID NO: 5; (374) a targeting sequence consisting of amino acids 23-34 of SEQ ID NO: 5; (375) a targeting sequence consisting of amino acids 24-36 of SEQ ID NO: 5; (376) a targeting sequence consisting of amino acids 26-34 of SEQ ID NO: 5; (377) a targeting sequence consisting of amino acids 13-26 of SEQ ID NO: 7; (378) a targeting sequence consisting of amino acids 13-24 of SEQ ID NO: 7; (379) a targeting sequence consisting of amino acids 14-26 of SEQ ID NO: 7; (380) a targeting sequence consisting of amino acids 16-24 of SEQ ID NO: 7; (381) a targeting sequence consisting of amino acids 9-22 of SEQ ID NO: 9; (382) a targeting sequence consisting of amino acids 9-20 of SEQ ID NO: 9; (383) a targeting sequence consisting of amino acids 10-22 of SEQ ID NO: 9; (384) a targeting sequence consisting of amino acids 12-20 of SEQ ID NO: 9; (385) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 105; (386) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 105; (387) a targeting sequence consisting of amino acids 18-31 of SEQ ID NO: 11; (388) a targeting sequence consisting of amino acids 18-29 of SEQ ID NO: 11; (389) a targeting sequence consisting of amino acids 19-31 of SEQ ID NO: 11; (390) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 98; (391) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 98; (392) a targeting sequence consisting of amino acids 18-31 of SEQ ID NO: 13; (393) a targeting sequence consisting of amino acids 18-29 of SEQ ID NO: 13; (394) a targeting sequence consisting of amino acids 19-31 of SEQ ID NO: 13; (395) a targeting sequence consisting of amino acids 21-29 of SEQ ID NO: 13; (396) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 99; (397) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 99; (398) a targeting sequence consisting of amino acids 28-41 of SEQ ID NO: 15; (399) a targeting sequence consisting of amino acids 28-39 of SEQ ID NO: 15; (400) a targeting sequence consisting of amino acids 29-41 of SEQ ID NO: 15; (401) a targeting sequence consisting of amino acids 31-39 of SEQ ID NO: 15; (402) a targeting sequence consisting of amino acids 12-25 of SEQ ID NO: 17; (403) a targeting sequence consisting of amino acids 13-25 of SEQ ID NO: 17; (404) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 100; (405) a targeting sequence consisting of amino acids 18-31 of SEQ ID NO: 19; (406) a targeting sequence consisting of amino acids 18-29 of SEQ ID NO: 19; (407) a targeting sequence consisting of amino acids 19-31 of SEQ ID NO: 19; (408) a targeting sequence consisting of amino acids 21-29 of SEQ ID NO: 19; (409) a targeting sequence consisting of amino acids 18-31 of SEQ ID NO: 21; (410) a targeting sequence consisting of amino acids 18-29 of SEQ ID NO: 21; (411) a targeting sequence consisting of amino acids 19-31 of SEQ ID NO: 21; (412) a targeting sequence consisting of amino acids 21-29 of SEQ ID NO: 21; (413) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 101; (414) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 101; (415) a targeting sequence consisting of amino acids 9-22 of SEQ ID NO: 23; (416) a targeting sequence consisting of amino acids 9-20 of SEQ ID NO: 23; (417) a targeting sequence consisting of amino acids 10-22 of SEQ ID NO: 23; (418) a targeting sequence consisting of amino acids 12-20 of SEQ ID NO: 23; (419) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 102; (420) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 102; (421) a targeting sequence consisting of amino acids 9-22 of SEQ ID NO: 25; (422) a targeting sequence consisting of amino acids 9-20 of SEQ ID NO: 25; (423) a targeting sequence consisting of amino acids 10-22 of SEQ ID NO: 25; (424) a targeting sequence consisting of amino acids 12-20 of SEQ ID NO: 25; (425) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 103; (426) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 103; (427) a targeting sequence consisting of amino acids 15-28 of SEQ ID NO: 27; (428) a targeting sequence consisting of amino acids 15-26 of SEQ ID NO: 27; (429) a targeting sequence consisting of amino acids 16-28 of SEQ ID NO: 27; (430) a targeting sequence consisting of amino acids 18-26 of SEQ ID NO: 27; (431) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 104; (432) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 104; (433) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 33; (434) a targeting sequence consisting of amino acids 1-11 of SEQ ID NO: 33; (435) a targeting sequence consisting of amino acids 3-11 of SEQ ID NO: 33; (436) a targeting sequence consisting of amino acids 1-14 of SEQ ID NO: 35; (437) a targeting sequence consisting of amino acids 1-12 of SEQ ID NO: 35; (438) a targeting sequence consisting of amino acids 2-14 of SEQ ID NO: 35; (439) a targeting sequence consisting of amino acids 14-27 of SEQ ID NO: 43; (440) a targeting sequence consisting of amino acids 14-25 of SEQ ID NO: 43; (441) a targeting sequence consisting of amino acids 15-27 of SEQ ID NO: 43; (442) a targeting sequence consisting of amino acids 20-33 of SEQ ID NO: 45; (443) a targeting sequence consisting of amino acids 20-31 of SEQ ID NO: 45; (444) a targeting sequence consisting of amino acids 21-33 of SEQ ID NO: 45; (445) a targeting sequence consisting of amino acids 1-15 of SEQ ID NO: 106; (446) a targeting sequence consisting of amino acids 1-13 of SEQ ID NO: 106; (447) a targeting sequence consisting of amino acids 28-41 of SEQ ID NO: 47; (448) a targeting sequence consisting of amino acids 28-39 of SEQ ID NO: 47; (449) a targeting sequence consisting of amino acids 18-31 of SEQ ID NO: 53; (450) a targeting sequence consisting of amino acids 18-29 of SEQ ID NO: 53; (451) a targeting sequence consisting of amino acids 19-31 of SEQ ID NO: 53; (452) a targeting sequence comprising amino acids 18-31 of SEQ ID NO: 61; (453) a targeting sequence comprising amino acids 18-29 of SEQ ID NO: 61; (454) a targeting sequence comprising amino acids 19-31 of SEQ ID NO: 61; (455) a targeting sequence comprising amino acids 9-22 of SEQ ID NO: 65; (456) a targeting sequence comprising amino acids 9-20 of SEQ ID NO: 65; (457) a targeting sequence comprising amino acids 10-22 of SEQ ID NO: 65; (458) a targeting sequence comprising amino acids 1-15 of SEQ ID NO: 107; (459) a targeting sequence comprising amino acids 1-13 of SEQ ID NO: 107; (460) a targeting sequence comprising amino acids 12-25 of SEQ ID NO: 67; (461) a targeting sequence comprising amino acids 12-23 of SEQ ID NO: 67; (462) a targeting sequence comprising amino acids 13-25 of SEQ ID NO: 67; (463) a targeting sequence comprising amino acids 15-23 of SEQ ID NO: 67; (464) a targeting sequence comprising amino acids 23-36 of SEQ ID NO: 69; (465) a targeting sequence comprising amino acids 23-34 of SEQ ID NO: 69; (466) a targeting sequence comprising amino acids 24-36 of SEQ ID NO: 69; (467) a targeting sequence comprising amino acids 26-34 of SEQ ID NO: 69; (468) a targeting sequence comprising amino acids 27-40 of SEQ ID NO: 75; (469) a targeting sequence comprising amino acids 27-38 of SEQ ID NO: 75; (470) a targeting sequence comprising amino acids 9-22 of SEQ ID NO: 77; (471) a targeting sequence comprising amino acids 9-20 of SEQ ID NO: 77; (472) a targeting sequence comprising amino acids 10-22 of SEQ ID NO: 77; (473) a targeting sequence comprising amino acids 12-20 of SEQ ID NO: 77; (474) a targeting sequence comprising amino acids 23-36 of SEQ ID NO: 81; (475) a targeting sequence comprising amino acids 23-34 of SEQ ID NO: 81; (476) a targeting sequence comprising amino acids 24-36 of SEQ ID NO: 81; (477) a targeting sequence comprising amino acids 26-34 of SEQ ID NO: 81; (478) a targeting sequence comprising amino acids 13-26 of SEQ ID NO: 87; (479) a targeting sequence comprising amino acids 13-24 of SEQ ID NO: 87; or (480) a targeting sequence comprising amino acids 14-26 of SEQ ID NO: 87; (481) a targeting sequence comprising SEQ ID NO: 371; (482) a targeting sequence comprising SEQ ID NO: 372; (483) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 381; (484) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 382; (485) an exosporium protein fragment comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 383; (486) an exosporium protein comprising an amino acid sequence having at least 85% identity with SEQ ID NO: 384; or (487) an exosporium protein fragment comprising an amino acid sequence having at least 85% identity with SEQ ID NO:385.
For example, the targeting sequence can comprise an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%.
Alternatively, the targeting sequence can consist of an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%.
The targeting sequence can comprise an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%.
Alternatively, the targeting sequence can consist of an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%.
The targeting sequence can comprise an amino acid sequence having at least about 56% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%.
Alternatively, the targeting sequence can consist of an amino acid sequence having at least about 56% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%.
The targeting sequence can comprise an amino sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%.
Alternatively, the targeting sequence can consist of an amino sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%.
The targeting sequence can comprise an amino acid sequence having at least about 68% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 81%.
Alternatively, the targeting sequence can consist of an amino acid sequence having at least about 68% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 81%.
The targeting sequence can comprise an amino sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%.
Alternatively, the targeting sequence can consist of an amino sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%.
The targeting sequence can comprise an amino sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 81%.
Alternatively, the targeting sequence can consist of an amino sequence having at least about 75% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 81%.
The targeting sequence can comprise an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 81%.
Alternatively, the targeting sequence can consist of an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO:1, wherein the identity with amino acids 25-35 is at least about 81%.
The targeting sequence can comprise an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 90%.
Alternatively, the targeting sequence can consist of an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 90%.
For example, the targeting sequence can consist of: (a) an amino acid sequence consisting of 16 amino acids and having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 54%; (b) amino acids 1-35 of SEQ ID NO: 1; (c) amino acids 20-35 of SEQ ID NO: 1; (d) SEQ ID NO: 1; (e) SEQ ID NO: 96; or (f) SEQ ID NO: 120.
In any of the fusion proteins described herein, the fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121.
The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121.
The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121.
The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having at least 99% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121.
The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having 100% identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 44, 46, 48, 50, 52, 54, 56, 58, 95, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121.
In any of the fusion proteins described herein, the fusion protein can comprise an exosporium protein comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 122.
The fusion protein can comprise an exosporium protein comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 122.
The fusion protein can comprise an exosporium protein comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 122.
The fusion protein can comprise an exosporium protein comprising an amino acid sequence having at least 99% identity with SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 122.
The fusion protein can comprise an exosporium protein comprising an amino acid sequence having 100% identity with SEQ ID NO: 60, 62, 64, 66, 68, 70, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, or 122.
In any of the fusion proteins described herein, the fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs. 381-385.
The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having at least 95% identity with any one of SEQ ID NOs. 381-385
The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having at least 98% identity with any one of SEQ ID NOs. 381-385
The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having at least 99% identity with any one of SEQ ID NOs. 381-385
The fusion protein can comprise an exosporium protein or an exosporium protein fragment comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs. 381-385.
In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid.
In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment can comprise an alanine residue at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.
In any of the fusion proteins described herein, the targeting sequence, exosporium protein, or exosporium protein fragment can further comprise a methionine, serine, or threonine residue at the amino acid position immediately preceding the first amino acid of the targeting sequence, exosporium protein, or exosporium protein fragment or at the position of the targeting sequence that corresponds to amino acid 20 of SEQ ID NO: 1.
1. Fusion Proteins Comprising an Enzyme Having ACC Deaminase Activity
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and an enzyme having 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity are provided.
Where the fusion protein comprises an enzyme having ACC deaminase activity, the enzyme having ACC deaminase activity can comprise an amino acid sequence comprising at least one amino acid substitution relative to the sequence of a wild-type
For example, the amino acid sequence of the enzyme can comprise two amino acid substitutions relative to the sequence of the wild-type
The amino acid sequence of the enzyme can comprise a substitution of the threonine residue at position 290 of SEQ ID NO: 242 with a glutamic acid residue and a substitution of the serine residue at position 317 of SEQ ID NO: 242 with a leucine residue.
The amino acid sequence of the enzyme can comprise a substitution of the threonine residue at position 290 of SEQ ID NO: 243 with a glutamic acid residue and a substitution of the serine residue at position 317 of SEQ ID NO: 243 with a leucine residue.
The amino acid sequence of the enzyme can comprise a substitution of the threonine residue at position 290 of SEQ ID NO: 244 with a glutamic acid residue and a substitution of the serine residue at position 317 of SEQ ID NO: 244 with a leucine residue.
The amino acid sequence of the enzyme can comprise a substitution of the threonine residue at position 290 of SEQ ID NO: 245 with a glutamic acid residue and a substitution of the serine residue at position 317 of SEQ ID NO: 245 with a leucine residue.
The enzyme can comprise SEQ ID NO: 246, 247, 248, or 249.
For example, the enzyme can comprise SEQ ID NO: 249.
Alternatively, the enzyme can consist of SEQ ID NO: 246, 247, 248, or 249.
For example, the enzyme can consist of SEQ ID NO: 249.
Alternatively or in addition, the enzyme having ACC deaminase activity can comprise a Bacillus enzyme (e.g., a Bacillus thuringiensis enzyme or a Bacillus pseudomycoides enzyme).
Alternatively or in addition, the enzyme having ACC deaminase activity can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 242-245.
For example, the enzyme having ACC deaminase activity can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 242-245.
The enzyme having ACC deaminase activity can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 242-245.
The enzyme having ACC deaminase activity can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 242-245.
The enzyme having ACC deaminase activity can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 242-245.
The enzyme having ACC deaminase activity can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 242-245.
The enzyme having ACC deaminase activity can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 242-245.
The enzyme having ACC deaminase activity can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 242-245.
The enzyme having ACC deaminase activity can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 242-245.
For example, the enzyme can comprise SEQ ID NO: 245.
Alternatively, the enzyme can consist of SEQ ID NO: 245.
2. Fusion Proteins Comprising a Phospholipase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a phospholipase are provided.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a phospholipase B.
For example, the phospholipase B can comprise a phospholipase B1.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a phospholipase C. The phospholipase C comprises a phospholipase C beta 1, a phospholipase C beta 2, a phospholipase C beta 3, a phospholipase C beta 4, a phospholipase C delta 1, a phospholipase C delta 3, a phospholipase C delta 4, a phospholipase C epsilon 1, a phospholipase C gamma 1, a phospholipase C gamma 2, a phospholipase C eta 1, a phospholipase C eta 2, a phospholipase C zeta 1, or a combination of any thereof.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a phospholipase D. The phospholipase D comprises a phospholipase D1, a phospholipase D2, a phospholipase D member 3, a phospholipase D member 4, a phospholipase D member 5, a phospholipase D member 6, or a combination of any thereof.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a phospholipase A2. The phospholipase A2 comprises a Group IIA phospholipase A2, a Group IIC phospholipase A2, a Group IID phospholipase A2, a Group IIE phospholipase A2, a Group IIF phospholipase A2, a Group III phospholipase A2, a Group IVA phospholipase A2, a Group IVB phospholipase A2, a Group IVC phospholipase A2, a Group IVD phospholipase A2, a Group IVE phospholipase A2, a Group VIF phospholipase A2, a Group V phospholipase A2, a Group VI phospholipase A2, a Group VII phospholipase A2, a Group X phospholipase A2, a Group XIIA phospholipase A2, a Group XIIB phospholipase A2, a Group XV phospholipase A2, a Group XVI phospholipase A2, or a combination of any thereof.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a 1-alkyl-2-acetylglycerophosphocholine esterase.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a phosphatidylinositol deacylase.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a phosphatidylinoslitol-specific phospholipase C. For example, the phosphatidylinoslitol-specific phospholipase C can comprise a Bacillus thuringiensis phosphatidylinoslitol-specific phospholipase C.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a sphingomyelin phosphodiesterase.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a sphingomyelin phosphodiesterase D.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise an alkylglycerophosphoethanolamine phosphodiesterase.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a variant-surface-glycoprotein phospholipase C.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a glycosylphosphatidylinositol phospholipase D.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise an N-acetylphosphatidylethanolamine-hydrolysing phospholipase D.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a phosphatidylinositol diacylglycerol-lyase.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a glycosylphosphatidylinositol diacylglycerol-lyase.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a patatin-like phospholipase domain containing protein 2 (PNPLA2).
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a patatin-like phospholipase domain containing protein 3 (PNPLA3).
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a Streptomyces phospholipase.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a Clostridium phospholipase. For example, the phospholipase can comprise a Streptomyces chromofuscus phospholipase (e.g., a Streptomyces chromofuscus phospholipase).
Where the fusion protein comprises a phospholipase, the phospholipase can comprise an Acidovorax phospholipase. For example, the phospholipase can comprise a Clostridium perfringens phospholipase (e.g., a Clostridium perfringens phospholipase C).
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a Listeria phospholipase. For example, the phospholipase can comprise a Listeria monocytogenes phospholipase (e.g., a Listeria monocytogenes phospholipase C).
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a Bacillus cereus phospholipase. For example, the phospholipase can comprise a Bacillus cereus phosphatidylcholine-specific phospholipase C or a Bacillus cereus phosphatidylinositol-specific phospholipase C.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise a Bacillus licheniformis phospholipase.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 252-260 and 373-375.
For example, the phospholipase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 252-260 and 373-375.
The phospholipase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 252-260 and 373-375.
The phospholipase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 252-260 and 373-375.
The phospholipase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 252-260 and 373-375.
The phospholipase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 252-260 and 373-375.
The phospholipase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 252-260 and 373-375.
The phospholipase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 252-260 and 373-375.
The phospholipase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 252-260 and 373-375.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 373-375.
For example, the phospholipase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 373-375.
The phospholipase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 373-375.
The phospholipase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 373-375.
The phospholipase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs 373-375.
The phospholipase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 373-375.
The phospholipase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 373-375.
The phospholipase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 373-375.
The phospholipase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 373-375.
Where the fusion protein comprises a phospholipase, the phospholipase can consist essentially of an amino acid sequence having at least 70% identity to SEQ ID NO: 250 or 251.
For example, the phospholipase can consist essentially of an amino acid sequence having at least 75% identity to SEQ ID NO: 250 or 251.
The phospholipase can consist essentially of an amino acid sequence having at least 80% identity to SEQ ID NO: 250 or 251.
The phospholipase can consist essentially of an amino acid sequence having at least 85% identity to SEQ ID NO: 250 or 251.
The phospholipase can consist essentially of an amino acid sequence having at least 90% identity to SEQ ID NO: 250 or 251.
The phospholipase can consist essentially of an amino acid sequence having at least 95% identity to SEQ ID NO: 250 or 251.
The phospholipase can consist essentially of an amino acid sequence having at least 98% identity to SEQ ID NO: 250 or 251.
The phospholipase can consist essentially of an amino acid sequence having at least 99% identity to SEQ ID NO: 250 or 251.
The phospholipase can consist essentially of an amino acid sequence having 100% identity to SEQ ID NO: 250 or 251.
Where the fusion protein comprises a phospholipase, the phospholipase can consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 250.
For example, the phospholipase can consist of an amino acid sequence having at least 75% identity to SEQ ID NO: 250.
The phospholipase can consist of an amino acid sequence having at least 80% identity to SEQ ID NO: 250.
The phospholipase can consist of an amino acid sequence having at least 85% identity to SEQ ID NO: 250.
The phospholipase can consist of an amino acid sequence having at least 90% identity to SEQ ID NO: 250.
The phospholipase can consist of an amino acid sequence having at least 95% identity to SEQ ID NO: 250.
The phospholipase can consist of an amino acid sequence having at least 98% identity to SEQ ID NO: 250.
The phospholipase can consist of an amino acid sequence having at least 99% identity to SEQ ID NO: 250.
The phospholipase can consist of an amino acid sequence having 100% identity to SEQ ID NO: 250.
Where the fusion protein comprises a phospholipase, the phospholipase can consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 251.
For example, the phospholipase can consist of an amino acid sequence having at least 75% identity to SEQ ID NO: 251.
The phospholipase can consist of an amino acid sequence having at least 80% identity to SEQ ID NO: 251.
The phospholipase can consist of an amino acid sequence having at least 85% identity to SEQ ID NO: 251.
The phospholipase can consist of an amino acid sequence having at least 90% identity to SEQ ID NO: 251.
The phospholipase can consist of an amino acid sequence having at least 95% identity to SEQ ID NO: 251.
The phospholipase can consist of an amino acid sequence having at least 98% identity to SEQ ID NO: 251.
The phospholipase can consist of an amino acid sequence having at least 99% identity to SEQ ID NO: 251.
The phospholipase can consist of an amino acid sequence having 100% identity to SEQ ID NO: 251.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 256 or 260.
For example, the phospholipase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 256 or 260.
The phospholipase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 256 or 260.
The phospholipase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 256 or 260.
The phospholipase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 256 or 260.
The phospholipase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 256 or 260.
The phospholipase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 256 or 260.
The phospholipase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 256 or 260.
The phospholipase can comprise an amino acid sequence having at 100% identity to SEQ ID NO: 256 or 260.
Where the fusion protein comprises a phospholipase, the phospholipase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 256.
For example, the phospholipase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 256.
The phospholipase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 256.
The phospholipase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 256.
The phospholipase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 256.
The phospholipase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 256.
The phospholipase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 256.
The phospholipase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 256.
The phospholipase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 256.
3. Fusion Proteins Comprising a Lipase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a lipase are provided.
Where the fusion protein comprises a lipase, the lipase can comprise a carboxyl ester lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a diacylglycerol lipase alpha.
Where the fusion protein comprises a lipase, the lipase can comprise a diacylglycerol lipase beta.
Where the fusion protein comprises a lipase, the lipase can comprise a lipase A.
Where the fusion protein comprises a lipase, the lipase can comprise a hepatic lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a hormone-sensitive lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a gastric lipase.
Where the fusion protein comprises a lipase, the lipase can comprise an endothelial lipase.
Where the fusion protein comprises a lipase, the lipase can comprise member H lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a lipase family member I.
Where the fusion protein comprises a lipase, the lipase can comprise a lipase family member J.
Where the fusion protein comprises a lipase, the lipase can comprise a lipase family member K.
Where the fusion protein comprises a lipase, the lipase can comprise a lipase family member M.
Where the fusion protein comprises a lipase, the lipase can comprise a lipase family member N.
Where the fusion protein comprises a lipase, the lipase can comprise a lipoprotein lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a monoglyceride lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a pancreatic lipase-related protein 2.
Where the fusion protein comprises a lipase, the lipase can comprise a pancreatic lipase-related protein 3.
Where the fusion protein comprises a lipase, the lipase can comprise an acylglycerol lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a galactolipase.
Where the fusion protein comprises a lipase, the lipase can comprise a lipoprotein lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a Burkholderia cepacia lipase.
Where the fusion protein comprises a lipase, the lipase can comprise a Burkholderia stearothermophilus lipase.
Where the fusion protein comprises a lipase, the lipase can comprise Pseudomonas lipase.
Where the fusion protein comprises a lipase, the lipase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 262-266.
For example, the lipase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 262-266.
The lipase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 262-266.
The lipase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 262-266.
The lipase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 262-266.
The lipase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 262-266.
The lipase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 262-266.
The lipase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 262-266.
The lipase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 262-266.
4. Fusion Proteins Comprising a Xylanase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a xylanase are provided.
Where the fusion protein comprises a xylanase, the xylanase can comprise a beta-xylanase.
For example, the beta-xylanase can comprise a glucuronoarabinoxylan endo-1,4-beta-xylanase, an exo-1,4-beta-xylanase, an endo-1,4-beta-xylanase, or a combination of any thereof.
Where the fusion protein comprises a xylanase, the xylanase can comprise a Caldicellulosiruptor xylanase (e.g., a Caldicellulosiruptor saccharolyticus xylanase).
Where the fusion protein comprises a xylanase, the xylanase can comprise a Bacillus xylanase (e.g., a Bacillus subtilis or Bacillus stearothermophilus xylanase).
Where the fusion protein comprises a xylanase, the xylanase can comprise a Neocallimastix xylanase (e.g., a Neocallimastix patriciarum xylanase).
Where the fusion protein comprises a xylanase, the xylanase can comprise a Thermomyces xylanase (e.g., a Thermomyces lanuginosus xylanase).
Where the fusion protein comprises a xylanase, the xylanase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 267-273.
For example, the xylanase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 267-273.
The xylanase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 267-273.
The xylanase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 267-273.
The xylanase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 267-273.
The xylanase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 267-273.
The xylanase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 267-273.
The xylanase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 267-273.
The xylanase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 267-273.
Where the fusion protein comprises a xylanase, the xylanase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 268 or 273.
For example, the xylanase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 268 or 273.
The xylanase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 268 or 273.
The xylanase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 268 or 273.
The xylanase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 268 or 273.
The xylanase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 268 or 273.
The xylanase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 268 or 273.
The xylanase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 268 or 273.
The xylanase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 268 or 273.
Where the fusion protein comprises a xylanase, the xylanase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 268.
For example, the xylanase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 268.
The xylanase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 268.
The xylanase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 268.
The xylanase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 268.
The xylanase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 268.
The xylanase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 268.
The xylanase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 268.
The xylanase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 268.
5. Fusion Proteins Comprising a Xylosidase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a xylosidase are provided.
Where the fusion protein comprises a xylosidase, the xylosidase can comprise a Caldicellulosiruptor saccharolyticus xylosidase.
Where the fusion protein comprises a xylosidase, the xylosidase can comprise a Bacillus pumilus xylosidase.
Where the fusion protein comprises a xylosidase, the xylosidase can comprise a Bacillus subtilis xylosidase.
Where the fusion protein comprises a xylosidase, the xylosidase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 274-276.
For example, the xylosidase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 274-276.
Where the fusion protein comprises a xylosidase, the xylosidase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 275.
For example, the xylosidase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 275.
The xylosidase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 275.
The xylosidase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 275.
The xylosidase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 275.
The xylosidase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 275.
The xylosidase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 275.
The xylosidase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 275.
The xylosidase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 275.
Where the fusion protein comprises a xylosidase, the xylosidase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 276.
For example, the xylosidase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 276.
The xylosidase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 276.
The xylosidase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 276.
The xylosidase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 276.
The xylosidase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 276.
The xylosidase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 276.
The xylosidase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 276.
The xylosidase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 276.
6. Fusion Proteins Comprising a Lactonase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a lactonase are provided.
Where the fusion protein comprises a lactonase, the lactonase can comprise a Bacillus lactonase (e.g., a Bacillus thuringiensis lactonase, a Bacillus pseudomycoides lactonase, or a combination thereof).
For example, the lactonase can comprise an AiiA lactonase.
Where the fusion protein comprises a lactonase, the lactonase can comprise an Agrobacterium lactonase.
Where the fusion protein comprises a lactonase, the lactonase can comprise a Rhodococcus lactonase.
Where the fusion protein comprises a lactonase, the lactonase can comprise a Streptomyces lactonase.
Where the fusion protein comprises a lactonase, the lactonase can comprise an Arthrobacter lactonase.
Where the fusion protein comprises a lactonase, the lactonase can comprise a Sphingomonas lactonase.
Where the fusion protein comprises a lactonase, the lactonase can comprise a Pseudomonas lactonase.
Where the fusion protein comprises a lactonase, the lactonase can comprise a Klebsiella lactonase.
For example, the lactonase can comprise an Agrobacterium lactonase, a Rhodococcus lactonase, a Streptomyces lactonase, an Arthrobacter lactonase, a Sphingomonas lactonase, a Pseudomonas lactonase, a Klebsiella lactonase, or a combination of any thereof.
Where the fusion protein comprises a lactonase, the lactonase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 277 or 278.
For example, the lactonase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 277 or 278.
The lactonase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 277 or 278.
The lactonase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 277 or 278.
The lactonase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 277 or 278.
The lactonase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 277 or 278.
The lactonase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 277 or 278.
The lactonase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 277 or 278.
The lactonase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 277 or 278.
Where the fusion protein comprises a lactonase, the lactonase can comprise a lactonase that is specific for a bacterial lactone homoserine signaling molecule.
7. Fusion Proteins Comprising a Chitosanase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a chitosanase are provided.
Where the fusion protein comprises a chitosanase, the chitosanase can comprise an exo-1,4-beta-D-glucosaminidase.
Where the fusion protein comprises a chitosanase, the chitosanase can comprise an endo-1,4-beta-d-glucosaminidase.
Where the fusion protein comprises a chitosanase, the chitosanase can comprise a Streptomyces chitosanase.
Where the fusion protein comprises a chitosanase, the chitosanase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 280.
For example, the chitosanase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 280.
The chitosanase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 280.
The chitosanase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 280.
The chitosanase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 280.
The chitosanase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 280.
The chitosanase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 280.
The chitosanase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 280.
The chitosanase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 280.
Where the fusion protein comprises a chitosanase, the chitosanase can consist essentially of an amino acid sequence having at least 70% identity to SEQ ID NO: 279.
For example, the chitosanase can consist essentially of an amino acid sequence having at least 75% identity to SEQ ID NO: 279.
The chitosanase can consist essentially of an amino acid sequence having at least 80% identity to SEQ ID NO: 279.
The chitosanase can consist essentially of an amino acid sequence having at least 85% identity to SEQ ID NO: 279.
The chitosanase can consist essentially of an amino acid sequence having at least 90% identity to SEQ ID NO: 279.
The chitosanase can consist essentially of an amino acid sequence having at least 95% identity to SEQ ID NO: 279.
The chitosanase can consist essentially of an amino acid sequence having at least 98% identity to SEQ ID NO: 279.
The chitosanase can consist essentially of an amino acid sequence having at least 99% identity to SEQ ID NO: 279.
The chitosanase can consist essentially of an amino acid sequence having at least 100% identity to SEQ ID NO: 279.
Where the fusion protein comprises a chitosanase, the chitosanase can consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 279.
For example, the chitosanase can consist of an amino acid sequence having at least 75% identity to SEQ ID NO: 279.
The chitosanase can consist of an amino acid sequence having at least 80% identity to SEQ ID NO: 279.
The chitosanase can consist of an amino acid sequence having at least 85% identity to SEQ ID NO: 279.
The chitosanase can consist of an amino acid sequence having at least 90% identity to SEQ ID NO: 279.
The chitosanase can consist of an amino acid sequence having at least 95% identity to SEQ ID NO: 279.
The chitosanase can consist of an amino acid sequence having at least 98% identity to SEQ ID NO: 279.
The chitosanase can consist of an amino acid sequence having at least 99% identity to SEQ ID NO: 279.
The chitosanase can consist of an amino acid sequence having at least 100% identity to SEQ ID NO: 279.
8. Fusion Proteins Comprising a Glucanase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a glucanase are provided.
Where the fusion protein comprises a glucanase, the glucanase can comprise an alpha-1,6-glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a beta 1,3/1,4 glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a xyloglucan:xyloglucosyl transferase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a cycloheptaglucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise an oligoxyloglucan beta-glycosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a cyclohexaglucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a xyloglucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a cellulose 1,4-beta-cellobiosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a glucan endo-1,3-beta-D-glucosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a cyclomaltodextrinase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a glucan 1,3-beta-glucosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a glucan endo-1,3-alpha-glucosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise an endo-1,3(4)-beta-glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a lichenase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a laminarinase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a glucan 1,4-beta-glucosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a glucan endo-1,6-beta-glucosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a glucan 1,3-alpha-glucosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise an amylopectinase.
Where the fusion protein comprises a glucanase, the glucanase can comprise an amyloglucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise an amyloglucosidase.
Where the fusion protein comprises a glucanase, the glucanase can comprise an Acidothermus glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise an Aspergillus glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a Paenibacillus glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a Helix glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a Bacillus circulans glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a Bacillus lichenformis glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a Clostridium glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a Trichoderma reesei beta-1,4-endoglucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise a Bacillus subtilis amylase.
For example, in any of the fusion proteins comprising a glucanase, the glucanase can comprise an Acidothermus glucanase, an Aspergillus glucanase, a Paenibacillus glucanase, a Helix glucanase, a Bacillus lichenformis glucanase, a Clostridium glucanase, a Trichoderma reesei beta-1,4-endoglucanase, a Bacillus subtilis amylase, or a combination of any thereof.
Where the glucanase comprises a Helix glucanase, the Helix glucanase can comprise a Helix pomatia glucanase (e.g., a Helix pomatia beta-1,3-endoglucanase).
Where the glucanase comprises an Acidothermus glucanase, the Acidothermus glucanase can comprise an Acidothermus cellulolyticus glucanase (e.g., an Acidothermus cellulolyticus beta-1,4 endoglucanase).
Where the glucanase comprises a Clostridium glucanase, the Clostridium glucanase can comprise a Clostridium thermocellum glucanase.
Where the fusion protein comprises a glucanase, the glucanase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 281-292 and 295-302.
For example, the glucanase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 281-292 and 295-302.
The glucanase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 281-292 and 295-302.
The glucanase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 281-292 and 295-302.
The glucanase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 281-292 and 295-302.
The glucanase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 281-292 and 295-302.
The glucanase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 281-292 and 295-302.
The glucanase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 281-292 and 295-302.
The glucanase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 281-292 and 295-302.
Where the fusion protein comprises a glucanase, the glucanase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
For example, the glucanase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
The glucanase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
The glucanase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
The glucanase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
The glucanase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
The glucanase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
The glucanase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
The glucanase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 281-292, 295-296 and 298-302.
Where the fusion protein comprises a glucanase, the glucanase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 299 or 300.
For example, the glucanase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 299 or 300.
The glucanase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 299 or 300.
The glucanase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 299 or 300.
The glucanase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 299 or 300.
The glucanase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 299 or 300.
The glucanase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 299 or 300.
The glucanase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 299 or 300.
The glucanase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 299 or 300.
Where the fusion protein comprises a glucanase, the glucanase can consist essentially of an amino acid sequence having at least 70% identity to SEQ ID NO: 293 or 294.
For example, the glucanase can consist essentially of an amino acid sequence having at least 75% identity to SEQ ID NO: 293 or 294.
The glucanase can consist essentially of an amino acid sequence having at least 80% identity to SEQ ID NO: 293 or 294.
The glucanase can consist essentially of an amino acid sequence having at least 85% identity to SEQ ID NO: 293 or 294.
The glucanase can consist essentially of an amino acid sequence having at least 90% identity to SEQ ID NO: 293 or 294.
The glucanase can consist essentially of an amino acid sequence having at least 95% identity to SEQ ID NO: 293 or 294.
The glucanase can consist essentially of an amino acid sequence having at least 98% identity to SEQ ID NO: 293 or 294.
The glucanase can consist essentially of an amino acid sequence having at least 99% identity to SEQ ID NO: 293 or 294.
The glucanase can consist essentially of an amino acid sequence having 100% identity to SEQ ID NO: 293 or 294.
Where the fusion protein comprises a glucanase, the glucanase can consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 293 or 294.
For example, the glucanase can consist of an amino acid sequence having at least 75% identity to SEQ ID NO: 293 or 294.
The glucanase can consist of an amino acid sequence having at least 80% identity to SEQ ID NO: 293 or 294.
The glucanase can consist of an amino acid sequence having at least 85% identity to SEQ ID NO: 293 or 294.
The glucanase can consist of an amino acid sequence having at least 90% identity to SEQ ID NO: 293 or 294.
The glucanase can consist of an amino acid sequence having at least 95% identity to SEQ ID NO: 293 or 294.
The glucanase can consist of an amino acid sequence having at least 98% identity to SEQ ID NO: 293 or 294.
The glucanase can consist of an amino acid sequence having at least 99% identity to SEQ ID NO: 293 or 294.
The glucanase can consist of an amino acid sequence having 100% identity to SEQ ID NO: 293 or 294.
Where the fusion protein comprises a glucanase, the glucanase suitably comprises a xyloglucanase.
Where the glucanase comprises a xyloglucanase, the xyloglucanase can comprise a xyloglucan-specific endo-beta-1,4-glucanase, a xyloglucan-specific exo-beta-1,4-glucanase, or a combination thereof.
Where the glucanase comprises a xyloglucanase, the xyloglucanase can comprise a Paenibacillus xyloglucanase (e.g., a Paenibacillus pabuli xyloglucanase) or a Bacillus xyloglucanase (e.g., a Bacillus lichenformis xyloglucanase).
Where the fusion protein comprises a glucanase, the glucanase suitably comprises a lichenase.
For example, where the glucanase comprises a lichenase, the lichenase can comprise a Bacillus subtilis lichenase or a Clostridium thermocellum lichenase.
Where the fusion protein comprises a glucanase, the glucanase suitably comprises a Bacillus subtilis amylase.
9. Fusion Proteins Comprising a Protease
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a protease are provided.
Where the fusion protein comprises a protease, the protease can comprise an asparagine protease.
Where the fusion protein comprises a protease, the protease can comprise a Bacillus protease.
For example, the protease can comprise a Bacillus subtilis protease.
Where the fusion protein comprises a protease, the protease can comprise an Aspergillus protease.
Where the fusion protein comprises a protease, the protease can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 303-306.
For example, the protease can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 303-306.
The protease can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 303-306.
The protease can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 303-306.
The protease can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 303-306.
The protease can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 303-306.
The protease can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 303-306.
The protease can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 303-306.
The protease can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 303-306.
Where the fusion protein comprises a protease, the protease can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 305 or 306.
For example, the protease can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 305 or 306.
The protease can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 305 or 306.
The protease can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 305 or 306.
The protease can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 305 or 306.
The protease can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 305 or 306.
The protease can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 305 or 306.
The protease can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 305 or 306.
The protease can comprise an amino acid sequence having 100% identity to SEQ ID NO: 305 or 306.
Where the fusion protein comprises a protease, the protease can comprise an amino acid sequence having 100% identity to SEQ ID NO: 304.
Where the fusion protein comprises a protease, the protease preferably does not consist of a methionine aminopeptidase.
Where the fusion protein comprises a protease, the protease preferably does not comprise a methionine aminopeptidase.
10. Fusion Proteins Comprising a Mannanase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a mannanase are provided.
For example, where the fusion protein comprises a mannanase, the mannanase can comprise a Bacillus mannanase.
Where the fusion protein comprises a mannanase, the mannanase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 307 or 308.
For example, the mannanase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 307 or 308.
11. Fusion Proteins Comprising a Pectinase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a pectinase are provided.
Where the fusion protein comprises a pectinase, the pectinase comprises a pectolyase.
For example, the pectolyase can comprise an Aspergillus pectolyase or a Bacillus pectolyase.
Alternatively or in addition, the pectolyase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 309 or 310.
For example, the pectolyase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 309 or 310.
The pectolyase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 309 or 310.
The pectolyase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 309 or 310.
The pectolyase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 309 or 310.
The pectolyase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 309 or 310.
The pectolyase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 309 or 310.
The pectolyase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 309 or 310.
The pectolyase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 309 or 310.
12. Fusion Proteins Comprising an Acid Phosphatase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and an acid phosphatase are provided.
Where the fusion protein comprises an acid phosphatase, the acid phosphatase can comprise a Triticum acid phosphatase. For example, the acid phosphatase can comprise a Triticum aestivum acid phosphatase.
Alternatively or in addition, where the fusion protein comprises an acid phosphatase, the acid phosphatase can comprise a Bacillus acid phosphatase. For example, the acid phosphatase can comprise a Bacillus thuringiensis acid phosphatase.
Alternatively or in addition, where the fusion protein comprises an acid phosphatase, the acid phosphatase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity, or 100% identity to any one of SEQ ID NOs: 311, 312, and 378.
For example, the acid phosphatase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 311.
The acid phosphatase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 312.
The acid phosphatase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 378.
The acid phosphatase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 378.
The acid phosphatase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 378.
The acid phosphatase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 378.
The acid phosphatase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 378.
The acid phosphatase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 378.
The acid phosphatase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 378.
The acid phosphatase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 378.
The acid phosphatase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 378.
13. Fusion Proteins Comprising a Phytase
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a phytase are provided.
Where the fusion protein comprises a phytase, the phytase can comprise a Triticum phytase. For example, the phytase can comprise a Triticum aestivum phytase.
Alternatively, or in addition, the phytase can comprise a Bacillus phytase. For example, the phytase can comprise a Bacillus thuringiensis phytase.
Alternatively or in addition, the phytase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 313-315 and 380.
For example, the phytase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 313-315 and 380.
The phytase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 313-315 and 380.
The phytase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 313-315 and 380.
The phytase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 313-315 and 380.
The phytase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 313-315 and 380.
The phytase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 313-315 and 380.
The phytase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 313-315 and 380.
The phytase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 313-315 and 380.
Alternatively or in addition, the phytase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO. 380.
For example, the phytase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO. 380.
The phytase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO. 380.
The phytase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO. 380.
The phytase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO. 380.
The phytase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO. 380.
The phytase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO. 380.
The phytase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO. 380.
The phytase can comprise an amino acid sequence having 100% identity to SEQ ID NO. 380.
14. Fusion Proteins Comprising an Expansin Protein
Fusion proteins comprising a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and an expansin protein are provided.
Where the fusion protein comprises an expansin protein, the expansin protein can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 316.
For example, the expansin can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 316.
The expansin can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 316.
The expansin can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 316.
The expansin can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 316.
The expansin can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 316.
The expansin can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 316.
The expansin can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 316.
The expansin can comprise an amino acid sequence having 100% identity to SEQ ID NO: 316.
15. Optional Inclusion of Signal Peptides in the Fusion Proteins
In any of the fusion proteins described herein, any of the signal peptides described herein can be present.
Where the signal peptide is present, it is preferably present at the amino terminus of the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the phytase, the acid phosphatase, the pectinase, the mannanase, or the expansin protein.
The signal peptide preferably immediately precedes the first amino acid of the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the phytase, the acid phosphatase, the pectinase, the mannanase, or the expansin protein.
Thus, for example, a fusion protein comprising the targeting sequence of SEQ ID NO: 96 and the lichenase of SEQ ID NO: 295 can have the following structures, shown with and without the signal sequence (e.g., the signal sequence of SEQ ID NO: 335):
Thus, in any of the fusion proteins described herein, the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the expansin protein, the phytase, the acid phosphatase, the pectinase, or the mannanase can further comprise a signal peptide.
Where the fusion protein comprises a signal peptide, the signal peptide can be present at the amino terminus of the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the expansin protein, the phytase, the acid phosphatase, the pectinase, or the mannanase.
In any of the fusion proteins described herein comprising a signal peptide, the signal peptide can comprise an amino acid sequence having at least 70% identity with any one of SEQ ID NOs. 317-360 and 376-379.
For example, the signal peptide can comprise an amino acid sequence having at least 75% identity with any one of SEQ ID NOs. 317-360 and 376-379.
The signal peptide can comprise an amino acid sequence having at least 80% identity with any one of SEQ ID NOs. 317-360 and 376-379.
The signal peptide can comprise an amino acid sequence having at least 85% identity with any one of SEQ ID NOs. 317-360 and 376-379.
The signal peptide can comprise an amino acid sequence having at least 90% identity with any one of SEQ ID NOs. 317-360 and 376-379.
The signal peptide can comprise an amino acid sequence having at least 95% identity with any one of SEQ ID NOs. 317-360 and 376-379.
The signal peptide can comprise an amino acid sequence having at least 98% identity with any one of SEQ ID NOs. 317-360 and 376-379.
The signal peptide can comprise an amino acid sequence having at least 99% identity with any one of SEQ ID NOs. 317-360 and 376-379.
The signal peptide can comprise an amino acid sequence having 100% identity with any one of SEQ ID NOs. 317-360 and 376-379.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 252. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 317 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 253. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 318 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 254. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 319 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 255. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 320 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 321 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 256. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 322 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 257. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 323 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 373. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 376 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
The fusion protein can comprise a phospholipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 375. The phospholipase further comprises a signal peptide comprising SEQ ID NO: 377 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a lipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 263. The lipase further comprises a signal peptide comprising SEQ ID NO: 324 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a lipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 264. The lipase further comprises a signal peptide comprising SEQ ID NO: 325 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a lipase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
The fusion protein can comprise a lipase comprising an amino acid sequence having 100% identity to SEQ ID NO: 265. The lipase further comprises a signal peptide comprising SEQ ID NO: 326 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a xylanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 100% identity to SEQ ID NO: 267. The xylanase further comprises a signal peptide comprising SEQ ID NO: 327 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a xylanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 268. The xylanase further comprises a signal peptide comprising SEQ ID NO: 328 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a xylanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 269. The xylanase further comprises a signal peptide comprising SEQ ID NO: 329 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a xylanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 270. The xylanase further comprises a signal peptide comprising SEQ ID NO: 330 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a xylanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
The fusion protein can comprise a xylanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 273. The xylanase further comprises a signal peptide comprising SEQ ID NO: 331 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having at least 70% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
The fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having at least 75% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
The fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having at least 80% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
The fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having at least 85% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
The fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having at least 90% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
The fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having at least 95% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
The fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having at least 98% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
The fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having at least 99% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
The fusion protein can comprise a chitosanase consisting essentially of an amino acid sequence having 100% identity to SEQ ID NO: 279. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 332 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a chitosanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
The fusion protein can comprise a chitosanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
The fusion protein can comprise a chitosanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
The fusion protein can comprise a chitosanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
The fusion protein can comprise a chitosanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
The fusion protein can comprise a chitosanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
The fusion protein can comprise a chitosanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
The fusion protein can comprise a chitosanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
The fusion protein can comprise a chitosanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 280. The chitosanase further comprises a signal peptide comprising SEQ ID NO: 333 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having at least 70% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
The fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having at least 75% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
The fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having at least 80% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
The fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having at least 85% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
The fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having at least 90% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
The fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having at least 95% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
The fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having at least 98% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
The fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having at least 99% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
The fusion protein can comprise a glucanase consisting essentially of an amino acid sequence having 100% identity to SEQ ID NO: 294. The glucanase further comprises a signal peptide comprising SEQ ID NO: 334 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a glucanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 295. The glucanase further comprises a signal peptide comprising SEQ ID NO: 335 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a glucanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 296. The glucanase further comprises a signal peptide comprising SEQ ID NO: 336 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a glucanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 297. The glucanase further comprises a signal peptide comprising SEQ ID NO: 337 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a glucanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 298. The glucanase further comprises a signal peptide comprising SEQ ID NO: 338 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a glucanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 299. The glucanase further comprises a signal peptide comprising SEQ ID NO: 339 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a glucanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 300. The glucanase further comprises a signal peptide comprising SEQ ID NO: 340 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a glucanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
The fusion protein can comprise a glucanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 302. The glucanase further comprises a signal peptide comprising SEQ ID NO: 341 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a protease comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having 100% identity to SEQ ID NO: 304. The protease further comprises a signal peptide comprising SEQ ID NO: 342 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a protease comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
The fusion protein can comprise a protease comprising an amino acid sequence having 100% identity to SEQ ID NO: 306. The protease further comprises a signal peptide comprising SEQ ID NO: 343 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a mannanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 307. The mannanase further comprises a signal peptide comprising SEQ ID NO: 344 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a mannanase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
The fusion protein can comprise a mannanase comprising an amino acid sequence having 100% identity to SEQ ID NO: 308. The mannanase further comprises a signal peptide comprising SEQ ID NO: 345 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having 100% identity to SEQ ID NO: 309. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 346 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
The fusion protein can comprise a pectolyase comprising an amino acid sequence having 100% identity to SEQ ID NO: 310. The pectolyase further comprises a signal peptide comprising SEQ ID NO: 347 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having 100% identity to SEQ ID NO: 311. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 348 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having 100% identity to SEQ ID NO: 312. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 349 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
The fusion protein can comprise an acid phosphatase comprising an amino acid sequence having 100% identity to SEQ ID NO: 378. The acid phosphatase further comprises a signal peptide comprising SEQ ID NO: 379 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phytase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having 100% identity to SEQ ID NO: 313. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phytase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having 100% identity to SEQ ID NO: 314. The phytase further comprises a signal peptide comprising SEQ ID NO: 350 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phytase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having 100% identity to SEQ ID NO: 315. The phytase further comprises a signal peptide comprising SEQ ID NO: 351 at its amino terminus.
Where the fusion protein comprises a signal peptide, the fusion protein can comprise a phytase comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus.
The fusion protein can comprise a phytase comprising an amino acid sequence having 100% identity to SEQ ID NO: 380. The phytase further comprises a signal peptide comprising SEQ ID NO: 354 at its amino terminus. Where the fusion protein comprises a signal peptide, the fusion protein can comprise an expansin protein comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
The fusion protein can comprise an expansin protein comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
The fusion protein can comprise an expansin protein comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
The fusion protein can comprise an expansin protein comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
The fusion protein can comprise an expansin protein comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
The fusion protein can comprise an expansin protein comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
The fusion protein can comprise an expansin protein comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
The fusion protein can comprise an expansin protein comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
The fusion protein can comprise an expansin protein comprising an amino acid sequence having 100% identity to SEQ ID NO: 316. The expansin protein further comprises a signal peptide comprising SEQ ID NO: 352 at its amino terminus.
D. Methods for Making the Fusion Proteins
Any of the fusion proteins described herein can be made using standard cloning and molecular biology methods known in the art. For example, a gene encoding a protein or peptide of interest (e.g., an enzyme having ACC deaminase activity, a phospholipase, a lipase, a xylanase, a xylosidase, a lactonase, a chitosanase, a protease, a glucanase, an expansin protein, a phytase, the an phosphatase, a pectinase, or a mannanase) can be amplified by polymerase chain reaction (PCR) and ligated to DNA coding for any of the targeting sequences, exosporium proteins, or exosporium protein fragments described herein, to form a DNA molecule that encodes the fusion protein. The DNA molecule encoding the fusion protein can be cloned into any suitable vector, for example a plasmid vector. The vector suitably comprises a multiple cloning site into which the DNA molecule encoding the fusion protein can be easily inserted. The vector also suitably contains a selectable marker, such as an antibiotic resistance gene, such that bacteria transformed, transfected, or mated with the vector can be readily identified and isolated. Where the vector is a plasmid, the plasmid suitably also comprises an origin of replication. Alternatively, DNA coding for the fusion protein can be integrated into the chromosomal DNA of the B. cereus family member or spore-forming bacterium host.
E. Tags, Markers, and Linkers that can be Included in the Fusion Proteins
Any of the fusion proteins described herein can also comprise additional polypeptide sequences that are not part of the targeting sequence, exosporium protein, exosporium protein fragment, or the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the expansin protein, the phytase, the acid phosphatase, the pectinase, or the mannanase. For example, the fusion protein can include tags or markers to facilitate purification or visualization of the fusion protein (e.g., a polyhistidine tag or a fluorescent protein such as GFP or YFP) or visualization of recombinant Bacillus cereus family member spores expressing the fusion protein.
Expression of fusion proteins on the exosporium of a Bacillus cereus family member using the targeting sequences, exosporium proteins, and exosporium protein fragments described herein is enhanced due to a lack of secondary structure in the amino-termini of these sequences, which allows for native folding of the fused proteins and retention of activity. Proper folding can be further enhanced by the inclusion of a short amino acid linker between the targeting sequence, exosporium protein, exosporium protein fragment, spore coat protein, and the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the expansin protein, the phytase, the acid phosphatase, the pectinase, or the mannanase.
Thus, any of the fusion proteins described herein can comprise an amino acid linker between the targeting sequence, the exosporium protein, or the exosporium protein fragment and the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the expansin protein, the phytase, the acid phosphatase, the pectinase, or the mannanase.
The linker can comprise a polyalanine linker or a polyglycine linker. A linker comprising a mixture of both alanine and glycine residues can also be used.
For example, in a fusion protein where the targeting sequence comprises SEQ ID NO: 1, a fusion protein can have one of the following structures:
Alternatively or in addition, the linker can comprise a protease recognition site. Inclusion of a protease recognition site allows for targeted removal, upon exposure to a protease that recognizes the protease recognition site, of the enzyme having ACC deaminase activity, the phospholipase, the lipase, the xylanase, the xylosidase, the lactonase, the chitosanase, the protease, the glucanase, the expansin protein, the phytase, the acid phosphatase, the pectinase, or the mannanase.
Where the fusion protein comprises both a linker and signal peptide, the linker would typically be amino-terminal to the signal peptide. For example, where the fusion protein comprises SEQ ID NO: 96, a polyalanine linker, the signal sequence of SEQ ID NO: 335, and the lichenase of SEQ ID NO: 295, these elements would typically be arranged in the following order within the fusion protein, going from the amino-terminus of the fusion protein to the carboxy-terminus:
The invention further relates to recombinant Bacillus cereus family members that express a fusion protein. The fusion protein can be any of the fusion proteins described in Section I above.
The recombinant Bacillus cereus family member can comprise any Bacillus species that is capable of producing an exosporium. For example, the recombinant Bacillus cereus family member can comprise Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus samanii, Bacillus gaemokensis, Bacillus weihenstephensis, Bacillus toyoiensis, or a combination of any thereof. The recombinant Bacillus cereus family member suitably comprises Bacillus thuringiensis or Bacillus mycoides.
To generate a recombinant Bacillus cereus family member expressing a fusion protein, any Bacillus cereus family member can be conjugated, transduced, or transformed with a vector encoding the fusion protein using standard methods known in the art (e.g., by electroporation). The bacteria can then be screened to identify transformants by any method known in the art. For example, where the vector includes an antibiotic resistance gene, the bacteria can be screened for antibiotic resistance. Alternatively, DNA encoding the fusion protein can be integrated into the chromosomal DNA of a B. cereus family member host. The recombinant Bacillus cereus family member can then exposed to conditions which will induce sporulation. Suitable conditions for inducing sporulation are known in the art. For example, the recombinant Bacillus cereus family member can be plated onto agar plates, and incubated at a temperature of about 30° C. for several days (e.g., 3 days).
Thus, the recombinant Bacillus cereus family member can be in the form of a spore.
Inactivated strains, non-toxic strains, or genetically manipulated strains of any of the above species can also suitably be used. For example, a Bacillus thuringiensis that lacks the Cry toxin can be used. Alternatively or in addition, once the recombinant B. cereus family member spores expressing the fusion protein have been generated, they can be inactivated to prevent further germination once in use. Any method for inactivating bacterial spores that is known in the art can be used. Suitable methods include, without limitation, heat treatment, gamma irradiation, x-ray irradiation, UV-A irradiation, UV-B irradiation, chemical treatment (e.g., treatment with glutaraldehyde, formaldehyde, hydrogen peroxide, acetic acid, bleach, or any combination thereof), or a combination thereof. Alternatively, spores derived from nontoxigenic strains, or genetically or physically inactivated strains, can be used.
Thus, the recombinant Bacillus cereus family member can be in the form of a spore, wherein the spore is inactivated.
The recombinant Bacillus cereus family member can coexpress two or more of any of the fusion proteins described herein. For example, the recombinant Bacillus cereus family member can coexpress at least one fusion protein that comprises a xyloglucanase together with a fusion protein that comprises a mannanase.
Many Bacillus cereus family member strains have inherent beneficial attributes. For example, some strains have plant-growth promoting effects. Other strains are endophytic. Some strains are both endophytic and have plant-growth promoting effects.
Thus, any of the recombinant Bacillus cereus family members described herein can comprise a plant-growth promoting strain of bacteria, an endophytic strain of bacteria, or a strain of bacteria that is both plant-growth promoting and endophytic.
The plant-growth promoting strain of bacteria can comprise a strain of bacteria that produces an insecticidal toxin (e.g., a Cry toxin), produces a fungicidal compound (e.g., a β-1,3-glucanase, a chitosanase, a lyticase, or a combination of any thereof), produces a nematocidal compound (e.g., a Cry toxin), produces a bacteriocidal compound, is resistant to one or more antibiotics, comprises one or more freely replicating plasmids, binds to plant roots, colonizes plant roots, forms biofilms, solubilizes nutrients, secretes organic acids, or any combination thereof.
For example, the recombinant Bacillus cereus family member can comprise: (a) Bacillus mycoides BT155 (NRRL No. B-50921), (b) Bacillus mycoides EE118 (NRRL No. B-50918), (c) Bacillus mycoides EE141 (NRRL No. B-50916), (d) Bacillus mycoides BT46-3 (NRRL No. B-50922), (e) Bacillus cereus family member EE128 (NRRL No. B-50917), (f) Bacillus thuringiensis BT013A (NRRL No. B-50924), (g) Bacillus cereus family member EE349 (NRRL No. B-50928), (h) Bacillus cereus family member EE-B00377 (NRRL B-67119), (i) Bacillus pseudomycoides EE-B00366 (NRRL B-67120), (j) Bacillus mycoides EE-B00363 (NRRL B-67121), (k) Bacillus cereus family member EE439 (NRRL B-50979); (l) Bacillus thuringiensis EE417 (NRRL B-50974); (m) Bacillus cereus EE444 (NRRL B-50977); (n) Bacillus thuringiensis EE319 (NRRL B-50983); (o) Bacillus mycoides EE116 (NRRL No. B-50919); or (p) Bacillus thuringiensis EE-B00184 (NRRL B-67122). For example, the recombinant Bacillus cereus family member can comprise Bacillus thuringiensis BT013A (NRRL No. B-50924) or Bacillus cereus family member EE349 (NRRL No. B-50928).
Each of the strains (a) through (g) and (o) was deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), having the address 1815 North University Street, Peoria, Illinois 61604 U.S.A., on Mar. 10, 2014, and is identified by the NRRL deposit number provided in parentheses. Bacillus thuringiensis BT013A is also known as Bacillus thuringiensis 4Q7. Each of the strains (h) through (j) was deposited with the USDA ARS on Aug. 19, 2015, and is identified by the NRRL deposit number provided in parentheses. Each of the strains (k), (l), and (m) were deposited with the USDA ARS on Sep. 10, 2014, and is identified by the NRRL deposit number provided in parentheses. Strain (n) was deposited with the USDA ARS on Sep. 17, 2014, and is identified by the NRRL deposit number provided in parentheses. Strain (p) was deposited with the USDA ARS on Aug. 19, 2015, and is identified by the NRRL deposit number provided in parentheses. It is hereby certified that the deposits were made in compliance with the terms of the Budapest Treaty and that: (i) during the pendency of this application, access to the deposited organisms will be afforded to the Commissioner upon request; (ii) all restrictions upon availability to the public of the deposited materials will be irrevocably removed upon granting of the patent, subject to 37 C.F.R. § 1.808(b); (iii) the deposit will be maintained for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer; and (iv) the deposit will be replaced if it should ever become non-viable.
These strains were isolated from the rhizospheres of various vigorous plants or from the inside of plants (for endophytic strains) and were identified by their 16S rRNA sequences (listed below in Table 18), and through biochemical assays. The strains were identified at least to their genus designation by means of conventional biochemistry and morphological indicators. Biochemical assays for confirmed Gram-positive strains such as Bacillus included growth on PEA medium and nutrient agar, microscopic examination, growth on 5% and 7.5% NaCl medium, growth at pH 5 and pH 9, growth at 42° C. and 50° C., the ability to produce acid upon fermentation with cellobiose, lactose, glycerol, glucose, sucrose, d-mannitol, and starch; fluorescent pigment production; gelatin hydrolysis; nitrate reduction; catalase production, starch hydrolysis; oxidase reaction, urease production and motility. Identification of these strains and demonstration of their plant-growth promoting effects and/or ability to grow endophytically are described further in the Examples hereinbelow.
Bacillus mycoides EE118
Bacillus mycoides EE141
Bacillus mycoides BT46-3
Bacillus cereus family member EE128
Bacillus cereus family member EE349
Bacillus mycoides BT155
Bacillus cereus family member EE439
Bacillus thuringiensis EE417
Bacillus cereus EE444
Bacillus thuringiensis EE319
Bacillus thuringiensis EE-B00184
Bacillus mycoides EE-B00363
Bacillus pseudomycoides EE-B00366
Bacillus cereus family member EE-B00377
Bacillus thuringiensis BT013A
Bacillus mycoides EE116
The recombinant Bacillus cereus family member can comprises an endophytic strain of bacteria. For example, the endophytic strain of bacteria can comprise Bacillus cereus family member EE349, Bacillus cereus family member EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, or Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B00184, Bacillus cereus family member EE-B00377; Bacillus pseudomycoides EE-B00366; or Bacillus mycoides EE-B00363.
Bacillus cereus family member EE349 is also a plant growth promoting strain of bacteria and is described above. As discussed further in the Examples below, Bacillus cereus family member EE349 has also been found to be endophytic.
The recombinant Bacillus cereus family member can comprise an inactivating mutation in its BclA gene, its CotE gene, or its CotO gene (e.g., a knock-out of the BclA gene, CotE gene, or CotO gene). For example, the recombinant Bacillus cereus family member can comprise an inactivating mutation in its BclA gene (e.g., a knock-out of the BclA gene). It has been found that expression of fusion proteins in a recombinant Bacillus cereus family member having such a mutation results in increased expression levels of the fusion protein.
As described further below in Section IV, the recombinant Bacillus cereus family member can comprise a mutation or other modification that allows for collection of exosporium fragments comprising the fusion proteins from spores of the recombinant Bacillus cereus family member.
III. Promoters for Expression of Fusion Proteins in Recombinant Bacillus cereus Family Members
The DNA encoding the fusion proteins used in the recombinant Bacillus cereus family members, exosporium fragments, formulations, plant seeds, and methods, described herein is suitably under the control of a sporulation promoter which will cause expression of the fusion protein on the exosporium of a B. cereus family member endospore (e.g., a native bclA promoter from a B. cereus family member).
Thus, any of the fusion proteins described above in Section I can be expressed in the recombinant Bacillus cereus family member under the control of a sporulation promoter that is native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein, or a portion of such a promoter.
Any of the fusion proteins can be expressed under the control of a high-expression sporulation promoter.
The high-expression sporulation promoter can comprise a sigma-K sporulation-specific polymerase promoter sequence.
For ease of reference, illustrative nucleotide sequences for promoters that can be used to express any of the fusion proteins in a recombinant Bacillus cereus family member are provided in Table 19 below, together with their SEQ ID NOs. Table 19 also provides illustrative minimal promoter sequences for many of the promoters. In Table 19, sigma-K sporulation-specific polymerase promoter sequences in the promoters are indicated by bold and underlined text. Several of the sequences have multiple sigma K sequences that overlap with one another. The overlaps are indicated by double underlining in the table. The promoter sequences are immediately upstream of the start codon for each of the indicated genes. In other words, in the sequences shown in Table 19 below, the last nucleotide of the promoter sequence immediately precedes the first nucleotide of the start codon for the coding region of the gene encoding the indicated protein.
Bacillus cereus family members
ATATCTTA
TTAATGTAAATACAAACAAGAAGATAAGGA
CATTAACTG
AAAGTAGAAACAGATAAAGGAGTGAAAAAC
A
GTAGTGTATGTCAGAACTCACGAGAAGGAGTGAACATA
TTA
TATTGTGCTTGTCCAAAACATGTGGTTATTACTCACGCGAT
TG
ACTAAATATTGAAAAAATATTGAATTTTTAATAAAATTTAA
AATT
AATATGTGAGTCATGAACATAATTAAATAATGTTTTCAA
GATA
TTGGAGTTGAGGAGATTTGGTCACAATCTCAAGACCTTT
ATA
TTTTCATAATACGAGAAAAAGCGGAGTTTAAAAGAATGAG
AATA
TTAACTGATTTTCTAACATCAAGGAGGGTACAT
ATATAATA
TTAACTGATTTTCTAACATCAAGGAGGGTACAT
CATAACGGA
GTAGAATATATGCTCGTGTCTAAAGGTTTAGAGC
AAAA
GAATAAGCTTCGCATATCGTGTATAAGGAAGTGTATTT
ATATTGAA
AAGTATTATATTGTAAAAGGTCATATGAAACGTG
CTA
AACTTTCCATTTTTTTAAATTGTTCAAGTAGTTTAAGATTT
ATCTACTA
TATAATGAACGCTTTATGGAGGTGAATTT
thuringiensis serovar
CTA
GGTGGTAGGCATCATCATGAAGGAGGAATAGAT
ACATATTTTC
TTGTCCGCCCATACACTAGGTGGTAGGCATCAT
TTAATA
ATTATGACAAGCAATCATAGGAGGTTACTAC
CATACTATA
AAATGAAAGAAATGAAATGAAAAATAGCGGAAA
CTTT
AGGTATTGTTTTGTTTTTTTATTGTGACAGTTCAAGAACG
AATTTA
AATGGTTTCCCGAAAGATAAAGAGCTGTTGGATCGAA
A
AAAACAGGTAAGATGGCCTAACGAGTTTGGAGGTAGGAGA
thuringiensis serovar
CGATA
TTATGTAATGTAACGTATAGGTGGGGATACTACT
CTACTA
TATAATGAACGCTTTATGGAGGTGAATTT
TTTACTA
TTAGTAATATGAATTTTTCAGAGGTGGATTTTATT
A
TAACGATAAATGAAACTTATGTATATGTATGGTAACTGTATA
CATAAGATG
AGTCATATGTTTTAAATTGTAGTAATGAAAAAC
ATA
TTGGAGTTGAGGAGATTTGGTCACAATCTCAAGACCTTTTT
ATTAACTG
AAAGTAGAAACAGATAAAGGAGTGAAAAAC
A
AGTACCGGAAAGTGTTTGTAGAACATTACAAAGATATATTAT
TAATGTT
GTATGACATTCCGTAGGAGGCACTTATA
The sigma-K sporulation-specific polymerase promoter sequences in the promoter sequences shown in Table 19 result in high expression levels of the fusion protein during late sporulation. The consensus sequence for the sigma-K sporulation-specific polymerase promoter sequence is CATANNNTN (SEQ ID NO: 361); however, this sequence can comprise up to two mutations and still be functional. The sigma-K sporulation-specific polymerase promoter sequence is generally found upstream of the ribosome binding site (RB S).
Promoters having a high degree of sequence identity to any of the sequences shown above in Table 19 can also be used to express the fusion proteins.
For example, the fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 80% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37-42 and 123-191.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 85% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37-42 and 123-191.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 90% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37-42 and 123-191.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 95% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37-42 and 123-191.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 98% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37-42 and 123-191.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 99% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37-42 and 123-191.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having 100% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37-42 and 123-191.
For example, fusion protein can be expressed under the control of a BclA promoter (e.g., SEQ ID NO: 149, 150, 175, 189, or 190), a CotY promoter (e.g., SEQ ID NO: 41, 42, or 181), an ExsY promoter (e.g., SEQ ID NO: 37, 38, or 180), or a rhamnose promoter (e.g., SEQ ID NO: 185), or a promoter having a high degree of sequence identity to any of these promoters.
Thus, for example, the fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 80% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37, 38, 41, 42, 149, 150, 175, 180, 181, 185, 189, or 190.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 85% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37, 38, 41, 42, 149, 150, 175, 180, 181, 185, 189, or 190.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 90% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37, 38, 41, 42, 149, 150, 175, 180, 181, 185, 189, or 190.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 95% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37, 38, 41, 42, 149, 150, 175, 180, 181, 185, 189, or 190.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 98% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37, 38, 41, 42, 149, 150, 175, 180, 181, 185, 189, or 190.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having at least 99% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37, 38, 41, 42, 149, 150, 175, 180, 181, 185, 189, or 190.
The fusion protein can be expressed under the control of a promoter comprising a nucleic acid sequence having 100% identity with a nucleic acid sequence of any one of SEQ ID NOs: 37, 38, 41, 42, 149, 150, 175, 180, 181, 185, 189, or 190.
The fusion protein can be expressed under the control of a promoter comprising a sigma-K sporulation specific polymerase promoter sequence, wherein the sigma-K sporulation-specific polymerase promoter sequence or sequences have 100% identity with the corresponding nucleotides of any of SEQ ID NOs: 37-42 and 123-191.
The fusion proteins can be expressed under the control of a promoter that is native to the targeting sequence, exosporium protein, or exosporium protein fragment of the fusion protein. Thus, for example, where the targeting sequence is derived from BclA, the fusion protein can be expressed under the control of a native BclA promoter (e.g., SEQ ID NO: 149, 150, 175, 189 or 190).
Table 19 also provides illustrative minimal promoter sequences. The fusion proteins can be expressed under any of these minimal promoter sequences.
Furthermore, the fusion protein can be expressed under a portion of any of the promoters listed above in Table 19, so long as the portion of the promoter includes a sigma-K sporulation-specific polymerase promoter sequence. For example, the fusion protein can be expressed under a promoter region that comprises the first 25, 50, 100, 150, 200, 250, or 300 nucleotides upstream of the start codon, so long as that region comprises a sigma-K sporulation-specific polymerase promoter sequence.
IV. Mutations and Other Genetic Alterations to Recombinant Bacillus cereus Family Members that Allow for Collection of Free Exosporium and Exosporium Fragments Derived from Such Recombinant Bacillus cereus Family Members
As is described further hereinbelow, the recombinant Bacillus cereus family members that express fusion proteins comprising a protein or peptide of interest (e.g., an enzyme having ACC deaminase activity, a phospholipase, a lipase, a xylanase, a xylosidase, a lactonase, a chitosanase, a protease, a glucanase, a phytase, an acid phosphatase, a pectinase, a mannanase, and/or an expansin protein) and a targeting sequence, an exosporium protein, or an exosporium protein fragment that targets the fusion protein to the exosporium of the recombinant Bacillus cereus family member can be used for various purposes, including delivering the proteins or peptides of interest plants, seeds, a plant growth medium, or an area surrounding a seed or a plant (e.g., via soil drench, foliar application, or as a seed treatment). However, in some cases, the presence of the living microorganisms may not be desirable, and instead, it would be desirable to separate the living spore from the fusion proteins in the exosporium on the outside surface of the spore. For example, in some applications it will be desirable to increase enzyme activity without concern for spore integrity. In such situations, use of exosporium fragments that have been separated from the spores may be preferred over the use of living microorganisms having the enzyme on their exosporium.
In addition, for some uses, it may be desirable to reduce the density of the product. In such instances, it would be desirable to separate the dense spore from the exosporium (containing the fusion proteins). Furthermore, under some circumstances the presence of live spores would lead to potential for bacterial growth in a product, which would be undesirable for some applications.
Mutations or other genetic alterations (e.g., overexpression of a protein) can be introduced into the recombinant Bacillus cereus family members that allow free exosporium to be separated from spores of the recombinant Bacillus cereus family member. This separation process yields exosporium fragments that contain the fusion proteins but that are substantially free of the spores themselves. By “substantially free of spores” it is meant that once the free exosporium is separated from the spores, a preparation is obtained that contains less than 5% by volume of spores, preferably less than 3% by volume of spores, even more preferably less than 1% by volume of spores, and most preferably contains no spores or if spores are present, they are undetectable. These exosporium fragments can be used in place of the recombinant Bacillus cereus family members themselves in any of the formulations, plant seeds, and methods described herein.
Exosporium fragments derived from spores of a recombinant Bacillus cereus family member can be used in any of the formulations, plant seeds, and methods described herein. The recombinant Bacillus cereus family member expresses any of the fusion proteins described herein. The recombinant Bacillus cereus family member also comprises a mutation or expresses a protein, wherein the expression of the protein is increased as compared to the expression of the protein in a wild-type Bacillus cereus family member under the same conditions. The mutation or the increased expression of the protein results in Bacillus cereus family member spores having an exosporium that is easier to remove from the spore as compared to the exosporium of a wild-type spore.
The recombinant Bacillus cereus family member: (i) can comprise a mutation in a CotE gene; (ii) can express an ExsY protein, wherein the expression of the ExsY protein is increased as compared to the expression of the ExsY protein in a wild-type Bacillus cereus family member under the same conditions, and wherein the ExsY protein comprises a carboxy-terminal tag comprising a globular protein; (iii) can express a BclB protein, wherein the expression of the BclB protein is increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions; (iv) can express a YjcB protein, wherein the expression of the YjcB protein is increased as compared to the expression of the YjcB protein in a wild-type Bacillus cereus family member under the same conditions; (v) can comprise a mutation in an ExsY gene; (vi) can comprise a mutation in a CotY gene; (vii) can comprise a mutation in an ExsA gene; or (viii) can comprise a mutation in a CotO gene.
The recombinant Bacillus cereus family member can comprise a mutation in the CotE gene, such as a knock-out of the CotE gene or a dominant negative form of the CotE gene. The mutation in the CotE gene can partially or completely inhibit the ability of CotE to attach the exosporium to the spore.
The recombinant Bacillus cereus family member can express an ExsY protein. The ExsY protein comprises a carboxy-terminal tag comprising a globular protein (e.g., a green fluorescent protein (GFP) or a variant thereof), and the expression of the ExsY protein is increased as compared to the expression of the ExsY protein in a wild-type Bacillus cereus family member under the same conditions. The globular protein can have a molecular weight of between 25 kDa and 100 kDa. Expression of the ExsY protein comprising the carboxy-terminal tag comprising a globular protein can inhibit binding of the ExsY protein to its targets in the exosporium.
The recombinant Bacillus cereus family member can express a BclB protein. Expression of the BclB protein can result in the formation of a fragile exosporium. The expression of the BclB protein can be increased as compared to the expression of the BclB protein in a wild-type Bacillus cereus family member under the same conditions.
The recombinant Bacillus cereus family member can express a YjcB protein. Expression of the YjcB protein can cause the exosporium to form in pieces rather than in a complete structure. The expression of the YjcB protein can be increased as compared to the expression of the YjcB protein in a wild-type Bacillus cereus family member under the same conditions.
The recombinant Bacillus cereus family member can comprise a mutation an ExsY gene, such as a knock-out of the ExsY gene. The mutation in the ExsY gene can partially or completely inhibit the ability of ExsY to complete the formation of the exosporium or attach the exosporium to the spore.
The recombinant Bacillus cereus family member can comprise a mutation a CotY gene, such as a knock-out of the CotY gene. The mutation in the CotY gene can result in the formation of a fragile exosporium.
The recombinant Bacillus cereus family member can comprise a mutation an ExsA gene, such as a knock-out of the ExsA gene. The mutation in the ExsA gene can result in the formation of a fragile exosporium.
The recombinant Bacillus cereus family member can comprise a mutation a CotO gene, such as a knock-out of the CotO gene or a dominant negative form of the CotO gene. The mutation in the CotO gene can cause the exosporium to form in strips.
For ease of reference, descriptions of illustrative sequences for CotE, ExsY, BclB, YjcB, CotY, ExsA, and CotO are provided in Table 20 below.
Exosporium fragments can be prepared from any of these recombinant Bacillus cereus family members and used for various purposes as described further hereinbelow. Where the recombinant Bacillus cereus family member expresses a fusion protein, the exosporium fragments will comprise the fusion proteins. Upon purification of the exosporium fragments that contain the fusion proteins from the spores, a cell-free protein preparation is obtained in which the fusion proteins are stabilized and supported through covalent bonds to the exosporium fragments.
To remove the exosporium from spores of the recombinant Bacillus cereus family members that have mutations or other genetic alterations that allow for collection of free exosporium, a suspension of the spores can be subjected to centrifugation or filtration to produce fragments of exosporium that are separated from the spores. Where the recombinant Bacillus cereus family member expresses a fusion protein, the exosporium fragments will comprise the fusion protein.
A suspension comprising the spores can be subjected to centrifugation, followed by collection of the supernatant. The supernatant comprises the fragments of the exosporium and is substantially free of spores.
Alternatively, a suspension comprising the spores can be subjected to filtration, followed by collection of the filtrate. The filtrate comprises the fragments of the exosporium and is substantially free of spores.
The suspension of spores can be agitated or mechanically disrupted prior to centrifugation or filtration.
The exosporium fragments can also be separated from the spores by gradient centrifugation, affinity purification, or by allowing the spores to settle out of the suspension.
Due to the strong covalent bonds between the fusion proteins and the exosporium fragments, the fusion proteins become resistant to heat. The heat resistance of the fusion proteins bound to the exosporium fragments allows them to be used for applications that require heat-resistant proteins or enzymes.
Exosporium fragments derived from a recombinant Bacillus cereus family member are provided.
The exosporium fragments can be derived from any of the recombinant Bacillus cereus family members that comprise any of the mutations or other genetic alterations described herein that allow for collection of free exosporium.
The exosporium fragments can comprise any of the fusion proteins described above in Section I.
V. Formulations
A formulation is provided. The formulation comprises any of the recombinant Bacillus cereus family members described herein. The formulation further comprises an agriculturally acceptable carrier.
Another formulation is provided. The formulation comprises exosporium fragments derived from any of the recombinant Bacillus cereus family members described herein. The formulation further comprises an agriculturally acceptable carrier.
In any of the formulations, the fusion protein can comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and the formulation can further comprise a mannanase or a xyloglucanase.
In any of the formulations, the fusion protein can comprise a mannanase and the formulation can further comprise a Bacillus cereus phosphatidylcholine-specific phospholipase C.
In any of the formulations, the fusion protein can comprise a xyloglucanase and the formulation can further comprise a Bacillus cereus phosphatidylcholine-specific phospholipase C.
In any of the formulations wherein the formulation comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and a mannanase or a xyloglucanase, the Bacillus cereus phosphatidylcholine-specific phospholipase C can comprise SEQ ID NO: 257.
In any of the formulations wherein the formulation comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and a mannanase or a xyloglucanase, the mannanase can comprise SEQ ID NO: 307.
In any of the formulations wherein the formulation comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and a mannanase or a xyloglucanase, the xyloglucanase can comprise SEQ ID NO: 299.
Amino acid sequences having a high degree of sequence identity to SEQ ID NO: 257, 307, and/or 299 can also be used. Thus, for example, the Bacillus cereus phosphatidylcholine-specific phospholipase C can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 257; the mannanase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 307; and/or the xyloglucanase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NP: 299.
In any of the formulations wherein the fusion protein comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and the formulation further comprises a mannanase or a xyloglucanase, the mannanase or the xyloglucanase can also be part of a fusion protein. Where the mannanase or xyloglucanase is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the phosphatidylcholine-specific phospholipase C. Alternatively or in addition, the formulation can comprise a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the mannanase or the xyloglucanase.
Alternatively or in addition, in any of the formulations where the fusion protein comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and the formulation further comprises a mannanase or a xyloglucanase, the mannanase or xyloglucanase can be in the form of a free enzyme.
In any of the formulations wherein the fusion protein comprises a mannanase and the formulation further comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C, the Bacillus cereus phosphatidylcholine-specific phospholipase C can also be part of a fusion protein. Where the Bacillus cereus phosphatidylcholine-specific phospholipase C is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the mannanase. Alternatively or in addition, the formulation can comprise a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the Bacillus cereus phosphatidylcholine-specific phospholipase C.
Alternatively or in addition, in any of the formulations wherein the fusion protein comprises a mannanase and the formulation further comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C, the Bacillus cereus phosphatidylcholine-specific phospholipase C can be in the form of a free enzyme.
In any of the formulations wherein the fusion protein comprises a xyloglucanase and the formulation further comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C, the Bacillus cereus phosphatidylcholine-specific phospholipase C can also be part of a fusion protein. Where the Bacillus cereus phosphatidylcholine-specific phospholipase C is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the xyloglucanase. Alternatively or in addition, the formulation can comprise a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the Bacillus cereus phosphatidylcholine-specific phospholipase C.
Alternatively or in addition, in any of the formulations wherein the fusion protein comprises a xyloglucanase and the formulation further comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C, the Bacillus cereus phosphatidylcholine-specific phospholipase C can be in the form of a free enzyme.
In any of the formulations wherein the formulation comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and a mannanase, the phospholipase C and the mannanase are preferably present in synergistically effective amounts.
In any of the formulations wherein the formulation comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and a xyloglucanase, the phospholipase C and the xyloglucanase are preferably present in synergistically effective amounts.
In any of the formulations, the fusion protein can comprise a xyloglucanase and the formulation can further comprise a mannanase.
In any of the formulations, the fusion protein can comprise a mannanase and the formulation can further comprise a xyloglucanase.
In any of the formulations wherein the formulation comprises a xyloglucanase and a mannanase, the xyloglucanase can comprise SEQ ID NO: 299.
In any of the formulations wherein the formulation comprises a xyloglucanase and a mannanase, the mannanase can comprise SEQ ID NO: 307.
Amino acid sequences having a high degree of sequence identity to SEQ ID NO: 299 and/or 307 can also be used. Thus, the mannanase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 307; and/or the xyloglucanase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NP: 299.
In any of the formulations wherein the fusion protein comprises a xyloglucanase and the formulation further comprises a mannanase, the mannanase can also be part of a fusion protein. Where the mannanase is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the xyloglucanase. Alternatively or in addition, the formulation can comprise a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the mannanase.
Alternatively or in addition, in any of the formulations wherein the fusion protein comprises a xyloglucanase and the formulation further comprises a mannanase, the mannanase can be in the form of a free enzyme.
In any of the formulations wherein the fusion protein comprises a mannanase and the formulation further comprises a xyloglucanase, the xyloglucanase can also be part of a fusion protein. Where the xyloglucanase is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the mannanase. Alternatively or in addition, the formulation can comprise a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the xyloglucanase.
Alternatively or in addition, in any of the formulations wherein the fusion protein comprises a mannanase and the formulation further comprises a xyloglucanase, the xyloglucanase can be in the form of a free enzyme.
In any of the formulations comprising a xyloglucanase and a mannanase, the xyloglucanase and the mannanase are preferably present in synergistically effective amounts.
In any of the formulations, the fusion protein can comprise an acid phosphatase comprising SEQ ID NO: 311, and the formulation can further comprise an acid phosphatase comprising SEQ ID NO: 312.
In any of the formulations wherein the fusion protein comprises an acid phosphatase comprising SEQ ID NO: 311 and the formulation further comprises an acid phosphatase comprising SEQ ID NO: 312, the acid phosphatase comprising SEQ ID NO: 312 can also be part of a fusion protein. Wherein the acid phosphatase comprising SEQ ID NO: 312 is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the acid phosphatase comprising SEQ ID NO: 311. Alternatively or in addition, the formulation can comprise a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the acid phosphatase comprising SEQ ID NO: 312.
Alternatively or in addition, in any of the formulations wherein the fusion protein comprises an acid phosphatase comprising SEQ ID NO: 311 and the formulation further comprises an acid phosphatase comprising SEQ ID NO: 312, the acid phosphatase comprising SEQ ID NO: 312 can be in the form of a free enzyme.
In any of the formulations, the fusion protein can comprise an acid phosphatase comprising SEQ ID NO: 312, and the formulation can further comprise an acid phosphatase comprising SEQ ID NO: 311.
In any of the formulations wherein the fusion protein comprises an acid phosphatase comprising SEQ ID NO: 312 and the formulation further comprises an acid phosphatase comprising SEQ ID NO: 311, the acid phosphatase comprising SEQ ID NO: 311 can also be part of a fusion protein. Wherein the acid phosphatase comprising SEQ ID NO: 311 is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the acid phosphatase comprising SEQ ID NO: 312. Alternatively or in addition, the formulation can comprise a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the acid phosphatase comprising SEQ ID NO: 311.
Alternatively or in addition, in any of the formulations wherein the fusion protein comprises an acid phosphatase comprising SEQ ID NO: 312 and the formulation further comprises an acid phosphatase comprising SEQ ID NO: 311, the acid phosphatase comprising SEQ ID NO: 311 can be in the form of a free enzyme.
Another formulation is provided. The formulation comprises a recombinant Bacillus cereus family member that expresses a fusion protein. Alternatively or in addition, the formulation comprises exosporium fragments derived from a Bacillus cereus family member that expresses a fusion protein. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises an acid phosphatase. The formulation further comprises a second enzyme.
The second enzyme suitably comprises a lipase, a phospholipase, a glucanase, a xylanase, a pectinase, a mannanase, a lichenase, or a combination of any thereof. The lipase, phospholipase, glucanase, xylanase, pectinase, mannanase, or lichenase can comprise any of the lipases, phospholipases, glucanases, xylanases, pectinases, mannanases, or lichenases described herein.
The acid phosphatase suitably comprises a Triticum aestivum acid phosphatase.
For example, the acid phosphatase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 311 or 312.
Another formulation is provided. The formulation comprises a recombinant Bacillus cereus family member that expresses a fusion protein. Alternatively or in addition, the formulation comprises exosporium fragments derived from a Bacillus cereus family member that expresses a fusion protein. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a phospholipase C. The formulation further comprises a second enzyme.
In any of the formulations wherein the fusion protein comprises a phospholipase C, the phospholipase C can comprise a Bacillus thuringiensis phospholipase C.
In any of the formulations wherein the fusion protein comprises a phospholipase C, the phospholipase C can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having 100% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
In any of the formulations wherein the fusion protein comprises a phospholipase C, the phospholipase C can consist essentially of SEQ ID NO: 250 or SEQ ID NO: 251.
In any of the formulations wherein the fusion protein comprises a phospholipase C, the phospholipase C can consist of SEQ ID NO: 250 or SEQ ID NO: 251.
The second enzyme suitably comprises a xylogluconase or a mannanase.
When the second enzyme comprises a xylogluconase, the xylogluconase can comprise a Paenibacillus pabuli xylogluconase or a Bacillus licheniformis xylogluconase.
When the second enzyme comprises a xylogluconase, the xylogluconase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 299 or 300.
The phospholipase C can comprise SEQ ID NO: 250 and the xylogluconase can comprise SEQ ID NO: 299. Alternatively, the phospholipase C can comprise SEQ ID NO: 250 and the xylogluconase can comprise SEQ ID NO: 300.
In any of the formulations described herein comprising a phospholipase C and a second enzyme comprising a xylogluconase, the phospholipase C and the xylogluconase can be present in synergistically effective amounts.
When the second enzyme comprises a mannanase, the mannanase can comprise a Bacillus circulans mannanase or a Bacillus subtilis mannanase.
Alternatively or in addition, when the second enzyme comprises a mannanase, the mannanase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 307 or 308.
In any of the formulations described herein comprising a phospholipase C and a second enzyme comprising a mannanase, the phospholipase C can comprise SEQ ID NO: 250 and the mannanase can comprise SEQ ID NO: 308. Alternatively, the phospholipase C can comprise SEQ ID NO: 250 and the mannanase can comprise SEQ ID NO: 307.
In any of the formulations described herein comprising a phospholipase C and a second enzyme comprising a mannanase, the phospholipase C and the mannanase can be present in synergistically effective amounts.
Any of the formulations described herein can further comprise an agrochemical. Suitable agrochemicals are described below in Section IX.
Any of the formulations described herein can further comprise a fertilizer, a biostimulant, or a combination thereof. Suitable fertilizers and biostimulants are described below in Section IX.
VI. Treated Seeds
A treated plant seed is provided. The plant seed can be treated with any of the recombinant Bacillus cereus family members described herein. The recombinant Bacillus cereus family member can express any of the fusion proteins described herein.
Another treated plant seed is provided. The plant seed can be treated with any of the exosporium fragments described herein. The exosporium fragments can be derived from any of the Bacillus cereus family members described herein. The exosporium fragments can comprise any of the fusion proteins described herein.
Yet another treated plant seed is provided. The plant seed can be treated with any of the formulations described herein.
In any of the treated plant seeds, the plant seed can be coated with the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation.
The recombinant Bacillus cereus family members, exosporium fragments, or formulations can used as seed treatments, e.g., seed coatings or dressings. Seed coating or dressing formulations may be in the form of a liquid carrier formulation, a slurry formulation, or a powder formulation.
Seed coating or dressing formulations can be applied with conventional additives that are provided to make the seed treatment have sticky qualities to stick to and coat the seeds. Suitable additives comprise: talcs, graphites, gums, stabilizing polymers, coating polymers, finishing polymers, slip agents for seed flow and plantability, cosmetic agents, and cellulosic materials such as carboxymethyl cellulose and the like.
The seed treatments formulations can further comprise colorant agents and/or other additives.
The recombinant Bacillus cereus family members, exosporium fragments, or formulations of the invention can be applied as seed treatments by themselves or can be applied in with other agrochemicals such as fungicides, insecticides, inoculants, plant growth regulators, plant growth promoting microbes, fertilizers, fertilizer enhancers, seed nutrients, biocontrol agents, herbicidal antidotes and seedling disease treatments and with other conventional seed treatments.
The seed treatment formulations(s) may be applied to seeds in a suitable carrier such as water or a powder. The seeds can then be allowed to dry and planted in conventional fashion. The recombinant Bacillus cereus family members or exosporium fragments can be applied directly to the seed as a solution or in combination with other commercially available additives. For example, the recombinant Bacillus cereus family members or exosporium fragments can be applied in combination with seedling-acceptable carrier(s) (e.g., a liquid carrier or a solid carrier).
Solutions containing the recombinant Bacillus cereus family members or exosporium fragments can be sprayed or otherwise applied to the seed (e.g., in a seed slurry or a seed soak).
Solid or dry materials containing recombinant Bacillus cereus family members or exosporium fragments are also useful to promote effective seedling germination, growth, and protection during early seedling establishment.
The recombinant Bacillus cereus family members or exosporium fragments can be used with a solubilizing carrier such as water, a buffer (e.g., citrate or phosphate buffer), other treating agents (e.g., alcohol or another solvent), and/or any soluble agent.
In addition, small amounts of drying agent enhancers, such as lower alcohols, etc. can be used in seed coating formulations.
Surfactants, emulsifiers and preservatives can also be added at relatively low (e.g., about 0.5% w/v or less) levels in order to enhance the stability of the seed coating product.
Seeds can be treated using a variety of methods including, but not limited to, pouring, pumping, drizzling, or spraying an aqueous solution containing the recombinant Bacillus cereus family members or exosporium fragments on or over a seed; or spraying or applying the recombinant Bacillus cereus family members or exosporium fragments onto a layer of seeds either with or without the use of a conveyor system.
Mixing devices useful for seed treatment include but are not limited to tumblers, mixing basins, mixing drums, and fluid application devices that include basins or drums used to contain the seed while coating.
After seed treatment, the seed may be air-dried or a stream of dry air may be optionally used to aid in the drying of the seed coatings.
Seed treatments containing the recombinant Bacillus cereus family members or exosporium fragments can be applied using any commercially available seed treatment machinery or can also be applied using any acceptable non-commercial method(s) such as the use of syringes or any other seed treatment device.
VII. Methods for Stimulating Plant Growth and/or Promoting Plant Health
A method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a recombinant Bacillus cereus family member to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The recombinant Bacillus cereus family member can comprise any of the recombinant Bacillus cereus family members described herein. The recombinant Bacillus cereus family member can express any of the fusion proteins described herein.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying exosporium fragments to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The exosporium fragments can comprise exosporium fragments derived from any of the recombinant Bacillus cereus family members described herein. The exosporium fragments can comprise any of the fusion proteins described herein.
Yet another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a formulation to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The formulation can comprise any of the formulations described herein.
In any of the methods for stimulating plant growth and/or promoting plant health, the fusion protein can comprise a Bacillus cereus phosphatidylcholine-specific phospholipase C and the method can further comprise applying a mannanase or a xyloglucanase to the plant growth medium, plant, plant seed, or area surrounding the plant or the plant seed.
In any of the methods for stimulating plant growth and/or promoting plant health, the fusion protein can comprise a mannanase and the method can further comprise applying a Bacillus cereus phosphatidylcholine-specific phospholipase C to the plant growth medium, plant, plant seed, or area surrounding the plant or the plant seed.
In any of the methods for stimulating plant growth and/or promoting plant health, the fusion protein can comprise a xyloglucanase and the method can further comprise applying a Bacillus cereus phosphatidylcholine-specific phospholipase C to the plant growth medium, plant, plant seed, or area surrounding the plant or the plant seed.
In any of the methods that comprise applying a Bacillus cereus phosphatidylcholine-specific phospholipase C and a mannanase or a xyloglucanase, the Bacillus cereus phosphatidylcholine-specific phospholipase C can comprise SEQ ID NO: 257.
In any of the methods that comprise applying a Bacillus cereus phosphatidylcholine-specific phospholipase C and a mannanase or a xyloglucanase, the mannanase can comprise SEQ ID NO: 307.
In any of the methods that comprise applying a Bacillus cereus phosphatidylcholine-specific phospholipase C and a mannanase or a xyloglucanase, the xyloglucanase can comprise SEQ ID NO: 299.
Amino acid sequences having a high degree of sequence identity to SEQ ID NO: 257, 307, and/or 299 can also be used. Thus, for example, the Bacillus cereus phosphatidylcholine-specific phospholipase C can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 257; the mannanase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 307; and/or the xyloglucanase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NP: 299.
In any of the methods wherein the fusion protein comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and the method further comprises applying a mannanase or a xyloglucanase, the mannanase or the xyloglucanase can also be part of a fusion protein. Where the mannanase or xyloglucanase is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the phosphatidylcholine-specific phospholipase C. Alternatively or in addition, the method can comprise applying a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the mannanase or the xyloglucanase.
Alternatively or in addition, in any of the methods wherein the fusion protein comprises a Bacillus cereus phosphatidylcholine-specific phospholipase C and the method further comprises applying a mannanase or a xyloglucanase, the mannanase or xyloglucanase can be in the form of a free enzyme.
In any of the methods wherein the fusion protein comprises a mannanase and the method further comprises applying a Bacillus cereus phosphatidylcholine-specific phospholipase C, the Bacillus cereus phosphatidylcholine-specific phospholipase C can also be part of a fusion protein. Where the Bacillus cereus phosphatidylcholine-specific phospholipase C is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the mannanase. Alternatively or in addition, the method can comprise applying a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the Bacillus cereus phosphatidylcholine-specific phospholipase C.
Alternatively or in addition, in any of the methods wherein the fusion protein comprises a mannanase and the method further comprises applying a Bacillus cereus phosphatidylcholine-specific phospholipase C, the Bacillus cereus phosphatidylcholine-specific phospholipase C can be in the form of a free enzyme.
In any of the methods wherein the fusion protein comprises a xyloglucanase and the method further comprises applying a Bacillus cereus phosphatidylcholine-specific phospholipase C, the Bacillus cereus phosphatidylcholine-specific phospholipase C can also be part of a fusion protein. Where the Bacillus cereus phosphatidylcholine-specific phospholipase C is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the xyloglucanase. Alternatively or in addition, the method can comprise applying a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the Bacillus cereus phosphatidylcholine-specific phospholipase C.
Alternatively or in addition, in any of the methods wherein the fusion protein comprises a xyloglucanase and the method further comprises applying a Bacillus cereus phosphatidylcholine-specific phospholipase C, the Bacillus cereus phosphatidylcholine-specific phospholipase C can be in the form of a free enzyme.
In any of the methods comprising applying a Bacillus cereus phosphatidylcholine-specific phospholipase C and a mannanase, the phospholipase C and the mannanase are preferably present in synergistically effective amounts.
In any of the methods comprising applying a Bacillus cereus phosphatidylcholine-specific phospholipase C and a xyloglucanase, the phospholipase C and the xyloglucanase are preferably present in synergistically effective amounts.
In any of the methods for stimulating plant growth and/or promoting plant health, the fusion protein can comprise a xyloglucanase and the method can further comprise applying a mannanase to the plant growth medium, plant, plant seed, or area surrounding the plant or the plant seed.
In any of the methods for stimulating plant growth and/or promoting plant health, the fusion protein can comprise a mannanase and the method can further comprise applying a xyloglucanase to the plant growth medium, plant, plant seed, or area surrounding the plant or the plant seed.
In any of the method comprising application of a xyloglucanase and a mannanase, the xyloglucanase can comprise SEQ ID NO: 299.
In any of the method comprising application of a xyloglucanase and a mannanase, the mannanase can comprise SEQ ID NO: 307.
Amino acid sequences having a high degree of sequence identity to SEQ ID NO: 299 and/or307 can also be used. Thus, the mannanase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 307; and/or the xyloglucanase can comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 299.
In any of the methods wherein the fusion protein comprises a xyloglucanase and the method further comprises applying a mannanase, the mannanase can also be part of a fusion protein. Where the mannanase is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the xyloglucanase. Alternatively or in addition, the method can comprise applying a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the mannanase.
Alternatively or in addition, in methods wherein the fusion protein comprises a xyloglucanase and the method further comprises applying a mannanase, the mannanase can be in the form of a free enzyme.
In any of the methods wherein the fusion protein comprises a mannanase and the method further comprises applying a xyloglucanase, the xyloglucanase can also be part of a fusion protein. Where the xyloglucanase is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the mannanase. Alternatively or in addition, the method can comprise applying a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the xyloglucanase.
Alternatively or in addition, in any of the methods wherein the fusion protein comprises a mannanase and the method further comprises applying a xyloglucanase, the xyloglucanase can be in the form of a free enzyme.
In any of the methods comprising applying a xyloglucanase and a mannanase, the xyloglucanase and the mannanase are preferably present in synergistically effective amounts.
In any of the methods for stimulating plant growth and/or promoting plant health, the fusion protein can comprise an acid phosphatase comprising SEQ ID NO: 311, and the method can further comprise applying an acid phosphatase comprising SEQ ID NO: 312 to the plant growth medium, plant, plant seed, or area surrounding the plant or the plant seed.
In any of the methods wherein the fusion protein comprises an acid phosphatase comprising SEQ ID NO: 311 and the method further comprises applying an acid phosphatase comprising SEQ ID NO: 312, the acid phosphatase comprising SEQ ID NO: 312 can also be part of a fusion protein. Wherein the acid phosphatase comprising SEQ ID NO: 312 is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the acid phosphatase comprising SEQ ID NO: 311. Alternatively or in addition, the method can comprise applying a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the acid phosphatase comprising SEQ ID NO: 312.
Alternatively or in addition, in any of the methods wherein the fusion protein comprises an acid phosphatase comprising SEQ ID NO: 311 and the method further comprises applying an acid phosphatase comprising SEQ ID NO: 312, the acid phosphatase comprising SEQ ID NO: 312 can be in the form of a free enzyme.
In any of the methods for stimulating plant growth and/or promoting plant health, the fusion protein can comprise an acid phosphatase comprising SEQ ID NO: 312, and the method can further comprise applying an acid phosphatase comprising SEQ ID NO: 311 to the plant growth medium, plant, plant seed, or area surrounding the plant or the plant seed.
In any of the methods wherein the fusion protein comprises an acid phosphatase comprising SEQ ID NO: 312 and the method further comprises applying an acid phosphatase comprising SEQ ID NO: 311, the acid phosphatase comprising SEQ ID NO: 311 can also be part of a fusion protein. Wherein the acid phosphatase comprising SEQ ID NO: 311 is part of a fusion protein, the fusion protein can be co-expressed in the same recombinant Bacillus cereus family member as the acid phosphatase comprising SEQ ID NO: 312. Alternatively or in addition, the method can comprise applying a second recombinant Bacillus cereus family member that expresses a fusion protein comprising the acid phosphatase comprising SEQ ID NO: 311.
Alternatively or in addition, in any of the methods wherein the fusion protein comprises an acid phosphatase comprising SEQ ID NO: 312 and the method further comprises applying an acid phosphatase comprising SEQ ID NO: 311, the acid phosphatase comprising SEQ ID NO: 311 can be in the form of a free enzyme.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a recombinant Bacillus cereus family member expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a xylosidase, a histidine protease, an acid phosphatase, or a combination of any thereof.
Yet another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying exosporium fragments derived from spores of a recombinant Bacillus cereus family member expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a xylosidase, a histidine protease, an acid phosphatase, or a combination of any thereof.
For example, the fusion protein can comprise a xylosidase.
Where the fusion protein comprises a xylosidase, the xylosidase can comprises a Caldicellulosiruptor saccharolyticus xylosidase, a Bacillus pumilus xylosidase, Bacillus subtilis xylosidase, or a combination thereof.
Where the fusion protein comprises a xylosidase, the xylosidase can comprise an amino acid sequence having at least 70% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 75% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 80% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 85% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 90% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 95% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 98% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having at least 99% identity to any one of SEQ ID NOs. 274-276.
The xylosidase can comprise an amino acid sequence having 100% identity to any one of SEQ ID NOs. 274-276.
The fusion protein can comprise a histidine protease.
The fusion protein can comprise an acid phosphatase.
A further method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a recombinant Bacillus cereus family member expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises an acid phosphatase. The method further comprises applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying exosporium fragments derived from spores of a recombinant Bacillus cereus expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises an acid phosphatase. The method further comprises applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
In any of the methods comprising applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed, the second enzyme suitably comprises a lipase, a phospholipase, a glucanase, a xylanase, a pectinase, a mannanase, a lichenase, a xylogluconase or a combination of any thereof.
In any of the methods wherein the fusion protein comprises an acid phosphatase, the acid phosphatase can comprise a Triticum aestivum acid phosphatase.
In any of the methods wherein the fusion protein comprises an acid phosphatase, the acid phosphatase can comprise amino acid sequence having at least 70% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise amino acid sequence having at least 75% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise amino acid sequence having at least 80% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise amino acid sequence having at least 85% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise amino acid sequence having at least 90% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise amino acid sequence having at least 95% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise amino acid sequence having at least 98% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise amino acid sequence having at least 99% identity to SEQ ID NO: 311 or 312.
The acid phosphatase can comprise amino acid sequence having at 100% identity to SEQ ID NO: 311 or 312.
A further method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying a recombinant Bacillus cereus family member expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a phospholipase C. The method further comprises applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
Another method for stimulating plant growth and/or promoting plant health is provided. The method comprises applying exosporium fragments derived from spores of a recombinant Bacillus cereus expressing a fusion protein to a plant growth medium, a plant, a plant seed, or an area surrounding a plant or a plant seed. The fusion protein comprises a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member. The fusion protein further comprises a phospholipase C. The method further comprises applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
In any of the methods wherein the fusion protein comprises a phospholipase C, the phospholipase C can comprise a Bacillus thuringiensis phospholipase C.
In any of the methods wherein the fusion protein comprises a phospholipase C, the phospholipase C can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
The phospholipase C can comprise an amino acid sequence having 100% identity to SEQ ID NO: 250 or SEQ ID NO: 251.
In any of the methods wherein the fusion protein comprises a phospholipase C, the phospholipase C can consist essentially of SEQ ID NO: 250 or SEQ ID NO: 251.
In any of the methods wherein the fusion protein comprises a phospholipase C, the phospholipase C can consist of SEQ ID NO: 250 or SEQ ID NO: 251.
In any of the methods comprising applying a second enzyme to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed, the second enzyme can comprise a xylogluconase or a mannanase.
When the second enzyme comprises a xylogluconase, the xylogluconase can comprise a Paenibacillus pabuli xylogluconase or a Bacillus licheniformis xylogluconase.
When the second enzyme comprises a xylogluconase, the xylogluconase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 299 or 300.
The xylogluconase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 299 or 300.
In the methods, the phospholipase C can comprise SEQ ID NO: 250 and the xylogluconase can comprise SEQ ID NO: 299. Alternatively, the phospholipase C can comprise SEQ ID NO: 250 and the xylogluconase can comprise SEQ ID NO: 300.
In any methods or formulations described herein comprising a phospholipase C and a second enzyme comprising a xylogluconase, the phospholipase C and the xylogluconase can be present in synergistically effective amounts.
When the second enzyme comprises a mannanase, the mannanase can comprise a Bacillus circulans mannanase or a Bacillus subtilis mannanase.
Alternatively or in addition, when the second enzyme comprises a mannanase, the mannanase can comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 307 or 308.
The mannanase can comprise an amino acid sequence having 100% identity to SEQ ID NO: 307 or 308.
In any of the formulations or methods described herein comprising a phospholipase C and a second enzyme comprising a mannanase, the phospholipase C can comprise SEQ ID NO: 250 and the mannanase can comprise SEQ ID NO: 308. Alternatively, the phospholipase C can comprise SEQ ID NO: 250 and the mannanase can comprise SEQ ID NO: 307.
In any of the formulations or methods described herein comprising a phospholipase C and a second enzyme comprising a mannanase, the phospholipase C and the mannanase can be present in synergistically effective amounts.
In any of the methods described herein, the method can further comprise inactivating the recombinant Bacillus cereus family member prior to applying the recombinant Bacillus cereus family member to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
In any of the methods described herein, the method can comprise applying the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation to the plant growth medium.
In any of the methods described herein involving the use of a plant growth medium, the plant growth medium can comprise soil, water, an aqueous solution, sand, gravel, a polysaccharide, mulch, compost, peat moss, straw, logs, clay, soybean meal, yeast extract, or a combination thereof.
The plant growth medium can comprise a fertilizer.
The plant growth medium can consist essentially of a fertilizer.
Any of the methods described herein can further comprise supplementing the plant growth medium with a substrate for an enzyme. Suitable substrates include, but are not limited to tryptophan, an adenosine monophosphate, an adenosine diphosphate, an adenosine triphosphate (e.g., adenosine-3-triphosphate), a polyphosphate, a protein meal, a trimetaphosphate, a cellulose, a methylcellulose, a chitin, a chitosan, a cellulose derivative, a phosphate, a fat, a wax, a phospholipid, a phytic acid, or a combination of any thereof.
In any of the methods described herein, the method can comprise applying the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation to the plant.
For example, the method can comprise applying the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation to roots of the plant.
Alternatively or in addition, the method can comprise applying the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation foliarly.
In any of the methods described herein, the method can comprise applying the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation to the plant seed.
Where the method comprises applying the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation to the plant seed, applying the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation to the plant seed can comprise: (a) applying recombinant Bacillus cereus family member, the exosporium fragments, or the formulation to the plant seed at the time of planting; or (b) coating the plant seed with the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation.
In any of the methods described herein, the method can further comprise applying an agrochemical to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed. Suitable agrochemicals are described below in Section IX.
In any of the methods described herein, the method can further comprise applying a fertilizer, a biostimulant, or a combination thereof to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed. Suitable fertilizers and biostimulants are described in Section IX below.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation can exhibit increased growth as compared to plants grown in the absence of the enzyme, the expansin protein, or the microorganism, under the same conditions.
In any of the methods described herein, seeds to which the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation has been applied can exhibit increased germination rates as compared to seeds to which the enzyme or microorganism has not been applied, under the same conditions.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation can exhibit increased nutrient uptake as compared to plants grown in the absence of the enzyme, the expansin protein, or the microorganism, under the same conditions.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation can exhibit decreased susceptibility to a pathogen as compared to plants grown in the absence of the enzyme or the microorganism, under the same conditions.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation can exhibit decreased susceptibility to an environmental stress (e.g., drought, flood, heat, freezing, salt, heavy metals, low pH, high pH, or a combination of any thereof) as compared to plants grown in the absence of the enzyme or the microorganism, under the same conditions.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation can exhibit increased nutrient content as compared to plants grown in the absence of the enzyme, the expansin protein, or the microorganism, under the same conditions.
The nutrient can comprise, for example, a polysaccharide, a protein, phytic acid, a phosphatate, a phospholipid, or a combination of any thereof.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation exhibit can increased root nodulation as compared to plants grown in the absence of the enzyme or the microorganism, under the same conditions.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation can exhibit slower fruit ripening as compared to plants grown in the absence of the enzyme or the microorganism, under the same conditions.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation can exhibit greater crop yield as compared to plants grown in the absence of the enzyme, the expansin protein, or the microorganism, under the same conditions.
In any of the methods described herein, plants grown in the presence of the recombinant Bacillus cereus family member, the exosporium fragments, or the formulation can exhibit altered leaf senescence as compared to plants grown in the absence of the enzyme or the microorganism, under the same conditions.
VIII. Animal Feed Additives, Animal Feed Compositions, and Methods for Delivering an Enzyme to an Animal
Feed enzymes provide a means to increase feed utilization and nutrition in agricultural animals. Enzymes can be used to increase animal performance and health benefits. In addition, the utilization of enzymes as feed additives can improve the utilization of feed by increasing the nutritive value of feed raw materials. The enzymes described herein, including, but not limited to phytases, xylanases, glucanases, xyloglucanases, mannanases, phospholipases, lichenases, xylosidases, pectolyases, phosphatases and lactonases can provide plant growth promoting properties to plants. These select enzymes are also useful feed additive enzymes that can be provided as a supplement to animal feed to increase animal performance and health benefits. Feed additive enzymes as described can be used to improve feed digestibility, thereby enhancing the efficiency of feed utilization. Improved feed utilization delivers to the animals a means to better utilize the plant nutrients obtained from feed sources such as cereals (barley, wheat, rye, oats, corn) and pasture forage grass or hays, such as alfalfa.
The enzymes described herein are particularly useful as feed additives because of their stability, which allows them to stay with the feed throughout the digestive process. The enzymes are also are not as easily absorbed across the epithelium (which would reduce their ability to continue to digest target substrates throughout the stomach and intestines as the conditions become more favorable to enzyme activity).
Any of the enzymes described herein can be used as feed additives provided on crop plants as a foliar treatment pre-harvest or as an over post-harvest spray treatment applied directly to the plant or plant part. Alternatively or in addition, any of the enzymes can be applied directly to vitamin and/or mineral premixes that are added to animal feed. Alternatively or in addition, the enzymes can be added in directly to the feed, either before, during, or after the feed has been processed. Supplied in any of these treatment types, the enzymes are useful for supporting gut health and performance in agricultural and companion animals including but not limited to poultry, swine, dairy and beef cattle, horses, goats, sheep, dogs, cats, fish, rabbits, rodents, and other, animal feed. Some of the advantages provided are lower feed costs and increased nutrient utilization and uptake, including the uptake of essential minerals and nutrients (e.g., calcium, zinc, cobalt, magnesium, iron). Increased nutrient utilization and uptake can inturn support increases in daily weight gain and overall animal health. Using enzymes as feed additives can also reduce feed costs and also generally improve the environmental impact for animal production.
One or more of the enzymes described herein can be used sequentially or simultaneously to provide enhanced nutritive benefits to an agricultural or companion animal. The enzymes as described are classified by the types of substrates that they work on. For instance, proteases break down proteins into amino acids making them more available for utilization and uptake by the animal if provided on a plant or feed derived from a plant or plant part.
Individual classes of enzymes that can be used as feed additives or in animal feed are further described below. These enzymes are useful to overcome limitations imposed on animal digestion due to the insufficiency of digestive enzymes that break down specific chemical linkages that bind and prevent the release of nutrients.
A. Phytases
Phytase is an enzyme that breaks down phytate molecules, a form of phosphorous found in plants that is not naturally available for the animal. Phytases therefore provide benefits by enhancing the release of available phosphorous, which in turn leads to increased animal growth. Phytase (e.g., the E246 phytase) allows the animal to absorb more phosphorus when provided on a plant or the plant part used for feed or in a feed additive.
B. Xylanases and Glucanases
Xylanase and glucanase break down non-starch polysaccharides, thereby releasing energy from the fibrous portion of grains and grain by-products. Glucanases break down the insoluble fraction of a feed crop, such as stalk tissues of corn fed to ruminant livestock such as dairy and beef cattle. Such corn stalk bales are usually provided as a supplemental feed to cattle during the winter months. Xylanases (e.g., the E100, E146, or E232 xylanases) and glucanases (e.g., the E94 glucanase) can be used to increase the feed quality of the bales by increasing the energy and nutrition that is provided to the animal. In addition, such enzymes can be added to corn stalk bales and used to reduce overall winter feed costs. Similarly, xylanases and glucanases can be used as feed additive enzymes on pasture forages (such as alfalfa) and other pasture grasses and hays.
C. Xyloglucanases
Xyloglucanse is an enzyme that breaks down xylgolucans (endo-hydrolysis of the 1,4-β-D-glucosidic linkages in xyloglucan). Xyloglucanases can also improve the overall hydrolysis of lignocellulosic materials. Xyloglucanases (e.g., the E149 and D381 xyloglucanases) can be used in the breakdown of polysaccharides thus releasing carbohydrates that are more utilizable to the animal.
D. Mannanases
Beta-mannanase is involved in the hydrolysis of terminal, non-reducing beta-D-mannose residues in beta-D-mannosides. Beta-mannanases (e.g., the E177 and E196 mannanases) can be used to improve production and performance when added to poultry and swine diets. Mannanase enzymes can be used as feed additive enzymes to enhance performance as measured by weight gain, feed conversion, and the overall health of poultry, swine and other animals.
E. Phospholipases
Phospholipases comprise a class of membrane-associated enzymes that cleave phospholipids just before the phosphate group. They can be used as dairy feed additives and supporting gut health and performance. In addition, phospholipases can be used to support the productivity of animals under stress, thus providing increased profitability.
Phospholipase C and phospholipase D enzymes are examples of phospholipases that are useful as feed additives. Phospholipase C (PLC) enzymes are capable of hydrolyzing phosphatidylinositol, resulting in a release of diacyl glycerol and inositol 1,4,5-trisphosphate (IP3). Two examples of PLC enzymes are the E143 and E95 phosphilipases derived from Bacillus thuringiensis described herein. Additional examples of PLC enzymes include those from Listeria described herein. In particular, Listeria monocytogenes secretes a phosphatidylinositol-specific phospholipase C (PI-PLC). This enzyme can be expressed using a secretory signal and a propeptide sequence (ID Code D473), can be expressed with only with a propeptide (ID Code D474), or can be expressed without either the signal or propetide (ID Code D475). PI-PLC (e.g., E258) binds tightly to anionic phospholipids (such as those containing phosphomethyl head groups) and can form aggregated complexes with those anionic lipids and provided to animals for improved fat digestibility and as a nutritional supplement. Other phospholipases, such as phospholipase D (e.g., E229 and D409) where the principal substrate is phosphatidylcholine, hydrolyze phospholipids to produce the signal molecule phosphatidic acid (PA) and choline, which is a water-soluble vitamin-like nutrient and can be used to readily benefit animal nutrition.
F. Lichenases
Lichenase is an enzyme that hydrolyzes (1→4)-beta-D-glucosidic linkages in beta-D-D-glucans containing (1→3)- and (1→4)-bonds. Lichenases act on lichenin and cereal beta-D-glucans. Lichenin is a complex glucan or starch. Cereal feeds such as corn and other grains are used as a common feed source for many agricultural and companion animals. Lichenases (e.g., the E111 and D436 lichenases) that hydrolyze forms of beta glucans from cereals not only can provide a greater prebiotic quality to the cereal grains, but also can contribute an immunostimulating quality to the grain making it a beneficial food additive.
G. Xylosidase
Xylosidases catalyze the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. More specifically, xylosidases (e.g., the E175 and E194 xylosidases) hydrolyze (1→4)-beta-D-xylans to remove the D-xylose residues from the non-reducing termini of the oligosaccharide. Xylosidases can also hydrolyzse xylobiose, a disaccharide of xylose monomers with a beta 1,4-bond between them. The resulting D-xylose is a pentose sugar obtained from the xylan-rich portion of hemicellulose, present in plant cell walls and fiber, can be used in animal feed and pet food to increase weight gain in animals under managed dietary regimes.
H. Pectolyase
Pectolyase is a pectinase, a type of enzyme that degrades pectin. Pectolyase (e.g., the E176 and D405 pectolyases) catalyzes the eliminative cleavage of alpha (α-(1-4)-D galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducing ends. Pectolyases are the only known pectinases capable of degrading highly esterified pectins (like those found in fruits) into small molecules and are capable of breaking down cell walls thus releasing the more energy-available polysaccharides that have functional roles in animal nutrition.
I. Acid Phosphatases
Acid Phosphatase (AcPC) is an enzyme that hydrolyzes organic phosphates at an acidic pH. Hydrolysis of phosphate esters is an important step in metabolism. Secreted AcPCs (e.g., the E252 AcPC) are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of phosphorus from extracellular phosphomonoesters. Such enzymes can be used as a supplement for livestock and companion feed that is more nutritionally efficient by freeing up phosphorus.
J. Lactonase
Lactonases (also referred to as acyl-homoserine lactonases) are metalloenzymes. Lactonase (e.g., the H51 lactonase) hydrolyze the ester bond of the homoserine lactone ring of acylated homoserine lactones. In hydrolyzing the lactone bond, lactonase can prevent such signaling molecules from bacteria from binding to their target transcriptional regulators. Increasing the lactonase activity in feed additives can be beneficial for controlling bacterial infections in animals by inhibiting quorum-sensing activity.
K. Animal Feed Additives and Animal Feed Compositions Comprising Recombinant Bacillus cereus Family Members
An animal feed additive is provided. The animal feed additive comprises any of the recombinant Bacillus cereus family members that expresses a fusion protein described herein. Alternatively, or in addition, the animal feed additive comprises exosporium fragments derived from any of the recombinant Bacillus cereus family member that expresses a fusion protein described herein.
An animal feed composition is provided. The animal feed composition comprises animal feed and any of the recombinant Bacillus cereus family members that expresses a fusion protein described herein. Alternatively, or in addition, the animal feed composition comprises animal feed and exosporium fragments derived from any of the recombinant Bacillus cereus family members that expresses a fusion protein described herein.
The animal feed additive or animal feed composition can further comprise a carrier suitable for injestion by a livestock or companion animal.
The carrier can comprise starch, polyvinyl alcohol, a dextrin, a limestone, sucrose, talc, cellulose, or a combination of any thereof.
When the carrier comprises dextrin, the dextrin can comprise maltodextrin, cyclodextrin, or a combination thereof.
For example, the carrier can comprise polyvinyl alcohol and cellulose.
The animal feed in the animal feed composition can comprise fodder.
When the animal feed comprises fodder, the fodder can comprise hay, straw, silage, a compressed feed, a pelleted feed, an oil, a grain, a sprouted grain, a legume (e.g., soybean), an insoluble fraction of a feedcrop (e.g., corn stalks), a crop residue, or a combination of any thereof.
“Crop residue” refers to materials left in an agricultural field or orchard after teh crop has been harvested (field residue) and materials left after the crop is processed (process residue). Crop residue includes, but is not limited to, stalks, stems, leaves, seed pods, husks, bagasse, molasses, roots, peels (e.g., banana peels), spent brewery material, and spent biofuel material. One example of crop residue that can be used in animal feed composition is the tops and leaves of sugarcane.
Alternatively, or in addition, the animal feed can comprise a cereal. For example, the cereal can comprise barley, wheat, rye, oats, corn, rice, millet, sorghum, or a combination of any thereof.
L. Methods for Delivering Enzymes to an Animal
A method for delivering an enzyme to an animal is provided. The method comprises feeding to the animal any of the recombinant Bacillus cereus family members that express any of the fusion proteins described herein. Alternatively or in addition, the method comprises feeding to the animal exosporium fragments derived from any of the recombinant Bacillus cereus family members that expresses any of the fusion proteins described herein.
The recombinant Bacillus cereus family member that expresses any fusion protein described herein can be provided in an animal feed additive or an animal feed composition. For example, the method can comprise feeding any of the animal feed additives or animal feed compositions described herein to the animal.
Alternatively, or in addition, the method can comprise applying the recombinant Bacillus cereus family member or the exosporium fragments to a plant, to a plant seed, or to a crop residue and feeding the plant, plant seed, or crop residue to the animal.
The method can comprise applying the recombinant Bacillus cereus family member or the exosporium fragments to the plant or plant seed prior to harvest. Alternatively, the method can comprise applying the recombinant Bacillus cereus family member or the exosporium fragments to the plant or plant seed after harvest.
The plant or plant seed can comprise a soybean, barley, wheat, rye, oat, rice, millet, sorghum, grass, or alfalfa plant or plant seed, or a combination of any thereof.
The animal can comprise a livestock animal or a companion animal. For example, the animal can comprise poultry, swine, cattle, a horse, a goat, a sheep, a dog, a cat, a fish, a rabbit, or a rodent.
IX. Carriers and Agrochemicals
As described above, the formulations described herein comprise an agriculturally acceptable carrier.
The agriculturally acceptable carrier can comprise a dispersant, a surfactant (e.g., a heavy petroleum oil, a heavy petroleum distillate, a polyol fatty acid ester, a polyethoxylated fatty acid ester, an aryl alkyl polyoxyethylene glycol, an alkyl amine acetate, an alkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, or a combination of any thereof), an additive (e.g., an oil, a gum, a resin, a clay, a polyoxyethylene glycol, a terpene, a viscid organic, a fatty acid ester, a sulfated alcohol, an alkyl sulfonate, a petroleum sulfonate, an alcohol sulfate, a sodium alkyl butane diamate, a polyester of sodium thiobutane dioate, a benzene acetonitrile derivative, a proteinaceous material, or a combination of any thereof), water, a thickener (a long chain alkylsulfonate of polyethylene glycol, a polyoxyethylene oleate, or a combination of any thereof), an anti-caking agent (e.g., sodium salt, a calcium carbonate, diatomaceous earth, or a combination of any thereof), a residue breakdown product, a composting formulation, a granular application, diatomaceous earth, an oil, a coloring agent, a stabilizer, a preservative, a polymer, a coating, or a combination of any thereof.
Where the agriculturally acceptable carrier comprises a surfactant, the surfactant can comprise a non-ionic surfactant.
Where the agriculturally acceptable carrier comprises an additive and the additive comprises a proteinaceous material, the proteinaceous material can comprise a milk product, wheat flour, soybean meal, blood, albumin, gelatin, alfalfa meal, yeast extract, or a combination of any thereof.
Where the agriculturally acceptable carrier comprises an anti-caking agent and the anti-caking agent comprises a sodium salt, the sodium salt can comprise a sodium salt of monomethyl naphthalene sulfonate, a sodium salt of dimethyl naphthalene sulfonate, a sodium sulfite, a sodium sulfate, or a combination of any thereof.
The agriculturally acceptable carrier can comprise vermiculite, charcoal, sugar factory carbonation press mud, rice husk, carboxymethyl cellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, a starch, talc, polyvinyl pyrrolidone, or a combination of any thereof.
Any of the formulations described herein can comprise a seed coating formulation (e.g., an aqueous or oil-based solution for application to seeds or a powder or granular formulation for application to seeds), a liquid formulation for application to plants or to a plant growth medium (e.g., a concentrated formulation or a ready-to-use formulation), or a solid formulation for application to plants or to a plant growth medium (e.g., a granular formulation or a powder agent).
As described above, any of the formulations described herein can comprise an agrochemical.
Similarly, any of the methods described herein can further comprise applying an agrochemical to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
The agrochemical can comprise a fertilizer, a micronutrient fertilizer material, an insecticide, a nematicide, an herbicide, a plant growth amendment, a fungicide, an insecticide, a molluscicide, an algicide, a bacterial inoculant, a fungal inoculant, a plant hormone, or a combination of any thereof.
Where the agrochemical comprises a bacterial inoculant, the bacterial inoculant can comprise a plant-growth promoting strain of bacteria, an endophytic strain of bacteria, or a strain of bacteria that is both plant-growth promoting and endophytic. For example, the strain of bacteria can produce an insecticidal toxin (e.g., a Cry toxin), can produce a fungicidal compound (e.g., a β-1,3-glucanase, a chitosanase, a lyticase, or a combination of any thereof), can produce a nematicidal compound (e.g., a Cry toxin), can produce a bacteriocidal compound, can be resistant to one or more antibiotics, can comprise one or more freely replicating plasmid, can bind to plant roots, can colonize plant roots, can form biofilms, can solubilize nutrients, can secrete organic acids, or a combination of any thereof.
Where the agrochemical comprises a bacterial inoculant, the bacterial inoculant can comprise:
Each of these strains has been deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), having the address 1815 North University Street, Peoria, Illinois 61604 U.S.A., and are identified by the NRRL deposit numbers provided in parentheses. Strains (a)-(d), (f), and (g) were deposited on Mar. 7, 2013. Strains (e), (h)-(q), (w), and (hh) were deposited on Mar. 10, 2014. Strains (x)-(ff) were deposited on Sep. 10, 2014. Strain (gg) was deposited on Sep. 17, 2014. Strains (r)-(v) were deposited on Aug. 19, 2015. Bacillus thuringiensis BT013A is also known as Bacillus thuringiensis 4Q7.
It is hereby certified that the deposits were made in compliance with the terms of the Budapest Treaty and that: (i) during the pendency of this application, access to the deposited organisms will be afforded to the Commissioner upon request; (ii) all restrictions upon availability to the public of the deposited materials will be irrevocably removed upon granting of the patent, subject to 37 C.F.R. § 1.808(b); (iii) the deposit will be maintained for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer; and (iv) the deposit will be replaced if it should ever become non-viable.
The isolation and characterization of these strains is described hereinbelow in the Examples. Partial 16S ribosomal RNA sequences for each of strains (e), (h), (k)-(m), (o), (r)-(t), (v)-(x), (aa), (cc), (gg), and (hh) are provided above in Table 18. Partial 16S ribosomal RNA sequences for each of strains (a)-(d), (f), (g), (i), (j), (n), (p), (q), (u), (y), (z), (bb), and (dd)-(ff) are provided in Table 21 below.
Bacillus megaterium EE385
Bacillus sp. EE387
Bacillus circulans EE38
Bacillus subtilis EE405
Lysinibacillus fusiformis EE442
Lysinibcaillus sphaericus EE443
Bacillus aryabhattai CAP53
Bacillus aryabhattai CAP56
Bacillus flexus BT054
Paracoccus kondratievae NC35
Enterobacter cloacae CAP12
Bacillus nealsonii BOBA57
Bacillus subtilis EE148
Alcaligenes faecalis EE107
Paenibacillus massiliensis BT23
Bacillus subtilis EE218
Bacillus megaterium EE281
Bacillus pumilus EE-B00143
Where the agrochemical comprises a fertilizer, the fertilizer can comprises a liquid or a dry fertilizer.
Where the agrochemical comprises a micronutrient fertilizer material, the micronutrient fertilizer material can comprises boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate decahydrate, an iron sulfate, an iron oxide, iron ammonium sulfate, an iron frit, an iron chelate, a manganese sulfate, a manganese oxide, a manganese chelate, a manganese chloride, a manganese frit, a sodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc carbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combination of any thereof.
Where the agrochemical comprises a fertilizer, the fertilizer can comprise ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcitic limestone, calcium oxide, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K2SO4-2MgSO4, kainite, sylvinite, kieserite, Epsom salts, elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, super phosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, humic acid, or a combination of any thereof.
Where the agrochemical comprises an insecticide, the insecticide can comprises an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or a combination of any thereof.
Where the agrochemical comprises an herbicide, the herbicide can comprise a chlorophenoxy compound, a nitrophenolic compound, a nitrocresolic compound, a dipyridyl compound, an acetamide, an aliphatic acid, an anilide, a benzamide, a benzoic acid, a benzoic acid derivative, anisic acid, an anisic acid derivative, a benzonitrile, benzothiadiazinone dioxide, a thiocarbamate, a carbamate, a carbanilate, chloropyridinyl, a cyclohexenone derivative, a dinitroaminobenzene derivative, a fluorodinitrotoluidine compound, isoxazolidinone, nicotinic acid, isopropylamine, an isopropylamine derivative, oxadiazolinone, a phosphate, a phthalate, a picolinic acid compound, a triazine, a triazole, a uracil, a urea derivative, endothall, sodium chlorate, or a combination of any thereof.
Where the agrochemical comprises an herbicide, the herbicide can comprise 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazon, bromacil, bromoxynil, butylate, carfentrazone, chlorimuron, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam, cycloate, DCPA, desmedipham, dicamba, dichlobenil, diclofop, diclosulam, diflufenzopyr, dimethenamid, diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron, MCPA, MCPB, mesotrione, metolachlor-s, metribuzin, metsulfuron, molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithiobac, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin, triflusulfuron, or a combination of any thereof.
Where the agrochemical comprises a fungicide, the fungicide can comprise a substituted benzene, a thiocarbamate, an ethylene bis dithiocarbamate, a thiophthalidamide, a copper compound, an organomercury compound, an organotin compound, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione, metlaxyl, thiamimefon, triforine, or a combination of any thereof.
Where the agrochemical comprises a fungicide, the fungicide can comprise aldimorph, ampropylfos, ampropylfos potassium, andoprim, anilazine, azaconazole, azoxystrobin, benalaxyl, benodanil, benomyl, benzamacril, benzamacryl-isobutyl, bialaphos, binapacryl, biphenyl, bitertanol, blasticidin-S, boscalid, bromuconazole, bupirimate, buthiobate, calcium polysulphide, capsimycin, captafol, captan, carbendazim, carvon, quinomethionate, chlobenthiazone, chlorfenazole, chloroneb, chloropicrin, chlorothalonil, chlozolinate, clozylacon, cufraneb, cymoxanil, cyproconazole, cyprodinil, cyprofuram, debacarb, dichlorophen, diclobutrazole, diclofluanid, diclomezine, dicloran, diethofencarb, dimethirimol, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinocap, diphenylamine, dipyrithione, ditalimfos, dithianon, dodemorph, dodine, drazoxolon, edifenphos, epoxiconazole, etaconazole, ethirimol, etridiazole, famoxadon, fenapanil, fenarimol, fenbuconazole, fenfuram, fenitropan, fenpiclonil, fenpropidin, fenpropimorph, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, flumetover, fluoromide, fluquinconazole, flurprimidol, flusilazole, flusulfamide, flutolanil, flutriafol, folpet, fosetyl-aluminium, fosetyl-sodium, fthalide, fuberidazole, furalaxyl, furametpyr, furcarbonil, furconazole, furconazole-cis, furmecyclox, guazatine, hexachlorobenzene, hexaconazole, hymexazole, imazalil, imibenconazole, iminoctadine, iminoctadine albesilate, iminoctadine triacetate, iodocarb, iprobenfos (IBP), iprodione, irumamycin, isoprothiolane, isovaledione, kasugamycin, kresoxim-methyl, copper preparations, such as: copper hydroxide, copper naphthenate, copper oxychloride, copper sulphate, copper oxide, oxine-copper and Bordeaux mixture, mancopper, mancozeb, maneb, meferimzone, mepanipyrim, mepronil, metconazole, methasulfocarb, methfuroxam, metiram, metomeclam, metsulfovax, mildiomycin, myclobutanil, myclozolin, nickel dimethyldithiocarbamate, nitrothal-isopropyl, nuarimol, ofurace, oxadixyl, oxamocarb, oxolinic acid, oxycarboxim, oxyfenthiin, paclobutrazole, pefurazoate, penconazole, pencycuron, phosdiphen, pimaricin, piperalin, polyoxin, polyoxorim, probenazole, prochloraz, procymidone, propamocarb, propanosine-sodium, propiconazole, propineb, prothiocinazole, pyrazophos, pyrifenox, pyrimethanil, pyroquilon, pyroxyfur, quinconazole, quintozene (PCNB), sulphur and sulphur preparations, tebuconazole, tecloftalam, tecnazene, tetcyclasis, tetraconazole, thiabendazole, thicyofen, thifluzamide, thiophanate-methyl, tioxymid, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazbutil, triazoxide, trichlamide, tricyclazole, tridemorph, trifloxystrobin, triflumizole, triforine, uniconazole, validamycin A, vinclozolin, viniconazole, zarilamide, zineb, ziram and also Dagger G, OK-8705, OK-8801, a-(1,1-dimethylethyl)-(3-(2-phenoxyethyl)-1H-1,2,4-triazole-1-ethanol, a-(2,4-dichlorophenyl)-[3-fluoro-3-propyl-1H-1,2,4-triazole-1-ethanol, a-(2,4-dichlorophenyl)-[3-methoxy-a-methyl-1H-1,2,4-triazole-1-ethanol, a-(5-methyl-1,3-dioxan-5-yl)[3-[[4-(trifluoromethyl)-phenyl]-methylene]-1H-1,2,4-triazole-1-ethanol, (5RS,6RS)-6-hydroxy-2,2,7,7-tetramethyl-5-(1H-1,2,4-triazol-1-yl)-3-octanone, (E)-a-(methoxyimino)-N-methyl-2-phenoxy-phenylacetamide, 1-isopropyl {2-methyl-1-[[[1-(4-methylphenyl)-ethyl]-amino]-carbonyl]-propyl}carbamate, 1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-ethanone-O-(phenyl methyl)-oxime, 1-(2-methyl-1-naphthalenyl)-1H-pyrrole-2,5-dione, 1-(3,5-dichlorophenyl)-3-(2-propenyl)-2,5-pyrrolidindione, 1-[(diiodomethyl)-sulphonyl]-4-methyl-benzene, 1-[[2-(2,4-dichlorophenyl)-1, 3-dioxolan-2-yl]-methyl]-1H-imidazole, 1-[[2-(4-chlorophenyl)-3-phenyloxiranyl]-methyl]-1H-1,2,4-triazole, 1-[1-[2-[(2,4-dichlorophenyl)-methoxy]-phenyl]-ethenyl]-1H-imidazole, 1-methyl-5-nonyl-2-(phenylmethyl)-3-pyrrolidinole, 2′,6′-dibromo-2-methyl-4′-trifluoromethoxy-4′-trifluoro-methyl-1, 3-thiazole-carboxanilide, 2,2-dichloro-N-[1-(4-chlorophenyl)-ethyl]-1-ethyl-3-methyl-cyclopropanecarboxamide, 2,6-dichloro-5-(methylthio)-4-pyrimidinyl-thiocyanate, 2,6-dichloro-N-(4-trifluoromethylbenzyl)-benzamide, 2,6-dichloro-N-[[4-(trifluoromethyl)-phenyl]-methyl]-benzamide, 2-(2,3,3-triiodo-2-propenyl)-2H-tetrazole, 2-[(1-methylethyl)-sulphonyl]-5-(trichloromethyl)-1,3,4-thiadiazole, 2-[[6-deoxy-4-O-(4-0-methyl-(3-D-glycopyranosyl)-a-D-glucopyranos yl]-amino]-4-methoxy-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile, 2-aminobutane, 2-bromo-2-(bromomethyl)-pentanedinitrile, 2-chloro-N-(2,3-dihydro-1,1,3-trimethyl-1H-inden-4-yl)-3-pyridinecarboxamide, 2-chloro-N-(2,6-dimethylphenyl)-N-(isothiocyanatomethyl)-acetamide, 2-phenylphenol (OPP), 3,4-dichloro-1-[4-(difluoromethoxy)-phenyl]-pyrrole-2,5-dione, 3,5-dichloro-N-[cyano[(1-methyl-2-propynyl)-oxy]-methyl]-benzamide, 3-(1,1-dimethylpropyl-1-oxo-1H-indene-2-carbonitrile, 3-[2-(4-chlorophenyl)-5-ethoxy-3-isoxazolidinyl]-pyridine, 4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)-1H-imidazole-1-sulphonamide, 4-methyl-tetrazolo[1,5-a]quinazolin-5(4H)-one, 8-(1,1-dimethylethyl)-N-ethyl-N-propyl-1,4-dioxaspiro[4, 5]decane-2-methanamine, 8-hydroxyquinoline sulphate, 9H-xanthene-2-[(phenylamino)-carbonyl]-9-carboxylic hydrazide, bis-(1-methylethyl)-3-methyl-4-[(3-methylbenzoyl)-oxy]-2,5-thiophenedicarboxylate, cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-cycloheptanol, cis-4-[3-[4-(1,1-dimethylpropyl)-phenyl-2-methylpropyl]-2,6-dimethyl-morpholine hydrochloride, ethyl [(4-chlorophenyl)-azo]-cyanoacetate, potassium bicarbonate, methanetetrathiol-sodium salt, methyl 1-(2,3-dihydro-2,2-dimethyl-inden-1-yl)-1H-imidazole-5-carboxylate, methyl N-(2,6-dimethylphenyl)-N-(5-isoxazolylcarbonyl)-DL-alaninate, methyl N-(chloroacetyl)-N-(2,6-dimethylphenyl)-DL-alaninate, N-(2,3-dichloro-4-hydroxyphenyl)-1-methyl-cyclohexanecarboxamide, N-(2,6-dimethyl phenyl)-2-methoxy-N-(tetra hydro-2-oxo-3-furanyl)-acetamide, N-(2,6-dimethyl phenyl)-2-methoxy-N-(tetrahydro-2-oxo-3-thienyl)-acetamide, N-(2-chloro-4-nitrophenyl)-4-methyl-3-nitro-benzenesulphonamide, N-(4-cyclohexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine, N-(4-hexylphenyl)-1,4,5,6-tetrahydro-2-pyrimidinamine, N-(5-chloro-2-methylphenyl)-2-methoxy-N-(2-oxo-3-oxazolidinyl)-acetamide, N-(6-methoxy)-3-pyridinyl)-cyclopropanecarboxamide, N-[2,2,2-trichloro-1-[(chloroacetyl)-amino]-ethyl]-benzamide, N-[3-chloro-4,5-bis(2-propinyloxy)-phenyl]-N′-methoxy-methanimidamide, N-formyl-N-hydroxy-DL-alanine-sodium salt, 0,0-diethyl [2-(dipropylamino)-2-oxoethyl]-ethylphosphoramidothioate, O-methyl S-phenyl phenylpropylphosphoramidothioate, S-methyl 1,2,3-benzothiadiazole-7-carbothioate, and spiro[2H]-1-benzopyrane-2,1′(3′H)-isobenzofuran]-3′-one, N-trichloromethyl)thio-4-cyclohexane-1,2-dicarboximide, tetramethylthioperoxydicarbonic diamide, methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alaninate, 4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1-H-pyrrol-3-carbonitril, or a combination of any thereof.
Where the agrochemical comprises a fungal inoculant, the fungal inoculant can comprise a fungal inoculant of the family Glomeraceae, a fungal inoculant of the family Claroidoglomeraceae, a fungal inoculant of the family Gigasporaceae, a fungal inoculant of the family Acaulosporaceae, a fungal inoculant of the family Sacculosporaceae, a fungal inoculant of the family Entrophosporaceae, a fungal inoculant of the family Pacidsporaceae, a fungal inoculant of the family Diversisporaceae, a fungal inoculant of the family Paraglomeraceae, a fungal inoculant of the family Archaeosporaceae, a fungal inoculant of the family Geosiphonaceae, a fungal inoculant of the family Ambisporaceae, a fungal inoculant of the family Scutellosporaceae, a fungal inoculant of the family Dentiscultataceae, a fungal inoculant of the family Racocetraceae, a fungal inoculant of the phylum Basidiomycota, a fungal inoculant of the phylum Ascomycota, a fungal inoculant of the phylum Zygomycota, or a combination of any thereof.
Where the agrochemical comprises a bacterial inoculant, the bacterial inoculant can comprise a bacterial inoculant of the genus Rhizobium, a bacterial inoculant of the genus Bradyrhizobium, a bacterial inoculant of the genus Mesorhizobium, a bacterial inoculant of the genus Azorhizobium, a bacterial inoculant of the genus Allorhizobium, a bacterial inoculant of the genus Sinorhizobium, a bacterial inoculant of the genus Kluyvera, a bacterial inoculant of the genus Azotobacter, a bacterial inoculant of the genus Pseudomonas, a bacterial inoculant of the genus Azospirillium, a bacterial inoculant of the genus Bacillus, a bacterial inoculant of the genus Streptomyces, a bacterial inoculant of the genus Paenibacillus, a bacterial inoculant of the genus Paracoccus, a bacterial inoculant of the genus Enterobacter, a bacterial inoculant of the genus Alcaligenes, a bacterial inoculant of the genus Mycobacterium, a bacterial inoculant of the genus Trichoderma, a bacterial inoculant of the genus Gliocladium, a bacterial inoculant of the genus Glomus, a bacterial inoculant of the genus Klebsiella, or a combination of any thereof.
Where the agrochemical comprises a bacterial inoculant and the bacterial inoculant comprises a bacterial inoculant of the genus Bacillus, the bacterial inoculant of the genus Bacillus can comprise Bacillus argri, Bacillus aizawai, Bacillus albolactis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus coagulans, Bacillus endoparasiticus, Bacillus endorhythmos, Bacillus kurstaki, Bacillus lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus laterosporus, Bacillus lentimorbus, Bacillus licheniformis, Bacillus megaterium, Bacillus medusa, Bacillus metiens, Bacillus natto, Bacillus nigrificans, Bacillus popillae, Bacillus pumilus, Bacillus siamensis, Bacillus sphearicus, Bacillus spp., Bacillus subtilis, Bacillus thuringiensis, Bacillus unifagellatu, or a combination of any thereof.
Where the agrochemical comprises a plant hormone, the plant hormone can comprise a gibberellin, an auxin, a kinetin, or a combination of any thereof.
Any of the formulations described herein can comprise a fertilizer, a biostimulant, or a combination thereof.
Likewise, any of the methods described herein can further comprise applying a fertilizer, a biostimulant, or a combination thereof to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed.
The fertilizer can comprise nitrogen, phosphate (e.g., monoammonium phosphate, diammonium phosphate, orthophosphate, orthopolyphosphate, or a combination of any thereof), potassium (e.g., potassium acetate), zinc, iron, selenium, boron, copper, or a combination of any thereof.
For example, the fertilizer can comprise 12% ammoniacal nitrogen and 58% available phosphate.
The biostimulant can comprises a gibberellic acid, an indole-3-butyric acid, a kinetin, an auxin, an auxin homolog or derivative, or a combination of any thereof.
Where the formulation comprises a fertilizer, a biostimulant, or a combination thereof, the fusion protein suitably comprises an acid phosphatase, a phospholipase, a mannanase, a glucanase, or a combination of any thereof.
Similarly, where the method further comprises applying a fertilizer, a biostimulant, or a combination thereof to the plant growth medium, the plant, the plant seed, or the area surrounding the plant or the plant seed, the fusion protein suitably comprises an acid phosphatase, a phospholipase, a mannanase, a glucanase, or a combination of any thereof.
X. Free Enzymes
Any of the enzymes described herein can also be used as free enzymes or as enzymes expressed in recombinant microorganisms in any of the methods, formulations, compositions, and plant seeds, described in U.S. Patent Application Publication No. US 2017/0356002 the contents of which are hereby incorporated by reference in their entirety.
XI. Plants
In any of the above methods relating to plants, the plant can be a dicotyledon, a monocotyledon, or a gymnosperm.
Likewise, for any of the seeds described herein the seed can be a seed of a dicotyledon, a monocotyledon, or a gymnosperm.
For example, where the plant is a dicotyledon or the seed is a seed of a dicotyledon, the dicotyledon can be selected from the group consisting of bean, pea, tomato, pepper, squash, alfalfa, almond, aniseseed, apple, apricot, arracha, artichoke, avocado, bambara groundnut, beet, bergamot, black pepper, black wattle, blackberry, blueberry, bitter orange, bok-choi, Brazil nut, breadfruit, broccoli, broad bean, Brussels sprouts, buckwheat, cabbage, camelina, Chinese cabbage, cacao, cantaloupe, caraway seeds, cardoon, carob, carrot, cashew nuts, cassava, castor bean, cauliflower, celeriac, celery, cherry, chestnut, chickpea, chicory, chili pepper, chrysanthemum, cinnamon, citron, clementine, clove, clover, coffee, cola nut, colza, corn, cotton, cottonseed, cowpea, crambe, cranberry, cress, cucumber, currant, custard apple, drumstick tree, earth pea, eggplant, endive, fennel, fenugreek, fig, filbert, flax, geranium, gooseberry, gourd, grape, grapefruit, guava, hemp, hempseed, henna, hop, horse bean, horseradish, indigo, jasmine, Jerusalem artichoke, jute, kale, kapok, kenaf, kohlrabi, kumquat, lavender, lemon, lentil, lespedeza, lettuce, lime, liquorice, litchi, loquat, lupine, macadamia nut, mace, mandarin, mangel, mango, medlar, melon, mint, mulberry, mustard, nectarine, niger seed, nutmeg, okra, olive, opium, orange, papaya, parsnip, pea, peach, peanut, pear, pecan nut, persimmon, pigeon pea, pistachio nut, plantain, plum, pomegranate, pomelo, poppy seed, potato, sweet potato, prune, pumpkin, quebracho, quince, trees of the genus Cinchona, quinoa, radish, ramie, rapeseed, raspberry, rhea, rhubarb, rose, rubber, rutabaga, safflower, sainfoin, salsify, sapodilla, Satsuma, scorzonera, sesame, shea tree, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, swede, sweet pepper, tangerine, tea, teff, tobacco, tomato, trefoil, tung tree, turnip, urena, vetch, walnut, watermelon, yerba mate, wintercress, shepherd's purse, garden cress, peppercress, watercress, pennycress, star anise, laurel, bay laurel, cassia, jamun, dill, tamarind, peppermint, oregano, rosemary, sage, soursop, pennywort, calophyllum, balsam pear, kukui nut, Tahitian chestnut, basil, huckleberry, hibiscus, passionfruit, star apple, sassafras, cactus, St. John's wort, loosestrife, hawthorn, cilantro, curry plant, kiwi, thyme, zucchini, ulluco, jicama, waterleaf, spiny monkey orange, yellow mombin, starfruit, amaranth, wasabi, Japanese pepper, yellow plum, mashua, Chinese toon, New Zealand spinach, bower spinach, ugu, tansy, chickweed, jocote, Malay apple, paracress, sowthistle, Chinese potato, horse parsley, hedge mustard, campion, agate, cassod tree, thistle, burnet, star gooseberry, saltwort, glasswort, sorrel, silver lace fern, collard greens, primrose, cowslip, purslane, knotgrass, terebinth, tree lettuce, wild betel, West African pepper, yerba santa, tarragon, parsley, chervil, land cress, burnet saxifrage, honeyherb, butterbur, shiso, water pepper, perilla, bitter bean, oca, kampong, Chinese celery, lemon basil, Thai basil, water mimosa, cicely, cabbage-tree, moringa, mauka, ostrich fern, rice paddy herb, yellow sawah lettuce, lovage, pepper grass, maca, bottle gourd, hyacinth bean, water spinach, catsear, fishwort, Okinawan spinach, lotus sweetjuice, gallant soldier, culantro, arugula, cardoon, caigua, mitsuba, chipilin, samphire, mampat, ebolo, ivy gourd, cabbage thistle, sea kale, chaya, huauzontle, Ethiopian mustard, magenta spreen, good king henry, epazole, lamb's quarters, centella plumed cockscomb, caper, rapini, napa cabbage, mizuna, Chinese savoy, kai-lan, mustard greens, Malabar spinach, chard, marshmallow, climbing wattle, China jute, paprika, annatto seed, spearmint, savory, marjoram, cumin, chamomile, lemon balm, allspice, bilberry, cherimoya, cloudberry, damson, pitaya, durian, elderberry, feijoa, jackfruit, jambul, jujube, physalis, purple mangosteen, rambutan, redcurrant, blackcurrant, salal berry, satsuma, ugli fruit, azuki bean, black bean, black-eyed pea, borlotti bean, common bean, green bean, kidney bean, lima bean, mung bean, navy bean, pinto bean, runner bean, mangetout, snap pea, broccoflower, calabrese, nettle, bell pepper, raddichio, daikon, white radish, skirret, tat soi, broccolini, black radish, burdock root, fava bean, broccoli raab, lablab, lupin, sterculia, velvet beans, winged beans, yam beans, mulga, ironweed, umbrella bush, tjuntjula, wakalpulka, witchetty bush, wiry wattle, chia, beech nut, candlenut, colocynth, mamoncillo, Maya nut, mongongo, ogbono nut, paradise nut, and cempedak.
Where the plant is a monocotyledon or the seed is a seed of a monocotyledon, the monocotyledon can be selected from the group consisting of corn, wheat, oat, rice, barley, millet, banana, onion, garlic, asparagus, ryegrass, millet, fonio, raishan, nipa grass, turmeric, saffron, galangal, chive, cardamom, date palm, pineapple, shallot, leek, scallion, water chestnut, ramp, Job's tears, bamboo, ragi, spotless watermeal, arrowleaf elephant ear, Tahitian spinach, abaca, areca, bajra, betel nut, broom millet, broom sorghum, citronella, coconut, cocoyam, maize, dasheen, durra, durum wheat, edo, Pique, formio, ginger, orchard grass, esparto grass, Sudan grass, guinea corn, Manila hemp, henequen, hybrid maize, jowar, lemon grass, maguey, bulrush millet, finger millet, foxtail millet, Japanese millet, proso millet, New Zealand flax, oats, oil palm, palm palmyra, sago palm, redtop, sisal, sorghum, spelt wheat, sweet corn, sweet sorghum, taro, teff, timothy grass, triticale, vanilla, wheat, and yam.
Where the plant is a gymnosperm or the seed is a seed of a gymnosperm, the gymnosperm can be from a family selected from the group consisting of Araucariaceae, Boweniaceae, Brassicaceae, Cephalotaxaceae, Cupressaceae, Cycadaceae, Ephedraceae, Ginkgoaceae, Gnetaceae, Pinaceae, Podocarpaceae, Taxaceae, Taxodiaceae, Welwitschiaceae, and Zamiaceae.
When the plant is from the family Brassicaceae, the plant can comprise a plant of the genus Brassica. For example, the plant of the family Brassicaceae can comprise Brassica napus, Brassica rapa, Brassica juncea, Brassica hirta, Brassica oleracea, Raphanus sativus, Sinapus alba, or Lepidium sativum.
The plants and plant seeds described herein may include transgenic plants or plant seeds, such as transgenic cereals (wheat, rice), maize, soybean, potato, cotton, tobacco, oilseed rape and fruit plants (fruit of apples, pears, citrus fruits and grapes. Preferred transgenic plants include corn, soybeans, potatoes, cotton, tobacco and oilseed rape.
Suitable transgenic plants and seeds can be characterized by the plant's formation of toxins, especially from the Bacillus thuringiensis genetic material (e.g., by gene CryIA (a), CryIA (h), CryIA (c), CryIIA, CryIIIA, CryIIIB2, Cry9c, Cry2Ab, Cry3Bb, CryIF or a combination thereof). The formation of toxins in plants increases the plant's resistance to insects, arachnids, nematodes and slugs and snails (hereinafter referred to as “Bt plants”). Bt plants, for example, are commercially available under the tradename YIELD GARD (for example maize, cotton, soybeans), Knock0ut® (for example maize), StarLink® (for example maize), Bollgard® (cotton), Nucotn® (cotton) and NewLeaf® (potato) maize varieties, cotton varieties, soybean varieties and potato varieties. Herbicide tolerance plants include plants under the trade names Roundup Ready® (a glyphosate tolerance, such as corn, cotton, soybeans), Clearfield® (for example maize), Liberty Link (tolerance with glufosinate, for example oilseed rape), IMI® (with imidazolinone tolerance) and STS® (tolerance to a sulfonylurea, such as maize).
Plant seeds as described herein can be genetically modified (e.g., any seed that results in a genetically modified plant or plant part that expresses herbicide tolerance, tolerance to environmental factors such as water stress, drought, viruses, and nitrogen production, or resistance to bacterial, fungi or insect toxins). Suitable genetically modified seeds include those of tole crops, vegetables, fruits, trees, fiber crops, oil crops, tuber crops, coffee, flowers, legume, cereals, as well as other plants of the monocotyledonous and dicotyledonous species. Preferably, the genetically modified seeds include peanut, tobacco, grasses, wheat, barley, rye, sorghum, rice, rapeseed, sugarbeet, sunflower, tomato, pepper, bean, lettuce, potato, and carrot. Most preferably, the genetically modified seeds include cotton, soybean, and corn (sweet, field, seed, or popcorn).
Particularly useful transgenic plants which may be treated according to the invention are plants containing transformation events, or a combination of transformation events, that are listed for example in the databases from various national or regional regulatory agencies (see for example gmoinfo.jrc.it/gmp_browse.aspx and www.agbios.com/dbase.php).
Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.
The following non-limiting examples are provided to further illustrate the present invention. Additional examples, demonstrating the ability of various enzymes as described herein to stimulate plant growth and/or promote plant health when applied to plants as free enzymes, are included in U.S. Patent Application Publication No. US 2017/0356002 the contents of which are hereby incorporated by reference in their entirety.
A wide variety of targeting sequences that that have a high degree of homology with amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) can be used to display enzymes, proteins, and peptides on the surface of Bacillus cereus family members. Several targeting sequences were compared by making fusion proteins containing the targeting sequences linked to Bacillus subtilis lipase (the signal peptide of SEQ ID NO: 324, directly linked to the lipase of SEQ ID NO: 263). Fusion constructs were synthesized using the promoters native to the targeting sequence, cloned into the replicating plasmid pMK4, and introduced into Bacillus thuringiensis BT013A. Bacillus thuringiensis BT013A was deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS), having the address 1815 North University Street, Peoria, Illinois 61604 U.S.A., on Mar. 10, 2014, and assigned NRRL deposit number B-50924. Bacillus thuringiensis BT013A is also known as Bacillus thuringiensis 4Q7.
Strains were taken into sporulation by incubation at 30° C. on nutrient agar plates containing chloramphenicol 10 μg/ml for 3 days. Spores were collected, washed, and resuspended in PBS at a rate of 1×108/ml. 1×105 spores for each fusion construct were suspended in 400 μl dH2O. The reactions were warmed with the reaction components to the desired reaction temperature (40° C.). 200 μl working buffer was added (9:1 Solution A: Solution B). Solution A was 50 mM Tris pH 10 and 13.6 mM deoxycholic acid and Solution B was 3 mg/ml p-nitrophenyl palmitate in isopropanol. The reaction was incubated at 40° C. for 10 minutes and placed on ice, centrifuged to remove spores, and absorbance at 420 nm was recorded. The results are shown in Table 22 below. Activity was normalized to a control fusion protein comprising amino acids 1-35 of SEQ ID NO: 1 fused to Bacillus subtilis lipase.
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
Several targeting sequences linked to lipase result in higher expression levels and activity of enzyme on the surface of spores. In particular, SEQ ID NOs. 96, 98, and 100, each containing a shorter targeting sequence, resulted in enhanced fusion expression on the surface of the BEMD spores. All the fusion proteins containing targeting sequences tested resulted in surface display of lipase.
A wide variety of exosporium proteins can be used to display enzymes, proteins, and peptides on the surface of Bacillus cereus family members. Several different exosporium proteins were compared by making fusion proteins containing the exosporium proteins linked to Bacillus subtilis lipase as described in Example 1. Fusion constructs were synthesized using the promoter native to the exosporium protein indicated in Table 23 below, cloned into the replicating plasmid pMK4, and introduced into Bacillus thuringiensis BT013A. Spores displaying the various exosporium protein—Bacillus subtilis 168 lipase fusions were made by growing the transformed bacteria in brain heart infusion broth with selective pressure from 10 μg/ml chloramphenicol, plating onto nutrient agar plates, and incubating at 30° C. for 3 days. After 3 days, the spores were washed off the plates, purified by centrifugation, and resuspended in PBS at 1×108 CFU/ml.
1×105 spores for each fusion construct were resuspended in 400 μl dH2O. The reactions were warmed with the reaction components to the desired reaction temperature (40° C.). 200 μl of working buffer was added (9:1 Solution A: Solution B). Solution A was 50 mM Tris pH 10 and 13.6 mM deoxycholic acid and Solution B was 3 mg/ml p-nitrophenyl palmitate in isopropanol. The reaction was incubated at 40° C. for 10 minutes and placed on ice, centrifuged to remove spores and absorbance at 420 nm was recorded. Results are shown in Table 23 below. Activity was normalized to SEQ ID NO: 109 linked to lipase.
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
B. thuringiensis
Use of the exosporium proteins of SEQ ID NOs. 109 and 110 resulted in the highest enzyme activity on the spore. All the fusion proteins containing exosporium proteins resulted in surface display of active Bacillus subtilis 168 lipase, albeit at different levels.
Additional exosporium proteins were demonstrated to result in targeting of fusion proteins to the exosporium using the fluorescent reporter mCherry. Fusion constructs were created that contained the exosporium proteins of SEQ ID NOs. 111, 120, and 110 linked to the mCherry reporter. Spores were grown for 1.5 days, collected, and resuspended as described above. 7 μl of fluorescent spores were put under a Nikon E1000 microscope and imaged during late sporulation. Circular localization in a ring is indicative of outer spore layer localization, and the appearance matches that of an exosporium protein. Fluorescent microscopy results are shown in
BEMD spores expressing Bacillus subtilis EE148 Phosphatase A4 (PhoA4) were created by gene synthesis of the genes coding for various targeting sequences and exosporium proteins under the control of their native promoters linked to PhoA4. The synthesized genes were cloned into pMK4 and introduced into Bacillus thuringiensis BT013A. Spores displaying the various exosporium protein—Bacillus subtilis EE148 PhoA4 fusions were made by growing the transformed bacteria in brain heart infusion broth with selective pressure from 10 μg/ml chloramphenicol, plating onto nutrient agar plates, and incubating at 30° C. for three days. After three days, the spores were washed off the plates, purified by centrifugation, and resuspended in PBS at 1×108 CFU/ml.
Soybeans were planted 2.54 cm deep in 10 cm deep pots filled with standard loam topsoil. BEMD spores expressing PhoA4 were diluted to a concentration of 1×104/ml in 50 ml of water and applied to each plant at planting. A water-only control was also included. Polyphosphate was added to pots in liquid at a rate of 0.5 mg/pot. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 13 hours of light a day under controlled temperature conditions between 15.5-25.5° C. Plants were watered to saturation every three days over the two week trial. At the end of two weeks, the height of each plant was measured, and measurements were normalized to control water-only plants.
Results are shown in Table 24 below. Soy grown in the presence of BEMD spores expressing fusion proteins containing PhoA4 linked to various targeting sequences and exosporium proteins with different fusion partners with PhoA4 all exhibited enhanced growth, but the extent of the effect varied depending on the targeting sequence or exosporium protein used.
Bacillus species
100%
Bacillus thuringiensis
100%
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
The pSUPER plasmid was generated through fusion of the pUC57 plasmid (containing an ampicillin resistance cassette) with the pBC16-1 plasmid from Bacillus (containing a tetracycline resistance). This 5.5 kbp plasmid can replicate in both E. coli and Bacillus spp. The pSUPER plasmid was modified by cloning of a PCR generated fragment through homologous recombination that fused the BclA promoter, start codon, and amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) in frame with Bacillus subtilis 168 endoglucanase (the signal peptide of SEQ ID NO: 334, directly linked to the β-1,4-endoglucanase of SEQ ID NO: 293; pSUPER-BclA 20-35-Endo). PCR fragments were generated that contained the BclA promoter (SEQ ID NO: 149), start codon, and amino acids 20-35 of BclA fused in frame to Bacillus subtilis 168 endoglucanase. These PCR fragments were digested with XhoI and ligated into the SalI site of the pSUPER plasmid to generate the plasmid pSUPER-BclA 20-35-Endoglucanase. This plasmid was then subjected to inverse PCR to amplify the entire plasmid backbone, but leaving out the sequence corresponding to amino acids 20-35 of BclA. This inverse PCR product was combined with a PCR product that amplified the equivalent region from each of SEQ ID NOs. 5, 15, 25, 81, 85, 87, or amino acids 20-33 of SEQ ID NO: 1. Thus, constructs were created that contained each of the following targeting sequences fused in frame with Bacillus subtilis 168 endoglucanase: (1) amino acids 20-35 of SEQ ID NO: 1; (2) amino acids 23-38 of SEQ ID NO: 5; (3) amino acids 28-43 of SEQ ID NO: 15; (4) amino acids 9-24 of SEQ ID NO: 25; (5) amino acids 23-38 of SEQ ID NO: 81; (6) amino acids 13-28 of SEQ ID NO: 85; (7) amino acids 13-28 of SEQ ID NO: 87; and (8) amino acids 20-33 of SEQ ID NO: 1. Each construct contained the wildtype BclA promoter and a methionine at the start codon, followed by the targeting sequence fused in frame to the Bacillus subtilis endoglucanase gene. Each of these constructs was transformed into E. coli and plated to obtain single colonies on Luria plates plus ampicillin (100 μg/ml). Plasmids from each single colony were grown up in overnight cultures in Luria broth plus ampicillin, and purified using a WIZARD SV miniprep kit, and sequences were verified by Sanger sequencing. DNA was also quantified via spectrophotometry, and the DNA was introduced into Bacillus thuringiensis BT013A. In addition, the pSUPER-BclA-20-35 Endo construct was introduced into Bacillus thuringiensis BT013A which had the native BclA protein removed from its genome through homologous recombination (BclA knockout, “BclA KO”). Correct colonies were screened by plating on nutrient broth plate containing antibiotic (tetracycline at 10 μg/ml). Each positive colony was grown up in brain heart infusion broth at 30° C. overnight at 300 rpm, with antibiotic, and genomic DNA was purified and re-sequenced to verify genetic purity. Verified colonies were grown overnight in brain heart infusion broth with 10 μg/ml tetracycline, and induced to sporulate through sporulation in a yeast extract-based media.
Each of the production runs in the yeast extract-based media were collected at 48 hours post production of spores, and subjected to enzyme comparison of the resultant spores. The assay for endoglucanase activity was performed by determining cellulase activity using a carboxymethylcellulose (CMC) substrate and a dinitrosalicylic acid (DNS reagent). A commercial source of cellulase enzyme was used to prepare standards in 50 mM citrate buffer, pH 4.8. 1% CMC (carboxymethylcellulose sodium salt) was prepared in 50 mM citrate buffer, pH 4.8, to serve as the substrate for the reaction. 250 μl of spore preparation was pelleted and the spores were resuspended in 150 μL of 50 mM citrate buffer, pH 4.8. The reaction was carried out with a reagent composed of 1% DNS, 1% NaOH, 0.05% Na2SO4, 0.2% phenol, and 18.2% Rochelle salts. 150 μl of the sample was mixed with 250 μl of the 1% CMC substrate and incubated in a water bath at 50° C. for 15 minutes. 300 μl of DNS reagent was added and the samples boiled at 100° C. for 10 minutes and then cooled on ice. The solution was centrifuged for 5 minutes at 14,000×g to remove the spores from the absorbance reading. The absorbance was determined at 540 nm using an IMPLEN nanospectrophotometer model P330. Samples were performed in triplicate with a blank for each reaction. The results from the enzyme readings are shown in Table 25.
For corn, 1 μl of each of the whole broth for each of the constructs was placed onto each seed. For summer squash, 2 μl of whole broth for each construct was placed onto each seed. To accomplish this, 50 seeds were placed in a 50 ml conical bottom polypropylene tube and vortexed lightly using a vortex mixer. To this swirling of seeds, 50 μl (for corn) or 100 μl (for squash) of broth containing the recombinant spores was slowly pipetted into the tube, and the vortexing action coated the seeds with an even coating of the whole cell broth from each construct. These seeds were then planted at 1″ deep into native soil using a 39.6 cm3 (15.6 in3) planting pot, with two seeds per pot. The pots were then watered to saturation, and the plants allowed to germinate. The plants were grown in a controlled growth room, set to 70° F. during the day, and 60° F. during the evening, with a light period of 14 hours/day, under artificial light conditions, for 14 days. After 14 days, the plants were measured for height, and results were normalized to a control group that received only water as treatment on the seeds.
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112%
The above data show that each of these constructs was able to stimulate plant growth and show that the use of different targeting sequences allows for control of the expression level of the enzyme on the outside of the spore.
Use of amino acids 20-35 of SEQ ID NO: 1 or AA 13-28 of SEQ ID NO: 85 as the targeting sequence resulted in the highest levels of enzyme production. This is surprising considering the low degree of identity between these targeting sequences (43.8% identity over the entire length of the targeting sequence). Use of amino acids 28-43 of SEQ ID NO: 15 or amino acids 9-24 of SEQ ID NO: 25 resulted in the largest plant response across the two plant types. Expression of the fusion protein containing amino acids 20-25 of SEQ ID NO: 1 as the targeting sequence in the BT013A BclA KO host led to very large (263.8%) increase in the amount of enzyme activity on the surface of the spores as compared to expression of the same fusion protein in the wild-type strain.
The pSUPER plasmid was modified by cloning of a PCR generated fragment (XhoI digestion and ligation) that fused the BclA promoter, start codon, and amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) followed by a six alanine linker sequence in frame with either Bacillus thuringiensis phosphatidylcholine-specific phospholipase C gene (PC-PLC) (the signal peptide of SEQ ID NO: 317, directly linked to the PLC of SEQ ID NO: 250; pSUPER-BclA 20-35-PL) or Bacillus subtilis lipase LipA (the signal peptide of SEQ ID NO: 324, directly linked to the lipase of SEQ ID NO: 263; pSUPER-BclA-20-35-Lipase), or Bacillus subtilis endoglucanase eglS (the signal peptide of SEQ ID NO: 334, directly linked to the β-1,4-endoglucanase of SEQ ID NO: 293; pSUPER-BclA-20-35-Endo) as described above in Example 4. These plasmids were then subjected to inverse PCR to amplify the entire plasmid backbone, but without the sequence corresponding to amino acids 20-35 of BclA. This inverse PCR product was combined with a PCR product that amplified the equivalent region from each of SEQ ID NOs. 5 (i.e., amino acids 23-38 of SEQ ID NO: 5), 15 (i.e., amino acids 28-43 of SEQ ID NO: 15), and 25 (i.e., amino acids 9-24 of SEQ ID NO: 25); the full-length exosporium proteins of SEQ ID NOs. 120, 111, 121, 108, and 114; or amino acids 20-33, 20-31, 21-33, 23-33, or 23-31 of SEQ ID NO: 1. Each of these constructs contained the wild-type BclA promoter, a methionine at the start codon, followed by the targeting sequence or exosporium protein fused in frame to the Bacillus cereus phosphatidylcholine-specific phospholipase C, Bacillus subtilis 168 Lipase LipA, or Bacillus subtilis 168 eglS endoglucanase gene. Each of these constructs was screened for correct transformants as described in Example 4 above.
Each of the production runs in the yeast extract-based media were collected at 48 hours post production of spores, and subjected to enzyme comparison of the resultant spores. Determination of enzyme data for endoglucanase was performed as described above in Example 4. For the phospholipase C enzyme assay, 1 ml of recombinant spores was pelleted at 10,000×g for 3 minutes, and supernatant removed and discarded. The spore pellet was then resuspended in 500 μl reaction buffer (0.25 mM Tris-HCL, 60% glycerol, 20 mM o-nitrophenyl phosphorylcholine, pH 7.2). A negative control for enzyme assays contained BT013A spores with no enzyme expression. Each sample was incubated at 37° C. for 18 hours, centrifuged again to remove the spores, diluted 1:1 in water, and the Abs540 read using a spectrophotometer. This was compared to a standard curve against commercially purchased phospholipase and lipase controls to establish the U/ml of activity. The results from the enzyme readings are shown in Tables 26 and 27.
Many of the targeting sequences and exosporium proteins were able to display a large amount of active enzymes on the surface of the spores, including SEQ ID NOs. 108, 111, 114, 120, and 121. Amino acids 20-31, 21-33, and 23-31 of SEQ ID NO: 1 provided similar enzyme expression levels to amino acids 20-35 of SEQ ID NO: 1, indicating that smaller fragments are adequate for the display of enzymes on the surface of the spores. Only amino acids 23-33 of SEQ ID NO: 1 exhibited a diminished enzyme display level on the spores.
Similar to the results shown above in Table 26, the highest levels of phospholipase or lipase on the spore surface were observed when amino acids 20-35 of SEQ ID NO: 1, amino acids 23-38 of SEQ ID NO: 5, or the exosporium protein sequence of SEQ ID NO: 120 were used.
The effects of spores expressing the BclA 20-35-PL construct on nodulation in soybeans are shown below in Table 28.
Bacillus thuringiensis BT013A
Spores expressing the 20-35-PL construct and non-transformed spores were applied to soybean seed using 1 μL volumes of spore preparations and a spore concentration of 1.8-1.9×108 spores per mL (from cultures that resulted in approximately 99% sporulation). Seeds were treated before planting by pipetting 1 μL of the spore preparation directly onto each seed and allowing it to dry. Nodulation was examined after about three weeks, at the V3 stage of development. Plants were carefully removed, dirt washed gently off of the roots, and nodules counted for each plant. As shown in Table 28, addition of spores displaying phospholipase onto the seeds of soybean allows for an accelerated number of nodules on the plants, which is a positive indication for both early growth as well as eventual increases in yield in soybeans.
The BEMD system can display a wide range of proteins, peptides, and enzymes using one or more of the targeting sequences described herein. Some of these targeting sequences have a high affinity for the exosporium which would be beneficial for fusion protein expression, but their low fusion protein expression level limits their use on the BEMD system. For such fusion proteins and sequences, alternative high-expression sporulation promoters can be used instead of the native promoters.
For example, SEQ ID NO: 13 (amino acids 1-39 of B. weihenstephensis KBAB4 gene 3572) provides a very effective N-terminal sequence for the delivery of proteins to the exosporium of Bacillus cereus family members, as shown in Table 29 below. All genes were synthesized in their complete form (including promoter regions and regions coding for fusion proteins) as described herein. When the native promoter elements for B. weihenstephensis KBAB4 gene 3572 (SEQ ID NO: 177) were used to express a fusion protein comprising the targeting sequence of SEQ ID NO: 13 fused to a β-galactosidase enzyme (from E. coli), a low level of fusion protein was expressed, leading to a reduction in enzyme activity on the surface of the spore. Enzyme activity was measure by the conversion of 0.5M o-nitrophenylgalactoside in solution over 10 minutes. Enzyme conversion was measured with a spectrophotometer at ABS540. Replacement of the native promoter elements of the B. weihenstephensis KBAB4 gene 3572 with the high-expression promoters of SEQ ID NO: 157 (B. anthracis BetA/BAS3290) or SEQ ID NO: 178 (B. weihenstephensis KBAB4 YVTN β-propeller protein) led to a dramatic increase in the enzymatic activity of the spores. On the other hand, replacement of the native promoter elements for B. weihenstephensis KBAB4 gene 3572 with the promoter native to B. anthracis Sterne BAS1882 (SEQ ID NO: 176) led to a decrease in the enzymatic activity of the spores. The expression level of the targeting sequence of SEQ ID NO: 13 fused to β-galactosidase was much lower (0.38×) when driven by the promoter of BAS1882 (SEQ ID NO: 176), and was greatly improved when driven from the BetA promoter (SEQ ID NO: 197) or YVTN protein promoter (SEQ ID NO: 178).
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As shown in Example 6 above, replacing the native promoter of a targeting sequence, exosporium protein, or exosporium protein fragment can greatly affect the level of fusion protein expressed on the exosporium of a Bacillus cereus family spore. For example, replacing the native BclA promoter with the BclB promoter greatly reduces the level of fusion protein on the surface of Bacillus cereus family member spores. Alternatively, replacement of native BclB promoter with the BclA promoter increases fusion protein levels on the exosporium dramatically.
Relative promoter expression levels for various exosporium proteins under the control of their native sporulation promoters were obtained from microarray data from Bergman et al., Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation, J. Bacteriol. 188(17): 6092-6100 (2006). The relative expression levels were determined during late sporulation timing (300 minutes after the start of the experiment), when sigma K promoters are most active. Sigma K promoters are important promoters for expression of exosporium localized genes and associated proteins. Relative expression is the increase in a gene's expression level when compared to the average of all other genes of the chromosome at all given times. Table 30 below shows the relative expression levels of a variety of sigma K driven genes in Bacillus cereus family members.
The plasmid pUCpE was constructed that contained the pUC19 backbone, which is able to replicate in E. coli, as well as the origin of replication erythromycin resistance cassette from pE194. This construct is able to replicate in both E. coli and Bacillus spp. A 1 kb DNA region corresponding to the upstream region of the CotE gene and a 1 kb region corresponding to the downstream region of the gene CotE were PCR amplified from Bacillus anthracis ΔSterne. The two 1 kb regions were then spliced together using splicing by overlapping extension via 15 bp homologous overhangs that corresponded to the opposing PCR amplicons. This 2 kb fragment was digested with XhoI (in external primers) and ligated into the SalI site of pUCpE. This plasmid construct was verified by digestion and DNA sequencing. A Gram-positive omega-kanamycin resistance gene was digested with BamHI and placed between the two 1-kb regions. The final construct was again PCR verified and sequenced, and the final plasmid was introduced into Bacillus anthracis ΔSterne. Correct clones were screened by looking for both erythromycin resistance and kanamycin resistance.
Clones were passaged under high temperature (40° C.) in brain heart infusion broth in the presence of kanamycin (25 μg/ml) and were routinely struck for isolation onto LB agar plates containing kanamycin and grown at 30° C. Individual colonies were toothpicked onto LB agar plates containing erythromycin 5 μg/ml and grown at 30° C. Clones that maintained kanamycin resistance but lost erythromycin resistance (signifying loss of the plasmid but recombination and removal of the CotE gene) were grown in brain heart infusion broth plus kanamycin, and chromosomal DNA was isolated using a Qiagen Chromosomal DNA isolation kit. Proper deletion of the CotE gene was determined by PCR amplification of the CotE gene region and loss of CotE, and gain of the kanamycin resistance cassette.
A construct was generated (pHP13-AcpC-eGFP) that encoded the exosporium protein AcpC (acid phosphatase) fused in frame to the fluorescent reporter protein eGFP (enhanced green fluorescent protein). The pHP13-AcpC-eGFP construct included the native AcpC promoter, ribosomal binding site, and coding sequence for AcpC (from B. anthracis ΔSterne), fused in frame to eGFP (from pGFPuv). This construct was generated by PCR amplification of the individual AcpC and eGFP genes with corresponding primers that contained a 15 bp overlapping region corresponding to the alternate amplicons. The two PCR amplicons were then purified, and combined into a second PCR reaction using external primers that contained XhoI sites. The two amplicons prime each other with their compatible ends, and create fusion PCR amplicons. The fusion PCR amplicons were purified and digested with XhoI for 1 hour at 37° C. The spliced PCR product was cloned into the SalI site of pHP13, and correct clones were sequence verified and transformed into SCS110 E. coli. The plasmid DNA was subsequently isolated from the E. coli and introduced into B. anthracis ΔSterne CotE::Kan, generated as described above, which was grown in brain heart infusion broth containing 10 μg/ml chloramphenicol overnight at 30° C. One milliliter of this culture was inoculated into nutrient broth (50 ml) in a baffled flask and grown at 30° C. for 3 days. Spores were collected via centrifugation at 10,000×g for 5 minutes, and the supernatant (containing the broken exosporium fragments) was filtered through a 100,000 Da membrane filter to obtain purified exosporium fragments containing the fusion proteins.
A transmission electron micrograph showing the CotE knockout spores is provided in
Purification of the exosporium fragments was performed as follows: CotE::kan spores were grown in brain heart infusion broth overnight at 30° C. and swabbed onto nutrient agar plates and grown at 30° C. for 3 days. After 3 days, the spores were collected by swabbing the plates with cotton swabs wetted with PBS and resuspended into 1 ml of PBS in a microcentrifuge tube. The spores were separated from the culture by centrifugation, and the supernatant containing the exosporium fragments was filtered through a 0.22 μM filter to remove any residual spores. The filtrate was then filtered through a 100 kDa filter to collect exosporium fragments but allow free proteins to pass through the filter. The 100 kDa filter was washed, and the collected exosporium fragments boiled in SDS buffer for 5 minutes and separated by SDS-PAGE electrophoresis.
Ten microliters of the exosporium fragment preparation containing the AcpC-eGFP fusion protein was tested for activity in a phosphatase assay against pNPP (p-nitrophenyl polyphosphate). Acid phosphatase activity was detected by spectrophotometry based on release of p-nitrophenol from phosphate through phosphatase activity. Briefly, 1 ml of 10 mM pNPP in phosphate buffer at pH 6.0 was incubated with exosporium fragments in a 1 ml microcentrifuge tube and allowed to incubate at 37° C. for 10 minutes. After 10 minutes, the tube was centrifuged for 1 minute to remove excess spores, and the supernatant read on a spectrophotometer at 420 nm for free p-nitrophenol. It was found that the purified exosporium fragments were able to effectively release the phosphate groups from pNPP, demonstrating that the AcpC was present in the exosporium fragments. The results of this assay are shown in
These results demonstrate that mutations that disrupt the exosporium, such as a knock-out mutation in the CotE gene, can be used to generate exosporium fragments that are substantially free of spores, and demonstrates that these exosporium fragments contain fusion proteins that are targeted to the exosporium.
The Gallus gallus ovalbumin gene from GenScript ORF clone OGa28271C and the Bacillus anthracis protective antigen (pagA) gene were amplified via polymerase chain reaction (PCR) using the primers shown below in Table 31. The amino acid sequences encoded by the ovalbumin gene and pagA genes are provided in Table 32 below.
The resulting PCR fragments were cloned into one of three expression plasmids (pSUPER-BclA-FL, pSUPER-BclA 20-35, or pSUPER-AcpC) using the splicing by overlapping extension (SOE) technique. The pSUPER-BclA-FL plasmid was generated through fusion of a PCR fragment which contained the BclA promoter (SEQ ID NO: 149), start codon, and coding sequence for full-length (FL) BclA fused in frame into the pSUPER plasmid. The pSUPER-BclA-20-35 plasmid was generated through fusion of a PCR fragment which contained the BclA promoter (SEQ ID NO: 149), start codon, and a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) fused in frame into the pSUPER plasmid. The pSUPER-AcpC plasmid was generated through fusion of a PCR fragment with included the native AcpC promoter (SEQ ID NO: 141), ribosomal binding site, and coding sequence for AcpC (from B. thuringiensis BT013A; SEQ ID NO: 120), fused in frame into the pSUPER plasmid. The pSUPER plasmid was generated through fusion of the pUC57 plasmid (containing an ampicillin resistance cassette) with the pBC16-1 plasmid from Bacillus (containing a tetracycline resistance). This 5.5 kbp plasmid can replicate in both E. coli and Bacillus spp.
The pSUPER-BclA-FL-OVAL construct generated using these methods encodes a fusion protein comprising full-length BclA and ovalbumin, and the pSUPER-BclA-FL-PAG encodes a fusion protein comprising full-length BclA and the B. anthracis protective antigen. These constructs were transformed into and propagated in E. coli strains. The sequences of the plasmids were verified by DNA sequencing.
In order to remove the E. coli-derived portions of the pSUPER plasmids and create smaller plasmids for expression in Bacillus, sequence-verified pSUPER constructs were amplified with primers that amplify the Bacillus-derived segment of the plasmid backbone. The resulting PCR products were self-ligated to generate the pBC plasmids (pBC-BclA-FL-OVAL plasmid and pBC-BclA-FL-PAG) that were used to transform various Bacillus strains in Example 10 below.
Knock Out (KO) Mutants: To make exsY and cotE knockout (KO) mutant strains of Bacillus thuringiensis BT013A, the plasmid pKOKI shuttle and integration vector was constructed that contained the pUC57 backbone, which is able to replicate in E. coli, as well as the origin of replication erythromycin resistance cassette from pE194. This construct is able to replicate in both E. coli and Bacillus spp. A 1 kb DNA region that corresponded to the upstream region of the cotE gene and a 1 kb region that corresponded to the downstream region of the gene cotE were PCR amplified from Bacillus thuringiensis BT013A. A second construct was made that contained the 1 kb DNA region that corresponded to the upstream region of the exsY gene and a 1 kb region that corresponded to the downstream region of the gene exsY, both of which were PCR amplified from Bacillus thuringiensis BT013A. For each construct, the two 1 kb regions were then spliced together using homologous recombination with overlapping regions with the pKOKI plasmid. The plasmid constructs were verified by digestion and DNA sequencing. Clones were screened by looking for erythromycin resistance.
Clones were passaged under high temperature (40° C.) in brain heart infusion (BHI) broth. Individual colonies were toothpicked onto LB agar plates containing erythromycin 5 μg/ml, grown at 30° C., and screened for the presence of the pKOKI plasmid as a free plasmid by colony PCR. Colonies that had an integration event were continued through passaging to screen for single colonies that lost erythromycin resistance (signifying loss of the plasmid but recombination and removal of the exsY or cotE gene). Verified deletions were confirmed by PCR amplification and sequencing of the target region of the chromosome. The pBC-BclA-FL-OVAL plasmid was transformed into the exsY knockout mutant and the pBC-BclA-FL-PAG plasmid was transformed into the cotE KO mutant. The pBC-BclA-FL-OVAL and pBC-BclA-FL-PAG plasmids are described above in Example 9.
Exosporium Fragment Creation: For each of the two KO mutants, overnight cultures were grown in BHI media at 30° C., 300 rpm, in baffled flasks with antibiotic selection. One milliliter of this overnight culture was inoculated into a yeast extract-based media (50 ml) in a baffled flask and grown at 30° C. for 3 days. An aliquot of spores was removed, 1% Tween was added, and the spores were agitated by vortexing for one minute. The spores were collected via centrifugation at 10,000×g for 5 minutes, and supernatant containing the exosporium fragments was filtered through a 0.22 μM filter to remove any residual spores. The supernatant (containing the exosporium fragments) was filtered through a 100,000 Da membrane filter to obtain purified exosporium fragments containing the fusion proteins. Smaller molecular weight proteins were removed by passaging through the 100 kDa filter. No spores were found in the filtrate or retentate of the supernatant.
Transmission electron micrographs are provided in
Presence of BclA-FL-OVAL or BclA-FL-PAG Protective Antigen in Exosporium Fragments Collected from the CotE and ExsY Knockout Mutants: Exosporium fragments were created and purified as described above from spores that contained the pBC-BclA-FL-OVAL or pBC-BclA-FL-PAG plasmid. These spores create an exosporium that contains fusion proteins comprising full-length BclA and ovalbumin or protective antigen A. Exosporium fragments containing these constructs were created from the cotE knockout mutant spores and the exsY knockout mutant spores. The ovalbumin or protective antigen A protein concentration was determined by dot blot. Table 33 below summarizes the dot blot results as compared to purified protein. Briefly, an enriched exosporium fragment fraction generated as described above, whole cell broth, or purified ovalbumin or protective antigen was blotted onto nitrocellulose and then probed with commercially available rabbit polyclonal antibodies against full-length ovalbumin or Bacillus anthracis protective antigen. Whole cell broth was taken from the overnight cultures as described above and not subjected to any vortexing, filtration, or centrifugation steps. The blots were then developed with horseradish peroxidase (HRP)-conjugated secondary antibodies. The ovalbumin and protective antigen antibodies were verified by Western blot for size and specificity. Western blots were performed using purified ovalbumin and protective antigen proteins. The ovalbumin and protective antigen antibodies recognized bands of the correct size and did not cross-react with untransformed whole cell broth from Bacillus thuringiensis BT013A.
Dot blot results are shown below in Table 33. The results show that the protein of interest (OVAL or PAG) was present in both the whole cell broth and exosporium fragment-enriched fractions. Most of the protein of interest was retained in the exosporium fragment-enriched fractions, demonstrating that the proteins of interest were present on the exosporium fragments.
To provide a further demonstration that exosporium fragments containing fusion proteins can be generated using the CotE knockout and ExsY knockout strains, and to demonstrate that exosporium fragments can be generated using CotO dominant negative strains, PCR fragments were generated that contained the BclA promoter (SEQ ID NO: 149), start codon, and amino acids 20-35 of BclA fused in frame to Bacillus subtilis 168 endoglucanase (the signal peptide of SEQ ID NO: 334, directly linked to the β-1,4-endoglucanase of SEQ ID NO: 293). These PCR fragments were digested with XhoI and ligated into the SalI site of the pSUPER plasmid to generate the plasmid pSUPER-BclA 20-35-Endoglucanase.
The CotE and ExsY knockout mutants were generated as described above in Example 10.
Dominant Negative Mutants: To create dominant negative mutants, PCR amplification was performed on the N-terminal half and the C-terminal half of CotO (SEQ ID NO: 199), containing amino acids 1-81 and 81-199 respectively. These fragments were cloned into the pHP13 E coli/Bacillus shuttle vector using homologous recombination. Correct clones were verified by Sanger sequencing. Each of the two CotO dominant negative mutants was introduced into Bacillus thuringiensis BT013A.
Presence of BclA 20-35 Endoglucanase in Exosporium Fragments Collection from the CotE and ExsY Knockout and CotO Dominant Negative Mutants: Exosporium fragments were created and purified as described in Example 10 from spores that contained the pSUPER BclA 20-35-Endo plasmid. These spores create an exosporium that displays fusion proteins comprising full-length BclA linked to endoglucanase. Exosporium fragments containing this construct were created from the cotE knockout mutant spores, exsY knockout mutant spores, CotO N-terminal dominant negative mutant spores, or CotO C-terminal dominant negative mutant spores. In each of these experiments, the amount of activity for the endoglucanase on the exosporium fragments was quantified as a percentage of the total enzyme levels. These results were compared against results generated using wild-type Bacillus thuringiensis BT013A that did not contain any mutations, but did contain the pSUPER BclA 20-35-Endo plasmid. The results are shown in Table 34 below.
Effects of Exosporium Fragments on Plant Growth: These exosporium fragments were then delivered as a seed treatment onto soybean seeds (as described in Example 5 above). A wild-type control (B. thuringiensis BT013A expressing the BclA 20-35 Endo construct) was also coated onto soybeans seeds. For each experiment, 1 μl of exosporium fragments from each construct, or a 1:2, a 1:4, or a 1:8 dilution of the fragments was applied to each seed. Results are shown in Table 34 below.
These results demonstrate that mutations that disrupt the exosporium, such as a knock-out mutation in the cotE or exsY gene, or a dominant negative mutation in the CotO protein, can be used to generate exosporium fragments that are substantially free of spores, and demonstrates that these exosporium fragments contain fusion proteins that are targeted to the exosporium. There was a small amount of background endoglucanase activity in the exosporium fragment preparation from the BT013 strain having no mutations and expressing the BclA 20-25 Endo construct (BT013A BclA 20-35 Endo). This was unexpected and may represent a low level of unstable exosporium that is being released from spores and captured during the exosporium fragment collection process. CotE and ExsY KO strains contained the highest amount of enzyme in the exosporium fragment fraction. The CotO dominant negative mutants that express a fusion protein also have an elevated level of enzyme in the exosporium fragment fraction.
The exosporium fragments from the CotE and ExsY mutants (not expressing BclA 20-35 Endo) applied directly to plants had a negative effect on growth and were removed from this experiment. When the exosporium fragments from BT013A BclA 20-35 Endo were applied to soybeans, there was a negative growth phenotype. When exosporium fragments from the CotE or ExsY mutants expressing the BclA 20-35 Endo fusion protein were added to soybeans, a substantial increase in growth rate occurred (+28.3% and +14.8% over BT013A BclA 20-35 Endo fragments). The CotE mutant exosporium fragments were still active at the 1:4 dilution, but the ExsY exosporium fragments were no longer giving a growth benefit to the soybeans at this dilution. The CotO dominant negative mutants expressing the BclA 20-35 Endo fusion protein gave a small increase in soybean growth compared to the fragments from BT013A BclA 20-35 Endo, giving +6.5% and +2.7% increases in growth, respectively.
Soil samples from rhizospheres of the healthiest and most resistant potato (Solanum tuberosum), yellow summer squash (Cucurbita pepo), tomato (Solanum lycopersicum), and pole bean (Phaseolus coccineus) plants were collected, diluted in sterile water, and spread onto nutrient agar plates. Bacterial isolates that demonstrated high growth rates and were able to be passaged and propagated were selected for further study. The selected strains were grown in minimal media (KH2PO4 3 g, Na2HPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H2O 0.15 g, CaCl2 2H2O 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30° C.) of selected strains were spun down, media decanted off, and resuspended in an equal amount of distilled water. Ten butterhead lettuce seeds per treatment were planted at a depth of 1 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting in 4 cm pots with 0.5 μl of resuspended bacteria in water mixed into 10 ml of H2O. Ten ml of H2O was sufficient to deliver the bacteria into the 3 in3 (49.16 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3 days. After one week, plant heights and leaf diameters, as well as overall health of the plants were collected. Initial screening of rhizosphere isolates resulted in obtaining greater than 200 distinct species of bacteria and fungi from the rhizosphere of the four plants. Some of the bacterial species are described in Table 35 below. Identified strains are indicated by their proper bacterial identifications. Other strains are indicated by their unknown identification number. Inoculants giving results near control (+/−2%) were not included in the table.
Paracoccus kondratiavae NC35
B. aryabhattai CAP53
B. flexus BT054
Bacillus mycoides strain BT155
B. aryabhattai CAP56
B. nealsonii BOBA57
E. cloacae CAP12
Bacterial strains that produced the greatest effect on the overall plant health and plant height in the initial lettuce trial were subjected to further identification. Bacterial strains were grown overnight in Luria Bertani broth at 37° C., and overnight cultures were spun down in a centrifuge. Media was decanted and the remaining bacterial pellet was subjected to chromosomal DNA isolation using the Qiagen Bacterial Chromosomal DNA Isolation kit. Chromosomal DNA was subjected to PCR amplification of the 16S rRNA coding regions using the primers E338F 5′-ACT CCT ACG GGA GGC AGC AGT-3′ (SEQ ID NO: 368), E1099R A 5′-GGG TTG CGC TCG TTG C-3′ (SEQ ID NO: 369), and E1099R B 5′-GGG TTG CGC TCG TTA C-3′ (SEQ ID NO: 370). PCR amplicons were purified using a Promega PCR purification kit, and the resultant amplicons were diluted and sent to the University of Missouri DNA Core for DNA sequencing. DNA sequences were compared to the NCBI BLAST database of bacterial isolates, and genus and species were identified by direct comparison to known strains. Top identified species are indicated in Table 35 above. In many cases, 16S rRNA DNA sequences were only able to delineate the genus of the selected bacterial strain. In cases where a direct identification was not forthcoming, additional biochemistry analyses, using methods standard in the field, were performed to differentiate strains at the species and strain levels, and are listed in Table 36 below.
E. cloacae
P. kondratiavae
B. aryabhattai
B. flexus
B. mycoides
B. aryabhattai
B. nealsonii
Soil samples from agricultural fields near Gas, Kansas were collected, diluted in sterile water, and spread onto nutrient agar plates. Bacterial isolates that demonstrated high growth rates and were able to be passaged and propagated were selected for further study. The selected strains were grown in minimal media (KH2PO4 3 g, Na2HPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H2O 0.15 g, CaCl2 2H20 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30° C.) of selected strains were spun down, media decanted off, and resuspended in an equal amount of distilled water. Corn seeds were coated with commercial seed polymer mixed with water alone (1.6 μl per seed total) or commercial seed polymer containing selected bacterial strains (1.6 μl per seed total). Coated seeds were planted in 3 inch (7.62 cm) diameter pots at a depth of 1 inch (2.54 cm) in loam top soil (Columbia, MO) that was sieved to remove large debris. Plants were grown at temperatures between 18-24° C. (65-75° F.) with 11 hours of light/day, and 50 ml of watering at planting and every 3 days. After two weeks, plant heights and leaf diameters, as well as overall health of the plants were collected. For germination assays and determining 3 day root length, seeds were coated as indicated above and evenly dispersed at 10 seeds per paper towel. The paper towels were wetted with 10 ml of water, rolled up, placed in a small plastic bag and incubated at 30° C. or placed on a germination heat mat at 27-30° C. (80-85° F.). Root measurements were recorded after 3 days. Initial screening of rhizosphere isolates resulted in obtaining greater than 100 distinct species of bacteria and fungi from the rhizosphere. Some of the bacterial species are described in Table 37 below. Identified strains are indicated by their proper bacterial identifications.
B. mycoides EE118
B. subtilis EE148
Alcaligenes faecalis EE107
B. mycoides EE141
B. mycoides BT46-3
B. cereus family
B. thuringiensis BT013A
Paenibacillus massiliensis
B. cereus family
B. subtilis EE218
B. megaterium EE281
Bacterial strains that produced the greatest effect on plant health are described in Table 37 above. Bacterial strains were grown overnight in Luria Bertani broth at 37° C., and overnight cultures were spun down in a centrifuge. Media was decanted and the remaining bacterial pellet was subjected to chromosomal DNA isolation using the Qiagen Bacterial Chromosomal DNA Isolation kit. Chromosomal DNA was subjected to PCR amplification of the 16S rRNA coding regions using the primers E338F 5′-ACT CCT ACG GGA GGC AGC AGT-3′ (SEQ ID NO: 368), E1099R A 5′-GGG TTG CGC TCG TTG C-3′ (SEQ ID NO: 369), and E1099R B 5′-GGG TTG CGC TCG TTA C-3′ (SEQ ID NO: 370). PCR amplicons were purified using a Promega PCR purification kit, and the resultant amplicons were diluted and sent to the University of Missouri DNA Core for DNA sequencing. DNA sequences were compared to the NCBI BLAST database of bacterial isolates, and genus and species were identified by direct comparison to known strains. Top identified species are indicated in Table 37 above. In many cases, 16S rRNA DNA sequences were only able to delineate the genus of the selected bacterial strain. In cases where a direct identification was not forthcoming, additional biochemistry analyses, using methods standard in the field, were performed to differentiate strains at the species and strain levels, and the differentiated strains are listed in Table 38 below.
B. cereus
Paeni-
B. cereus
B.
B.
B.
B.
bacillus
B.
Alcaligenes
B.
B.
thuringiensis
subtilis
subtilis
megaterium
massiliensis
mycoides
faecalis
mycoides
mycoides
The selected strains were grown in minimal media (KH2PO4 3 g, Na2HPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H2O 0.15 g, CaCl2 2H2O 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30° C.) of selected strains were spun down, media decanted off, and bacteria resuspended in an equal amount of distilled water. Ten ZEBA-coated alfalfa seeds were planted for each treatment at a depth of 0.6 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. ZEBA is a superabsorbent cornstarch based polymer used as a moisture-retention seed coating. Seeds were inoculated at planting with 0.5 μl of resuspended bacteria in water mixed into 10 ml of H2O. Ten ml of H2O was sufficient to deliver the bacteria into the 3 in3 (49.16 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3 days. Alfalfa was allowed to grow for 1 week to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and final height data are listed in Table 39 below.
B. aryabhattai CAP56
B. nealsonii BOBA57
E. cloacae CAP12
The selected strains were grown in minimal media (KH2PO4 3 g, Na2HPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H2O 0.15 g, CaCl2 2H2O 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30° C.) of selected strains were spun down, media decanted off, and resuspended in equal amount of distilled water. Ten cucumber seeds were planted for each treatment at a depth of 1 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 μl of resuspended bacteria in water mixed into 10 ml of H2O. Ten ml of H2O was sufficient to deliver the bacteria into the 3 in3 (49.16 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3 days. Cucumbers were allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and final height data are listed in Table 40 below.
B. aryabhattai CAP53
B. aryabhattai CAP56
B. nealsonii BOBA57
The selected strains were grown in minimal media (KH2PO4 3 g, Na2HPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H2O 0.15 g, CaCl2 2H2O 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30° C.) of selected strains were spun down, media decanted off, and resuspended in an equal amount of distilled water. Ten yellow squash seeds were planted for each treatment at a depth of 1 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 μl of resuspended bacteria in water mixed into 10 ml of H2O. Ten ml of H2O was sufficient to deliver the bacteria into the 3 in3 (49.16 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3 days. Squash was allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications, final height data, and final leaf diameter (by span of the two leaves) data are listed in Table 41 below.
B. aryabhattai
B. flexus BT054
Bacillus mycoides
B. aryabhattai
B. nealsonii
E. cloacae CAP12
The selected strains were grown in minimal media (KH2PO4 3 g, Na2HPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H2O 0.15 g, CaCl2 2H2O 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30° C.) of selected strains were spun down, media decanted off, and resuspended in an equal amount of distilled water. Thirty ryegrass seeds were planted for each treatment at a depth of 0.3 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 μl of resuspended bacteria in water mixed into 10 ml of H2O. Ten ml of H2O was sufficient to deliver the bacteria into the 3 in3 (49.16 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3 days. Ryegrass was allowed to grow for 1.5 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and height data are listed in Table 42 below.
B. aryabhattai CAP53
B. flexus BT054
Bacillus mycoides BT155
B. aryabhattai CAP56
B. nealsonii BOBA57
E. cloacae CAP12
The selected strains were grown in minimal media (KH2PO4 3 g, Na2HPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H2O 0.15 g, CaCl2 2H2O 0.013 g, and glucose 1 g, per L dry weight). Overnight cultures (30° C.) of selected strains were spun down, media decanted off, and resuspended in an equal amount of distilled water. Ten corn seeds were planted for each treatment at a depth of 2.5 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 μl of resuspended bacteria in water mixed into 10 ml of H2O. Ten ml of H2O was sufficient to deliver the bacteria into the 3 in3 (49.16 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3 days. Corn was allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and final height data are listed in Table 43 below.
B. aryabhattai CAP53
B. flexus BT054
Bacillus mycoides strain BT155
B. aryabhattai CAP56
B. nealsonii BOBA57
The selected strains were grown in minimal media (KH2PO4 3 g, Na2HPO4 6 g, NH4Cl 1 g, NaCl 0.50 g, MgSO4 7H2O 0.15 g, CaCl2 2H2O 0.013 g, and glucose 1 g, per L dry weight, or for Bradyrhizobium or Rhizobium on yeast mannitol media). Overnight cultures (30° C.) of selected strains were spun down, media decanted off, and resuspended in equal amount of distilled water. Ten soybean seeds were planted for each treatment at a depth of 2.5 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 μl of resuspended bacteria in water mixed into 10 ml of H2O. When testing two bacterial strains, 0.5 μl of each resuspended bacteria was mixed into 10 ml of H2O. Ten ml of H2O was sufficient to deliver the bacteria into the 3 in3 (49.16 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65-75° F. (18-24° C.) with 11 hours of light/day, and 5 ml of watering every 3 days. Soybeans were allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and final height data are listed in Table 44 below. Co-inoculation of bacteria strains in the present invention with members of the Bradyrhizobium sp. or Rhizobium sp. lead to an increase in plant growth compared to either inoculant alone.
B. aryabhattai CAP53
B. flexus BT054
Bacillus mycoides
B. aryabhattai CAP56
B. aryabhattai CAP53
B. aryabhattai CAP53
B. aryabhattai CAP53
Bradyrhizobium sp.
Rhizobium sp.
Bacillus mycoides strain BT155, Bacillus mycoides strain EE118, Bacillus mycoides strain EE141, Bacillus mycoides strain BT46-3, Bacillus cereus family member strain EE349, Bacillus thuringiensis strain BT013A, and Bacillus megaterium strain EE281 were grown in Luria Bertani broth at 37° C. and overnight cultures were spun down, media decanted off, and resuspended in equal amount of distilled water. Twenty corn seeds were planted for each treatment at a depth of 2.5 cm in loam top soil (Columbia, MO) that was sieved to remove large debris. Seeds were inoculated at planting with 0.5 μl of resuspended bacteria in water mixed into 50 ml of H2O. Fifty ml of H2O was sufficient to deliver the bacteria into the 29 in3 (475.22 cm3) of soil as well as saturate the soil for proper germination of seeds. Plants were grown at temperatures between 65-72° F. with 13 hours of light/day, and 5 ml of watering every 3 days. Seedlings were allowed to grow for 2 weeks to analyze emergence and initial outgrowth of plants under described conditions. Identified strains indicated by their proper bacterial identifications and final height data are listed in Table 45 below.
100%
B. mycoides EE118
B. mycoides EE141
B. mycoides BT46-3
Bacillus thuringiensis BT013A
Bacillus cereus family member EE128
Bacillus mycoides BT155
Bacillus megaterium EE281
All plant-growth promoting bacteria tested had a beneficial effect on corn height at two weeks under the described conditions. The Bacillus cereus family member EE128 strain had the greatest effect in this trial, giving a greater than at 14% boost in corn height.
The BEMD system can be used to display a wide range of proteins, peptides, and enzymes using any of the targeting sequences described herein to provide beneficial agricultural effects. Additional beneficial effects can be obtained by selecting an expression host (a Bacillus cereus family member) having inherent beneficial attributes. Many strains of members of the Bacillus cereus family have plant-growth promoting benefits. Additionally, many Bacillus cereus family member strains provide have protective effects, through direct fungicidal, insecticidal, nematocidal, or other protective activities. By using such strains these as the expression host for the BEMD system, the end spore product would have a combination of positive benefits in agriculture.
Table 46 below provides results for an experiment wherein a fusion protein was expressed in various Bacillus cereus family member strains. All strains expressed a fusion protein comprising amino acids 1-35 of SEQ ID NO: 1 and the phosphatase PhoA4 from Bacillus subtilis, a beneficial enzyme for enhanced phosphate uptake in corn. The gene was synthesized, cloned into the pMK4 vector, and introduced into each of the Bacillus spp. indicated in Table 46 below. Strains were taken into sporulation by incubation at 30° C. on nutrient agar plates containing chloramphenicol 10 μg/ml for three days. Spores were collected, washed, and applied to corn at planting at a rate of 1×105 CFU/ml in 50 ml of water per 7.62 cm diameter pot with 5 mg polyphosphate per pot. Corn was grown in silt loam soil for two weeks. Plants were grown under ideal light using T5 lamps, 54 watts, and exposed to 13 hours of light a day under controlled temperature conditions between 15.5-25.5° C. Plants were watered to saturation every three days over a two week trial. At the end of two weeks, the height of each plant was measured and measurements were normalized to control Bacillus thuringiensis spores. Expression of the amino acids 1-35 of SEQ ID NO: 1—Phosphatase fusion protein led to an increase in corn height at 2 weeks regardless of the expression host strain selected. As shown in Table 46, use of a plant-growth promoting Bacillus cereus family member further increased corn height
Bacillus Species
B. thuringiensis
100%
B. thuringiensis
B. mycoides
B. mycoides
B. cereus family
B. cereus family
B. mycoides
B. mycoides
Bacillus cereus family member EE349 was found to have the ability to grow endophytically and to be capable as serving as a host strain for the BEMD system. To demonstrate the ability of Bacillus cereus family member EE349 to grow endophytically and to serve as a host strain for the BEMD system, Bacillus cereus family member EE349 was transformed with the pSUPER-BclA 20-35-endoglucanase plasmid (described above in Example 4). The transformed bacteria were then induced to sporulate by swabbing the bacteria onto nutrient agar plates and incubating the plates at 30° C. for 72 hours. After 72 hours, the bacterial spores were collected from the plate by swabbing into sterile phosphate buffered saline (PBS), and were purified by density centrifugation three times.
These spores were diluted to a concentration of 1×105 spores/50 ml water, and the 50 ml of water was then added to commercial hybrid corn seed in potting soil at planting. The corn seeds were coated with a fungicide and a biological inoculant. The corn hybrid variety was BECK 5475RR, which contains the ROUNDUP READY glyphosate resistance gene and AQUAMAX drought resistance gene. Plants were grown under artificial light for 14 hours a day and plant growth over a ten day period was determined. Plants were watered every three days over the course of the experiment. After ten days, the plants were measured for height and normalized against the height of untreated corn plants. The results of these experiments are shown in Table 47 below.
100%
Bacillus cereus family
Bacillus cereus family
As can be seen from the data shown in Table 47, expression of the pSUPER-BclA 20-35-endoglucanase in the endophytic strain Bacillus cereus family member EE349 resulted in increased corn growth as compared to untreated plants, or plants treated with Bacillus cereus family member EE349 alone.
Bacillus cereus family member EE349 expressing the BclA 20-35-endoglucanase was then isolated from the inside of the corn plants. The ten day old plants were extracted from the soil and washed to remove excess debris. The plants were then inverted, exposed to 5% bleach for ten minutes, washed in water, exposed to hydrogen peroxide (10%) for ten minutes, washed again in water, and the stalks split with a sterile razor blade. The split halves of the stalks were placed face down onto nutrient agar plates for two hours. After two hours, the stalks were removed, and the agar plates incubated at 30° C. for 48 hours. After 48 hours, the plates were examined for colony morphology, and Bacillus cereus family member colonies found internal to the plant were toothpicked onto nutrient agar and nutrient agar plus tetracycline plates (to select for bacteria containing the pSUPER-20-35 BclA-endoglucanase plasmid). The resultant increase in Bacillus cereus family member EE349 colony numbers is indicated shown in Table 48 below. These results demonstrate the ability of the BEMD system to be introduced into the target plant by expression in an endophytic strain of the Bacillus cereus family.
Bacillus cereus
Bacillus cereus
Bacillus cereus
Tetracycline resistant Bacillus clones were grown overnight at 30° C. in brain heart infusion broth plus tetracycline, and spun down at 10,000×g for 5 minutes. The supernatant was removed, and the pellet frozen overnight at −20° C. Chromosomal DNA was then extracted from each clone, and the presence of the pSUPER-20-35 BclA-endoglucanase plasmid determined by transformation of the chromosomal DNA (containing the plasmid) into DH5α E. coli cells and plating on LB plus ampicillin plates. Correct clones were subjected to DNA sequence analysis, which verified that Bacillus cereus family member EE349 was internal to the plant (endophytic) and contained the plasmid.
Many endophytic bacteria were found in the corn seedlings, with a number of different strains and species within the Bacillus cereus family found inside both the control and the EE349 treated plants. The tetracycline resistant Bacillus cereus family members (indicating the presence of the pSUPER-20-35 BclA-endoglucanase plasmid) were only found in the treated corn seedlings, and all had the same colony morphology of the original expression host, Bacillus cereus family members EE349. The presence of the pSUPER 20-35 BclA-endoglucanase plasmid was verified by PCR amplification using unique primers.
In addition to the endophytic strain Bacillus cereus family member EE349 discussed above in the immediately preceding example, several other Bacillus cereus family members that have the ability to grow endophytically were also identified: Bacillus cereus family member EE439, Bacillus thuringiensis EE417, Bacillus cereus EE444, Bacillus thuringiensis EE319, Bacillus thuringiensis EE-B 00184, Bacillus mycoides EE-B 00363, Bacillus pseudomycoides EE-B00366, and Bacillus cereus family member EE-B00377.
To obtain these additional Bacillus cereus family members, commercial hybrid corn seed was planted in potting soil and allowed to grow. The corn seeds were coated with a fungicide and a biological inoculant. Plants were grown under artificial light for 14 hours a day and plant growth over a 14 day period was determined. Plants were watered every three days over the course of the experiment. After 14 days, the plants were extracted from the soil and washed to remove excess debris. The plants were then inverted, exposed to 5% bleach for ten minutes, washed in water, exposed to hydrogen peroxide (10%) for ten minutes, washed again in water, and the stalks split with a sterile razor blade. The split halves of the stalks were placed face down onto nutrient agar plates for two hours. After two hours, the stalks were removed, and the agar plates incubated at 30° C. for 48 hours. After 48 hours, the plates were examined for colony morphology, and Bacillus cereus family member colonies found internal to the plant were toothpicked onto nutrient agar. These were then were grown overnight at 30° C. in brain heart infusion broth, and spun down at 10,000× g for 5 minutes. The supernatant was removed, and the pellet frozen overnight at −20° C. Chromosomal DNA was then extracted from each clone, the identity of each colony was verified by PCR using 16S rRNA primers, and amplicons were sent for DNA sequencing and identification. The 16S rRNA sequences for these strains are provided above in Table 18.
The endophytic bacterial strains Bacillus megaterium EE385, Bacillus sp. EE387, Bacillus circulans EE388, Bacillus subtilis EE405, Lysinibacillus fusiformis EE442, Lysinibacillus spp. EE443, and Bacillus pumilus EE-B00143 were isolated from corn seedlings. Two week old corn seedlings were first sterilized. The plants were extracted them from the soil and washed them to remove excess debris. The plants were then inverted, exposed to 5% bleach for ten minutes, washed in water, exposed to hydrogen peroxide (10%) for ten minutes, and washed again in water. The stalks were then split with a sterile razor blade. The split halves of the stalks were placed face down onto nutrient agar plates for two hours. After two hours, the plant stems were removed from the plates, and the plates were then incubated at 30° C. for 48 hours. Bacilli colonies that were endophytic were selected for further analysis. These strains were grown up in brain heart infusion broth overnight at 30° C., and the cultures subjected to extraction of DNA using a Qiagen Chromosomal DNA Kit. The DNA was PCR amplified to obtain the 16S rRNA gene, which was sent for DNA sequencing. The resultant sequences BLAST searched using the NCBI databases to establish the identity of the Bacilli species. The 16S rRNA sequences are provided above in Table 21.
Bacillus thuringiensis EE417, Bacillus thuringiensis EE-B00184, and Bacillus cereus EE439 were found to have the ability to grow endophytically (see Examples 23 and 24 above) and to be capable as serving as host strains for the BEMD system. To demonstrate the ability of these Bacilli to grow endophytically and to serve as a host strain for the BEMD system, each of these strains was transformed with the pMK4-BclA 20-35-eGFP plasmid, which encodes a fusion protein comprising amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) fused in frame to eGFP. The transformed bacteria were then induced to sporulate by swabbing the bacteria onto nutrient agar plates and incubating the plates at 30° C. for 72 hours. After 72 hours, the bacterial spores were collected from the plate by swabbing into sterile phosphate buffered saline (PBS), and were purified by density centrifugation three times.
These spores were diluted to a concentration of 1×108/ml, and 1 μl of whole cell broth was then added to commercial hybrid corn seed in potting soil at planting. The corn seeds were coated with a fungicide and a biological inoculant. The corn hybrid variety was BECK 6175YE, which contains the ROUNDUP READY glyphosate resistance gene and AQUAMAX drought resistance gene. Plants were grown under artificial light for 14 hours a day and plant growth over a ten day period was determined. Plants were watered every three days over the course of the experiment.
Bacillus thuringiensis EE417, Bacillus thuringiensis EE-B00184, and Bacillus cereus EE439 expressing the BclA 20-35-eGFP fusion protein were then isolated from the inside of the corn plants. The ten day old plants were extracted from the soil and washed to remove excess debris. The plants were then inverted, washed in water, exposed to 5% bleach for ten minutes, washed in water, exposed to 70% ethanol for ten minutes, washed again in water, and the stalks split with a sterile razor blade. The split halves of the stalks were placed face down onto nutrient agar plates for two hours at 30° C. After two hours, the stalks were removed, and the agar plates incubated at 30° C. for 48 hours. After 48 hours, the plates were examined for colony morphology, and Bacillus colonies found internal to the plant were toothpicked onto nutrient agar and nutrient agar plus chloramphenicol plates (to select for bacteria containing the pMK4-20-35 BclA-eGFP plasmid). Results are shown in Table 49 below. These results demonstrate the ability of the BEMD system to be introduced into the target plant by expression in an endophytic strain of the Bacillus cereus family.
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus cereus EE439
To further demonstrate the ability of endophytic strain Bacillus thuringiensis EE-B00184 to express proteins on the surface of the spores, the pSUPER BclA-20-35-Endo construct described above in Example 4 was also introduced into Bacillus thuringiensis EE-B00184. Transformed cells were screen by PCR and Sanger sequencing. Spores were made by growing up an overnight culture in BHI plus selection (chloramphenicol), and 500 μl of each culture was swabbed onto nutrient broth agar plates and allowed to incubate at 30° C. for 3 days. After 3 days, the spores were swabbed off into PBS, diluted to a concentration of 1×108/ml, and spun down to recover the spores. Enzyme measurement of the spores was performed as described above in Example 4 and calculated as mU/ml for each construct. Results are shown in Table 50 below.
Endophytic strains can be administered to the plant through addition into the plant growth medium, including soil, irrigation, and granular formulations. Endophytic strains can also enter the target plant through the aerial portions of the plants. These strains create a unique and effective delivery mechanism for delivering proteins and peptides of interest into the plant.
Two amino acids of the wild-type
The wild-type (SEQ ID NO: 245) and mutant (SEQ ID NO: 249) enzymes were cloned into the pSUPER-BclA20-35 plasmid using the splicing by overlapping extension (SOE) technique. The pSUPER-BclA20-35 plasmid contains a PCR fragment which includes the BclA promoter (SEQ ID NO: 149), a start codon, and a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1, the targeting sequence), fused in frame into the pSUPER plasmid. Thus, the following constructs were generated: pSUPER-BclA20-35-SEQ ID NO: 245 and pSUPER-BclA20-35-SEQ ID NO: 249. A six alanine linker was also included between the targeting sequence (amino acids 20-35 of BclA) and the
In order to remove the E. coli-derived portions of the pSUPER plasmids and create smaller plasmids for expression in Bacillus, sequence-verified pSUPER constructs were amplified with primers that amplify the Bacillus-derived segment of the plasmid backbone. The resulting PCR products were self-ligated to generate the pBC plasmids (pBC-BclA20-35-SEQ ID NO: 245 and pBC-BclA20-35-SEQ ID NO: 249) that were used to transform the Bacillus thuringiensis BT013A exsY knock-out mutant strain described above in Example 10. Transformants were grown in broth overnight with tetracycline selection, and total DNA was collected using standard procedures. DNA was then subjected to Sanger sequencing to verify that each transformant contained the desired construct.
Non-transformed Bacillus thuringiensis BT013A exsY knockout spores and Bacillus thuringiensis BT013A exsY knockout spores transformed with each of the pBC-BclA20-35-SEQ ID NO: 245 and pBC-BclA20-35-SEQ ID NO: 249 constructs were grown at 30° to 1.8-1.9×108 spores per mL in yeast extract-based media for 64 hours. These sporulation cultures resulted in approximately 99% sporulation.
In order to standardize (ensure approximately equal enzyme activity for both constructs) the enzyme activities of the
In the second step of the reaction, α-KB is combined with 2,4-dinitrophenyl (DNP) hydrazine to form α-ketobutyric acid (2,4-dinitrophenyl) hydrazone:
This hydrazone was then quantified spectrophotometrically by measuring its absorption at 540 nm. Conversion of amounts of α-KB to enzymatic activity was achieved by quantification using a α-KB standard curve (see
100 μL of each spore sample (whole cell broth) was mixed with 100 μL 0.1 M Tris-HCl, pH 8.5 A dilution series of α-KB standards (50, 100, 500, 1000, and 2000 nM) was also prepared from a 10 mM solution of α-KB in 0.1 M Tris-HCl (pH 8.5). α-KB was purchased from Sigma-Aldrich (St. Louis, MO).
Spore samples, blanks and KB standards were then subjected to the two step ACCD activity assay described above. For the first reaction, 20 μL of 500 mM ACC or milliQ purified grade water was added to each spore sample or α-KB standard, respectively. The reaction tubes were vortexed and incubated at 30° C. in a heat block for 1 hour. The reaction was stopped by the addition of 1 mL of 0.56 M HCl (hydrochloric acid) to the reaction tubes followed by vortexing and centrifugation at maximum speed for 5 minutes.
The second reaction was initiated by transferring 1 mL volumes of the first reaction mixtures to 800 μL volumes of 0.56 M HCl in new 15 mL conical tubes. A volume of 300 μL of 0.2% (w/v) DNP hydrazine in 2.0 M HCl was added to each of the reaction tubes and the tubes were vortexed and incubated at 30° C. in a static incubator for 30 min. After the incubation, 1.0 mL of 2 M NaOH was added to each of the tubes and the tubes were vortexed again. The reactions were then allowed to stabilize for 10-20 seconds to ensure the colorimetric reaction ran to completion. A volume of 1 mL from each of the reactions was transferred to a polystyrene cuvette and read on a spectrophotometer at a wavelength of 540 nm. The absorbance reading of a true “blank” (0.1 M Tris buffer, pH 8.5) was subtracted from each reaction sample to generate the final absorbance used in data analysis.
A standard curve was generated from the absorbance read from the α-KB standards and is shown in
Enzyme activity for the non-transformed spores (background, spore control) and for spores transformed with the D-cysteine desulfhydrase (E195; SEQ ID NO: 245) and ACC deaminase (D406; SEQ ID NO: 249) constructs was measured using the ACCD activity assay. Results are shown in
Error bars in
Non-transformed spores and spores transformed with the ACC deaminase construct pBC-BclA20-35-SEQ ID NO: 249 (D406) were prepared as described above in Example 26. These spores were applied as foliar treatments to two-week old corn plants (Beck's corn hybrid 5828 YH) at the V2 to V3 stage of development. Corn plants were grown from seed in an environmentally controlled growth room. Seed was planted directly into 39.7 cm3 pots containing Timberline #13 top soil at a depth of 2.54 cm, with 2 seeds per pot. After planting, 50 mL of room temperature water was added to each pot to allow for germination. The pots were kept in an artificial lighted growth room receiving approximately 300 μmol m−2 s1 (light photons) for a 12/12 light/day cycle and a 21° C. day/15° C. night temperature range. Plants received the same watering and fertilizer regimes.
At two weeks, spore preparations containing 1.8-1.9×108 total spores in 1 mL of water with 0.5% non-ionic surfactant (ALLIGARE SURFACE, Alligare LLC) were applied via foliar spray. The ALLIGARE SURFACE surfactant contains a blend of alkylpolyoxethylene, glycol derivatives, humectant, and formulation aids. Plants were treated with equal volumes of non-transformed spores (BEMD control solution) or spores expressing the D406 ACC deaminase. After treatment, the plants were placed under the same environmental growth conditions and allowed to grow an additional two weeks before growth parameters (shoot mass, root mass, and root length) were measured. Table 51 below summarizes the results and shows average shoot and root mass and root length normalized to water-treated controls for two trials (18 plants per trial). ACC deaminase (D406) applied as a foliar treatment modestly increased shoot mass (+2%), and substantially increased root mass (+10%) and root length (+11%).
Non-transformed spores, as well as spores expressing either the ACC deaminase construct pBC-BclA20-35-SEQ ID NO: 249 (D406) or the
Corn plants (Beck's corn hybrid 5828 YH) were grown as described in Example 27 and were treated at two weeks (at the V2 to V3 stage of development) via foliar spray with exosporium fragments derived from non-transformed spores, exosporium fragments derived from spores expressing the
At four weeks, growth parameters (root mass and root length) were measured and compared to control plants treated with exosporium fragments derived from non-transformed spores. Table 52 below summarizes the normalized shoot length, root mass, and length averaged across two trials. Data are normalized to results from plants treated with exosporium fragments derived from non-transformed spores. Foliar applications of exosporium fragments containing
The examples provided hereinbelow describe the use of the BEMD system to deliver various enzymes to plants via seed, in-furrow, or foliar treatment. Enzyme activities can be determined using substrate-specific spectrophotometric bioassays. Within each enzyme class, enzyme activities can be standardized so that approximately the same activity will be applied to the seeds, plants, or soil surrounding the plants. Illustrative enzyme activity assays that can be used for this purpose are described below.
β-1,3-Endoglucanase
β-1,3-Endoglucanase activity can be assessed by measuring the hydrolytic cleavage of the soluble β-1,3-glucan derivative carboxymethyl curdlan (CM-Curdlan). The cleavage of β-1,3 bonds liberates the reducing end of one of the two sugar monomers originally involved in the linkage. Liberated reducing ends are subsequently quantified by their reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino-5-nitrosalicylic acid, which can then be quantified spectrophotometrically by measuring its absorption at 540 nm. Conversion of the amounts of liberated reducing ends to enzymatic activity is achieved by quantification and comparison to a glucose standard curve that is generated using a pre-quantified, commercially available endoglucanase preparation as a standard.
β-1,4-Endoglucanase
β-1,4-Endoglucanase activity can be assessed by measuring the hydrolytic cleavage of the soluble β-1,4-glucan derivative carboxymethyl cellulose (CMC). The cleavage of β-1,4 bonds liberates the reducing end of one of the two sugar monomers originally involved in the linkage. Liberated reducing ends are subsequently quantified by their reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino-5-nitrosalicylic acid, which can then be quantified spectrophotometrically by measuring its absorption at 540 nm. Conversion of the amounts of liberated reducing ends to enzymatic activity is achieved by quantification and comparison to a glucose standard curve that is generated using a pre-quantified, commercially available endoglucanase preparation as a standard.
Chitosanase
Chitosanase activity can be assessed by measuring the hydrolytic cleavage of chitosan. The cleavage of β-1,4 bonds liberates the reducing end of one of the two glucosamine monomers originally involved in the linkage. Liberated reducing ends are subsequently quantified by their reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino-5-nitrosalicylic acid, which can then be quantified spectrophotometrically by measuring its absorption at 540 nm. Conversion of the amounts of liberated reducing ends to enzymatic activity is achieved by quantification and comparison to a glucose standard curve generated using a pre-quantified, commercially available chitosanase preparation as a standard.
Mannanase
Mannanase activity can be assessed by measuring the hydrolytic cleavage of galactomannan from carob (locust bean), which has a 22:78 ratio of galactose:mannose, with the mannose residues having β-1,4 linkages, and an α-1,6-linked galactose on every third mannose (in the middle mannose of the subunit). The cleavage of β-1,4 bonds liberates the reducing end of one of the mannose residues originally involved in the linkage. Liberated reducing ends are subsequently quantified by their reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino-5-nitrosalicylic acid, which can then be quantified spectrophotometrically by measuring by its absorption at 540 nm. Conversion of amounts of liberated reducing ends to enzymatic activity is achieved by quantification and comparison to a mannose standard curve generated using a pre-quantified commercially available mannanase enzyme.
Acid Phosphatase
Acid phosphatase activity can be assessed by measuring by the release of p-nitrophenol (pNP) from bis(4-nitrophenyl) phosphate [bNPP]. The cleavage of the phosphate bonds liberates two molecules of pNP from phosphate. The bNPP compound is colorless, whereas pNP is bright yellow and can be quantified spectrophotometrically by measuring its absorption at 405 nm under basic conditions. To optimize colorimetric detection, hydroxide can be added at the end of the enzymatic reaction in all enzyme standards, product standards, and samples. Conversion of absorbance to enzymatic activity is achieved by quantification and comparison to a pNP standard curve generated using a pre-quantified, commercially available acid phosphatase preparation as a standard.
Phospholipase C
Phospholipase C activity can be assessed by measuring the release of p-nitrophenol (pNP) from p-nitrophenylphosphorylcholine (NPPC). The cleavage of the phosphate bond liberates pNP from phosphorylcholine. The NPPC compound is colorless, whereas pNP is a bright yellow-green that can be quantified spectrophotometrically by measuring its absorption at 405-410 nm. Conversion of amounts of pNP to enzymatic activity is achieved by quantification and comparison to a pNP standard curve generated using a pre-quantified, commercially available phospholipase C preparation as a standard.
Lipase
Lipase activity can be assessed by measuring using p-nitrophenyl acyl esters (e.g. p-nitrophenyl palmitate) as chromogenic substrate analogs to provide for a continuous spectrophotometric assay. The lipolysis of p-nitrophenyl esters (laurates, palmitates, oleates) gives rise to the yellow colored p-nitrophenol, which can be quantified spectrophotometrically by measuring its absorbance of 405-410 nm. This assay provides a rapid and sensitive measurement to determine lipase activity (mU mL−1) by measuring the yellow chromogen (p-nitrophenol) resulting from the hydrolysis of the substrate.
Xyloglucanase
Xyloglucanase activity can be assessed by measuring the cleavage or endo-hydrolysis of xyloglucan (tamarind). Liberated reducing ends are subsequently quantified by their reduction of 3,5-dinitrosalicylic acid (DNS) to 3-amino-5-nitrosalicylic acid, which can be quantified spectrophotometrically by measuring its absorption at 540 nm. Conversion of the amounts of liberated reducing ends to enzymatic activity is achieved by quantification and comparison to a glucose standard curve generated using a pre-quantified, commercially available xyloglucanase preparation (Paenibacillus, GH5) as a standard.
Other methods can also be used to determine xyloglucanase activity using substrate-specific assays. For example, as described in Ariza et al. (Structure and activity of Paenibacillus polymyxa xyloglucanase from glycoside hydrolase Family 44, J. Biol. Chem. 286(39): 33890-33900, 2011), xyloglucanase activity can be measured using the chromogenic substrate azurine cross-linked xyloglucan. The absorbance of the enzyme-reacted substrate is measured at 650 nm to quantify enzyme activity. Other substrate-specific assays of xyloglucanase activity use tamarind xyloglucan, carboxymethylcellulose, barley glucan, wheat arabinoxylan, or carob galactomannan as a substrate, with absorbance measurements of the enzyme-reacted substrates taken at 650 nm to quantify xyloglucanase activity.
Constructs encoding fusion proteins containing β-1,4-endoglucanases were prepared using the same methods as described above in Example 26. Each of the constructs contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a β-1,4-endoglucanase. The β-1,4-endoglucanases used were as follows:
The construct containing the signal peptide of SEQ ID NO: 334 directly linked to the β-1,4-endoglucanase of SEQ ID NO: 293 was cloned into a tetracycline resistance-conferring plasmid. This plasmid (pBCm-BclA20-35) was derived from the pBC16 Bacillus cereus plasmid and contains the origin of replication and tet resistance gene from pBC16. The resulting plasmid and the host strain expressing the plasmid were given the ID code E94.
The construct containing the β-1,4-endoglucanase of SEQ ID NO: 293, without any signal peptide, was cloned into the tetracycline-resistance conferring pBCm-BclA20-35 plasmid. The resulting plasmid and the host strain expressing the plasmid were given the ID code E112.
The construct containing the signal peptide of SEQ ID NO: 334, directly linked to the β-1,4-endoglucanase of SEQ ID NO: 294, was cloned into the tetracycline-resistance conferring pBCm-BclA20-35 plasmid. The resulting plasmid and the host strain expressing the plasmid were given the ID code E113.
Each of the constructs in the tetracycline-resistance conferring pBCm-BclA20-35 plasmid was transformed into wild-type Bacillus thuringiensis BT013A and spores were generated as described above in Example 26.
Spores expressing each of the constructs were applied to corn (Beck's corn hybrid 5828 NR) in-furrow treatments using 1 μL volumes of spore preparations and a spore concentration of 1.8-1.9×108 spores per mL (from cultures that resulted in approximately 99% sporulation). For in-furrow treatment, 1 μL of the spore preparation was diluted into 1 mL of water, which was then applied to the soil surrounding the seed at the time of planting.
Plants were grown in an environmentally controlled growth room under the same conditions described above in Example 27. Plant height was measured at two weeks and normalized to control plants treated with water alone. Results are provided in Table 53 below as plant height normalized to control averaged across three trials (18 plants per trial).
In-furrow treatment with the β-1,4-endoglucanase enzymes (E94, E112, and E113) all increased plant height over control, by +11%, +20% and +19%, respectively (Table 53).
A construct encoding a fusion protein containing a lichenase was prepared using the same methods as described above in Example 26. The construct contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a lichenase. The lichenase contained the signal peptide of SEQ ID NO: 335, directly linked to the lichenase of SEQ ID NO: 295. This construct was cloned into the tetracycline-resistance conferring pBCm-BclA20-35 plasmid described above in Example 30 and given the ID code E111. This plasmid was transformed into wild-type Bacillus thuringiensis BT013A and spores were generated as described above in Example 26.
In two separate experiments, corn seeds (Beck's corn hybrid 5828 NR) were treated with 1 μL of spores expressing the lichenase E111 enzyme or β-1,4-endoglucanase E94 (Experiment 1) or with only with spores expressing the lichenase E111 (Experiment 2). The β-1,4-endoglucanase E94 constructs is described above in Example 30. Seed treatment was conducted as described above in Example 30.
Plants were grown in a controlled growth environment under the same conditions as described above in Example 27. Plant height was measured at two weeks. Results are shown in Tables 54 and 55 below as average plant height normalized to a water-only control for each trial in each experiment. Each experiment involved two replicate trials using 18 plants per trial.
The lichenase enzyme applied as a seed treatment increased plant height by 5-6% in both experiments as compared to control (water treated) plants (Tables 54 and 55).
Spores expressing the E111 lichenase described above in Example 31 or the 3-1,4-endoglucanase E94 described above in Example 30 were applied in-furrow to corn (Beck's corn hybrid 5828 NR). One microliter of each spore preparation was diluted into 1 mL of water. The diluted spores were then applied to the soil surrounding the seeds at the time of planting. Plants were grown in an environmentally controlled growth room under the same conditions as described above in Example 27. After two weeks of growth, plant height was measured and compared to water-treated controls. Results are provided in Table 56 below as plant height normalized to control averaged across three trials (18 plants per trial).
In-furrow treatments using lichenase (E111) and β-1,4-endoglucanase (E94) both increased plant growth. In-furrow application of lichenase (E111) increased plant height by +13% while β-1,4-endoglucanase E94 increased plant height by +11% as compared to water-treated controls.
Constructs encoding fusion proteins containing xyloglucanases were prepared as described above in Example 26. Each of the constructs contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a xyloglucanase. The xyloglucanase used were as follows:
Corn seed (Beck's corn hybrid 5828 NR) was treated with 1 μL of the spore preparations using the method described above in Example 30. Seeds were planted and plants were grown in an environmentally controlled growth room under the same conditions as described above in Example 27. Plant height was measured at two weeks and compared to water-treated controls. Results are summarized in Table 57 below as plant height normalized to control averaged across three trials (18 plants per trial). Both xyloglucanase enzymes increased plant height, by +5% for E149 and +3% for D381, compared to control (Table 57).
Constructs encoding fusion proteins containing xylanases were prepared as described above in Example 26. Each of the constructs contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a xylanase. The xylanases used were as follows:
Corn seed (Beck's corn hybrid 5828 NR) was treated as described in Example 30 using 1 μL of each of the spore preparations. Seeds were planted and plants were grown in an environmentally controlled growth room under the same conditions as described above in Example 27. Plant height was measured at two weeks and compared to water-treated controls. Results are summarized in Table 58 below as normalized plant height averaged across three trials (18 plants per trial). Seed treatment with the two xylanase (XynA) enzymes resulted in different effects on plant growth. Xylanase E100 showed a +7% increase in plant height over the control corn plants.
Constructs encoding fusion proteins containing xylosidases were prepared as described above in Example 26. Each of the constructs contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a xylosidase. The xylosidases used were as follows:
These xylosidase constructs and the β-1,4-endoglucanase E94 construct described above in Example 30 were tested for their effects as seed treatments on soybean. Soybean seeds (MorSoyXtra 38X52) were treated with 1 μL of each of the spore preparations by pipetting 1 μL of the spore preparation directly onto each seed and allowing it to dry. Treated seeds were then grown in an environmentally controlled room. Seeds were planted directly into 39.7 cm3 pots containing Timberline #13 top soil at a depth of 2.54 cm, with 2 seeds per pot. After planting, 50 mL of room temperature water was added to each pot to allow for germination. The pots were kept in an artificial lighted growth room receiving approximately 300 μmol m−2 s1 (light photons) for a 12/12 light/day cycle and a 21° C. day/15° C. night temperature range. Plants received the same watering and fertilizer regimes. After 4-5 weeks, plants were measured for changes in dry biomass parameters (total weight and above ground weight). Biomass results are summarized in Table 59 as normalized to control plants grown from water-treated seeds, averaged across two trials (18 plants per trial).
When compared to control plants, β-1,4-endoglucanase (E94) enzyme increased the average total dry weight by +45% and the above ground dry soybean biomass by +66%. Xylosidase (E175), on average, increased total dry biomass by +15% and the above ground dry biomass by +23%.
The xylosidase constructs described above in Example 35 were also tested for their effects on fractional green canopy cover (FGCC) in soybeans. FGCC is a key diagnostic image analysis measure used to estimate canopy development, and provides a means to predict light interception and evapotranspiration from plant leaf surfaces. The Canopeo FGCC mobile application provides a comparison of red to green (R/G) and blue to green (B/C) color ratios and provides a nondestructive measure of green canopy cover. The result of the analysis is a binary image where white pixels correspond to the pixels that satisfy the selection criteria (green canopy) and black pixels correspond to the pixels that did not match the criteria (not green canopy). Fractional green canopy (green band ˜500-570 nm) covers ranges from 0 (no green canopy) to 1 (100% green canopy cover) (Patrignani A. et al., Canopeo: A powerful new tool for measuring fractional green canopy cover, B
Soybean seeds (MorSoyXtra 38X52) were treated as described in Example 35 with 1 μL of each xylosidase spore preparations or with the β-1,4-endoglucanase E94 spore preparation described above in Example 30. Plants were grown in an environmentally controlled growth room under the same conditions as described above in Example 35. At the trifoliate leaf stage, FGCC was measured to quantify canopy size and overall “greenness.” Results are summarized in Table 60. Table 60 provides the FGCC parameters and normalized responses compared to water-treated controls averaged across two trials (18 plants per trial). Xylosidase (E175) seed treatment enhanced greenness compared to water-treated controls. Xylosidase (E175) seed treatment resulted in increases of +13% (Trial 1) and +7% (Trial 2) of the FGCC-calculated parameter. Thus, xylosidase (E175) may have positive impacts for increasing a “stay green” phenotype in soybean especially during the grain and pod filing stages of development, enabling more assimilation and carbohydrate transfer during these two stages to increase overall yield.
The xylosidase constructs described above in Example 35 and the β-1,4-endoglucanase E94 described above in Example 30 were also tested for their effects on nodulation in soybeans. Soybean seeds (MorSoyXtra 38X52) were treated as described in Example 35 with 1 μL of each of the xylosidase spore preparations or with the β-1,4-endoglucanase E94 spore preparation. At 4-5 weeks, nodules were counted and normalized to results from control plants grown from water-treated seeds. The results are summarized in Table 61 below as the normalized nodule count averaged across two trials (18 plants per trial). Seed treatment with xylosidase (E175) or β-1,4-endoglucanase (E94) increased the number of nodules per plant by +5% and +9%, respectively, as compared to control plants.
Constructs encoding fusion proteins containing mannanases were prepared as described above in Example 26. Each of the constructs contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a mannanases. The mannanases used were as follows:
Corn seeds (Beck's hybrid 5828 YH) were treated as described in Example 30 with 1 μL of each of the mannanase spore preparations. Seeds were planted and plants were grown in an environmentally controlled growth room under the same conditions as described above in Example 27. Plant height was measured at two weeks and compared plants grown from to water-treated control seeds. Results are summarized in Table 62 as normalized plant height averaged across two trials (18 plants per trial).
Seed treatment with the mannanase enzymes E177 and E196 promoted plant growth as measured by plant height. Mannanase E177 resulted in a +14% increase and mannanase E196 resulted in a +5% increase in plant height as compared to the control.
The mannanase constructs described above in Example 38 and the β-1,4-endoglucanase E94 construct described in Example 30 above were also tested for their effects on plant growth in soybean when applied as seed treatments.
Soybean seeds (MorSoyXtra 38X52) were treated as described in Example 35 with 1 μL of each of the mannanase spore preparations or with the β-1,4-endoglucanase E94 spore preparation. Seeds were planted and plants were grown in an environmentally controlled growth room under the same conditions as described above in Example 35. Growth measurements (dry biomass and nodulation) were taken at 4-5 weeks and normalized to control plants grown from water-treated seeds. Tables 63 and 64 below summarize normalized results averaged from two trials (12 plants per trial)
As shown in Table 63, seed treatment with β-1,4-endoglucanase (E94) enzyme increased total dry weight by +45% and above ground dry soybean biomass by +66% compared to the control treatment. Mannanase (E177) increased total dry biomass by +10% and increased the total dry above ground biomass by +13% compared to the control plants. Mannanase (E196) increased total dry biomass by +21% and the total dry above ground biomass by +30% compared to controls.
As shown in Table 64, seed treatment with mannanase E177 resulted in a +9% increase in nodule count per plant and treatment with mannanase E196 resulted in a +6% increase in nodule count per plant, as compared to the negative control. Mannanase E177 resulted in equivalent increases in nodule count per plant relative to β-1,4-endoglucanase E94.
Constructs encoding fusion proteins containing pectin-degrading enzymes were prepared as described above in Example 26. Each of the constructs contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a pectin-degrading enzyme. The pectin-degrading enzyme used were as follows:
Corn seeds (Beck's hybrid 5828 YH) were treated as described in Example 30 with 1 μl of each of the pectin-degrading enzyme spore preparations. Seeds were planted and plants were grown in an environmentally controlled growth room under the same conditions as described above in Example 27. At two weeks, plant height was measured and normalized to control plants grown from water-treated seeds. The results are summarized in Table 65 as the average plant height normalized to control across two trials (18 plants per trial). Both of the pectin degrading enzymes, E176 and D405, showed similar effects as seed treatments in corn, resulting in a +1% increase in plant height as compared to the control treatment.
The constructs containing the pectin-degrading enzymes were also tested for their effects on soybeans when applied as a seed treatment. Soybean seeds (MorSoy Xtra 38X52) were treated as described in Example 35 with 1 μl of each of the pectin-degrading enzyme spore preparations or with the β-1,4-endoglucanase E94 spore preparation described above in Example 30. Seeds were planted and plants were grown in an environmentally controlled growth room under the same conditions as described above in Example 35. At 4-5 weeks, measurements of dry biomass and nodulation were obtained and compared to control plants grown from water-treated seeds. Tables 66 and 67 summarize normalized biomass and nodulation measurements averaged across 3 trials (18 plants per trial).
Dry weight differences for total dry weight and above ground dry weight or biomass (grams) were increased over the control treatment for both of the pectin-degrading enzymes (E176 and D405). Pectin lyase (E176) increased total dry biomass by +16% and increased total above ground dry biomass by +25%, whereas endopolygalacturonase (D405) resulted in average increases of +25% for total dry biomass, and a +42% increase for total above ground dry biomass as compared to the control treatment (Table 66). The β-1,4-endoglucanase (E94) enzyme applied as a seed treatment to soybean resulted in a positive increase in total dry biomass (+45%) and also in above-ground dry soybean biomass (+66%) as compared to the control treatment.
Soybean plants grown from seeds treated with β-1,4-endoglucanase E94 had on average a +9% increase in nodules per plant compared to the control plants grown from water-treated seeds (Table 67). Seed treatment with the pectin lyase E176 resulted in an average +2% increase in nodules per plant compared to control plants.
Constructs encoding fusion proteins containing phospholipase C (PLC) enzymes were prepared as described above in Example 26. Each of the constructs contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a PLC. The PLC enzymes used were as follows:
Table 68 below shows the domains included in these constructs and Table 69 provides a depiction of the arrangement of the domains within each construct.
The E95, E143, and E144 constructs were cloned into the tetracycline-resistance conferring pBCm-BclA20-35 plasmid described above in Example 30. The E62 construct was cloned into pBC-BclA20-35 plasmid, which contains a 200 bp additional piece of DNA that is removed in pBCm and does not add any value to the plasmid. The resulting plasmids were then transformed into Bacillus thuringiensis BT013A and spores were generated as described above in Example 26.
The activity of the PLC enzymes on the surface of the spores was then evaluated by measuring the release of p-nitrophenol (pNP) from a p-nitrophenylphosphorylcholine (NPPC) substrate (Carbosynth, product code #EN26341). The NPPC compound is colorless, whereas pNP is a bright yellow-green and can be quantified spectrophotometrically by measuring its absorption at 405-410 nm. Production of one mol of pNP in one minute is equivalent to 1 unit of PLC activity.
A standard curve was first generated using commercially available pNP (Acros Organics, product code #157050050). To generate the standard curve, solutions containing varying pNP concentrations ranging from 0.025 mM to 0.50 mM in 100 mM HEPES buffer pH 7.0 were prepared. The absorption of each sample at 405 nm was then measured using a spectrophotometer. The standard curve is shown in
Spores expressing each of the PLC constructs were then incubated with 2.8 μmol of the NPPC substrate in 100 mM HEPES, pH 7.0 for 180 minutes at 37° C. Each spore preparation contained approximately 2×108 spores. The NPPC substrate alone was used as a negative control. The reaction was then stopped by adding 2004, of 0.1 M sodium bicarbonate. The absorption of each sample at 405 nm was then measured using a spectrophotometer. Results are shown in
The PLC constructs described in Example 42 above were also evaluated for their effects on soybean when applied as a seed treatment. Soybean seeds (Beck's soybean hybrid 297NR) were treated as described above in Example 35, except that a 0.5 μl volume of each of the E62, E143, and E144 spore preparations was used. The spore preparations contained 1.8-1.9×108 spores per mL (from cultures that resulted in approximately 99% sporulation). Control seeds were treated with non-transformed Bacillus spores with no enzymes (E0) or with water alone. Seeds were planted and plants were grown in an environmentally controlled growth room. Seeds was planted directly into 39.7 cm3 pots containing Timberline #13 top soil combined with MIRACLE-GRO potting soil at a ratio of 1:4 and planted at a depth of 2.54 cm, with 2 seeds per pot. After planting, 50 mL of room temperature water was added to each pot to allow for germination. The pots were kept in an artificial lighted growth room receiving approximately 300 μmol m−2 s1 (light photons) for a 12/12 light/day cycle and a 21° C. day/15° C. night temperature range. Plants received the same watering and fertilizer regimes.
Plant height was measured at 4-5 weeks. Soybean growth from seeds that received the phospholipase C (E62, E143, E144) was compared with growth from seeds treated with Bacillus spores alone (E0). Results are summarized in Table 70 as the average plant height normalized to control plants grown from seeds treated with water alone. All treatments including the spore without enzyme (E0) treatment are reported as normalized to plants that received the water-control alone seed treatment. Soybean plants grown from seeds treated with the phospholipase C enzymes (E62, E143, E144) or with Bacillus spores alone had substantial increases in plant height compared to control plants grown from the water treated seeds. The phospholipase C E143 treatment had the greatest effect, showing a +88% increase in plant height over the water-only control (Table 70).
Bacillus Spores (EO no enzyme)
A construct encoding a fusion protein containing a phospholipase D (PLD) enzyme was prepared as described above in Example 26. The construct contained the BclA promoter (SEQ ID NO: 149), a start codon, a coding sequence for amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1), and a coding sequence for a PLD (SEQ ID NO: 256; ID CODE: D409). The construct was cloned into the tetracycline-resistance conferring pBCm-BclA20-35 plasmid described above in Example 30. This plasmid was then transformed into wild-type Bacillus thuringiensis BT013A and spores were generated as described above in Example 26.
Corn seeds (hybrid 5828 YH) and soybean seeds (hybrid MorSoyXtra 38X52) were treated as described above in Examples 30 and 35 using a 1 μL (1× standard application) or 2 μL (2× standard application) of the spore preparation. As in preceding examples, the spore preparations contained 1.8-1.9×108 spores/mL (from cultures that result in approximately 99% sporulation). Seeds were treated as described in Examples 27 and 35. Seeds were planted and grown as previously described in Example 27 (for corn) or Example 35 (for soybean) and growth measurements (plant height, dry biomass, nodulation) were taken at 2 weeks (for corn) or at 4-5 weeks (soybean). Two or three replicate trials were conducted using on average 18 plants per trial and results are summarized on Tables 71-74. Results were normalized to water treated controls.
Phospholipase D409 applied at a 1× use rate to corn seed provided an increase in for corn growth as measured by a +3% increase in plant height over control plants grown from water-treated seeds (Table 71).
Phospholipase D409 applied at the higher 2× use rate (2 μL/seed) to soybean seeds resulted in an incremental increase in total fresh weight, root fresh weight, and above ground fresh weight as compared to the seed treated with the water control. The greatest effects were seen with above ground fresh weight (a 17% increase over control) (Table 72).
Phospholipase D (D409) applied to soybean seed at the 1× and 2× use rates resulted in similar overall increases in plant height (+16% or +17%, respectively) as compared to the seed treated with the water control. Data shown is an average of three trials (Table 73).
Changes in early nodulation as measured by average nodules per soybean plant were compared using seed treatments with phospholipase D (D409) at a 1× (1 μL/seed) and a 2× (2 μL/seed) use rates. Nodules were counted on 12 separate plants per trial at 4-5 weeks. Plants grown from seeds treated with phospholipase D (D409) at either use rate showed an increase in nodules per plant compared to control. On average, the 1× and 2× use rates resulted in a +23% and +18% increase in nodulation, respectively, over control plants (Table 74).
Spore preparations as described in the above examples containing the enzymes described below were applied to soybean (hybrid MorSoyXtra 38X52) (Tables 75-77) or corn (Beck's corn hybrids 5828 YH) (Tables 78-80). Spores were treated with 1 μL of the spore preparations. All seeds were treated as described in Example 30 (for corn) or 35 (for soybean). All plants were grown as described in Example 27 (for corn) or 35 (for soybean). Corn plant height was measured at two weeks (Tables 78-80). Growth measurements (biomass, height, nodulation) were taken at 4-5 weeks for soybean (Tables 75-77). All measurements are reported in Tables 75-80 as averages or as normalized to control plants grown from water treated seeds. Two or three replicate trials were conducted using on average 18 plants per trial.
Table 75 summarizes the average number of nodules per plant recorded at 4-5 weeks in soybean plants grown from seeds treated with β-1,4-endoglucanase E94, xylosidase E175, xylosidase E194, mannanase E177, mannanase E196, or endopolygalacturonase D405. The table averages data taken from 3 separate trials with 16 replicate plants per trial, a total of 48 plants. Seed treatment using β-1,4-endoglucanase (E94) and mannanase enzymes (E177 and E196) increased nodule count per plant by +10%, +8% and +6%, respectively, as compared to control plants grown from water-treated seeds.
Table 76 summarizes the total dry weight, above ground dry weight, and root dry weight for soybean (hybrid MorSoyXtra 38X52) plants grown from seeds treated with β-1,4-endoglucanase E94, xylosidase E175, xylosidase E194, mannanase E177, mannanase E196 or endopolygalacturonase D405. The data in Table 76 are averages of data recorded from 2 separate trials with 16 replicate plants per trial, a total of 32 plants. All of the enzymes tested showed improvements in the above ground biomass (weight) compared to control plants. In addition, the ratio of above ground dry weight to root dry weight was determined to investigate changes in above ground biomass to root biomass in response to individual treatments. This ratio for the plants that received the water control seed treatment is equivalent to 2.5. All of the enzymes in Table 76 (E94, E175, E194, E196 and D405) resulted in an increased ratio of above ground dry weight to root dry weight compared to water-treated controls. The largest effects were seen with treatment with β-1,4-endoglucanase E94 and endopolygalacturonase D405, which showed ratios of 5.6 and 5.5 respectively, over two-fold higher than controls (2.5).
Table 77 summarizes the total dry weight and above ground dry weight normalized to controls for soybean (hybrid MorSoyXtra 38X52) plants grown from seeds treated with β-1,4-endoglucanase E94, xylosidase E175, xylosidase E194, mannanase E177, mannanase E196, or endopolygalacturonase D405. Differences in the amount of total plant dry weight and above ground dry weight are reported for 2 trials with 18 replicate plants per trial. The greatest comparative increases across the classes of enzymes were observed for β-1,4-endoglucanase E94 and endopolygalacturonase D405. These results further support the ratio increases described above in Table 76.
Table 78 summarizes average plant height recorded at two weeks for corn plants (Beck's hybrid 5828 YH) grown from seeds treated with β-1,4-endoglucanase E94, xylosidase E175, xylosidase E194, mannanase E177, mannanase E196. The data in the table are averages from 2 trials with 18 replicate plants per trial. Corn seed treatment with the mannanase enzymes E177 and E196 resulted in substantial increases in plant height, +26% and +15%, respectively, as compared to corn plants grown from the water-treated seeds.
Table 79 summarizes another study comparing plant height at two weeks for corn plants (Beck's hybrid 5828) grown from seeds treated with xylosidase E175 and mannanases E177 and E196. The table shows average plant height normalized to water treated controls from 3 trials with 18 replicate plants per trial. Corn seed treatment for all the enzymes tested resulted in increased plant height compared to control. Seed treatments with the mannanase class of enzymes E177 and E196 showed the largest effects, with respective increases of +26% and +15% in plant height as compared to control plants grown from water treated seed.
The effect of seed treatment, foliar application, or in-furrow treatment on corn and soybean yield in the field will be tested. Field seed beds at a variety of US Midwest locations will be prepared using conventional or conservation tillage methods for corn and soybean plantings. Fertilizer will be applied as recommended by conventional farming practices and will remain consistent between the US Midwest locations. Herbicides will be applied for weed control and supplemented with cultivation when necessary.
Corn and soybean hybrids will be treated as seeds, in-furrow, or via foliar spray with spore preparations prepared as described in the above Examples and containing a phospholipase, a β-1,4-endoglucanase, an ACC deaminase, a lichenase, a xyloglucanase, a xylanase, a xylosidase, a mannanase, or a pectin-degrading enzyme.
Seed treatment will be conducted with a WINTERSTEIGER HEGE 11 (Wintersteiger AG, Austria). The capacity of this seed treatment machinery can accommodate a large number of different seed types, sizes and amounts of seed (20-3000 grams). The seeds will be loaded into bowls of the seed treater machinery. The bowl selection depends on the treatment seed amount required and the size of the bowl selected: large 14.5 L bowl (500-3000 g seed per coating); medium 7 L bowl (80-800 g seed per coating); and small 1 L bowl (20-100 g seed per coating). During operation, the seeds will be distributed toward the radial peripheries of the rotatable bowls via an application of centrifugal force with the centrifugal coating device. The spinning disc located at the bottom of the bowl will distribute the seed treatment evenly over the seed. At this point, the spin cycle will start, which will cause the seeds to revolve around the bowl center in a circle to evenly coat the seeds. The process of seed providing a seed treatment, coating or dressing will be initiated after the seed is evenly dispersed around the spreader. Seed treatment sample material (such as a powdered, semi-liquid, liquid or a slurry) may be applied onto the rotatable disk as the disks are spinning within the rotatable bowls used to distribute the seed treatment evenly to provide a uniform coat and dress the surface of the seed.
An equivalent amount of each spore preparation of approximately 1 μL (standard application) will be applied per each seed. A constant air flow will be delivered using compressed air (2-6 bars) during seed coating to assist with uniformly coating the seeds in the bowl. The amount of time for the coating of the seed will depends on the amount of the seed, the viscosity of the seed treatment and the type of the seed used in the treatment. A seed treatment calculator will be used to adjust for all volumes, for most major and commercially grown crops, and the type of seed treatment being applied. After the seed has been treated and dried, the seeds will be distributed into a larger storage container(s). Seeds will be either air dried or dried with a continuous air stream that passes over the seeds. Seeds will then be transferred into a separate container or bag for shipment, transfer or storage.
After treatment, corn and soybean seeds will be planted in properly prepared fields. Corn seed will be planted 4-5 cm deep to assure normal root development. Corn seed will be planted at 28,000 to 32,000 plants per acre with row widths of 30 inch rows with seed spacing of approximately 1.6 to 1.8 seeds per foot. Soybean seeds will be planted 1.5 to 2 inches deep to assure normal root development. Soybean seed will be planted at approximately on average 140,000 plants per acre with row widths of 30 inch (76.2 cm) rows with seed spacing of approximately 7 to 8 seeds per foot (per 30.5 cm). Two-row plots, 25 feet (7.62 m) long will be planted at all (10-20) locations. Each hybrid will be grown in at least three separate plots (replicates) at each location to account for field variability.
Parallel experiments will treat plants with foliar application or in-furrow treatment using the same spore preparations. The yield (Bu/Ac) of each crop treated with the growth promoting enzymes delivered as seed treatments, foliar or in-furrow treatments will be reported on a per location and across location basis. Yield will be reported as normalized to the non-treated control plants and show as an increase or a decrease in Bu/Ac compared to the control plants that did not receive treatment with an enzyme.
The pSUPER plasmid was generated through fusion of the pUC57 plasmid (containing an ampicillin resistance cassette and a ColE1 origin of replication) with the pBC16-1 plasmid from Bacillus cereus (containing a tetracycline resistance gene, repU replication gene and oriU origin of replication). This 5.8 kb plasmid can replicate in both E. coli and Bacillus spp. and can be selected by conferring resistance to β-lactam antibiotics in E. coli and resistance to tetracycline in Bacillus spp. The pSUPER plasmid was modified by insertion of a PCR-generated fragment that fused the BclA promoter (SEQ ID NO: 149), a start codon, amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1) and a polyalanine linker sequence (AAAAADLE; SEQ ID NO: 386) in frame with Bacillus subtilis 168 β-1,4-endoglucanase (the signal peptide of SEQ ID NO: 334, directly linked to the β-1,4-endoglucanase of SEQ ID NO: 293) resulting in a plasmid termed pSUPER-BclA 20-35-Endo. For replacement of the targeting sequence the pSUPER-BclA 20-35-Endo plasmid was subjected to inverse PCR to amplify the entire plasmid backbone, but leaving out the sequence corresponding to amino acids 20-35 of BclA. This inverse PCR product was combined with a PCR product containing the equivalent region from each of SEQ ID NOs: 5, 15, 25, 81, 85, 87, or amino acids 20-33 of SEQ ID NO: 1. Thus, constructs were created that contained each of the following targeting sequences fused in frame with Bacillus subtilis 168 endoglucanase: (1) amino acids 20-35 of SEQ ID NO: 1; (2) SEQ ID NO: 381; (3) SEQ ID NO: 383; or (4) SEQ ID NO: 382. Each of these constructs was transformed into E. coli and plated on Lysogeny broth petri plates plus ampicillin (100 μg/ml) to obtain single colonies. Individual colonies were used to inoculate Lysogeny broth plus ampicillin and incubated overnight at 37° C., 300 rpm. Plasmids from resulting cultures were extracted using the Wizard Plus SV Minipreps DNA Purification System (Promega Corporation). DNA concentrations of these plasmid extracts were determined via spectrophotometry and obtained plasmids subjected to analytical digests with appropriate combinations of restriction enzymes and then visualized by agarose gel electrophoresis to investigate plasmid size and presence of distinct plasmid features. Relevant sections, such as the 3-1,4-endoglucanase expression cassette, of the purified pSUPER derivatives were further investigated by Sanger sequencing. Verified pSUPER plasmids were introduced by electroporation into Bacillus thuringiensis BT013A or a BT013A derivative in which the native bclA gene (BclA knockout, “BclA KO”) had been removed from the genome through homologous recombination. Single transformed colonies were isolated by plating on nutrient broth petri plates containing tetracycline (10 μg/ml). Individual positive colonies were used to inoculate brain heart infusion broth containing tetracycline (10 μg/ml) and incubated overnight at 30° C., 300 rpm. Genomic DNA of resulting cultures was purified and sections of the pSUPER plasmid were re-sequenced to verify genetic purity of the closed sequences. Verified colonies were grown overnight in brain heart infusion broth with 10 μg/ml tetracycline and induced to sporulate through incubation in a minimal yeast extract-based media.
The pSUPER-BclA 20-35-Endo plasmid was subjected to inverse PCR to amplify the entire plasmid backbone, but leaving out the sequences corresponding to the signal peptide and the β-1,4-endoglucanase open reading frame. This inverse PCR product was combined with a PCR product containing a nucleotide sequence encoding for one of the following xylanases SEQ ID NO: 268, SEQ ID NO: 273 (E232), or a fusion of the signal peptide of SEQ ID NO: 328 directly linked to the xylanase of SEQ ID NO: 268 (E100). Thus, constructs were created that contained open reading frames for the following amino acid sequences fused in frame with a start codon, amino acids 20-35 of BclA (SEQ ID NO: 1), a polyalanine linker (AAAAADLE; SEQ ID NO: 386) sequence and (1) SEQ ID NO: 268 (pSUPER-BclA 20-35-XynA_nosec); (2) SEQ ID NO: 273 (pSUPER-BclA 20-35-XynC); or (3) SEQ ID NO: 328 directly linked to SEQ ID NO: 268 (pSUPER-BclA 20-35-XynA) under transcriptional control of the BclA promoter. These constructs were transformed into E. coli and plated on Lysogeny broth petri plates plus ampicillin (100 μg/ml) to obtain single colonies. Individual colonies were used to inoculate Lysogeny broth plus ampicillin and incubated overnight at 37° C., 300 rpm. Plasmids from resulting cultures were extracted using the Wizard Plus SV Minipreps DNA Purification System (Promega Corporation). DNA concentrations of these plasmid extracts were determined via spectrophotometry. The plasmids were then subjected to analytical digests with appropriate combinations of restriction enzymes and visualized by agarose gel electrophoresis to investigate plasmid size and presence of distinct plasmid features. Relevant sections, such as the xylanase expression cassette, of the purified pSUPER derivatives were further verified by Sanger sequencing. pBC fragments (pBC16-1-derived section of pSUPER including BclA/xylanase expression cassette) of the created pSUPER plasmids were amplified by PCR and subsequently circularized by blunt-end ligation. Resulting pBC ligations were introduced by electroporation into Bacillus thuringiensis BT013A. Single transformed colonies were isolated by plating on nutrient broth petri plates containing tetracycline (10 μg/ml). Individual positive colonies were used to inoculate brain heart infusion broth containing tetracycline (10 μg/ml) and incubated overnight at 30° C., 300 rpm. Genomic DNA of resulting cultures was purified and the pBC plasmid re-sequenced to verify the genetic purity of the cloned sequences. Verified colonies were grown overnight in brain heart infusion broth with 10 μg/ml tetracycline and induced to sporulate through incubation in a minimal yeast extract-based media.
The pSUPER-BclA 20-35-Endo plasmid (see above) was subjected to inverse PCR to amplify the entire plasmid backbone, but leaving out the sequence for the tetracycline resistance gene. This inverse PCR product was combined with a PCR product containing a non-antibiotic selectable marker expression cassette. The resulting plasmid was subjected to another inverse PCR adjusted to amplify the entire plasmid backbone, but leaving out the sequences corresponding to the signal peptide and the β-1,4-endoglucanase open reading frame. This inverse PCR product was combined with a PCR product containing a nucleotide sequence encoding for a fusion of SEQ ID NO: 317 and SEQ ID NO: 250 (designated as ID CODE: E80 when expressed with the non-antibiotic selectable marker system). Thus, a construct was created that contained a start codon, amino acids 20-35 of BclA (SEQ ID NO: 1), an alanine linker (AAAAAALE (SEQ ID NO: 387) as described in Table 80 below) sequence fused in frame with Bacillus thuringiensis phospholipase C (the signal peptide of SEQ ID NO: 317, directly linked to the phospholipase C of SEQ ID NO: 250) under transcriptional control of the BclA promoter. This construct was transformed into E. coli and plated on Lysogeny broth petri plates plus ampicillin (100 μg/ml) to obtain single colonies. Individual colonies were used to inoculate Lysogeny broth plus ampicillin and incubated overnight at 37° C., 300 rpm. Plasmids from resulting cultures were extracted using the Wizard Plus SV Minipreps DNA Purification System (Promega Corporation). DNA concentrations of these plasmid extracts were determined via spectrophotometry. The plasmids were then subjected to analytical digests with appropriate combinations of restriction enzymes and visualized by agarose gel electrophoresis to investigate plasmid size and presence of distinct plasmid features. Relevant sections, such as the phospholipase expression cassette, of the purified pSUPER derivatives were further verified by Sanger sequencing. The pBC fragment (pBC16-1-derived section of pSUPER including BclA/phospholipase expression cassette) of the created pSUPER plasmid was amplified by PCR and subsequently circularized by blunt-end ligation. The resulting pBC ligation was introduced using electroporation into a Bacillus thuringiensis BT013A derivative strain that had been modified to support the use of the non-antibiotic selectable marker. Single colonies of transformations were obtained by plating on minimal broth petri plates. Individual colonies were used to inoculate a minimal broth and incubated overnight at 30° C., 300 rpm. Genomic DNA of resulting cultures was purified and the pBC plasmid re-sequenced to verify genetic purity. Verified colonies were grown overnight in a minimal media and induced to sporulate through incubation in a minimal yeast extract-based media.
The activity level of the recombinant Bacillus cereus family members expressing fusion proteins containing endoglucanases produced in Example 47, the E100 xylanase produced in Example 48, or the E80 phospholipase C produced in the present example (Example 49) were measured and compared to the activity level of recombinant Bacillus cereus family members expressing fusion proteins containing lichenases; mannanases; xyloglucanases; the E143, D473, D474, D475, and E258 phospholipase C enzymes; phospholipase D enzymes; xylosidases; pectolyases; acid phosphatase; phytase; and lactonase, (all prepared using the same expression system as described in Example 48 and the linker sequences listed in Table 80 below); and an ACC deaminase prepared as exosporium fragments as described in Example 26 and using the linker sequence listed in Table 80 below. The activity assays for each of these enzyme types have been described in examples hereinabove (e.g., Example 29) except for those to measure phospholipase, xylanase, lichenase, xylosidase, pectic enzymes, phytase, lactonase, or ACC deaminase activity which are described below.
Phospholipase
Phospholipase C (PLC) activity was assayed by quantifying p-nitrophenol release using p-nitrophenylphosphorylcholine (NPPC; CAS #21064-69-7, Carbosynth EN26341) as a substrate. Aliquots (1 mL) of spore productions were centrifuged 5 min at 16,100×g in a tabletop microcentrifuge to pellet spores. Spore pellets were resuspended in 30 deionized water and transferred to a 300 μL/well 96-well PCR plate. Thirty microliters of reaction buffer (20 mM NPPC, 200 mM HEPES, pH 7.0, 60% w/v D-sorbitol) were added to each well and reactions were incubated at 37° C. in a PCR thermocycler for 45 min. Subsequently, 60 μL 0.5 M sodium bicarbonate buffer containing 25 mM EDTA was added to stop reactions and develop color. The PCR plate was centrifuged 5 min. at 2,272×g in a plate centrifuge to pellet spores. Supernatants were transferred to a flat bottom 350 μL/well 96-well microplate and absorbance at 405 nm was measured using a Synergy HTX (BioTek) microplate reader. PLC activity was quantified by comparison to a p-nitrophenol (CAS #100-02-7, Acros Organics 157050050) standard curve under consideration of incubation time. Samples were assayed in triplicate.
Xylanase
Xylanase activity was quantified by a reducing sugar assay using 4-O-methyl-D-glucurono-D-xylan (MGX; CAS #9062-57-1, Sigma M5144) as a substrate. Aliquots (0.5-1 mL) of spore productions were centrifuged 5 min at 16,100×g in a tabletop microcentrifuge to pellet spores. Spore pellets were resuspended in 60 μL 50 mM citrate buffer, pH 5.0 and transferred to a 300 μL/well 96-well PCR plate. Eighty microliters of 0.5% w/v MGX (in water) were added to each well and reactions were incubated at 50° C. in a PCR thermocycler for 40 min. The plate was placed on ice and 120 μL DNS reagent (1% w/v 3,5-dintrosalicylic acid, 1% NaOH, 0.05% Na2SO4, 0.2% phenol, 18.2% Rochelle salt) was added. Reactions were subsequently incubated at 100° C. for 10 min. in a PCR thermocycler. The PCR plate was centrifuged 5 min. at 2,272×g in a plate centrifuge to pellet spores. Supernatants were transferred to a flat bottom 350 μL/well 96-well microplate and absorbance at 540 nm was measured using a Synergy HTX (BioTek) microplate reader. Xylanase activity was quantified by comparison to a glucose standard curve under consideration of incubation time. Samples were assayed in triplicate.
Lichenase
Lichenase activity was quantified by a reducing sugar assay using β-D-glucan from barley (CAS #9041-22-9, Sigma G6513) as a substrate. Aliquots (1 mL) of spore productions were centrifuged 5 min. at 16,100×g in a tabletop microcentrifuge to pellet spores. Spore pellets were resuspended in 60 μL 50 mM HEPES buffer, pH 7.0 and transferred to a 300 μL/well 96-well PCR plate. Eighty microliters of 0.5% w/v β-D-glucan (in water) were added to each well and reactions were incubated at 50° C. in a PCR thermocycler for 25 min. The plate was placed on ice and 120 μL DNS reagent (1% w/v 3,5-dintrosalicylic acid, 1% NaOH, 0.05% Na2SO4, 0.2% phenol, 18.2% Rochelle salt) was added. Reactions were subsequently incubated at 100° C. for 10 min in a PCR thermocycler. The PCR plate was centrifuged 5 min at 2,272×g in a plate centrifuge to pellet spores. Supernatants were transferred to a flat bottom 350 μL/well 96-well microplate and absorbance at 540 nm was measured using a Synergy HTX (BioTek) microplate reader. Lichenase activity was quantified by comparison to a glucose standard curve under consideration of incubation time. Samples were assayed in triplicate.
Xylosidase
Xylosidase activity was assayed by quantifying p-nitrophenol release using p-nitrophenyl-β-D-xylopyranoside (PNPX; CAS #2001-96-9, Sigma N2132) as a substrate. Aliquots (50-200 μL) of spore productions were centrifuged 5 min at 16,100×g in a tabletop microcentrifuge to pellet spores. Spore pellets were resuspended in 50 μL 100 mM citrate buffer, pH 6.2 and transferred to a 300 μL/well 96-well PCR plate. Fifty microliters of 5 mM PNPX (in water) were added to each well and reactions were incubated at 37° C. in a PCR thermocycler for 20 min. Enzymatic activity was stopped by addition of 100 μL 95% ethanol. Subsequently, 50 μL 0.5 M sodium bicarbonate was added for color development. The PCR plate was centrifuged 5 min at 2,272×g in a plate centrifuge to pellet spores. Supernatants were transferred to a flat bottom 350 μL/well 96-well microplate and absorbance at 405 nm was measured using a Synergy HTX (BioTek) microplate reader. Xylosidase activity was determined by comparison to a p-nitrophenol (CAS #100-02-7, Acros Organics 157050050) standard curve under consideration of incubation time. Samples were assayed in triplicate.
Pectic Enzymes
Pectolyase/polygalacturonase activity was quantified by a reducing sugar assay using pectin from citrus peel (CAS #9000-69-5, Sigma P9135) as a substrate. Aliquots (0.5-1 mL) of spore productions were centrifuged 5 min at 16,100×g in a tabletop microcentrifuge to pellet spores. Spore pellets were resuspended in 70 μL 50 mM CHES buffer, pH 10.0 and transferred to a 300 μL/well 96-well PCR plate. Thirty microliters of deionized water and 40 μL 1% w/v pectin (in water) were added to each well and reactions were incubated at 50° C. in a PCR thermocycler for 45 min. The plate was placed on ice and 120 μL DNS reagent (1% w/v 3,5-dintrosalicylic acid, 1% NaOH, 0.05% Na2SO4, 0.2% phenol, 18.2% Rochelle salt) was added. Reactions were subsequently incubated at 75° C. for 10 min. in a PCR thermocycler. The PCR plate was centrifuged 5 min at 2,272×g in a plate centrifuge to pellet spores. Supernatants were transferred to a flat bottom 350 μL/well 96-well microplate and absorbance at 540 nm was measured using a Synergy HTX (BioTek) microplate reader. Pectolyase/polygalacturonase activity was quantified by comparison to a glucose standard curve under consideration of incubation time. Samples were assayed in triplicate.
Phytase
Phytase activity was assayed by quantifying p-nitrophenol release using p-nitrophenyl phosphate (pNPP; CAS #4264-83-9, Sigma 487600-M) as a substrate. The reactions were prepared on ice in 1.7 mL tubes, each reaction contained 100 μL of whole spore productions, 100 μL 200 mM Na-Acetate buffer (pH 5), and 30 μL 30 mM pNPP (in water). Then, the reactions were incubated at 37° C. with agitation (800 rpm) for 30 min. Subsequently, reactions were place on ice and 100 μL 1 M NaOH were added to develop the yellow color that p-nitrophenol have at alkaline pH. Tubes were then centrifuged 5 min at 16,100×g in a tabletop microcentrifuge to pellet spores, and 200 μL supernatants were transferred to a flat bottom 350 μL/well 96-well microplate and absorbance at 400 nm was measured using a Synergy HTX (BioTek) microplate reader. Phytase activity was determined using the Beer-Lambert law and extinction coefficient at 400 nm of 18000 M−1 cm−1. Samples were assayed at least in duplicate.
Lactonase
Lactonase activity was measured using a pigmentation bioassay using Chromobacterium violaceum strain CV026, a mutant that only produces the purple pigment violacein in response to exogenous homoserine lactone (HSL). HSL was reacted with lactonase enzyme samples (H51), along with BT013A E0 (background strain with no enzyme) control treatment for 2 hours at 37° C. The samples were frozen at −80° C. to stop the reaction. LB-kanamycin agar petri plates were inoculated evenly with a culture of CV026 that had been grown in LB-kanamycin liquid culture overnight. A volume of 300 μL of the liquid culture containing the enzyme production and BT013A control was spread over the reaction petri plates. Once the media had dried on petri plates, a 1 μL volume of the thawed reaction products was applied to pre-marked spots on the petri plates. Treatments were applied in triplicate. Following overnight incubation at 30° C., images were taken of the petri plates, and the area of purple pigmentation produced by CV026 was quantified using ImageJ Software (National Institute of Health). A reduction in pigmentation indicated that the HSL in a given reaction had been degraded.
ACC Deaminase
ACC deaminase activity was assayed by measuring the amount of NADH (CAS #606-68-8, Sigma N8129) used by lactate dehydrogenase (EC number 1.1.1.27, Sigma 10127230001) when using a-ketobutyrate, made by ACC deaminase after a reaction with ACC (CAS #22059-21-8, Sigma 149101-M), as a substrate. 0.9-1 mL aliquots of spore productions were centrifuged for 5 minutes at 14,000 rpm in a tabletop microcentrifuge to pellet the spores. Spore pellets were resuspended in 1 mL of 100 mM TRIS buffer pH 8.0 then spun down again at 14,000 rpm for 5 minutes to pellet spores. The spore pellet was then resuspended in 600 μL 100 mM TRIS buffer pH 8.0, 10 mM ACC and 0.132 mM NADH and 200 μL was transferred to 3 wells in a 300 μL/well 96-well PCR plate. 5 U of lactate dehydrogenase was added to each well and the mixture was transferred to a flat bottom 350 μL/well 96-well microplate and allowed to react at 37° C. in a Synergy HTX (BioTek) microplate reader taking readings at 340 nm every 2 minutes for 1 hour. ACC deaminase activity was measured by subtracting background readings then using Beer-Lambert law and the NADH extinction coefficient. Samples were assayed in triplicate.
Table 80 lists the enzymatic activity measured from each production batch for these series of enzymes.
Bacillus subtilis strain 168
Bacillus subtilis strain 168
Bacillus subtilis strain 168
Bacillus licheniformis
Paenibacillus pabuli
Bacillus subtilis strain 168
Bacillus thuringiensis
Bacillus thuringiensis
Listeria monocytogenes
Listeria monocytogenes
Listeria monocytogenes
Bacillus cereus
Bacillus licheniformis
Bacillus subtilis strain 168
Bacillus subtilis strain 168
Bacillus subtilis strain 168
Bacillus subtilis strain 168
Clostridium thermocellum
Bacillus subtilis strain 168
Bacillus pumilis
Aspergillus niger strain
Bacillus subtilis strain 168
Bacillus thuringiensis
Bacillus thuringiensis
Bacillus thuringiensis
Recombinant Bacillus cereus family members expressing fusion proteins containing a xyloglucanase, a mannanase, a xylanase, or a xylosidase were, prepared using the methods described above in Example 48 and were delivered as seed treatments onto corn seeds (Beck's corn hybrid 5828 YH) using the volumes (μL) applied to the outer coats of each seed as a seed treatment and indicated in Table 81-84. Each enzyme was prepared in whole cell broth which was collected and utilized in the experiments. A wild-type control (B. thuringiensis BT013A untransformed host strain (E0; no-enzyme control) was also applied to seed using a volume of 1 μL per seed and used as a control comparison for the enzyme treatments Corn seed that received the seed treatments with xyloglucanase, mannanase, xylanase, xylosidase and the E0 control treatments was then planted in 4×4 cm pots filled with Timberline 18 top soil with two seeds each planted per pot and initially watered with 50 mL of water to promote germination. Two replicates using 18 seeds per each treatment per replicate were placed in an environmentally controlled growth room and randomized using a complete randomized block design. Plants were grown under light levels of approximately 300 μmol m−2 s−1 and a 13 hour photoperiod (13/11 day/night cycle). Plants were maintained at 23.9° C. during the light hours and 18.3° C. during the dark hours. Plant height (an indicator of plant growth rate) was measured at 15 days after planting (DAP) or approximately at the V3 developmental growth stage to capture early plant vigor. An average plant height per each enzyme treatment was normalized to plants grown from seed that received and the E0 control provided to the seed using a 1 μL volume. Results of the xyloglucanase, mannanase, xylanase and xylosidase enzyme seed treatments on corn growth rate as indicated by changes in plant height are compared to the E0 (no enzyme) control shown in Table 81-84.
Xyloglucanases D381 and E149 applied as a seed treatment on corn resulted in an increased growth rates during early stages of corn development as indicated by an increased percentage averaged plant height normalized to plants grown from seed that received a water control treatment (Table 81). In general, plant height increased with increasing volumes of the xyloglucanase enzymes applied at 1 μL to 3 μL to the seed prior to planting. Seed treatments that received the 3 μL of the xyloglucanase enzymes (either D381 or E149) resulted in the greatest increases in plant height as normalized to the water control and compared to the E0 control which was generally comparable to the water control over the two replicates used in the trials. Seed treatment of corn using xyloglucanase D381 (SEQ ID NO: 299) applied using the 3 μL seed treatment volume per seed exhibited slightly greater increases in plant height at V3 (15 DAP) as compared to the xyloglucanase E149 (SEQ ID NO: 300), an 2.4% increase.
Corn seed treatment using mannanases (E196 and E177) resulted in increased growth rates as indicated by an increase in plant height when compared to plants grown from seed that received the water control treatment or the BT013A E0 control (no enzyme) as shown in Table 82. Plants that were grown from seed treated with the E0 control did not show any substantial differences in plant height (cm) as compared to those grown from seed that received the water control for the two replicate treatments within the trial. Seed treatment with mannanase E196 (SEQ ID NO: 307) resulted in plants with increased plant height seen with the increasing dose of the enzyme from 1 μL to 3 μL with the 3 μL volume applied as a seed treatment yielded the greatest increase in height, a +7% increase over the control (grown from water-treated seed) plants. Mannanase E177 (SEQ ID NO: 308) applied as a seed treatment also resulted in corn plants with an increased growth rate or plant height as compared to the control plants (grown from water-treated seed). The greatest overall increases in plant height for plants were from seed treated with mannanase E177 (1 μL enzyme applied to the seed) an increase of +8% over the control plants.
Seed treatments applied to corn seed using xylanase enzymes (E100, E146 and E232) also resulted in corn plants with increased plant height as compared or normalized to those plants grown from seed that the E0 control (1 μL) and assessed using two replicates of the same trial (Table 83). Generally, the enzyme volumes applied to the seed using either 1 μL or 2 μL resulted in similar increases in plant height. Seeds that received the xylanase E232 (SEQ ID NO: 273) resulted in the greatest increased plant height, +4% (1 μL) and +5% (2 μL) increase over or normalized to the plants grown from the E0 treated seed.
In a separate experiment using xylosidase as a seed treatment on corn (Beck's corn hybrid 5828 YX), a similar approach was followed related to growth conditions and randomization of treatments within trials as previously described. In this experiment, corn seed that received the seed treatment with xylosidase and the corresponding E0 control treatment was then planted in 4×4 cm pots filled with a 2:1 Timberline 20 top soil to sand mixture to simulate early plant growth effects of corn grown in a sandy loam type soil. A three replicate trial using 18 plants per each treatment per replicate were grown under control environmental conditions as described above. Plant height (an indicator of plant growth rate) was measured at 15 days after planting (DAP) to capture early plant vigor. An average plant height (cm) was calculated per each enzyme treatment and normalized to a water control (provided using a 1 μL volume). Results of the xylosidase enzyme seed treatments and the E0 control on corn growth rates are represented as the average change in plant height compared to the water control averaged for the three replicates and shown in Table 84.
Seed treatments on corn using xylosidase (E194 and E175) enzymes applied using varying volumes of 1 μL to 3 μL resulted in positive growth benefits to V3 (15 DAP) corn plants. Seed treatment using xylosidase E194 did result in plants with increased heights compared to plants grown from seed that received the water or E0 control treatments, however changing the volume of the enzyme used as a seed treatment did not have an overall effect on the height increase at the V3 stage of development. Seed treatment using xylosidase E175 did however result in plants with increased height with the higher volume enzyme treatment of 3 μL and resulted in an +6% increase in plant height over the water control plants compared across the three replicates in the trial.
Recombinant Bacillus cereus family members expressing fusion proteins containing enzymes (xyloglucanase D381 (SEQ ID NO: 299)) and phospholipase C E143 (SEQ ID NO: 250) were prepared as described above in Examples 48 and Table 80 and were delivered as an in-furrow treatment to the area surrounding corn seeds (Beck's corn hybrid 5828 YH). Corn seed was planted in 4×4 cm pots filled with 2:1 Timberline 20 topsoil to sand with two seeds each planted per pot. Enzyme treatments as described in Tables 85-89 were applied using a 1× (3.5 μL enzyme in 1 mL water); 2× (7.0 μL enzyme in 2 mL water); or 3× (10.5 μL enzyme in 3 mL water) and compared to wild-type control (B. thuringiensis BT013A (E0; no enzyme) applied using a 1× treatment. Two to three replicates in an experimental trial as indicated were conducted for each in-furrow trial, with of 18 replicate plants per replicate. Seed was placed in an environmentally controlled growth room. The treatments were randomly assigned to location using a complete randomized block design. All treatments were applied to the soil area directly surrounding each seed after planting but prior to covering the seed with soil. Each pot was initially watered with 50 mL of water to promote germination. After germination, plants were watered using consistent regimes for all the in-furrow treatments. Plants were grown under light levels of approximately 300 μmol m−2 s1 (light photons) and a 13-hour photoperiod (13/11 day/night cycle). Plants were maintained at 23.9° C. during the light hours and 18.3° C. during the dark hours. Plant height (an indicator of plant growth rate) was measured at approximately 10 days after planting (DAP) or at the V2 stage of development to capture early plant vigor. An average plant height per each enzyme treatment was normalized to the plants grown from seed that received a 1× application treatment with water. Effects of the various enzyme in-furrow treatments on corn growth rate or plant height (cm) are shown for the individual enzymes and enzyme combinations in Tables 85-89.
Plant height differences for the in-furrow treatments using xyloglucanase enzymes (E149 and D381) and a phospholipase C (E143) enzyme are reported in Tables 85 and 86. Volumes of 1×, 2× and 3× application treatments of the production enzymes were applied to the soil area directly surrounding each seed. Plant growth measurements, plant height (cm) were compared for the in-furrow treatments using the xyloglucanase and phospholipase C enzymes or combinations of the two enzymes applied together. Plant height differences for the enzyme treatments were compared to plants grown from seed that received treatment using the wild-type control untransformed host strain (B. thuringiensis BT013A; E0).
Xyloglucanase (D381) and phospholipase C (E143) were applied individually and in combination treatments (Table 85). In-furrow applications of xyloglucanase and phospholipase C applied as combination resulted in greater increases in early plant growth as measured by change in plant height (cm) as compared to the wild-type control (B. thuringiensis BT013A; E0); (normalized to 100%). Increasing the concentration of xyloglucanase applied in combination with phospholipase C from 1× to 3× resulted in an almost +7% increased plant height over plants that were grown from seed treated with the E0 control. Likewise, increasing the concentration of in-furrow applied phospholipase C (2× and 3×) used in combination with xyloglucanase (1×) resulted in increases in plant growth rate or plant height compared to the 1× applications using both enzymes applied separately as in-furrow treatments or plants grown from control E0 seed (shown in Table 85).
In another experiment, treatments with xyloglucanase (E149) and phospholipase C (E143) were applied as in-furrow treatments to corn and compared to the wild-type control (B. thuringiensis BT013A (E0) control (normalized to 100%). The results are depicted in Table 86. Both the xyloglucanase and phospholipase C enzyme applications when applied individually resulted in increased early plant growth rates or increased plant height as compared to plants grown from seed that received the in-furrow treatment with the E0 control. In addition, select in-furrow applications using the combined xyloglucanase and phospholipase C treatments (1× E149:1× E143; 3× E149:1× E143; 1× E149:2× E143) resulted in increased average plant growth rate over either of the enzymes treatments applied individually and compared to the plant growth for plants grown from seed that received the E0 in-furrow treatment (1×). The greatest increase in plant height resulted in corn that received the in-furrow combination treatment of xyloglucanase E149 (1×) and phospholipase C E143 (2×), a +6% increase over the plants grown from the E0 control-treated seed.
Mannanase E177 (SEQ ID NO: 308) (prepared as described in Example 48) applied as an in-furrow treatment resulted in greater early season growth as characterized by an increase in plant height at the V3 stage of development in corn. The enzymes mannanase E177 and phospholipase C E143 applied as an individual enzyme treatment (1×) or in combinations with different application treatments resulted in increased early growth rate benefits as compared to plants grown from seed treated with the E0 control. Mannanases E177 (Table 87) and E196 (Table 88) and phospholipase C were applied individually as enzyme treatments and as combination treatments using mannanase and phospholipase C in-furrow applications to corn. Mannanase E196 (also prepared as described in Example 48) applied as an in-furrow treatment resulted in an average of +5% increase in plant height when tested in a 3 replicate trial and normalized to the plants grown from seed that received the E0 control treatment. The phospholipase C in-furrow treatment also resulted in plants with increased plant height (+2.3%) over the control plants (E0 control). However, the combination in-furrow treatment of mannanase E196 applied using a high dose (3×) and phospholipase C (1×) resulted in a synergistic effect on early season growth with an increased plant height of more than +9% over the plants grown from seed that received an in-furrow treatment with the E0 control.
Phosphatidylinositol phospholipase C E258 and Phospholipase C E80 (prepared as described in Examples 48 and 49, respectively), both which are capable of hydrolyzing phospholipids in the cell membrane and phytase E246 which functions as a phosphatase to mobilize phosphorous from the soil thus creating a useable form for plants were tested in an in-furrow replicated trials. Both of the phospholipase C enzymes (E80 and E258) were provided as an in-furrow treatment using a 1× application and resulted in plants that had an approximate +6% and +7% increase in plant height when compared to plants grown from seed that received the E0 in-furrow treatment at the time of planting. In-furrow treatment with Phytase E246 (1×; 2× and 3×) tested in the same three replicates per trial as phospholipase C also resulted in substantial increases in early seedling vigor and growth as measured by changes in plant height with respective increases of approximately of +7%, +5% and +9% as compared to the in-furrow treatment using the E0 control (Table 89).
Corn (Beck's hybrid 5828 YH) seed that contained seed treatments with neonicotinoid systemic pesticides, clothianidin and imidacloprid were tested for compatibility in in-furrow trials tested using two versions of phospholipase C enzymes. Both phospholipase C (E80 and E143) enzymes have the same core phospholipase C sequence (SEQ ID NO: 250). However, phospholipase C E80 also contains an additional secretory signal (SEQ ID NO: 317). In addition, the cloning and expression of phospholipase C E143 enzyme production batch was produced as previously described in Example 48 and the phospholipase C E80 enzyme production batch was produced as described in Example 49. Early plant growth (plant height) was measured using in-furrow comparisons of the phospholipase C enzymes E80 and E143 grown from seed that was treated with PONCHO 600 (clothianidin) and GAUCHO 600 (imidacloprid), neonicotinoid insecticides prior to applying the in-furrow application treatments. Plant growth using the phospholipase C treatments were compared to plants that were grown from seed that received only the seed treatments of PONCHO 600 (clothianidin) and GAUCHO 600 (imidacloprid) insecticides (no in-furrow treatment).
Both the phospholipase C enzymes (E143 and E80) applied as in-furrow treatments provided a growth advantage to plants grown from seed that had the neonicotinoid seed treatments either PONCHO 600 (clothianidin) or GAUCHO 600 (imidacloprid). Plants grown from seed that contained either seed treatment with exhibited an increased plant growth rate or increased overall plant height as compared to plants grown from water treated seed (1×), a +4.3% (PONCHO 600) and a +7.2% (GAUCHO 600) increase compared to plants grown from seed that received a water controls. In-furrow treatment with either phospholipase C (E143) and phospholipase C (E80) provided a growth benefit on top of what was seen with the neonicotinoid seed treatments alone for both combinations with either the PONCHO 600 or GAUCHO 600 neonicotinoid seed treatments (Table 90).
In another experiment, recombinant Bacillus cereus family members expressing fusion proteins containing phospholipase C or phospholipase D enzymes were each prepared as described in Example 48 and delivered as an in-furrow treatment to the area surrounding corn seeds (Beck's corn hybrid 5828 YH) in an environment modulating field conditions. Corn seed was planted in 4×4 cm pots filled with 2:1 Timberline 23 top soil to sand with two seeds each planted per pot. Enzyme treatments as described in Tables 90-92 were applied using a 1× (3.5 μL enzyme in 1 mL water) and 2× (7.0 μL enzyme in 2 mL water) treatments as indicated in Tables 91-93 and compared to wild-type control (B. thuringiensis BT013A; E0) applied using a 1× treatment. Two to three replicates in a trial as indicated were conducted for each in-furrow treatment, with 18 plants per replicate treatment. Each treatment was randomly assigned to location using a complete randomized block design. Plants were grown on a raised patio with exposure to outside conditions full sunlight (June, 2018) with an approximate 14/10 day/night cycle. All treatments were applied to the soil area directly surrounding each seed after planting but prior to covering the seed with soil. Each pot was initially watered with 50 mL of water to promote germination. After germination, plants were watered using consistent regimes for all treatments. Plant height (an indicator of plant growth rate) was measured at approximately 10 days after planting (DAP) or the transition from V2 to V3 stage of development to determine the effect on very early emergence and seedling vigor. An average plant height per each phospholipase enzyme treatment was normalized to plants grown from seed that received an in-furrow treatment with the BT013A E0 control provided using the 1× application as described (Tables 91-93). Effects of the phospholipase C and phospholipase D enzymes applied using an in-furrow application on corn growth rate (plant height) are shown in Tables 91-93.
Phospholipase D (D409 and E229) enzymes were compared to Phospholipase C (E143) treatments applied in-furrow to corn in plant growth trials grown under outdoor conditions as described above to assess their effect on very early seedling vigor and growth (Table 91). The phospholipase in-furrow treatments were normalized to plants grown from seed that received an in-furrow treatment with the E0 control (1×). Phospholipase D (D409 and E229) applied as in-furrow treatments to corn seed directly after planting resulted in increased growth rates seen in both the 1× and 2× in-furrow treatment applications. The 1× in-furrow treatment with both Phospholipase D D409 and phospholipase D E229 resulted in slightly increased plant height as compared to the 2× treatments with either enzyme. Both the 1× phospholipase D in-furrow treatments resulted in slight increases in corn plant height over the in-furrow treatment with phospholipase C E143 which showed a +5% increase as compared to plants grown from seed that received the E0 control in-furrow treatment.
Phospholipase C enzymes from Listeria monocytogenes (Lm. D473, D474 and D475) were compared to a phospholipase C from Bacillus thuringiensis (E80) that has been shown to have plant growth promoting effects. In-furrow treatments were applied to corn seed that was planted in two outdoor trials as previously described using different applications at a 1× and 2× concentration. The phospholipase C enzymes all contain the same core sequence (SEQ ID NO: 374) but differ with respect to the secretory signal and propeptide sequences. D473 contains the secretory signal (SEQ ID NO: 376) directly linked to SEQ ID NO: 373 (which contains a propeptide sequence). D474 is just SEQ ID NO: 373 without the secretory signal. D475 (SEQ ID NO: 374) lacks both the signal secretory sequence and the propeptide sequence.
Phospholipase C (E80) used as a 1× in-furrow treatment on corn seed resulted in plants that had a +12% increase in plant height over the plants grown from seed treated with the E0 control. In-furrow treatments using Lm phospholipase C (D473, D474 and D475) applied using the 1× or 2× concentrations all resulted in plants with increased plant height over the plants grown from seed that received the E0 control treatment. These in-furrow treatments with Lm phospholipase C (D473, D474 and D475) also resulted in plants with slight increased plant height compared to the in-furrow treatment using phospholipase C E80. All of the 1× in-furrow treatments with Lm phospholipase (D473, D474 and D475) resulted in plants with increased plant height compared to the 2× in-furrow treatments with the same phospholipase C enzymes. The 1× in-furrow treatment using Lm phospholipase C D475 enzyme resulted in corn plants with increased plant height, a more than +20% increase compared to plants grown from seed that received the in-furrow treatment using the E0 control.
Recombinant Bacillus cereus family members expressing fusion proteins containing pectolyase enzymes (D405 and D176), prepared as described in Example 48, were applied in-furrow directly after planting the seeds resulted in substantial increases in plant height and compared to plants grown from seed that received in-furrow treatments using the E0 control. Pectolyases (endopolygalacturonase (D405) and pectin lyase (E176)) applied as in-furrow treatments at 1× and 2× concentrations resulted in similar increases in average plant height averaged over the three trials, an average of +12% (D405) and +8% (E176) as compared to plants grown from seed that received the in-furrow E0 control treatment. Phospholipase C from Bacillus thuringiensis (E80) that has been shown to have plant growth promoting effects also resulted in positive increases in plant height, a +5% increase as compared to the E0 control plants (Table 93).
Fractional green canopy cover (FGCC) is a diagnostic analysis tool (Canopeo) used to estimate green canopy area. Canopeo (Matlab, Mathworks, Inc., Natick, MA) provides a measurement tool based on the selection criteria for green canopy area that is determined using color ratios of red to green (R/G) and blue to green (B/G) which compensates for excess green index or background. FGCC cover ranges from 0 (0% green canopy cover) to 1 (100% green canopy cover). The classification of green canopy is based on the following criteria:
R/G<P1 and B/G<P2 and 2G−R−B>P3
Where P1 and P2 are parameters that have a value near 1 to classify the pixels that are in the green wavelength band range (500-570 nm), and P3 is a parameter that sets the minimum excess green vegetation and allows for background subtraction. The default parameter values for Canopeo are P1=0.95, P2=0.95, and P3=20.
Soybean plants (variety 424L4) were grown from seed that received seed treatments with enzymes: xyloglucanase, mannanase, β-1,4-endoglucanase, xylanase, phospholipase, pectolyase and lichenase. Recombinant Bacillus cereus family members expressing fusion proteins containing various enzymes were prepared as described above in Example 48 (xyloglucanase, mannanase, xylanase, pectolyase, lichenase, and phospholipase) or Example 47 (endoglucanase), and were delivered as a seed treatment onto soybean seeds using the volumes (μL) applied to the outer coats of each seed as a seed treatment and indicated in Table 94-97. A wild-type control (B. thuringiensis; E0 no-enzyme) control was also applied to seed using a volume of 1 μL per seed and used as a control comparison for the enzyme treatments Soybean seed that received the seed treatments with the enzymes as described and the E0 control treatments were allowed to dry and then planted in 4×4 cm pots seeded into a mixture of 2:1 Timberline Top soil to sand with two seeds each planted per pot and initially watered with 50 mL of water to promote germination. Each enzyme treatment and a water control treatment (no seed treatment) were seeded into 3 trays that represented separate replicates per enzyme treatment and grown in an outdoor facility. Within each experiment, the same number replicate trays and total plants per treatment was held consistent between treatments. Each tray supported 18 plants per treatment. The trays were randomized between the treatments. Plants grown in an outdoor facility were exposed to similar environmental parameters that simulated a field planting (May, 2018). The soybean plants were grown for approximately 12 to 14 days until the unifoliate leaf stage or for 20 day until the trifoliate leaf stage. At both of these stages the leaves were fully expanded. FGCC was determined using the Canopeo measurement tool and the images were collected 50 cm above the bottom of the plant trays using a selection parameter of 0.91 resolution. Twenty representative images were collected per each treatment and analyzed to classify all the pixels in each image. A representative pixel classification assigned for the 20 images per treatment per tray. FGCC was then calculated for the soybean plants in each tray or treatment replicate and then further calculated on a per plant basis for each tray (trial). Results are shown for the different enzyme seed treatments in Tables 94-98. Differences in FGCC were reported as a percentage of green canopy and normalized to soybean plants grown from seed that only received a water control treatment in Tables 94-96. Differences in FGCC were reported as a percentage of green canopy and normalized to soybean plants grown from seed that received the E0 treatment control in Tables 97-98.
Xyloglucanase and Mannanase
The wild-type control (B. thuringiensis BT013A E0, no-enzyme control) resulted in plants with increased green canopy area normalized to the soybean plants grown from seeds that received the water only seed treatment. Xyloglucanase (SEQ ID NO: 299) and mannanase (SEQ ID NO: 307) provided as seed treatments both increased green canopy area in soybean measured at 14 DAP (unifoliate leaf stage). The amount of the seed treatment application (1 μL to 3 μL per seed) varied as to the effect on FGCC. All of the xyloglucanase D381 (SEQ ID NO: 299) provided as seed treatments applied at 1 μL, 2 μL or 3 μL per seed resulted in positive outcomes of an increase in green canopy area (FGCC) with a respective +18.1% (1 μL), +23.3% (2 μL) and +14.7% (3 μL) increase in FGCC over the plants grown from seed that received the water control (Table 94). Mannanase E196 (SEQ ID NO: 307) provided as a soybean seed treatment also showed increased FGCC but only with the 1 μL and 3 μL application doses, respective increases of +17.4% and +12.4% compared from plants grown from seed that received the water control (Table 94).
Pectolyase and Lichenase
Pectolyase enzymes comprising pectin lyase E176 (SEQ ID NO: 310) and endopolygalacturonase D405 (signal peptide of SEQ ID NO: 346 directly linked to SEQ ID NO: 309) were assessed as seed treatments in the same trials using 3 replicate trays per trial for effects on FGCC at the trifoliate leaf stage (20 DAP). Lichenase D436 (SEQ ID NO: 296) was also assessed as a soybean seed treatment for effect on FGCC at the trifoliate leaf stage (20 DAP). Each of the 3 trays per enzyme and E0 control treatments contained 18 plants with the entire tray consisting of one enzyme treatment. Calculations for green canopy area or FGCC was normalized to plants grown from water control treated seed. Seed treatment enzymes (pectin lyase E176, endopolygalacturonase D405 and lichenase) used seed treatment volumes which are described in Table 95 below. The E0 base control was applied using a 1 μL volume per seed.
Pectin lyase E176 (SEQ ID NO: 310) applied as a seed treatment using 1-3 μL volumes per seed resulted in plants that had the highest values for FGCC when compared to pants grown from seed that received a water control (Table 95). The highest FGCC values were obtained for plants that received the 3 μLand a +29% (trifoliate) average increase for FGCC over the 3 replicate trays (normalized to green canopy area for plants grown from seed treated with water alone). The endopolygalaturonase D405 seed treatment also resulted in plants with increased FGCC, which was higher in the 2 μL and 3 μL volumes of the production batch applied to the seed. The trifoliate leaves of soybean plants grown from seed treated with 2 μL of endopolygalacturonase D405 per seed had substantial increased FGCC, an almost +73% increase in plants grown to the trifoliate leaf stage (20 DAP) as compared or normalized to the same age plants grown from the water control treated seed. Lichenase D436 was applied using lower volumes as a seed treatment to soybean (0.25 μL, 0.5 μLand 1 μL volumes). The lower application of lichenase D436 (0.25 L) resulted in a greater increase in FGCC in plants at the trifoliate (20 DAP) leaf stage. An increase of FGCC of approximately +14% (trifoliate) is shown as the average of for the 3 trays and compared to plants from the water control treatment.
Phospholipase C
Phospholipase C (PLC) originally derived from Listeria monocytogenes was cloned using different versions with a secretory sequence or a propeptide or both attached to the core PLC sequence to examine if these accessory sequences contributed to increases in FGCC in soybean plants that received the seed treatments using PLC. Lm Phospholipase D473, D474, and D475 were described in Example 51, above. Each of the Lm. PLC treatments were applied using 2 μL of production enzyme which was applied directly to each soybean seed prior to planting and compared to the FGCC of plants grown from seed that received water (1 μL per seed). The FGCC values for the Lm. PLC D475 that had just the core PLC sequence (2 μL) resulted in a higher average FGCC, a +22.3% increase as measured for the trifoliate leaves as compared to the Lm. PLC D473 or Lm. PLC D473 (2 μL) treatments. However, all three Lm. PLC (D473, D474 and D475) all had higher increased green canopy area or FGCC at the trifoliate leaf stage when normalized to plants at the same stage that were grown from the water control treated seed.
Listeria monocytogenes
Listeria monocytogenes
Listeria monocytogenes
Xylanase and Endoglucanase
Xylanase E100 (signal sequence of SEQ ID NO: 328 directly linked to SEQ ID NO: 268) and xylanase E232 (SEQ ID NO: 273) were compared to each other as seed treatments to effect changes in green canopy area in soybean plants grown to the unifoliate leaf stage (14 DAP). Xylanase treatments were also compared to the plants grown from soybean seed treated with the β-1,4-endoglucanase E94 (signal sequence of SEQ ID NO: 334 directly linked to SEQ ID NO: 293) enzyme. FGCC values for all the enzyme treated seed are reported as normalized to plants grown from B. thuringiensis BT013A; E0, no-enzyme control) in Table 97. Soybean plants had comparable FGCC values from plants grown from seed that received a seed treatment with xylanase E100 provided at 1 μL and β-1,4-endoglucanase E94. Both resulted in plants that had approximately over a +4% increase in green canopy area when normalized to plants grown from seed that received the E0 control. Xylanase E232 used as a seed treatment also provided using a 1 μL volumes of the production batch enzyme per seed resulted in an almost +7% increase in FGCC values in plants (14 DAP) when compared to plants grown from the E0 control treated seed.
Other Phospholipases
Phospholipase D D409 (SEQ ID NO: 256) and phospholipase E229 (SEQ ID NO: 260) were compared to phospholipase C E143 (SEQ ID NO: 250) after plants grown from the enzyme treated seed was normalized to the B. thuringiensis BT013A (E0 control). The two phospholipase D enzymes applied as seed treatments using either a 1 μL or a 2 μL volume of the production batches per seed differed with respect to the FGCC values compared to plants grown from seeds that received the E0 control treatment as shown in Table 97. All of the plants grown from seed that received the phospholipase D (D409 and E229) seed treatments resulted in an average overall increased FGCC as compared to plants from seeds that received the phospholipase C (E143) treatment applied at the same volume (1 μL). An increase in FGCC of +5% for phospholipase D D409 and a +19% for phospholipase D E229 over the plants that were grown from seed that received the seed treatment with phospholipase C E143. However, all of the phospholipase seed treatments produced plants with higher FGCC values as compared to or normalized to plants grown from B. thuringiensis BT013A; E0, no-enzyme control) treated seed.
Replicated field trials were conducted using two soybean varieties, (Beck's 297R4 and 312R4) at U.S. Midwest locations. The soybean varieties were grown in three separate locations, variety Beck's 297R4 (OH, WI & IA) and Beck's 312R4 (IN, IL, IA) for yield trials conducted using lichenase and mannanase enzymes and in 3 separate locations in Illinois for Beck's 297R4 and Beck's 38X52N for yield trials conducted using ACC deaminase and lactonase enzymes. Seed from soybean varieties contained a base seed treatment containing EverGol Energy (65 mL per 100 kg of seed), GAUCHO 600 (0.12 mg active ingredient/per seed) and ILeVO (0.15 mg active ingredient fluopyram (48.4%) per seed). Recombinant Bacillus cereus family members (prepared as described above) were applied as seed treatments using the fluid ounce equivalent use rates as described in Tables 99-102 and applied on top of the base seed treatment. Enzymes applied as seed treatments were allowed to dry on the outer surface of the seed prior to planting.
Soybean seed was planted at 1.5 to 2 inches (3.8 to 5.1 cm) depth to assure normal root development. Soybean seed was planted at approximately 150,000 plants per acre with row widths of 30-inch (76.2 cm) rows with seed spacing of approximately 7 to 8 seeds per foot (0.3 meter). Field seed beds at each location were prepared using conventional or conservation tillage methods for the plantings. Fertilizer was applied as recommended by conventional farming practices and remained consistent between the US Midwest locations. Herbicides were applied for weed control and supplemented with cultivation when necessary.
Yield (Bu/Ac) from soybean plants that were grown from seed that received seed treatments with lichenase (E111) was compared to plants grown from seed that received the base seed treatment (control; no enzyme). Average yield and the change in yield over the control plants is reported individually for the two soybean varieties for the 3 locations harvested in Table 99.
Soybean grown from seeds that received lichenase as a seed treatment had greater yields as compared to soybean plants that were grown from seed that received the base seed treatment (control) for both Beck's 297R4 and Beck's 312R4 varieties. The average yield (Bu/Ac) increases for the 3 locations differed between varieties, yet both had positive increases in yield as compared to the yield from the control plants. Beck's variety 297R4 had on average a +3.34 Bu/Ac (224.6 kg/Ha) increase while Beck's 312R4 had a +1.45 Bu/Ac (97.5 kg/Ha) increase as compared to yield harvested from the control plants.
In another replicated field trial, yield (Bu/Ac) was collected from soybean plants that were grown from seed that received seed treatments with mannanase (E177) and compared to plants grown from seed that received the base seed treatment (control; no enzyme). Average yield and the change in yield over the control plants is reported in Table 100 for the 3 locations harvested.
Soybean (Beck's variety 297R4) that received the seed treatment with mannanase (E177) enzyme had increased yield as compared to soybean plants grown from seed that received only the base seed treatment control. Soybean plants grown from seed treated with mannanase had an average yield increase of +2.11 Bu/Ac (142 kg/Ha) across the 3 locations as compared to yield from the control plants.
Yield (Bu/Ac) from soybean plants that were grown from seed that received seed treatments with ACC deaminase was compared to plants grown from seed that received the base seed treatment (control; no enzyme). Average yield and change in yield over the control plants is reported in Table 101 individually for the two soybean varieties (Beck's 297R4 and Beck's 38X52N) for the 3 locations harvested.
Soybean that received the seed treatment with the ACC deaminase (D406) enzyme had an increased yield as compared to soybean plants grown from seed that received only the base seed treatment control. Soybean plants (Beck's variety 2974R4) grown from seed treated with ACC deaminase had an average yield increase of +0.91 Bu/Ac (61.2 kg/Ha) whereas (Beck's variety 38X52N) had an average yield increase of +0.71 Bu/Ac (47.7 kg/Ha) across the 3 locations as compared to yield from the control plants.
In another replicated field yield trial conducted at 3 U.S. Midwest locations (IL), yield (Bu/Ac) is reported from soybean plants that were grown from seed that received seed treatments with lactonase (H51) enzyme and compared to plants grown from seed that received the base seed treatment (control; no enzyme). Average yield and change in yield over the control plants is reported in Table 102 individually for the two soybean varieties (Beck's 297R4 and Beck's 38X52N) harvested.
Yield increase in soybean plants grown from seed that received a treatment with the lactonase (H51) enzyme differed between the 2 soybean varieties (Beck's 297R4 and Beck's 38X52) grown in the 3 locations (IL). Soybean plants (Beck's 38X52N) had a +3.79 Bu/Ac (254.9 kg/Ha) yield increase over soybean grown from seed that received the base seed treatment (no enzyme). Soybean plants (Beck's 297R4) had a slight +0.80 Bu/Ac (53.8 kg/Ha) yield increase over the control plants.
Enzymes, expressed on the exosporium of recombinant Bacillus cereus family members as described herein, that are useful as feed additives were characterized for activity using an in vitro test system that simulates gastrointestinal conditions to assess the accessibility of these enzymes as they enter and pass through the digestive juices found in the gut and the gastrointestinal track of a mammal, such as a bovine species. The mammalian model was adapted from Kaufman et. al., 2007 (Kaufman et al., “Lead bioaccessibility in food web intermediates and the influence on ecological risk characterization”, Environmental Science and Technology 2007; 41: 5902-5907).
A mammalian physiologically based extraction test (PBET) was conducted using the digestive and intestinal juices acquired from bovine and sampled using separate times including a time zero (T0) sampling to simulate the enzyme activity state during the consumption of feed. Two stages of the digestion process during bovine digestion were carried out using known amounts of each of the enzymes generated using batch production fermentation methods as previously described (Table 80) to achieve the desired activity levels. The first stage (Stage 1) digestion process used a 2-step collection process to simulate the gastric juices from a recently fed animal (T0) and juices during the digestion process “steady state” (T1). The second stage (Stage 2) digestion process used a 2-step collection process to simulate passage through the intestine and monitoring activity of the feed enzymes at two-time points; slow intestinal passage (T3) and just prior to the end of intestinal passage (T4).
For Stage 1: The PBET assay was conducted using a gastric digestive juice containing: pepsin (1.25 g/L), malic acid (0.5 g/L), citric acid (0.5 g/L), lactic acid (420 μL/L), acetic acid (500 μL/L) in deionized water and adjusted to pH 4.0 to simulate a just fed animal and pH 2.5 to simulate steady state conditions during food digestion. The initial (Stage 1) PBET reactions used 4 mL of the gastric digestive juice combined with the “feed additive” enzyme in to a 15 mL centrifuge tube placed at 37° C. for 10 min. After the 10 min. incubation time, the tubes were kept at 37° C. but shaken at 250 rpm at a 45° C. angle for 1 hour. Time points were taken during the reaction to simulate the consumption of food in the stomach. Multiple samples at each time points were collected during the hour incubation T0 (just fed); and T1 (digestion started reaching steady state levels) time intervals. At each collection point, the shaker was stopped an aliquot of 100 μL of gastric juice containing the “feed additive” enzyme was collected from each tube. The gastric juice samples were then transferred separately to a 1.5 mL sealed microcentrifuge tube and immediately centrifuged for 10 min. at 11,000×g. The supernatant from each sample was decanted into a fresh tube and the supernatant fraction was collected and stored at −20° C. until the time for the initiation of the enzyme-specific activity assays.
Once the Stage 1 digestive stimulation was completed, the digestive juice in each simulation reaction was adjusted to pH 7.0 using 300 μL of NaHCO3 solution. For Stage 2: After the pH had been adjusted, a 10× concentrated solution that simulated a digestive juice found in the bovine intestine was added to the reaction mixtures of each sample which contained bile extract (26 mg/mL) and pancreatin (7.5 mg/mL to achieve the desired 1× final concentrations (bile extract=2.6 mg/mL and pancreatin=0.75 mg/mL). This intestinal solution was added to each replicate sample prior to returning the sample tubes to the shaker at 37° C., 45° angle and shaking at 100 to 150 rpm to mimic the slow intestinal passage of the “feed additive” enzymes. A 100 μL aliquot of the intestinal solution was taken from each tube after a 1-hour incubation interval for the T3 time interval (slow intestinal passage). Tubes were then returned to the shaker and incubated for a total time of 3.5 hours (almost complete passage through the intestine: end of the intestinal simulation) collected for the T4 time interval. The samples were then transferred to a 1.5 mL sealed microcentrifuge tube an immediately centrifuged for 10 min. at 11,000×g. The supernatant was decanted into a fresh tube and the supernatant was stored at −20° C. until further assayed using enzyme-specific assays.
Enzymes selected for use as feed additives were compared to commercially available highly purified (free) enzymes with known activity levels; the positive controls (free enzyme) from each enzyme class as described in Table 103. The “feed additive” enzymes and the commercial enzymes were assayed with the digestive enzymes to confirm that the enzyme-specific assays were functional and could be used as comparisons for the “feed additive” enzymes to ensure that they maintained a functional activity as they passed through the digestive track of mammal, such as a bovine mammal.
Paenibacillus sp.
Bacillus subtilis
Bacillus sp.
Neocallimastix patriciarum
Acidothermus cellulolyticus
Enzyme-specific enzyme activity assays were conducted on each 100 μL aliquot as collected for the stage 1 (digestion through the stomach; T0 and T1) and stage 2 (movement through the intestine; T3 and T4) digestive assays. A representative standard curve for enzymes in Table 104 was obtained using glucose as a cleavable and detectable substrate and resulted in the following equation: y=12.511x3−398.76x2+448.51x+40.714 (R2=0.9994). The R2 value of 0.9994 for the standard curve was highly positively correlated to the individual enzyme specific activities and was used to determine the starting and ending activities for each of the feed additive enzymes in Stages 1 and 2 of the digestion assays.
Lichenase E111, mannanases E177 and E196, xylanases E232, E146, and E100, and xyloglucanases E149 and D381 (all prepared as described above in Example 48) were tested as feed additive enzymes and were compared to the background activity of the E0 control (Bacillus thuringiensis BT013A, the same strain as used to express the recombinant enzymes). In addition, the effectiveness of mannanase as a free enzyme to act as a feed additive and remain active during digestion was tested. Mannanase D448 and D449 were prepared as free enzymes using the pFEe4B vector (which contains ampicillin and tetracycline resistance genes) in Bacillus subtilis. The pFe4B vector carries segments enabling replication and selection in Bacillus and E. coli. The E. coli section (pUC57) contains the ColE1 origin of replication and bla, while the Bacillus section (pBC16) harbors repU, oriU and tetL. In this study, the pBC section was further augmented with expression cassettes for mannanase (D448 or D449) and lacI. The lacI cassette is driven by a Bacillus paralicheniformis penicillase promoter and results in constitutive expression of E. coli Lad needed to suppress expression of the cargo of interest prior to induction. The expression cassette for the mannanases contained a modified Bacillus subtilis groE operon promoter fused to the binding site for Lad. The mannanase was further fused to the amyQ secretion signal of Bacillus amyloliquefaciens to ensure its secretion outside of the producing cell. pFEe4B derivatives carrying mannanase were transformed into Bacillus subtilis and expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). The whole cell broth was filtered and the supernatant (filtrate) used as the final product in this example. Note that whole cell broth, centrifugates, or filtrates can be used as described herein.
All four enzyme types produced in the BT013A strain with a BclA20-35 targeting sequence (amino acids 20-35 of SEQ ID NO: 1) had activity at the start of the digestive simulation and were able to maintain some activity throughout the simulation (Table 104). This suggests that throughout the process the animal would have access to more freed carbohydrates and sugars to convert into energy. The free enzymes (D448 and D449), however, showed a drop in activity over time during the assay. Thus, this data demonstrate the stability of the sporulated enzymes in this assay, supporting their ability to stay with the feed throughout the process and not be readily absorbed across the epithelium, which would reduce their ability to continue to digest target substrates throughout the stomach and intestines as the conditions become more favorable to enzyme activity.
Enzyme-specific enzyme activity assays for endoglucanases were conducted on each 100 μL aliquot as collected for the stage 1 (digestion through the stomach; T0 and T1) and stage 2 (movement through the intestine; T3 and T4) digestive assays. A representative standard curve for enzymes in Table 105 was obtained using glucose as a cleavable and detectable substrate and resulted in the following equation: y=152.57x3−266.97x2+426.35x+42.75 (R2=0.9994). The R2 value of 0.9994 for the standard curve was highly positively correlated to the individual enzyme specific activities and was used to determine the starting and ending activities for each of the feed additive enzymes in Stages 1 and 2 of the digestion assays. Data for endoglucanases are summarized in Table 105.
(8.2%)
(8.0%)
The activities of the “feed additive” enzymes as described in Table 105 with differing targeting sequences and targeting sequence lengths were compared to the background activity of the E0 no enzyme control or the untransformed host strain or the (Bacillus thuringiensis BT013A) that was the same strain as used to express the recombinant enzymes. The activities of the “feed additive” enzymes were also compared to the commercially available Endoglucanase (endo-1,4-β-D-glucanase) from Acidothermus cellulolyticus and described in Table 105. The starting level of endoglucanase activity was highest for the short BclA 20-35 targeting sequence (E94) followed by the BclA targeting sequence lacking the globular domain (E107) and lowest for the full length BclA targeting sequence (E108). Endoglucanase activities were higher for each of the BclA-based targeting sequences than that of CotY. Interestingly, the BclA 1-166 targeting sequence with no globular domain lost less activity as it went through the digestive system than the shorter more active targeting sequence and in fact the endoglucanase activity was enhanced in the low pH high proteolytic environment suggesting there is a specific improvement for feed to have a targeting sequence that will allow more protection from the acidic environment of the gut allowed by the longer BclA targeting sequence or CotY.
In other assays, another model can be used to characterize the activity of the present “feed additive” enzymes of the present invention using an avian digestive track model that generally provides a gizzard digestion assay suitable for poultry industry. Similar to the mammalian PBET assay, “feed additive” enzymes can be assessed using an Avian PBET assay which provides an intestinal simulation model to capture the complexity of the avian digestive track using a dynamic avian gizzard-intestine system containing grit (Martinez-Haro et al., “Avian digestive tract simulation to study the effect of grit geochemistry and food on Pb shot bioaccessibility”, Environmental Science and Technology 2009; 43: 9480-9486).
In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.
As various changes could be made in the above products, formulations, and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
For further illustration, additional non-limiting embodiments of the present disclosure are set forth below.
The present application is a U.S. National Stage Application of PCT Application No. PCT/US2018/051995, filed Sep. 20, 2018, which claims the benefit of U.S. Provisional Application Ser. No. 62/560,876, filed Sep. 20, 2017. Each of the above-cited applications is incorporated herein by reference in its entirety.
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| PCT/US2018/051995 | 9/20/2018 | WO |
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|---|---|---|---|
| WO2019/060574 | 3/28/2019 | WO | A |
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| Number | Date | Country | |
|---|---|---|---|
| 20200216828 A1 | Jul 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62560876 | Sep 2017 | US |
