Patent Application
20240384284
This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831 and PCT Rule 13ter. The Sequence Listing XML file submitted in the USPTO Patent Center, “888689-0002-US02_sequence_listing_xml_14 May 2024.xml,” was created on May 14, 2024, contains 95 sequences, has a file size of 188 kilobytes (192,512 bytes), and is incorporated by reference in its entirety into the specification.
Ruminant animals, including cattle and sheep, have a unique digestive system involving a multichambered stomach where microbes perform essential steps in digestion via a process known as enteric fermentation. The animal provides its ruminal microbes with a specialized environment and a constant supply of plant matter, from which the microbes convert structural carbohydrates such as cellulose to simple sugars that the animal can use for energy. This symbiosis makes ruminants an important part of the agricultural system due to their unique ability to digest food sources that most other animals cannot. However, enteric fermentation also results in production of methane (CH4), a greenhouse gas (GHG), many times more powerful than CO2. Ruminant enteric fermentation is the largest single source of human-generated methane, making up about 5% of worldwide GHG emissions in recent years (GWP100). This is larger than the effect of worldwide deforestation, aviation, and shipping combined.
Leading scientists and conservation organizations have called for urgent focus on methane as a means to achieve immediate results in the transition to climate sustainability, and 150 countries have signed a pledge to reduce their methane emissions 30% by 2030. Elimination of enteric methane supports such worldwide efforts and offers large climate benefits, including a reduction in the GHG footprint of beef, dairy, and sheep products by 46-65%, and avoidance of up to 0.3° C. of warming within the next 20 years.
Production of methane is not an intrinsic requirement of enteric fermentation or ruminant agriculture, and the productivity of livestock is unchanged or even improved when methane emission is reduced. The proximate source of methane is a relatively small contingent of methanogenic microbe species in the rumen microbiome that use the Wolfe cycle to reduce CO2 to methane across a multistep process that produces necessary precursors for DNA and protein and drives the flow of cellular energy via ferredoxin. In the second-to-last step of this cycle, methyl-coenzyme M reductase (MCR) reduces methyl-coenzyme M (CH3—S-COM) with coenzyme B-thiol (CoB—SH) to the disulfide COM-S—S—CoB, while releasing CH4 as a byproduct, which subsequently makes its way into the atmosphere. When ruminants' feed is supplemented with a compound that blocks the activity of MCR or precursor enzymes, methane production drops dramatically, as does the population of methanogens in the rumen.
Chemicals exist that can impede methanogenesis in vivo with varying mechanisms and efficiency. 3-nitrooxypropanol is a commercially available feed supplement that inactivates MCR by oxidation of its active site. Bromoform (CHBr3) competitively inhibits methanogenesis by binding to both MCR and the enzyme that provides its substrate in the prior step of the Wolfe cycle, coenzyme M methyltransferase (CMM). Bromoform is one of the most effective antimethanogens characterized to date, effecting in vivo methane reduction of up to 98%. Bromoform and related haloalkanes occur naturally in marine organisms, most prominently the red macroalgae genus Asparagopsis which is farmed expressly for use as a bromoform-rich feed supplement to reduce enteric methane emissions from livestock.
Harnessing bromoform to reduce methane in form of the pure chemical or seaweed additives presents several challenges that impose additional costs and logistical requirements on farmers and the broader supply chain. Pure bromoform is expensive to synthesize, hard to formulate for delivery on farms, and presents health and environmental concerns when present as a concentrated point source. If relying on native biosynthesis in Asparagopsis or similar species, significant areas of ocean would need to be converted to aquaculture, or resource-intensive facilities built on land, to produce sufficient amounts of bromoform-bearing material to meaningfully reduce enteric methane emissions. This results in environmental impacts that infringe the benefits of any emission reduction achieved. Bromoform also volatilizes from seaweeds to the atmosphere, where it contributes to ionic degradation of ozone, and a lack of containment of this flux during the pre-harvest growth period in large-scale aquaculture is of environmental concern. Bromoform in the form of chemical additives or seaweed must also be transported long distances from production sites and specially formulated and packaged to manage volatilization of bromoform in storage, bioavailability, and practical use, adding further environmental costs and barriers to adoption.
A need exists for a system of integrating bromoform and similar molecules into animal feed without the barrier imposed by producing and delivering this chemical separately from the primary feed supply. There is also a need for the system to avoid the high land and resource demands associated with existing means of bromoform production via seaweed aquaculture. There is a need for the system to limit loss of bromoform from harvested plant material. Finally, there is a need for the system to contain or prevent bromoform leakage during the pre-harvest growth period and from unharvested material.
One embodiment described herein is a genetically modified feed crop for reducing methanogenesis in a livestock animal, the genetically modified feed crop comprising: a transgenic polynucleotide construct comprising a haloperoxidase (HPO) gene sequence encoding a HPO protein, wherein the HPO protein enables biosynthesis of bromoform (CHBr3), chloroform (CHCl3), iodoform (CHI3), or a combination thereof in the genetically modified feed crop. In another aspect, the HPO gene sequence encoding a HPO protein is a vanadium-dependent bromoperoxidase (VBPO), a vanadium-dependent iodoperoxidase (VIPO), vanadium-dependent chloroperoxidase (VCPO), or a nonspecific vanadium-dependent haloperoxidase (VHPO). In another aspect, the genetically modified feed crop is a monocot plant or a dicot plant. In another aspect, the HPO gene sequence has at least 90-99% identity to any one of SEQ ID NO: 1, 3, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91. In another aspect, the HPO gene sequence is selected from any one of SEQ ID NO: 1, 3, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91. In another aspect, the HPO protein comprises an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 2, 4-8, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92. In another aspect, the HPO protein comprises an amino acid sequence selected from any one of SEQ ID NO: 2, 4-8, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92. In another aspect, the HPO gene sequence comprises a whole-plant constitutive promoter and the HPO gene sequence is expressed under the whole-plant constitutive promoter. In another aspect, the whole-plant constitutive promoter comprises a polynucleotide sequence having at least 90-99% identity to any one of SEQ ID NO: 19-20, or 93. In another aspect, the whole-plant constitutive promoter comprises a polynucleotide sequence selected from any one of SEQ ID NO: 19-20, or 93. In another aspect, the HPO gene sequence comprises an endosperm-specific promoter or photosynthetic-tissue promoter and is expressed under the endosperm-specific promoter or photosynthetic-tissue promoter. In another aspect, the endosperm-specific promoter comprises a polynucleotide sequence having at least 90-99% identity to any one of SEQ ID NO: 24-25, or 94; and the photosynthetic-tissue promoter comprises a polynucleotide sequence having at least 90-99% identity to any one of SEQ ID NO: 21-23. In another aspect, the endosperm-specific promoter comprises a polynucleotide sequence selected from any one of SEQ ID NO: 24-25, or 94; and the photosynthetic-tissue promoter comprises a polynucleotide sequence selected from any one of SEQ ID NO: 21-23. The genetically modified feed crop of claim 1, further comprising: a second transgenic polynucleotide construct comprising: an acetoacetyl-CoA thiolase 2 (AACT2) gene sequence encoding an AACT2 protein. In another aspect, the AACT2 gene sequence has at least 90-99% identity to any one of SEQ ID NO: 9-10 or 12-13. In another aspect, the AACT2 gene sequence is selected from any one of SEQ ID NO: 9-10 or 12-13. In another aspect, the AACT2 protein comprises an amino acid sequence having at least 90-99% identity to SEQ ID NO: 11. In another aspect, the AACT2 protein comprises an amino acid sequence selected from SEQ ID NO: 11. In another aspect, the AACT2 gene sequence comprises a whole-plant constitutive promoter and the AACT2 gene sequence is expressed under the whole-plant constitutive promoter. In another aspect, the whole-plant constitutive promoter comprises a polynucleotide sequence having at least 90-99% identity to any one of SEQ ID NO: 19-20, or 93. In another aspect, the whole-plant constitutive promoter comprises a polynucleotide sequence selected from any one of SEQ ID NO: 19-20, or 93. In another aspect, the HPO gene sequence and AACT2 gene sequence are separated by a self-cleaving peptide gene sequence encoding for a self-cleaving peptide. In another aspect, the self-cleaving peptide gene sequence has at least 90-99% identity to any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the self-cleaving peptide gene sequence is selected from any one of SEQ ID NO: 29, 31, 33, 35, 37, or 39. In another aspect, the self-cleaving peptide comprises an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the self-cleaving peptide comprises an amino acid sequence selected from any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the HPO is a Vanadium-dependent bromoperoxidase (VBPO) gene sequence having at least 90-99% identity to any one of SEQ. ID NO: 1 or 3. In another aspect, the HPO is a Vanadium-dependent bromoperoxidase (VBPO) gene sequence selected from any one of SEQ ID NO: 1 or 3. In another aspect, the HPO is a Vanadium-dependent bromoperoxidase (VBPO) protein comprising an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 2 or 4-8. In another aspect, the HPO is a Vanadium-dependent bromoperoxidase (VBPO) protein comprising an amino acid sequence selected from any one of SEQ ID NO: 2 or 4-8. In another aspect, the HPO is a Vanadium-dependent iodoperoxidase (VIPO) gene sequence having at least 90-99% identity to any one of SEQ. ID NO: 41, 43, 45, 47, 49 or 51-60. In another aspect, the HPO is a Vanadium-dependent iodoperoxidase (VIPO) gene sequence selected from any one of SEQ ID NO: 41, 43, 45, 47, 49 or 51-60. In another aspect, the HPO is a Vanadium-dependent iodoperoxidase (VIPO) protein comprising an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 42, 44, 46, 48, or 50. In another aspect, the HPO is a Vanadium-dependent iodoperoxidase (VIPO) protein comprising an amino acid sequence selected from any one of SEQ ID NO: 42, 44, 46, 48, or 50. In another aspect, the HPO is a Vanadium-dependent chloroperoxidase (VCPO) gene sequence having at least 90-99% identity to any one of SEQ. ID NO: 61, 63, 65, 67, 69, 71, 73, or 75. In another aspect, the HPO is a Vanadium-dependent chloroperoxidase (VCPO) gene sequence selected from any one of SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, or 75. In another aspect, the HPO is a Vanadium-dependent chloroperoxidase (VCPO) protein comprising an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 62, 64, 66, 68, 70, 72, 74, or 76. In another aspect, the HPO is a Vanadium-dependent chloroperoxidase (VCPO) protein comprising an amino acid sequence selected from any one of SEQ ID NO: 62, 64, 66, 68, 70, 72, 74, or 76. In another aspect, the HPO is a nonspecific vanadium-dependent haloperoxidase (VHPO) gene sequence having at least 90-99% identity to any one of SEQ. ID NO: 77, 79, 81, 83, 85, 87, 89, or 91. In another aspect, the HPO is a nonspecific vanadium-dependent haloperoxidase (VHPO) gene sequence selected from any one of SEQ ID NO: 77, 79, 81, 83, 85, 87, 89, or 91. In another aspect, the HPO is a nonspecific vanadium-dependent haloperoxidase (VHPO) protein comprising an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, or 92. In another aspect, the HPO is a nonspecific vanadium-dependent haloperoxidase (VHPO) protein comprising an amino acid sequence selected from any one of SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, or 92.
In another aspect, the genetically modified feed crop further comprises: a third transgenic polynucleotide construct comprising: a Wrinkled 1 (WRI1) gene sequence encoding a WRI1 protein. In another aspect, the WRI1 gene sequence has at least 90-99% identity to any one of SEQ ID NO: 14-15 or 17-18. In another aspect, the WRI1 gene sequence is selected from any one of SEQ ID NO: 14-15 or 17-18. In another aspect, the WRI1 protein comprises an amino acid sequence having at least 90-99% identity to SEQ ID NO: 16. In another aspect, the WRI1 protein comprises an amino acid sequence selected from SEQ ID NO: 16. In another aspect, the HPO gene sequence and WRI1 gene sequence are separated by a self-cleaving peptide gene sequence encoding for a self-cleaving peptide. In another aspect, the self-cleaving peptide gene sequence has at least 90-99% identity to any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the self-cleaving peptide gene sequence is selected from any one of SEQ ID NO: 29, 31, 33, 35, 37, or 39. In another aspect, the self-cleaving peptide comprises an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the self-cleaving peptide comprises an amino acid sequence selected from any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40.
Another embodiment described herein is a method for reducing methanogenesis in a livestock animal, the method comprising: feeding a livestock animal a genetically modified feed crop comprising: a transgenic polynucleotide construct comprising a haloperoxidase (HPO) gene sequence encoding a HPO protein; wherein the HPO protein enables biosynthesis of bromoform (CHBr3), chloroform (CHCl3), iodoform (CHI3), or a combination thereof in the genetically modified feed crop. In another aspect, the HPO gene sequence encoding a HPO protein is a vanadium-dependent bromoperoxidase (VBPO), a vanadium-dependent iodoperoxidase (VIPO), vanadium-dependent chloroperoxidase (VCPO), or a nonspecific vanadium-dependent haloperoxidase (VHPO). In another aspect, the method reduces the level of methane produced and released from the livestock animal as compared to a livestock animal that is not fed the genetically modified feed crop. In another aspect, the livestock animal is a cow, a sheep, a goat, a buffalo, a camel, a deer, an elk, an antelope, a gazelle, or a giraffe.
Another embodiment described herein is a method of making a genetically modified feed crop for reducing methanogenesis in a livestock animal, the method comprising: transforming a monocot plant or a dicot plant with a transgenic polynucleotide construct comprising a haloperoxidase (HPO) gene sequence encoding a HPO protein, wherein the HPO protein enables biosynthesis of bromoform (CHBr3), chloroform (CHCl3), iodoform (CHI3), or a combination thereof in the genetically modified feed crop; and growing the transformed monocot plant or dicot plant comprising the transgenic polynucleotide construct to generate the genetically modified feed crop. In another aspect, the HPO gene sequence encoding a HPO protein is a vanadium-dependent bromoperoxidase (VBPO), a vanadium-dependent iodoperoxidase (VIPO), vanadium-dependent chloroperoxidase (VCPO), or a nonspecific vanadium-dependent haloperoxidase (VHPO). In another aspect, the genetically modified feed crop comprises an increased level of HPO protein as compared to a plant that is not transformed with the transgenic polynucleotide construct. In another aspect, the livestock animal is a cow, a sheep, a goat, a buffalo, a camel, a deer, an elk, an antelope, a gazelle, or a giraffe.
Another embodiment described herein is the use of the genetically modified feed crop as described herein for reducing methanogenesis in a livestock animal. In another aspect, the livestock animal is a ruminant animal. In another aspect, the livestock animal is a cow, a sheep, a goat, a buffalo, a camel, a deer, an elk, an antelope, a gazelle, or a giraffe.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.
As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.
As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements presented herein, whether explicitly set forth or not.
As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.
As used herein, the term “or” can be conjunctive or disjunctive.
As used herein, the term “and/or” refers to both the conjuctive and disjunctive.
As used herein, the term “substantially” means to a great or significant extent, but not completely.
As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to +10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “˜” means “about” or “approximately.”
All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to +10% of any value within the range or within 3 or more standard deviations, including the end points.
As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.
As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.
As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.
As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.
As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject's age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.
As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non-human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a ruminant animal. In another embodiment, the subject is a cow, goat, sheep, buffalo, camel, deer, moose, elk, antelope, gazelle, or giraffe.
As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.
For the purposes of this description, the names of genes and proteins given from a particular species are understood to refer to a representative homolog of the named gene. A homolog is any genomic DNA sequence having 70% or greater identity to the genomic region corresponding to the representative gene, including introns, untranscribed regions, and regulatory regions; any protein-coding RNA or cDNA sequence having 70% or greater identity to the protein-coding RNA or cDNA sequence of the representative gene; or any protein having 70% or greater identity to the representative protein or protein product of the representative gene. Percent identity for DNA and RNA homology is determined over any nucleotide sequence window of 500 base pairs or greater, and protein homology is determined over any amino acid sequence window of 100 residues or greater.
As used herein, the terms “land plant,” “land plants,” “plant,” “plants,” “feed crop,” “feed crops,” “crop,” or “crops” shall be understood to refer to any member of the phylogenetic plant grouping known in the art as the Embryophyta, comprising hornworts, liverworts, mosses, lycophytes, ferns, and other pteridophytes, gymnosperms, and angiosperms. The terms shall be understood to include plant species having aquatic or semiaquatic growth habits but originating in this phylogenetic grouping. For example, duckweeds, reeds, rushes, seagrasses, mangroves, water lilies, domestic rice, wild rice, and water lettuces are land plants.
Haloform is the common name of a compound having the formula CHX3, where X is a halogen atom, halogens comprising bromine, chlorine, iodine, or fluorine. A haloform may contain more than one type of halogen, for example, bromoiodochloroform is the common name of the compound CHBrICl, which is a haloform. Bromoform is the common name of the compound CHBr3. Iodoform is the common name of the compound CHI3. Chloroform is the common name of the compound CHCl3. Nevertheless, the plants described herein may result in production of additional organohalide compounds having antimethanogenic (e.g., methane-reducing) effects, in combination with or separately from one or more haloforms. An organohalide is any compound containing at least one carbon atom and at least one halogen atom. As used herein, the term “haloform” is understood to represent CHX3 and any other compounds containing carbon and a halogen whose formation results from activity of a haloperoxidase enzyme or that has the effect of impeding methanogenesis by a similar mechanism to a haloform. As used herein “haloform” refers to CHX3 where X is a halogen atom, as well as any small organohalide molecules comprising up to 9 halogen atoms and up to 30 carbon atoms and including nitrogen, oxygen, bromine, chlorine, fluorine, or iodine atoms formed by activity of a VHPO on a halogen and an organic substrate, as either an end product or an intermediate, particularly including the organohalides CH3Br, CH2Br2, CHBr3, CH3I, CH2I2, CHI3, CH3Cl, CH2Cl2, CHCl3; other methyl halides in which bromine, iodine, chlorine, or fluorine occur in any combination within the structure described for methyl halides, primary alkyl halides, meaning any molecule of the structure R—CH2X, R—CHX2, or R—CX3, where X is a halogen and R is an alkyl group or haloalkane (including haloalkanes of bromine, chlorine, fluorine, and iodine); secondary alkyl bromides, including molecules having the structure: R1—(CHX)—R2 or R1—(CX2)—R2, where X is a halogen and R1 and R2 are alkyl groups or haloalkanes; and haloketones, such as molecules containing at least one carbon-oxygen double bond and at least one halogen atom, including 1,1 dihaloacetones, X2(CH)(CO)CH2, where X is a halogen; 1,5-halogenated 2,4-diones; 1-dihalogenated ketones; or 1-trihalogenated ketones. As used herein, the term “bromoform” is understood to represent CHBr3 and any other compounds containing carbon and bromine whose formation results from activity of a bromoperoxidase enzyme or that has the effect of impeding methanogenesis by a similar mechanism to CHBr3. As used herein, the term “iodoform” is understood to represent CHI3 and any other compounds containing carbon and iodine whose formation results from activity of an iodoperoxidase enzyme or that has the effect of impeding methanogenesis by a similar mechanism to CHI3. As used herein, the term “chloroform” is understood to represent CHCl3 and any other compounds containing carbon and chlorine whose formation results from activity of a chloroperoxidase enzyme or that has the effect of impeding methanogenesis by a similar mechanism to CHCl3.
Biological processes and molecular activities of chemicals, enzymes, proteins, and nucleic acids are faithfully described as understood in the art at the time of the description. Nevertheless, this description is not intended to be limited by a particular model of molecular or metabolic mechanism of its components. Alternative modes of action, molecular substrates, and interactions by the components different from those described here but having the same or similar effect should be understood to remain within the scope of the embodiments described herein.
Described herein are systems enabling haloform biosynthesis in land plants that are commonly used as animal feed, including grains like corn, soybean, and canola, and pasture crops like alfalfa and ryegrass. By synthesizing haloform directly in these crops, the benefits of methane reduction are achieved with no additional land, energy, or resources beyond what is already needed for growing the crops themselves. The engineered plants make haloform in situ from soil halogens and native plant metabolites and do not require special facilities or supplementation, with the further benefit of enabling methane reduction in otherwise difficult-to-serve systems including smallholders in the developing world. Further, methods and composition described herein uniquely integrates this molecule with a common method of stabilizing and storing haloform—immersion in vegetable oil—by biosynthesizing the active molecule directly within oil-bearing cells.
The methods and composition described herein use several genetic components that work alone or in various permutations. In one embodiment, the transgenic expression of a Vanadium-dependent Bromoperoxidase (VBPO) gene derived from Asparagopsis red algae (seaweed) or marine cyanobacteria is used to produce haloform by catalyzing the reaction of bromide with native plant carbonyl compounds including acetoacetyl-S-CoA (AcAc-COA). In another embodiment, the transgenic expression of acetoacetyl-CoA thiolase 2 (AACT2) of Arabidopsis thaliana, increases the availability of the AcAc-COA precursors. In another embodiment, the transgenic expression of the Wrinkled 1 (WRI1) transcription factor of Arabidopsis thaliana upregulates fatty acid biosynthesis, increasing the availability of precursors for both AcAc-COA and cuticle polymers, and increasing accumulation of storage oil and cuticular wax, which stabilize accumulated haloform and prevent its evaporation. In another embodiment, specific promoters direct the previously listed metabolic functions to the portion of feed plants consumed by animals, which avoids unnecessary tradeoffs with plant growth and limits unwanted emission of haloform into the atmosphere.
Another embodiment is a polynucleotide sequence described herein. In one aspect, the polynucleotide has at least 85% to 99% identity, including all percentages within the specified range, to SEQ ID NO: 1, 3, 9, 10, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 94, or 95. In another aspect, the polynucleotide is selected from SEQ ID NO: 1, 3, 9, 10, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 94, or 95.
Another embodiment described herein is a polynucleotide vector comprising one or more nucleotide sequences described herein.
Another embodiment described herein is a cell comprising one or more nucleotide sequences described herein or a polynucleotide vector described herein.
Another embodiment is a polypeptide encoded by a nucleotide sequence described herein. In one aspect, the polypeptide has at least 85% to 99% identity, including all percentages within the specified range, to SEQ ID NO: 2, 4, 5, 6, 7, 8, 11, 16, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92. In another aspect, the polypeptide is selected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 11, 16, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92.
Another embodiment described herein is a process for manufacturing one or more of the nucleotide sequence described herein or a polypeptide encoded by the nucleotide sequence described herein, the process comprising: transforming or transfecting a cell with a nucleic acid comprising a nucleotide sequence described herein; growing the cells; optionally isolating additional quantities of a nucleotide sequence described herein; inducing expression of a polypeptide encoded by a nucleotide sequence of described herein; isolating the polypeptide encoded by a nucleotide described herein.
Another embodiment described herein is a means for manufacturing one or more of the nucleotide sequences described herein or a polypeptide encoded by a nucleotide sequence described herein, the process comprising: transforming or transfecting a cell with a nucleic acid comprising a nucleotide sequence described herein; growing the cells; optionally isolating additional quantities of a nucleotide sequence described herein; inducing expression of a polypeptide encoded by a nucleotide sequence of described herein; isolating the polypeptide encoded by a nucleotide described herein.
Another embodiment described herein is a nucleotide sequence or a polypeptide encoded by the nucleotide sequence produced by a method or means described herein Another embodiment described herein is the use of an effective amount of a polypeptide encoded by one or more of the nucleotide sequences described herein in SEQ ID NO: 1, 3, 9, 10, 12, 13, 14, 15, 17, 18, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91.
Another embodiment described herein is a research tool comprising a polypeptide encoded by a nucleotide sequence described herein.
Another embodiment described herein is a reagent comprising a polypeptide encoded by a nucleotide sequence described herein.
The polynucleotides described herein include variants that have substitutions, deletions, and/or additions that can involve one or more nucleotides. The variants can be altered in coding regions, non-coding regions, or both. Alterations in the coding regions can produce conservative or non-conservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions, which do not alter the properties and activities of the binding.
Further embodiments described herein include nucleic acid molecules comprising polynucleotides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and more preferably at least about 90-99% or 100% identity to (a) nucleotide sequences, or degenerate, homologous, or codon-optimized variants thereof, encoding polypeptides having the amino acid sequences in SEQ ID NO: 2, 4, 5, 6, 7, 8, 11, 16, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92; (b) nucleotide sequences, or degenerate, homologous, or codon-optimized variants thereof, encoding polypeptides having the amino acid sequences in SEQ ID NO: 2, 4, 5, 6, 7, 8, 11, 16, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92; and (c) nucleotide sequences capable of hybridizing to the complement of any of the nucleotide sequences in (a) or (b) above and capable of expressing functional polypeptides of amino acid sequences in SEQ ID NO: 2, 4, 5, 6, 7, 8, 11, 16, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92.
Further embodiments described herein include nucleic acid molecules comprising polynucleotides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and more preferably at least about 90-99% or 100% identity to (a) nucleotide sequences that are polycistronic sequences described herein in SEQ ID NO: 28 or 95.
Further embodiments described herein include nucleic acid molecules comprising polynucleotides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and more preferably at least about 90-99% or 100% identity to (a) nucleotide sequences that are promoter or terminator sequences described herein in SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 93, or 94.
By a polynucleotide having a nucleotide sequence, for example, at least 90-99% “identical” to a reference nucleotide sequence encoding a polypeptide is intended that the nucleotide sequence encoding the polynucleotide be identical to the reference sequence except that the polynucleotide sequence can include up to about 10-to-1 point mutations, additions, or deletions per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
In other words, to obtain a polynucleotide having a nucleotide sequence about at least 90-99% identical to a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence can be deleted, added, or substituted, with another nucleotide, or a number of nucleotides up to 10% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5′- or 3′-terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The same is applicable to polypeptide sequences about at least 90-99% identical to a reference polypeptide sequence.
As noted above, two or more polynucleotide sequences can be compared by determining their percent identity. Two or more amino acid sequences likewise can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or peptide sequences, is generally described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:4 82-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14 (6): 6745-6763 (1986).
For example, due to the degeneracy of the genetic code, one having ordinary skill in the art will recognize that a large number of the nucleic acid molecules having a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence shown in SEQ ID NO: 1, 3, 9, 10, 12, 13, 14, 15, 17, 18, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91, or degenerate, homologous, or codon-optimized variants thereof, will encode a polypeptide.
The polynucleotides described herein include those encoding mutations, variations, substitutions, additions, deletions, and particular examples of the polypeptides described herein. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.
Thus, fragments, derivatives, or analogs of the polypeptides of SEQ ID NO: 2, 4, 5, 6, 7, 8, 11, 16, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92 can be (i) ones in which one or more of the amino acid residues (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 residues, or even more) are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue). Such substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) ones in which one or more of the amino acid residues includes a substituent group (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues or even more), or (iii) ones in which the mature polypeptide is fused with another polypeptide or compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) ones in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives, and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
In addition, fragments, derivatives, or analogs of the polypeptides of SEQ ID NO: 2, 4, 5, 6, 7, 8, 11, 16, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92 can be substituted with one or more conserved or non-conserved amino acid residue (preferably a conserved amino acid residue). In some cases, these polypeptides, fragments, derivatives, or analogs thereof will have a polypeptide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide sequence shown in SEQ ID NO: 2, 4, 5, 6, 7, 8, 11, 16, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92 and will comprise functional or non-functional proteins or enzymes. Similarly, additions or deletions to the polypeptides can be made either at the N- or C-termini or within non-conserved regions of the polypeptide (which are assumed to be non-critical because they have not been photogenically conserved).
As described herein, in many cases the amino acid substitutions, mutations, additions, or deletions are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein or additions or deletions to the N- or C-termini. Of course, the number of amino acid substitutions, additions, or deletions a skilled artisan would make depends on many factors, including those described herein. Generally, the number of substitutions, additions, or deletions for any given polypeptide will not be more than about 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 5, 6, 4, 3, 2, or 1.
Described herein are modified land plants containing haloform at a concentration of at least about 10 μg per gram (parts per million) dry matter in the entire plant, or in any sub-part of the plant such as leaves, seeds, roots, tubers, or stems.
Also described herein are methods of producing plants containing desired levels of haloform by transgenic expression of a vanadium-dependent bromoperoxidase gene, with or without the use of additional complementary genetic modifications.
Also described herein are methods of adjusting haloform concentration in particular target plant species and tissues via altered biosynthesis of acetoacetyl as an organic precursor, altered biosynthesis of lipids to increase haloform stability, and the use of regulatory elements including codon optimization, introns, and constitutive promoters, as well as seed, endosperm, leaf, and photosynthetic tissue-specific promoters.
Also described herein are methods of reducing the enteric methane emissions of livestock animals, especially ruminants such as cows, by incorporation of plants having the desired characteristics (e.g., haloform levels present) into animal diets in a manner to provide a total haloform inclusion level of at least 2 μg haloform per gram total dry matter.
One embodiment described herein is a genetically modified feed crop for reducing methanogenesis in a livestock animal, the genetically modified feed crop comprising: a transgenic polynucleotide construct comprising a haloperoxidase (HPO) gene sequence encoding a HPO protein, wherein the HPO protein enables biosynthesis of bromoform (CHBr3), chloroform (CHCl3), iodoform (CHI3), or a combination thereof in the genetically modified feed crop. In another aspect, the HPO gene sequence encoding a HPO protein is a vanadium-dependent bromoperoxidase (VBPO), a vanadium-dependent iodoperoxidase (VIPO), vanadium-dependent chloroperoxidase (VCPO), or a nonspecific vanadium-dependent haloperoxidase (VHPO). In another aspect, the genetically modified feed crop is a monocot plant or a dicot plant. In another aspect, the HPO gene sequence has at least 90-99% identity to any one of SEQ ID NO: 1, 3, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91. In another aspect, the HPO gene sequence is selected from any one of SEQ ID NO: 1, 3, 41, 43, 45, 47, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, or 91. In another aspect, the HPO protein comprises an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 2, 4-8, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92. In another aspect, the HPO protein comprises an amino acid sequence selected from any one of SEQ ID NO: 2, 4-8, 42, 44, 46, 48, 50, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, or 92. In another aspect, the HPO gene sequence comprises a whole-plant constitutive promoter and the HPO gene sequence is expressed under the whole-plant constitutive promoter. In another aspect, the whole-plant constitutive promoter comprises a polynucleotide sequence having at least 90-99% identity to any one of SEQ ID NO: 19-20, or 93. In another aspect, the whole-plant constitutive promoter comprises a polynucleotide sequence selected from any one of SEQ ID NO: 19-20, or 93. In another aspect, the HPO gene sequence comprises an endosperm-specific promoter or photosynthetic-tissue promoter and is expressed under the endosperm-specific promoter or photosynthetic-tissue promoter. In another aspect, the endosperm-specific promoter comprises a polynucleotide sequence having at least 90-99% identity to any one of SEQ ID NO: 24-25, or 94; and the photosynthetic-tissue promoter comprises a polynucleotide sequence having at least 90-99% identity to any one of SEQ ID NO: 21-23. In another aspect, the endosperm-specific promoter comprises a polynucleotide sequence selected from any one of SEQ ID NO: 24-25, or 94; and the photosynthetic-tissue promoter comprises a polynucleotide sequence selected from any one of SEQ ID NO: 21-23. The genetically modified feed crop of claim 1, further comprising: a second transgenic polynucleotide construct comprising: an acetoacetyl-CoA thiolase 2 (AACT2) gene sequence encoding an AACT2 protein. In another aspect, the AACT2 gene sequence has at least 90-99% identity to any one of SEQ ID NO: 9-10 or 12-13. In another aspect, the AACT2 gene sequence is selected from any one of SEQ ID NO: 9-10 or 12-13. In another aspect, the AACT2 protein comprises an amino acid sequence having at least 90-99% identity to SEQ ID NO: 11. In another aspect, the AACT2 protein comprises an amino acid sequence selected from SEQ ID NO: 11. In another aspect, the AACT2 gene sequence comprises a whole-plant constitutive promoter and the AACT2 gene sequence is expressed under the whole-plant constitutive promoter. In another aspect, the whole-plant constitutive promoter comprises a polynucleotide sequence having at least 90-99% identity to any one of SEQ ID NO: 19-20, or 93. In another aspect, the whole-plant constitutive promoter comprises a polynucleotide sequence selected from any one of SEQ ID NO: 19-20, or 93. In another aspect, the HPO gene sequence and AACT2 gene sequence are separated by a self-cleaving peptide gene sequence encoding for a self-cleaving peptide. In another aspect, the self-cleaving peptide gene sequence has at least 90-99% identity to any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the self-cleaving peptide gene sequence is selected from any one of SEQ ID NO: 29, 31, 33, 35, 37, or 39. In another aspect, the self-cleaving peptide comprises an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the self-cleaving peptide comprises an amino acid sequence selected from any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the HPO is a Vanadium-dependent bromoperoxidase (VBPO) gene sequence having at least 90-99% identity to any one of SEQ. ID NO: 1 or 3. In another aspect, the HPO is a Vanadium-dependent bromoperoxidase (VBPO) gene sequence selected from any one of SEQ ID NO: 1 or 3. In another aspect, the HPO is a Vanadium-dependent bromoperoxidase (VBPO) protein comprising an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 2 or 4-8. In another aspect, the HPO is a Vanadium-dependent bromoperoxidase (VBPO) protein comprising an amino acid sequence selected from any one of SEQ ID NO: 2 or 4-8. In another aspect, the HPO is a Vanadium-dependent iodoperoxidase (VIPO) gene sequence having at least 90-99% identity to any one of SEQ. ID NO: 41, 43, 45, 47, 49 or 51-60. In another aspect, the HPO is a Vanadium-dependent iodoperoxidase (VIPO) gene sequence selected from any one of SEQ ID NO: 41, 43, 45, 47, 49 or 51-60. In another aspect, the HPO is a Vanadium-dependent iodoperoxidase (VIPO) protein comprising an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 42, 44, 46, 48, or 50. In another aspect, the HPO is a Vanadium-dependent iodoperoxidase (VIPO) protein comprising an amino acid sequence selected from any one of SEQ ID NO: 42, 44, 46, 48, or 50. In another aspect, the HPO is a Vanadium-dependent chloroperoxidase (VCPO) gene sequence having at least 90-99% identity to any one of SEQ. ID NO: 61, 63, 65, 67, 69, 71, 73, or 75. In another aspect, the HPO is a Vanadium-dependent chloroperoxidase (VCPO) gene sequence selected from any one of SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, or 75. In another aspect, the HPO is a Vanadium-dependent chloroperoxidase (VCPO) protein comprising an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 62, 64, 66, 68, 70, 72, 74, or 76. In another aspect, the HPO is a Vanadium-dependent chloroperoxidase (VCPO) protein comprising an amino acid sequence selected from any one of SEQ ID NO: 62, 64, 66, 68, 70, 72, 74, or 76. In another aspect, the HPO is a nonspecific vanadium-dependent haloperoxidase (VHPO) gene sequence having at least 90-99% identity to any one of SEQ. ID NO: 77, 79, 81, 83, 85, 87, 89, or 91. In another aspect, the HPO is a nonspecific vanadium-dependent haloperoxidase (VHPO) gene sequence selected from any one of SEQ ID NO: 77, 79, 81, 83, 85, 87, 89, or 91. In another aspect, the HPO is a nonspecific vanadium-dependent haloperoxidase (VHPO) protein comprising an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, or 92. In another aspect, the HPO is a nonspecific vanadium-dependent haloperoxidase (VHPO) protein comprising an amino acid sequence selected from any one of SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, or 92.
In another aspect, the genetically modified feed crop further comprises: a third transgenic polynucleotide construct comprising: a Wrinkled 1 (WRI1) gene sequence encoding a WRI1 protein. In another aspect, the WRI1 gene sequence has at least 90-99% identity to any one of SEQ ID NO: 14-15 or 17-18. In another aspect, the WRI1 gene sequence is selected from any one of SEQ ID NO: 14-15 or 17-18. In another aspect, the WRI1 protein comprises an amino acid sequence having at least 90-99% identity to SEQ ID NO: 16. In another aspect, the WRI1 protein comprises an amino acid sequence selected from SEQ ID NO: 16. In another aspect, the HPO gene sequence and WRI1 gene sequence are separated by a self-cleaving peptide gene sequence encoding for a self-cleaving peptide. In another aspect, the self-cleaving peptide gene sequence has at least 90-99% identity to any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the self-cleaving peptide gene sequence is selected from any one of SEQ ID NO: 29, 31, 33, 35, 37, or 39. In another aspect, the self-cleaving peptide comprises an amino acid sequence having at least 90-99% identity to any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40. In another aspect, the self-cleaving peptide comprises an amino acid sequence selected from any one of SEQ ID NO: 30, 32, 34, 36, 38, or 40.
Another embodiment described herein is a method for reducing methanogenesis in a livestock animal, the method comprising: feeding a livestock animal a genetically modified feed crop comprising: a transgenic polynucleotide construct comprising a haloperoxidase (HPO) gene sequence encoding a HPO protein; wherein the HPO protein enables biosynthesis of bromoform (CHBr3), chloroform (CHCl3), iodoform (CHI3), or a combination thereof in the genetically modified feed crop. In another aspect, the HPO gene sequence encoding a HPO protein is a vanadium-dependent bromoperoxidase (VBPO), a vanadium-dependent iodoperoxidase (VIPO), vanadium-dependent chloroperoxidase (VCPO), or a nonspecific vanadium-dependent haloperoxidase (VHPO). In another aspect, the method reduces the level of methane produced and released from the livestock animal as compared to a livestock animal that is not fed the genetically modified feed crop. In another aspect, the livestock animal is a cow, a sheep, a goat, a buffalo, a camel, a deer, an elk, an antelope, a gazelle, or a giraffe.
Another embodiment described herein is a method of making a genetically modified feed crop for reducing methanogenesis in a livestock animal, the method comprising: transforming a monocot plant or a dicot plant with a transgenic polynucleotide construct comprising a haloperoxidase (HPO) gene sequence encoding a HPO protein, wherein the HPO protein enables biosynthesis of bromoform (CHBr3), chloroform (CHCl3), iodoform (CHI3), or a combination thereof in the genetically modified feed crop; and growing the transformed monocot plant or dicot plant comprising the transgenic polynucleotide construct to generate the genetically modified feed crop. In another aspect, the HPO gene sequence encoding a HPO protein is a vanadium-dependent bromoperoxidase (VBPO), a vanadium-dependent iodoperoxidase (VIPO), vanadium-dependent chloroperoxidase (VCPO), or a nonspecific vanadium-dependent haloperoxidase (VHPO). In another aspect, the genetically modified feed crop comprises an increased level of HPO protein as compared to a plant that is not transformed with the transgenic polynucleotide construct. In another aspect, the livestock animal is a cow, a sheep, a goat, a buffalo, a camel, a deer, an elk, an antelope, a gazelle, or a giraffe.
Another embodiment described herein is the use of the genetically modified feed crop as described herein for reducing methanogenesis in a livestock animal. In another aspect, the livestock animal is a ruminant animal. In another aspect, the livestock animal is a cow, a sheep, a goat, a buffalo, a camel, a deer, an elk, an antelope, a gazelle, or a giraffe.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
Various embodiments and aspects of the inventions described herein are summarized by the following clauses:
Diversion of Soil-Derived Bromine from Native Methyl Bromide Biosynthesis to Bromoform Biosynthesis by Expression of VBPO
Normally, bromine taken up from soil in unmodified land plants accumulates in tissues or is converted to volatile methyl bromide (CH3Br) via enzymes encoded by HOL (Harmless to Ozone Layer) genes, followed by release to the atmosphere (
In Arabidopsis, VBPO (SEQ ID NO: 1) was transgenically expressed under control of a seed-specific Napin promoter derived from Brassica napus (SEQ ID NO: 24) and grown in commercial potting soil under typical conditions. Mature Arabidopsis seeds of 5 independent transgenic lines were manually harvested and cleaned of chaff. Seed samples weighing from 0.3951 to 0.1072 grams were frozen in liquid nitrogen and ground by mechanical shaking with steel beads. Methanol (0.8 or 1.6 mL) was added to each seed sample, and samples were blended with beads, vortexed thoroughly, allowed to incubate overnight at 4° C., and vortexed again. Samples were centrifuged to collect debris, and the supernatants were analyzed by Gas Chromatography using an Agilent 6890 instrument. GC analysis used a 30 meter long, 250 μm diameter 5% phenyl methyl siloxane column with hydrogen carrier gas running in 1:1 split mode with port temperature of 250° C. and continuous flow 0.9 mL/min. Accounting for original sample mass and extraction volume, this quantification revealed that VBPO results in expression of 159.02 micrograms of bromoform per gram of fresh weight of mature seed in one transgenic line, and 1,653.68 micrograms bromoform per gram of fresh weight of mature seed in another.
The VBPO transgene includes a protein sequence (SEQ ID NO: 2, 4-8) reverse-translated to DNA and codon-optimized for expression in dicots (represented by Arabidopsis thaliana) (SEQ ID NO: 1) or monocots (SEQ ID NO: 3) (represented by corn, Zea mays), and lacking introns.
In Arabidopsis, VBPO (SEQ ID NO: 1) was transgenically expressed under control of a seed-specific Napin promoter derived from Brassica napus (SEQ ID NO: 24), and the transgenically-modified Arabidopsis was grown in commercial potting soil under typical conditions. This resulted in the production of ˜159.02 μg bromoform per gram fresh weight of mature seed in one independent transgenic line, and ˜1,653.68 μg bromoform per gram in another transgenic line, as determined by gas chromatography with mass spectrometry (GC-MS). In comparison, the experimental controls of Arabidopsis with no transgenically expressed VBPO did not produce any bromoform.
To assess the genetic stability of seed-specific production of bromoform enabled by this invention Arabidopsis plants transgenically expressing VBPO under control of the Napin promoter were brought to homozygosity using selective marker segregation and grown for six generations. Bromoform was extracted in methanol from seed samples of the first and sixth generations of five independent transgenic lines and quantified by GC-MS analysis at Avazyme, Inc. By comparing bromoform levels in sixth-generation plant seeds to first-generation seeds of the corresponding line, this experiment is expected to reveal that bromoform production levels remain stable across generations and are not subject to genetic silencing or otherwise counteracted by native processes.
galactanivorans
galactanivorans
digitata
digitata
marincola
marincola
japonica
japonica
Laminaria digitata
Laminaria digitata
galactanivorans, codon-optimized for dicot plants
galactanivorans, codon-optimized for monocot plants
digitata, codon-optimized for dicot plants
digitata, codon-optimized for monocot plants
marincola, codon-optimized for dicot plants
marincola, codon-optimized for monocot plants
japonica, codon-optimized for dicot plants
japonica, codon-optimized for monocot plants
Laminaria digitata, codon-optimized for dicot plants
digitata, codon-optimized for monocot plants
didymospora
didymospora
inaequalis
inaequalis
tritici-repentis
tritici-repentis
Graciliariopsis chorda
chorda
Rhodopirellula baltica
baltica
Nitrosomonas sp. Nm84
Agrobacterium species RAC06
Bradyrhizobium yuanmingense
yuanmingense
Bradyrhizobium sp. B039
Fucus distichus
Fucus distichus
Phytomonospora endophytica
Phytomonospora endophytica
Cellulomonas xylanylitca
Cellulomonas xylanylitca
Nostoc sp. P10264
Nostoc sp. P10264
Skeletonema marinoi
Skeletonema marinoi
Actinarchaeum halophilum
Actinarchaeum halophilum
Another embodiment described herein is the transgenic expression of acetoacetyl-CoA thiolase 2 (AACT2) of Arabidopsis thaliana (“Arabidopsis”) or other organisms, which provides increased AcAc-COA availability (SEQ ID NO: 9-15). AcAc-COA is present throughout the plant due to the universal importance and wide-ranging functions of its derivatives. However, its local abundance is a limiting factor for bromoform synthesis which varies temporally and with tissue type. The AACT2 enzyme converts 2 acetyl-S-CoA (Ac-COA) molecules to AcAc-COA (
AACT2 may be expressed under constitutive or targeted promoters as described herein. The AACT2 transgene for use in dicots consists of either the native Arabidopsis protein coding sequence sans introns (e.g., the cDNA), or the native coding sequence in which the first intron is maintained but other introns are deleted (SEQ ID NO: 9-11). The variation in intron composition, in combination with promoter selection, provides a means of tuning the transgene expression level and resultant stoichiometry of AcAc-CoA synthesis in combination with other aspects described herein to achieve the desired level of bromoform across variation in target species and plant tissues. The AACT2 transgene for use in monocots consists of the Arabidopsis protein coding sequence codon-optimized for Zea mays with or without the addition of the 5′-untranslated region (UTR) of Oryza sativa Superoxide Dismutase (SOD) (SEQ ID NO: 12-13).
Another embodiment described herein is broadly increased lipid biosynthesis conferred by overexpression of Wrinkled 1 (WRI1) of Arabidopsis. WRI1 is a transcription factor that positively regulates fatty acid synthesis at multiple points in the metabolic pathway, and in its native setting controls storage oil accumulation during seed filling. Applied in seeds, transgenic expression of WRI1 increases storage oil accumulation. Applied in other tissues, WRI1 transgenic expression can cause plant parts that do not typically synthesize storage oil to do so. The accumulation of oil increases the holding capacity of plants for synthesized bromoform, which is lipid-soluble, and reduces loss to the atmosphere through volatilization. In plants where starch is the primary carbon storage molecule in endosperm, including corn and other monocots, the role of WRI1 to alter carbon allocation towards oil synthesis is more central, and this mechanism of increased oil accumulation differs from conventional high-oil corn produced through breeding for increased embryo (germ) size.
WRI1 overexpression also results in increased Ac-COA as a substrate for AACT2, and increased medium to long chain fatty acid synthesis provides increased material for formation of cuticular polymers, which provides a barrier to VOC emission and further sink material to which bromoform is adsorbed. A significant benefit is the containment provided by synthesis of bromoform in self-contained, oil-bearing plant organs. This benefit is compounded by the fact that engineered plant parts used for animal feed are living tissue (e.g., seeds or forage plants) in which bromoform biosynthesis remains active up to the moment of consumption. This maximizes the portion of synthesized bromoform that is received by animals rather than lost during harvest, shipping, and storage of dead material.
As is the case with AACT2, The WRI1 transgene may exist as several versions with varying intron composition to adjust expression level depending on target system and application. The WRI1 transgene for use in dicots consists of either the native Arabidopsis protein coding sequence sans introns (e.g., the cDNA), or the native coding sequence in which the first intron is maintained but other introns are deleted (SEQ ID NO: 14-16). WRI1 transgenes for use in monocots consist of a Zea mays codon-optimized coding sequence, with or without an appended 5′-untranslated region (UTR) derived from the first intron of rice (Oryza sativa) Cytosolic Superoxide Dismutase 2 (OsSODCc2) (SEQ ID NO: 17-18).
AACT2, WRI1, and VBPO were assembled into a tricistronic sequence joined by the P2A self-cleaving peptide (SEQ ID NO: 28) and transiently expressed under control of the Ubiquitin 10 promoter (SEQ ID NO: 19) in Nicotiana tabacum. Another construct containing VBPO alone under control of the Ubiquitin 10 promoter was also transiently expressed in Nicotiana tabacum. Leaf tissue samples from the transformed area were taken 3 days after transformation, frozen in liquid nitrogen and ground using steel or cubic zirconia beads, then methanol was added to each tissue sample, and samples were blended with beads, vortexed thoroughly, allowed to incubate overnight at 4° C., and vortexed again. Samples were centrifuged to collect debris, and the supernatants were analyzed for absolute bromoform concentration (accounting for original sample mass and extraction volume) using GC-MS at Avazyme, Inc. Comparison of bromoform content in this experiment between leaves expressing VBPO alone, and leaves expressing VBPO in combination with AACT2 and WRI1, is expected to quantify the percentage by which expression of the additional genes enhances total bromoform synthesis and/or accumulation.
Another embodiment described herein is the use of different promoters to direct expression of the transgenic enzymes to different parts of the plant according to the specifics of its use as animal feed. As with the coding sequences themselves, the use of promoters is combinatorial and varies with target species and application.
For any of the listed transgenes in dicots, expression covering most plant tissues is conferred by the Ubiquitin 10 promoter of Arabidopsis (pAtUBQ10) (SEQ ID NO: 19). In monocots constitutive expression can be conferred by the Ubiquitin 1 promoter of corn (pZmUbi1) or another constitutive promoter such as that of Alcohol Dehydrogenase 1 (pZmAdh1) (SEQ ID NO: 20).
Expression that is primarily in photosynthetic tissues consumed by animals in forage applications (e.g., leaves, stems) and which is low in root, seed, and reproductive tissue is conferred in dicots and monocots by a ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit promoter derived from Arabidopsis (pAtRbcS1a) or another plant such as sweet potato (Ipomoea batatas) (pIbRbcS1), or a chlorophyll a/b binding protein promoter derived from Arabidopsis (pAtCAB3) or another plant such as black pine (Pinus thunbergii) (pPtCAB6) (SEQ ID NO: 21-23).
Expression primarily within the internal seed tissues of monocots and dicots, including the endosperm and/or embryo, is conferred by promoters of seed storage proteins derived from Napin of rapeseed (Brassica napus) (pBnNapin) or Cruciferin of Arabidopsis thaliana (pAtCruciferin) in dicots, and additionally in monocots from an appropriate α-zein gene of corn (pZmZein) or other appropriate variety-specific zein (SEQ ID NO: 24-25). Promoters and coding sequences are used in combination with the terminators of Arabidopsis heat-shock protein (tAtHSP), Agrobacterium tumefaciens nopaline synthase (tNOS), or other appropriate terminators (SEQ ID NO: 26-27).
Leaves of Nicotiana tabacum and elite corn variety Zea mays LH244 were transiently transformed with two separate constructs expressing AACT2, WRI1, and VBPO assembled into a tricistron (SEQ ID NO: 28) under control of the 35S promoter (SEQ ID NO: 93) and Arabidopsis thaliana Ubiquitin 10 promoter (SEQ ID NO: 19). Bromoform was extracted from transformed leaf tissue as described in Example 3 and analyzed for absolute bromoform concentration at Avazyme, Inc. This experiment is expected to reveal the percentage by which expression of VBPO under the Ubiquitin promoter enhanced bromoform production and/or accumulation relative to expression under the 35S promoter.
A tricistronic sequence containing AACT2, WRI1, and VBPO as described in Example 3 was transgenically expressed in Arabidopsis and Setaria italica variety ‘Red Siberian’ in three different constructs. At least five Arabidopsis plants were transformed for each of 2 genetic constructs carrying the tricistronic transgene (SEQ ID NO: 28), in one of which the transgene was under control of the Napin promoter (SEQ ID NO: 24) and in the other of which the gene was under control of the 35S promoter (SEQ ID NO: 93). At least five Setaria plants were transformed for each of 2 genetic constructs, in one of which the transgene (SEQ ID NO: 28) was under control of the 35S promoter (SEQ ID NO: 93) and in the other of which the transgene was under control of the 19 kDa Zein promoter (SEQ ID NO: 94) and the VBPO gene sequence within the tricistron was codon-optimized for monocot expression using the maize genetic code (SEQ ID NO: 3 or 95). Mature seed of each species of transformed plants was harvested and screened for transformants using resistance to kanamycin, and transformant viability was assessed as the frequency of kanamycin resistant plants surviving to produce the first true leaf. These results revealed that in Arabidopsis, expression of the bromoform biosynthesis construct under the 35S promoter yielded no viable transformants, while seed-tailored expression under the Napin promoter yielded ˜1% viable transformants in the first generation. In Setaria, seed-tailored expression of the bromoform biosynthesis construct under the Zein promoter yielded ˜0.2% viable transformants, while expression under the 35S promoter yielded a single viable transformant from ˜8,000 seeds, or ˜0.01% viability; the surviving transformant of the 35S construct also had stunted growth.
Assessment of Methane Emissions from Livestock
Bromoform-bearing plants are used to reduce methane emissions from livestock by incorporation into feed. In this example, bromoform is delivered to livestock in any fresh, dried, or processed plant tissue. Efficacy of this method can be demonstrated by methane reduction from rumen fluid upon exposure to engineered Arabidopsis, crop plants, or other model plants such as Setaria. Rumen fluid studies are a widely accepted analyses for assessing animal nutrition. Typically, a small amount of fresh rumen material is extracted from living cows or other animals that are exposed to a given treatment and monitored via gas chromatography in a sealed system for detailed effects on emission of methane, hydrogen, volatile fatty acids, and other components of digestion.
Tailored expression of bromoform biosynthesis is used to create a common animal feed grain such as corn (Zea mays) that is calibrated according to its inclusion rate in a specific livestock diet to provide an overall effective dose to eliminate maximum methane emissions. For example, an effective dosage rate of bromoform for methane elimination in cows is ˜25 μg per gram total feed, and corn commonly makes up ˜1-80% of cattle diets. Thus, using the means provided herein to adjust multiple components of bromoform biosynthesis, corn seed is specifically engineered to produce bromoform at a rate of from ˜2500 μg per gram, when included at ˜1% of feed, to ˜31.25 μg per gram, when included at ˜80% of feed. The same tailored methods are applied for other feed components making up a large portion of livestock diets such as soybean grain, barley grain, silages, or hays.
Bromoform biosynthesis is used to provide a methane-reducing forage crop directly consumed by animals grazing in a pasture rather than mixed into a farmer-provided ration with other feed components. In this experimental example, the crop is a plant species commonly planted in a managed pasture including monocots such as ryegrass (Lolium sp.), sorghum (Sorghum sp.) and fescue (Festuca sp.), or dicots such as alfalfa (Medicago sp.), clover (Trifolium), and pea (Pisum sp.). The pasture crop may have a perennial or an annual cultivation cycle. Using the regulatory genetic components as described herein, bromoform biosynthesis may be constitutive, limited spatially to aboveground plant parts that grazing animals primarily eat, or limited temporally to daytime when grazing primarily takes place. Bromoform is made at a level corresponding to the proportion of pasture in the animal diet and the proportion of bromoform-bearing species in the pasture.
For example, if ˜25 μg per gram dry matter in total feed is an effective dosage rate for methane elimination in cows, and the forage crop is applied to cattle eating a 100% forage diet at a particular point in time, the forage crop may include a mixture of red clover (Trifolium pratense), perennial ryegrass (Lolium perenne), meadow brome (Bromus commutatus), tall fescue (Festuca arundinacea), and alfalfa (Medicago sativa), where each plant species is engineered to produce ˜25 μg bromoform per gram dry matter in aboveground plant parts.
Alternatively, the forage crop may include a single engineered pasture plant providing a higher level of bromoform that is consumed in a mixture with other unmodified food components. For example, if cattle are eating a diet of 50% forage and 50% farmer-provided rations, and the forage pasture is made up of ˜20% perennial ryegrass, the forage crop may include perennial ryegrass containing ˜250 μg bromoform per gram dry matter such that, when consumed alongside other forage plants and farmer-supplied rations, a sufficient dose of bromoform is provided to achieve maximum methane elimination from the total diet. In this experimental example, the bromoform-bearing plant may be consumed with spatial or temporal separation from other feed sources, while still providing sufficient total bromoform to achieve maximum methane reduction. The efficacy of a methane-reducing forage crop may be demonstrated in a model system comprising feeding engineered forage species to methane-generating insects such as termites, cockroaches, and beetles.
Leaf disks of the grass Setaria viridis A10.1 were transiently transformed with a genetic construct expressing a tricistronic, partially monocot-optimized sequence of AACT2, WRI1, and VBPO (SEQ ID NO: 95) under control of the Arabidopsis thaliana Ubiquitin 10 promoter (SEQ ID NO: 19). Three days after transformation, to simulate methane reduction in grazing livestock consuming a forage plant, transgenic Setaria leaf disks were fed to a highly methanogenic, leaf litter-feeding species of cockroach, Gromphadorhina portentosa, which is able to consume similar foods to cattle and in which methane production occurs in the gut via the same groups of microbes and through the same fermentation process as in cattle. Cockroaches were transferred into chambers containing a methane sensor. The chamber containing the sensor, cockroaches, and food source was sealed for a period of two hours, and methane accumulation at the end of this period was measured. The chamber as unsealed and cockroaches were maintained in the chamber for an additional 44 hours with the transgenic Setaria leaf disks as a sole food source, after which the chamber was again sealed for another 2-hour accumulation period and endpoint methane measurement. Cockroaches consuming untransformed Setaria leaf disks were used as a control, and each treatment group consisted of 3-4 cockroaches weighing ˜20 grams. Accumulated methane in cockroaches consuming the transgenic Setaria leaf disks decreased by 24% over 48 hours, from 3.95 parts per million methane per gram biomass to 3 parts per million methane per gram biomass. In contrast, accumulated methane in cockroaches consuming the control nontransgenic Setaria leaf disks increased by 2% over 48 hours, from 4.7 parts per million methane per gram biomass to 4.8 parts per million methane per gram biomass. The results of this experiment indicate that leaf tissues of a forage crop can be successfully engineered to reduce enteric methane emissions in animals that consume them.
A plant containing bromoform at a very high level is used as a supplement or small fraction of an animal diet to deliver a sufficient dose of bromoform to the animal to eliminate total methane emissions. In this experimental example, the plant may be an existing component of animal feed such as corn, ryegrass, beets, or canola/rapeseed. The plant may also be a species not commonly incorporated into animal feed, but nevertheless is suitable as a component of animal diets, such as duckweed (family Lemnoideae), coral bean (Erythrina beteroana), seepweed (Suaeda sp.), crambe (Crambe sp.), seashore mallow (Kosteletzkya virginica), or saltgrass (Distichlis sp.).
This experimental example may also use a processed bromoform-bearing plant product, for example, freeze-dried plant tissue or oil pressed from seeds. The plant product is included in animal diets according to the amount of bromoform synthesized. For example, if engineered canola seeds contain ˜2,500 μg bromoform per gram plant matter, they may be supplemented to livestock diets at ˜1% of total feed to provide an overall effective dose of ˜25 μg per gram bromoform in total feed.
To determine the methane-suppressing effect of a feed prepared in this manner, transgenic Arabidopsis seed of a variety documented to produce ˜1,653.68 μg per gram bromoform as described in Example 1 was incorporated into a synthetic cattle diet, The synthetic cattle diet consisted of (by mass) 25% ground corn; 40% Setaria hay; 10% ground soybeans; 10% fresh carrot; 5% vegetable oil; and 10% ground Arabidopsis seed made up of transgenic and nontransgenic portions calculated to achieve a specific dosage of bromoform. A version of this diet was prepared to include ˜25 μg per gram total bromoform as 1.5% transgenic Arabidopsis and 8.5% nontransgenic Arabidopsis and was compared to a control containing no bromoform (as 10% nontransgenic Arabidopsis). Each synthetic cattle diet was fed to a highly methanogenic, leaf litter-feeding species of cockroach, Gromphadorhina portentosa, which is able to consume similar foods to cattle and in which methane production occurs in the gut via the same groups of microbes and through the same fermentation process as in cattle. After consuming the control diet for at least 1 week, cockroaches were transferred into chambers containing a methane sensor assembly pre-calibrated against known concentrations of methane, with a restricted gas exchange rate to outside air. This system allowed enteric methane emitted by the cockroaches to accumulate in the chamber, where it was measured by the sensor. ˜ 20 grams of cockroach biomass, consisting of 3-5 individuals, was used as each experimental unit. Cockroaches were maintained in the methane measurement chamber for 1 week with constant access to the either the control or experimental diet, and methane was measured ˜5 times per minute for the duration. Results of this experiment revealed that methane emissions from cockroaches consuming the control diet accumulated to ˜97 parts per million, while emissions from cockroaches consuming the control diet began dropping within 6 hours after introduction of the experimental diet and continued dropping for 5 days until reaching a level of ˜18 parts per million. Accounting for the fact that ambient air contains ˜2 parts per million methane, this represented an 83% reduction in methane emissions from use of the invention described here.
Delivery via plant material within which the compound was natively biosynthesized provides for a novel means of incorporating and distributing bromoform within a livestock diet. Inclusion within plant tissues, as opposed to mixing or supplementation, may alter the way in which bromoform moves through the digestive system and comes in contact with methane-producing microbes during feed digestion. This in turn may reduce the required dosage of bromoform needed to achieve a given level of methane reduction. These factors may also improve the safety profile of bromoform by reducing side effects to the livestock animal, excretion of excess, or accumulation within animal tissue. They may also improve the utility of bromoform by improving its palatability as a component of animal feed, because the total dosage is reduced or because the total amount of bromoform consumed is distributed into the interior of food particles rather than mixed with or coated on the outside.
Experimental versions of the cattle diet as described in Example 8 were prepared as follows. Diet A included ˜25 μg per gram total bromoform (as 1.5% transgenic Arabidopsis and 8.5% nontransgenic Arabidopsis); Diet B included ˜50 μg per gram total bromoform (as 3% transgenic Arabidopsis and 7% nontransgenic Arabidopsis); and Diet C included ˜100 μg per gram total bromoform (as 6% transgenic Arabidopsis and 4% nontransgenic Arabidopsis). Four control diets were additionally prepared as follows. Diet D included no bromoform (as 10% nontransgenic Arabidopsis); Diet E included ˜25 μg per gram total bromoform (as 0.0025% synthetic bromoform diluted into the vegetable oil portion of the diet and 10% nontransgenic Arabidopsis); Diet F included ˜50 μg per gram total bromoform (as 0.005% synthetic bromoform diluted into the vegetable oil portion of the diet and 10% nontransgenic Arabidopsis); and Diet G included ˜100 μg per gram total bromoform (as 0.01% synthetic bromoform diluted into the vegetable oil portion of the diet and 10% nontransgenic Arabidopsis).
One experimental unit (as described in Example 8) per diets A-F was fed on control diet D for at least one week, then transferred to a methane chamber as described in Example 8 and fed on the respective experimental diet for an additional week. Methane accumulation was measured at 0, 6, 48, 96, and 168 hours after introduction of the experimental diet. This experiment was repeated three times.
As shown in
Use of synthetic or Asparagopsis-derived bromoform is significantly hampered by instability of bromoform in these forms, requiring specialized storage conditions and frequent precise dosage. The same limitation extends to non-bromoform based additives as well, including microbial products and synthetic chemicals, which must be delivered, stored, and mixed into cattle feed using special procedures. The ability to store and process grains and plant parts modified with the present invention through the existing agricultural supply chain and feed machinery as a convenient source of bromoform for methane reduction is a significant improvement.
Seed of transgenic Arabidopsis plants expressing the VBPO gene under control of the Napin promoter was subjected to a preliminary stability experiment mimicking supply chain conditions. Freshly harvested mature transgenic seed was divided into four parts. Part A was immediately ground and extracted in methanol as described in Example 1 for quantification of bromoform. Part B was immediately stored at −80° C. in an airtight container. Part C stored for 4 weeks at 25° C. and 70% ambient humidity, with air circulation, and then ground and extracted in methanol for quantification of bromoform as described in Example 1. Part D was stored for 4 weeks at 25° C. and 70% ambient humidity, with air circulation, and then used to prepare a simulated cattle diet containing 5% transgenic plant material as described in Example 9. At the same time as Part D, Part B was removed from cold storage and used to prepare an identical simulated cattle diet. Diets composed using Parts B and D were each fed to a single experimental unit of cockroaches as described in Example 8, and methane was measured at 0 and 72 hours. In cockroaches consuming diets containing 5% ambiently stored transgenic plant parts in Diet D, methane emissions were reduced by 72% after 72 hours.
Oil extracts of Asparagopsis seaweed have been reported to decrease in bromoform content by 44% after four weeks of exposure to air at the environmental conditions used in this experiment, and freeze-dried Asparagopsis decreases in bromoform content by 16% even inside of a sealed bag at the same conditions. Extracted bromoform in samples of Part A and C was quantified by GC-MS at Avazyme, Inc. The results of this experiment are expected to show the amount of bromoform present in transgenic seeds at the time of harvest sampled in Part A, and after a month of storage at ambient conditions sampled in Part C. By comparison of concentrations in Part C and part A, the results of the experiment are expected to reveal the extent to which bromoform is lost over time from transgenic seeds at ambient conditions, and the extent to which delivery of bromoform via plant tissues as described In the present invention offers practical advantages in bromoform stability relative to current alternatives including, for example, oil preparations of seaweed, oil or aqueous preparations of synthetic bromoform, or freeze-dried seaweed.
In some applications, production of particular haloforms is preferred. For example, iodoform may be preferred in some livestock feed applications for having lower volatility than bromoform, an improved safety profile, and/or a wider dosage range. The production of a particular haloform may also be preferred to enable increased accumulation and/or production of the haloform in plants due to lower phytotoxicity or differences in halogen substrate availability within plant tissues or improved species-specific suitability for a desired plant species. Certain haloforms are preferred due to co-benefits in agricultural production, such as antifungal activity during plant growth or storage, or silage quality. To produce iodoform in somatic tissues, Vanadium-dependent iodoperoxidase (VIPO; SEQ ID NO: 41, 43, 45, 47, 49 or 51-60) is expressed under a constitutive or photosynthetic promoter (SEQ ID NO: 19-23, 93) in a monocot or dicot plant. Iodoform is extracted as described for bromoform in Example 3 and quantified by GC-MS. To produce iodoform in seeds, VIPO is expressed under a seed-specific promoter (SEQ ID NO: 24-25, or 94) and quantified as described in Example 1. Seeds or transiently or stably transformed leaf tissue is fed to cockroaches or added to rumen fluid as described in Examples 5, 7, or 8 and used to assess the methane-reducing effect of iodoform biosynthesized within plant tissues.
The ability to synthesize a mixture of haloforms are desirable as a means to maximize usage of all available substrates within plants, improve safety to both plants and animals, improve palatability to animals, and/or improve efficacy of the methane-reducing properties of the transgenic plant. Production of a mixture of haloforms is enabled by expression of a nonspecific haloperoxidase (VHPO; SEQ ID NO: 77, 79, 81, 83, 85, 87, 89, or 91) which accommodates incorporation of any or all of bromine, chlorine, or iodine into haloforms. The VHPO enzyme enables transgenic plants to maintain their methane-reducing properties in diverse settings by using whichever halogen is in greatest abundance at a particular time, agricultural setting, or part of the plant. Production of a mixture of haloforms is enabled by expression of any combination of Vanadium-dependent bromoperoxidase (VBPO, SEQ ID NO: 1 or 3); Vanadium-dependent iodoperoxidase (VIPO; SEQ ID NO: 41, 43, 45, 47, 49, or 51-60); Vanadium-dependent chloroperoxidase (VCPO; SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, or 75); or nonspecific haloperoxidase (VHPO; SEQ ID NO: 77, 79, 81, 83, 85, 87, 89, or 91), and the selection of a particular combination of transgenes may be used to determine the profile of haloforms produced. To assess the production of haloform mixtures by particular combinations of haloperoxidase transgenes, tobacco (Nicotiana tabacum) leaves are transiently transformed with VBPO; VIPO; VCPO, and/or VHPO under control of a constitutive promoter and combined via assembly into a single construct or via co-transformation of multiple constructs. Haloforms may be extracted in methanol as described for bromoform in Example 3 and identified and quantified by GC-MS. To assess the methane-reducing effects of haloform mixtures biosynthesized by particular combinations of haloperoxidase transgenes, transiently or stably transformed leaf tissue or stably transformed seeds of Arabidopsis, Setaria, or corn are fed to cockroaches or added to rumen fluid as described in Examples 5, 7, or 8.
This application claims priority to U.S. Provisional Patent Application No. 63/503,222, filed May 19, 2023; U.S. Provisional Patent Application No. 63/471,102, filed Jun. 5, 2023; and U.S. Provisional Patent Application No. 63/512,776, filed Jul. 10, 2023, each of which are incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63503222 | May 2023 | US | |
| 63471102 | Jun 2023 | US | |
| 63512776 | Jul 2023 | US |
