Patent Application
20210378263
This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled “222107_2230_seq_listing_ST25.txt”, created on Oct. 25, 2019. The content of the sequence listing is incorporated herein in its entirety.
Losses in honey bee (Apis mellifera) colonies are estimated to be ˜40% annually, warranting robust research to curb the economic and ecological damage. According to the 2017 USDA Annual Report, honey bees “ . . . add at least $15 billion to the value of U.S. agriculture annually through increased yields and superior-quality harvests.”
Losses are believed to be in part attributed to colony collapse disorder (CCD), an umbrella name defining a syndrome more than a specific disease, as well as to other “disappearing diseases”. If left unattended, this can have significant impact in sustaining current agricultural practices as well as having negative environmental consequences.
There is debate regarding the responsible etiological agent(s), be these chemical or biological in nature. There is a substantial agreement, however, that the causes of the losses are attributable to three large categories of stressors: 1) the spread of pathogens and pests fostered by anthropic commercial activities; 2) the lack of a constant supply of food source throughout the year; and 3) the widespread use of pesticides both in domestic and agricultural settings. Strategies to mitigate and/or reverse losses in honey bees and other pollinators are currently lacking.
Accordingly, there is a need to address the aforementioned deficiencies and inadequacies.
Described herein are engineered cells. In certain aspects, engineered cells can comprise a bacterium containing therein one or more stably-expressing expression vectors for exogenous expression of one or more recombinant carboxylesterase enzymes, thereby providing the engineered cell an exogenous pathway for hydrolyzing ester bonds. The bacterium can be a bacteria of the genus Escherichia, Lactobacillus, Fructobacillus, Gilliamella, Snodgrassella, Actinobacteria, Parasaccharibacter. Pseudomonas, Stenotrophomonas, Sphigobacterium, or Bacillus.
In certain embodiments, one or more stably-expressing vectors according to the present disclosure can comprise one or more coding sequences for a recombinant carboxylesterase or one or more coding sequences for a neonicotinoid and/or neonicotinoid metabolite degradation recombinant enzyme.
In certain embodiments, the recombinant carboxylesterase enzymes can be Methyl Parathion Hydrolase (MPH), Pyrethroid Hydrolase (PytH), E3 carboxylesterase, E3Trp251Leu carboxylesterase, Deinococcus radiodurans phosphotriesterase, or Sulpholobus acidocaldarius phosphotriesterase. In certain aspects, recombinant carboxylesterase enzymes as described herein, such as Deinococcus radiodurans phosphotriesterase, or Sulpholobus acidocaldarius phosphotriesterase, are thermostable recombinant carboxylesterase enzymes. In certain embodiments, the recombinant carboxylesterase enzymes can comprise or be encoded by sequences comprising one or more of sequences 90% to 100% identical to SEQ ID NOs: 1-5.
Described herein is modified bee bread. In embodiments according to the present disclosure, modified bee bread as described herein can comprise isolated wild-type or synthetic bee bread, one or more purified recombinant carboxylesterases, one or more purified neonicotinoid and/or neonicotinoid metabolite degradation recombinant enzymes, one or more engineered cells, or any combination thereof. The one or more purified recombinant carboxylesterases, one or more purified neonicotinoid and/or neonicotinoid metabolite degradation recombinant enzymes, or both can be present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The one or more engineered cells can be present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The one or more insecticides can be one or more insecticides from the pyrethroid, synthetic pyrethroid, organophosphate, or neonicotinoid class of insecticides.
Described herein are methods of providing protection from insecticides and/or methods of providing a subject in need thereof protection from insecticides. Methods as described herein can comprise, in certain aspects, providing a bee bread (wild-type or synthetic); providing one or more purified recombinant carboxylesterases, one or more purified neonicotinoid and/or neonicotinoid metabolite degradation recombinant enzymes, one or more engineered cells, or any combination thereof; and introducing one or more purified recombinant carboxylesterases, one or more purified neonicotinoid and/or neonicotinoid metabolite degradation recombinant enzymes, one or more engineered cells, or any combination thereof into the bee bread. The one or more purified recombinant carboxylesterases, one or more purified neonicotinoid and/or neonicotinoid metabolite degradation recombinant enzymes, or both can be present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The one or more engineered cells can be present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The one or more insecticides can be one or more insecticides from the pyrethroid, synthetic pyrethroid, organophosphate, or neonicotinoid class of insecticides.
Also described herein are kits, comprising: one or more bacterial cells; and one or more expression vectors comprising one or more recombinant carboxylesterase coding sequences and/or one or more coding sequences for a neonicotinoid and/or neonicotinoid metabolite degradation recombinant enzyme.
In certain aspects, described herein is a method of creating an engineered cell, comprising: providing wild-type bee bread; isolating one or more bacteria from the wild-type bee bread; introducing one or more expression vectors comprising one or more recombinant carboxylesterase coding sequences and/or one or more coding sequences for a neonicotinoid and/or neonicotinoid metabolite degradation recombinant enzyme into the one or more bacteria; selecting one or more stably-expressing recombinant carboxylesterase bacteria after the one or more expression vectors are introduced; and isolating the stably-expressing recombinant carboxylesterase bacteria.
Described herein are methods of treating a subject for pesticide exposure, the methods comprising delivering a modified bee bread as described herein to a subject in need thereof. The modified bee bread can be delivered to the subject in an amount effective to reduce the active amount of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The one or more insecticides are one or more insecticides from the pyrethroid, synthetic pyrethroid, organophosphate, or neonicotinoid class of insecticides.
In further aspects, described herein are engineered cells, comprising a bacterium containing therein one or more stably-expressing expression vectors for exogenous expression of one or more recombinant oxalate decarboxylase enzymes, thereby providing the engineered cell an exogenous pathway for removing carboxyl groups. The bacterium can be a bacteria of the genus Escherichia, Lactobacillus, Fructobacillus, Gilliamella, Snodgrassella, Actinobacteria, Parasaccharibacter. Pseudomonas, Stenotrophomonas, Sphigobacterium, or Bacillus. The one or more stably-expressing vectors can comprise one or more coding sequences for a recombinant oxalate decarboxylase. The oxalate decarboxylase can be a B. subtilis, Burkholderia pseudomallei, or Pantoea allii oxalate decarboxylase. In an embodiment, the oxalate decarboxylase is a B. subtilis oxalate decarboxylase. In an embodiment, the oxalate decarboxylase is a Burkholderia pseudomallei oxalate decarboxylase. In an embodiment, the oxalate decarboxylase is a Pantoea allii oxalate decarboxylase.
The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NOs: 6-11. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO: 6. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO: 7. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO: 8. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO: 9. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO: 10. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO: 11.
In embodiments, described herein are modified bee breads, comprising bee bread; and one or more purified recombinant oxalate decarboxylases, one or more engineered cells, or both. In certain aspects, the bee bread can be wild-type bee bread. In certain aspects, the bee bread can be synthetic bee bread. The one or more purified recombinant oxalate decarboxlyases are B. subtilis, Burkholderia pseudomallei, or Pantoea allii oxalate decarboxylases. In an embodiment, the oxalate decarboxylase is a B. subtilis oxalate decarboxylase. In an embodiment, the oxalate decarboxylase is a Burkholderia pseudomallei oxalate decarboxylase. In an embodiment, the oxalate decarboxylase is a Pantoea allii oxalate decarboxylase.
In certain aspects, one or more purified recombinant oxalate decarboxylases are present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first.
In certain aspects, the one or more engineered cells are present in an amount effective to reduce the amount of active ingredient of oxalic acid from a first level to a second level, wherein the second level is less than the first.
Described herein are additional embodiments of methods of providing protection from insecticides, comprising providing a bee bread; providing one or more purified recombinant oxalate decarboxylases, one or more engineered cells, or both; and introducing the one or more purified recombinant oxalate decarboxylases, one or more engineered cells, or both into the bee bread. Such a bee bread can be provided to a subject in need thereof, a plurality of subjects in need thereof, or a hive or a subject in need thereof. In certain aspects, the bee bread can be wild-type bee bread. In certain aspects, the bee bread can be synthetic bee bread. One or more purified recombinant oxalate decarboxlyases according to methods as described herein can be B. subtilis, Burkholderia pseudomallei, or Pantoea allii oxalate decarboxylases. In an embodiment, the oxalate decarboxylase is a B. subtilis oxalate decarboxylase. In an embodiment, the oxalate decarboxylase is a Burkholderia pseudomallei oxalate decarboxylase. In an embodiment, the oxalate decarboxylase is a Pantoea allii oxalate decarboxylase. The one or more oxalate carboxylesterases are present in an amount effective to reduce the amount of active ingredient of of one or more insecticides from a first level to a second level, wherein the second level is less than the first level. The one or more engineered cells are present in an amount effective to reduce the amount of active ingredient of oxalic acid from a first level to a second level, wherein the second level is less than the first level.
Described herein are additional embodiments of kits, comprising one or more bacterial cells and one or more expression vectors comprising one or more recombinant oxalate decarboxylase coding sequences. The one or more coding sequences have about 90% to about 100% sequence identify with SEQ ID NOs 6-11. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO. 6. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO. 7. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO. 8. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO 9. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO. 10. The one or more stably-expressing vectors can comprise one or more coding sequences with about 90% to about 100% sequence identify with SEQ ID NO. 11.
Described herein are additional embodiments of methods of creating an engineered cell, comprising providing wild-type bee bread, isolating one or more bacteria from the wild-type bee bread, introducing one or more expression vectors comprising one or more recombinant oxalate decarboxylase coding sequences into the one or more bacteria, selecting one or more stably-expressing recombinant oxalate decarboxylase bacteria after the one or more expression vectors are introduced; and isolating the stably-expressing recombinant oxalate decarboxylase bacteria. Described herein are also cells created by this method.
Described herein are additional embodiments of methods of treating a subject for pesticide exposure, the method comprising delivering a modified bee bread as described herein to a subject in need thereof. The modified bee bread can be delivered to the subject in an amount effective to reduce the active amount of of one or more insecticides from a first level to a second level, wherein the second level is less than the first.
In further aspects, described herein are additional embodiments of engineered cells. In an embodiment, an engineered cells comprises a bacterium containing therein one or more stably-expressing expression vectors for exogenous expression of one or more recombinant 6-chloronicotinic acid degradation enzymes, thereby providing the engineered cell an exogenous pathway for degrading 6-chloronicotinic acid. In certain aspects, the bacterium can be a bacteria of the genus Escherichia, Lactobacillus, Fructobacillus, Gilliamella, Snodgrassella, Actinobacteria, Parasaccharibacter, Pseudomonas, Stenotrophomonas, Sphigobacterium, or Bacillus. The one or more stably-expressing vectors can comprise one or more coding sequences for a recombinant 6-chloronicotinic acid chlorohydrolase (cch2) or a cassette comprising a a recombinant 6-chloronicotinic acid chlorohydrolase (cch2) fused to a transporter. The recombinant 6-chloronicotinic acid chlorohydrolase can be a Bradyrhizobiaceae bacterium cch2. The one or more stably-expressing vectors can comprise one or more coding sequences encoding cch2 with about 90% to about 100% sequence identify with the cch2 sequence of SEQ ID NOs: 12-13.
In additional aspects, described herein are a modified bee bread, comprising bee bread; and one or more purified recombinant 6-chloronicotinic acid chlorohydrolases (cch2), one or more engineered cells, or both. The bee bread can be a wild-type bee bread. The bee bread can be a synthetic bee bread. The one or more purified recombinant 6-chloronicotinic acid chlorohydrolases can be a Bradyrhizobiaceae bacterium 6-chloronicotinic acid chlorohydrolase. The one or more purified recombinant 6-chloronicotinic acid chlorohydrolases can fused to a transporter on the n- or c-terminus.
The one or more purified recombinant 6-chloronicotinic acid chlorohydrolases can be present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first.
The one or more engineered cells can be present in an amount effective to reduce the amount of 6-CNA from a first level to a second level, wherein the second level is less than the first.
Described herein are embodiments of methods of providing protection from insecticides, comprising providing a bee bread, providing one or more purified recombinant 6-chloronicotinic acid chlorohydrolases, one or more engineered cells, or both, and introducing the one or more purified recombinant 6-chloronicotinic acid chlorohydrolases, one or more engineered cells, or both into the bee bread. The bee bread can be a wild-type bee bread. The bee bread can be a synthetic bee bread. The one or more purified recombinant 6-chloronicotinic acid chlorohydrolases can be a Bradyrhizobiaceae bacterium 6-chloronicotinic acid chlorohydrolase. The one or more 6-chloronicotinic acid chlorohydrolases can be present in an amount effective to reduce the amount of active ingredient of of one or more insecticides from a first level to a second level, wherein the second level is less than the first level. The one or more engineered cells can be present in an amount effective to reduce the amount of 6-CNA from a first level to a second level, wherein the second level is less than the first level.
Described herein are further embodiments of kits, comprising one or more bacterial cells, and one or more expression vectors comprising one or more recombinant 6-chloronicotinic acid chlorohydrolase coding sequences. The one or more coding sequences can have about 90% to about 100% sequence identify with the 6-chloronicotinic acid chlorohydrolase sequence of SEQ ID NOs: 12-13. A coding sequence for a transporter can also be contained therein.
Further described herein are methods of creating an engineered cell, comprising providing wild-type bee bread, isolating one or more bacteria from the wild-type bee bread, introducing one or more expression vectors comprising one or more recombinant 6-chloronicotinic acid chlorohydrolase coding sequences into the one or more bacteria, selecting one or more stably-expressing recombinant 6-chloronicotinic acid chlorohydrolase bacteria after the one or more expression vectors are introduced, and isolating the stably-expressing recombinant 6-chloronicotinic acid chlorohydrolase bacteria. Described herein are engineered cells created by methods as described herein.
Described herein are additional embodiments of methods of treating a subject for pesticide exposure, the method comprising delivering a modified bee bread as described herein to a subject in need thereof, or a hive of a subject in need thereof.
According to embodiments of the present disclosure, the modified bee bread can be delivered to the subject in an amount effective to reduce the active amount of of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The cell can further comprise a 6-HNA degradation operon. A cell with a functional 6-HNA degradation operon such as those described herein can also be added to bee bread as described herein. In an embodiment, the modified bee bread can further comprise one or more engineered cells expressing a 6-HNA degradation operon. In embodiments, the one or more engineered cells express a 6-HNA degradation operon or one or more genes of such according to the examples below. The one or more cells can comprise a 6-HNA degradation operon. Embodiments of methods can further comprise introducing one or more expression vectors encoding one or more recombinant gene products of a 6-HNA degradation operon. The one or more expression vectors encoding one or more recombinant gene products of a 6-HNA degradation operon comprise one or more recombinant nucleotide sequences encoding one or more or SEQ ID. NOs: 14-24.
Many aspects of the disclosed devices and methods can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the relevant principles. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.
Pseudomonas putida KT2440 engineered with a construct (pGnMM105) bearing the cassette borne on pGnMM83 (
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of melittology, apiology, microbiology, molecular biology, toxicology, organic chemistry, analytical chemistry, and biochemistry.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is in atmosphere. Standard temperature and pressure are defined as 25° C. and 1 atmosphere.
Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
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 of molecular biology, medicinal chemistry, and/or organic chemistry. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.
As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
As used herein, a “subject” can be a member of the Apidae family of bees. In certain embodiments, a subject is a bee of the Apis or Bombus genus. A “subject in need thereof” can be a subject as described herein (a bee of the Apis or Bombus genus) that is subjected to, or at risk for, pesticide exposure. In certain embodiments, a “subject in need thereof” is a honey bee (Apis mellifera) that is subjected to, or at risk for, pesticide exposure.
As used herein, “pesticide” or “insecticide” can refer to a compound of the organophostphate, pyrethroid, or neonicotinoid class of insecticides.
As used herein, “control” is an alternative subject or sample used in an experiment for comparison purposes and included to minimize or distinguish the effect of variables other than an independent variable.
As used herein, “overexpressed” or “overexpression” refers to an increased expression level of an RNA or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell.
As used herein, “expression” refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins.
As used herein, “nucleic acid” and “polynucleotide” generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. “Polynucleotide” and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. For instance, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. “Polynucleotide” and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acids” or “polynucleotide” as that term is intended herein.
As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” generally refer to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.
As used herein, “nucleic acid sequence” and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.
As used herein, “DNA molecule” includes nucleic acids/polynucleotides that are made of DNA.
As used herein, “wild-type” is the typical form of an organism, variety, strain, gene, protein, or characteristic as it occurs in nature, as distinguished from mutant forms that may result from selective breeding or transformation with a transgene.
As used herein, “identity,” is a relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math. 1988, 48: 1073. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch (J. Mol. Biol., 1970, 48: 443-453) algorithm (e.g., NBLAST, and XBLAST). The default parameters are used to determine the identity for the polypeptides or polynucleotides of the present disclosure.
As used herein, “heterologous” refers to compounds, molecules, nucleotide sequences (including genes), and polypeptide sequences (including peptides and proteins) that are different in both activity (function) and sequence or chemical structure. As used herein, “heterologous” can also refer to a gene or gene product that is from a different organism. For example, a human GTP cyclohydrolase or a synthase can be said to be heterologous when expressed in yeast.
As used herein, “homologue” refers to a polypeptide sequence that shares a threshold level of similarity and/or identity as determined by alignment of matching amino acids. Two or more polypeptides determined to be homologues are said to be homologues. Homology is a qualitative term that describes the relationship between polypeptide sequences that is based upon the quantitative similarity.
As used herein, “paralog” refers to a homologue produced via gene duplication of a gene. In other words, paralogs are homologues that result from divergent evolution from a common ancestral gene.
As used herein, “orthologues” refers to homologues produced by speciation followed by divergence of sequence but not activity in separate species. When speciation follows duplication and one homologue sorts with one species and the other copy sorts with the other species, subsequent divergence of the duplicated sequence is associated with one or the other species. Such species specific homologues are referred to herein as orthologues.
As used herein, “xenologs” are homologues resulting from horizontal gene transfer.
As used herein, “similarity” is a quantitative term that defines the degree of sequence match between two compared polypeptide sequences.
As used herein, “cell,” “cell line,” and “cell culture” include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.
As used herein, “culturing” refers to maintaining cells under conditions in which they can proliferate and avoid senescence as a group of cells. “Culturing” can also include conditions in which the cells also or alternatively differentiate.
As used herein, “organism”, “host”, and “subject” refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single isolated eukaryotic cell or cultured cell or cell line, or as complex as a mammal, including a human being, and animals (e.g., arthropods, vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans). “Subject” may also be a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.
As used herein, “gene” refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. As used herein, “synthetic gene” can refer to a recombinant gene comprising one or more coding sequences for a protein of interest, or a synthetically purified protein that is not naturally occurring in its purified state.
As used herein, the term “recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc.). Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.
As used herein, “plasmid” as used herein refers to a non-chromosomal double-stranded DNA sequence including an intact “replicon” such that the plasmid is replicated in a host cell.
As used herein, the term “vector” or “expression vector” is used in reference to a vehicle used to introduce an exogenous nucleic acid sequence into a cell. A vector may include a DNA molecule, linear or circular (e.g. plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of both. As one of skill in the art would understand, recombinant coding sequences as described herein, for example any of the nucleotide sequences of (or nucleotide sequences encoding peptide sequences of) SEQ ID NOs: 1-24, can be cloned into a host-appropriate expression vector (also referred to herein as a backbone, using molecular cloning methods as known in the art) that is suitable for driving expression of the desired coding sequence (for example any of SEQ ID NOs: 1-24, or recombinant sequences encoding the peptide sequences of any of the nucleotide sequences of SEQ ID NOs: 1-24) in a desired host under the control of appropriate genetic elements (promoters and terminators, which can readily be determined by the skilled artisan according to desired expression levels in said host). An example of such a suitable vector which can be used as a host expression vector to express sequences as described herein is plasmid pBBR1MCS-2, available from Addgene®, and coding sequences can be cloned into the vector as a HindIII-BamHI insert, for example. Other examples of suitable vectors include a pUC backbone cloning vector, or a pMAL vector (available from New England Biolabs®). Further examples include vectors for the transformation of Bacilli such as pNW33n (available from the Bacillus Genetic Stock Center, BGSC). Furthermore, it is noted that expression vectors can contain suitable constitutive or inducible promotors to drive expressing of coding sequences as described herein, examples of promoters being known in the art and described herein.
As used herein, “operatively linked” or “operatively coupled” indicates that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition can also be applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements), and/or selectable markers in an expression vector.
As used herein, “cDNA” refers to a DNA sequence that is complementary to a RNA transcript in a cell. It is a man-made molecule. Typically, cDNA is made in vitro by an enzyme called reverse-transcriptase using RNA transcripts as templates.
As used herein, the term “transfection” refers to the introduction of an exogenous and/or recombinant nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus, or chloroplast. The nucleic acid may be in the form of naked DNA or RNA, it may be associated with various proteins or regulatory elements (e.g., a promoter and/or signal element), or the nucleic acid may be incorporated into a vector or a chromosome.
As used herein, “transformation” or “transformed” refers to the introduction of a nucleic acid (e.g., DNA or RNA) into cells in such a way as to allow expression of the coding portions of the introduced nucleic acid.
As used herein, “stable expression,” “stable incorporation,” “stable transfection” and the like refer to the integration of an exogenous gene into the genome of a host cell, which can allow for long term expression of the exogenous gene.
As used herein, “transient expression,” “transient transfection,” and the like refer to the introduction of an exogenous gene into a host cell that does not result in stable incorporation of the gene into the host cell.
As used herein “chemical” refers to any molecule, compound, particle, or other substance that can be a substrate for an enzyme in the enzymatic pathway described herein and/or a carboxylesterase enzyme or biochemical pathway. A “chemical” can also be used to refer to a metabolite of a carboxylic ester. As such, “chemical” can refer to nucleic acids, proteins, organic compounds, inorganic compounds, metabolites etc. “Chemical” can also refer to the product produced by the carboxylesterase action.
As used herein “biologically coupled” refers to the association of or interaction between two or more physically distinct molecules, groups of molecules compounds, organisms, or particles where the association is directly or indirectly mediated between the two or more physically distinct molecules, groups of molecules compounds, organisms or particles via a biologic molecule or compound. This can include direct binding between two biologic molecules and signal transduction pathways.
As used herein, “biological communication” refers to the communication between two or more molecules, compounds, or objects that is mediated by a biologic molecule or biologic interaction.
As used herein, “biologic molecule,” “biomolecule,” and the like refer to any molecule that is present in a living organism and includes without limitation, macromolecules (e.g. proteins, polysaccharides, lipids, and nucleic acids) as well as small molecules (e.g. metabolites and other products produced by a living organism).
As used herein, “regulation” refers to the control of gene or protein expression or function.
As used herein, “promoter” refers to the DNA sequence(s) that control or otherwise modify transcription of a gene and can include binding sites for transcription factors, RNA polymerases, and other biomolecules and substances (e.g. inorganic compounds) that can influence transcription of a gene by interaction with the promoter. Typically these sequences are located at the 5′ end of the sense strand of the gene, but can be located anywhere in the genome.
As used herein, “native” refers to the endogenous version of a molecule or compound relative to the host cell or population being described.
As used herein, “non-naturally occurring” refers to a non-native version of a molecule or compound or non-native expression or presence of a molecule or compound within a host cell or other composition. This can include where a native molecule or compound is influenced to be expressed or present at a different location within a host, at a non-native period of time within a host, or is otherwise in an altered environment, even when considered within the host. Non-limiting examples include where a protein that is expressed only in the nucleus of a cell is expressed in the cytoplasm of the cell or when a protein that is only normally expressed during the embryonic stage of development is expressed during the adult stage.
As used herein, “encode” refers to the biologic phenomena of transcribing DNA into an RNA that, in some cases, can be translated into a protein product. As such, when a protein is said herein to be encoded by a particular nucleotide sequence, it is to be understood that this refers to this biologic relationship between DNA and protein. It is well established that RNA can be translated into protein based on the triplet code where 3 nucleotides represent an amino acid. This term also includes the idea that DNA can be transcribed into RNA molecules with biologic functions, such as ribozymes and interfering RNA species. As such, when a RNA molecule is said to be encoded by a particular nucleotide sequence it is to be understood that this is referring to the transcriptional relationship between the DNA and RNA species in question. As such “encoding nucleotide” refers to herein as the nucleotide which can give rise through transcription, and in the case of proteins, translation a functional RNA or protein.
As used herein “codon optimized” or “codon optimization” refers to a codon modification or making modifications to the codons for amino acids in a polypeptide such that they reflect the codon usage bias of the cell type that the polypeptide is expressed in. Modifications to the codons can be made using techniques generally known in the art.
As used herein, “derived” or “derived from” means copied from or modified from a starting material to a sequence or composition that is different than the starting material (not exactly the same as but retaining characteristics of, in terms of sequence, structure, concentration, quantity, etc), while maintaining the desired biological activity (for example enzymatic activity, nutritional function, etc). For example, a sequence that is derived from a parent sequence maintains sequences necessary for encoding or expression of a nucleotide that confers a biological function (such as enzymatic activity), but is altered in a way that the overall sequence of the derived sequence is not exactly identical to the parent sequence.
Described herein are compositions and methods relating to pesticide degradation. Compositions and methods relating to pesticide degradation can comprise the exogenous expression of one or more recombinant carboxylesterase enzymes, oxalate decarboxylase enzymes, and/or one or more enzymes related to neonicotinoid (or neonicotinoid metabolite, such as 6-chloronicotonic acid, 6-CNA) degradation.
Compositions and methods as described herein serve as improvements upon existing technologies in the state of the art, in part, by application of enzymatic remediation strategies to the hive environment (for example the hive environment of a bee of the Apis or Bombus genus) and modification of the bee gut microbiota. Such application of remediation strategies to the hive environment in particular is a practical application that provides pollinator insects with defenses against commonly used pesticides, the effects of which on pollinator insects have been devastating to pollinator colonies residing in such hives.
In additional aspects, such bacteria can also be selected and amplified from the milieu of the hive microorganisms and in some cases they can be molecularly bred to enhance their metabolic capabilities without genetic engineering and reintroduced to the hive at a level or amount significantly greater than the natural-occurring level or amount.
Compositions and methods as described herein comprise recombinant carboxylesterase enzymes, oxalate decarboxylase enzymes, or recombinant neonicotinoid (or neonicotinoid metabolite) degrading enzymes. Recombinant carboxylesterase enzymes, oxalate decarboxylase enzymes, or neonicotinoid (or neonicotinoid metabolite) degrading enzymes can be expressed in engineered cells as described herein, can be purified and introduced into or otherwise mixed with bee bread (synthetic or wild-type), or both. Such engineered cells can also be added to the hive directly. In certain aspects, recombinant carboxylesterase enzymes, oxalate decarboxylase enzymes, or neonicotinoid (or neonicotinoid metabolite) degrading enzymes are present in the engineered cell[s], the bee bread, or both in an amount effective to reduce the active ingredient of an insectide (for example a pyrethroid, synthetic pyrethroid, organophosphate, or neonicotinoid) from a first level to a second level, where the second level is less than the first level.
Recombinant carboxylesterase enzymes as described herein are enzymes that catalyze the hydrolysis of carboxylic esters. In certain aspects, recombinant carboxylases as described herein are derived from insects. In certain aspects, recombinant carboxylases as described herein are derived from thermophilic bacteria.
In certain embodiments as described herein, a recombinant carboxylesterase can be Methyl Parathion Hydrolase (MPH), Pyrethroid Hydrolase (PytH), or E3 carboxylesterase, or derivatives there.
In an embodiment, a carboxylesterase as described herein is Methyl Parathion Hydrolase (MPH), an OP carboxylesterase, originally isolated from Plesiomonas sp. M6 (33), GenBank accession No DQ677027).
In an embodiment, a carboxylesterase as described herein is Pyrethroid Hydrolase (PytH) (GenBank accession No FJ688006), encoded by the PytH gene from Sphingobium sp. JZ-1.
In an embodiment, a carboxylesterase as described herein is a naturally-occurring variant (Trp251Leu) of the E3 carboxylesterase (wild-type allele GenBank: AAB67728) from the blowfly Lucilia cuprina.
In an embodiment, a carboxylesterase as described herein is Deinococcus radiodurans phosphotriesterase (Genbank accession #3GTF_A). In an embodiment, a carboxylesterase is Sulpholobus acidocaldarius phosphotriesterase (Genbank accession #WP_011278935).
Embodiments of nucleotide coding sequences of recombinant carboxylesterases as described herein are shown in the examples below, for example in Example 3 and Example 4 below. Recombinant carboxylesterases as described herein can be codon-optimized according to the host in which they are to be expressed. Furthermore, in certain aspects, recombinant carboxylesterases can be have coding sequences that retain sequence identify of about 90% to about 100% to embodiments of sequences as described herein, for example SEQ. ID. NOs 1-5.
Recombinant oxalate decarboxylase (ODC) enzymes as described herein are enzymes are manganese-containing, multimeric enzymes of the cupin protein superfamily. ODC is one of the three enzymes identified to decompose oxalic acid and oxalate, and within ODC catalysis, oxalate is split into formate and CO2. In certain aspects, recombinant oxalate decarboxylases as described herein are derived from fungi and/or bacteria. In certain aspects, recombinant carboxylases as described herein are derived from thermophilic bacteria.
In certain embodiments as described herein, a recombinant oxalate decarboxylase can be B. subtilis, Burkholderia pseudomallei, or Pantoea allii oxalate decarboxylase, or derivatives thereof.
Embodiments of nucleotide coding sequences of recombinant oxalate decarboxylases as described herein are shown in the examples below, for example in Example 7 and 9 below. Recombinant oxalate decarboxylases as described herein can be codon-optimized according to the host in which they are to be expressed. Furthermore, in certain aspects, recombinant oxalate decarboxylases can be have coding sequences that retain sequence identify of about 90% to about 100% to embodiments of sequences as described herein, for example SEQ. ID. NOs 6-11.
Examples of neonicotinoid (or neonicotinoid metabolite, for example 6-CNA) degrading enzymes are described, for example, in Example 5 and
Described herein are expression vectors for the expression of recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes as described herein. In certain embodiments, expression vectors as described herein are plasmid expression vectors comprising a coding sequence for one or more recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes as described herein. Furthermore, in certain aspects, recombinant neonicotinoid (or neonicotinoid metabolite) degrading enzymes can be have coding sequences that retain sequence identify of about 90% to about 100% to embodiments of cch2 sequences as described herein, for example SEQ. ID. NOs 12-13.
In certain embodiments, coding sequences and/or expression vectors for the expression of recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes can also comprise a coding sequence for a signal peptide for enzyme secretion. In an embodiment, a recombinant cch2 is fused with a sequence encoding a transport sequence (at either the 5′ or 3′ end of the encoding mRNA or N- or C-terminus of the resultant peptide). Such examples are known in the art.
Described herein are engineered cells. Engineered cells as described herein can be gram positive (+) or gram negative bacteria (−). Engineered cells as described herein can comprise expression vectors for the expression of recombinant carboxylases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes as described herein. Engineered cells can express exogenous recombinant carboxylesterases (in other words, express recombinant carboxylesterases that are not expressed under wild-type normal physiological conditions), or can otherwise express recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes at a level above normal wild-type physiological expression. Engineered cells can be engineered with expression vectors comprising one or more coding sequences for recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes according to known methods of the art, for example transformation by electroporation and/or conjugation.
In certain aspects, engineered cells as described herein are derived from bacteria of the genus Lactobacillus. In an embodiment, an engineered cell as described herein can be a derivative of the species Lactobacillus kunkeei, L. lactis, L. kullabergensis, L. melliventris, L. kimbladii, L. helsinborgensis, L. mellis, L. sp. AcjLac18.
In certain aspects, engineered cells as described herein are derived from bacteria of the genus Fructobacillus. In an embodiment, an engineered cell as described herein can be a derivative of the species Fructobacillus fructosus.
In certain aspects, engineered cells as described herein are derived from bacteria of the genus Gilliamella. In an embodiment, an engineered cell as described herein can be a derivative of the species Gilliamella apicola.
In certain aspects, engineered cells as described herein are derived from bacteria of the genus Snodgrassella. In an embodiment, an engineered cell as described herein can be a derivative of the species Snodgrassella alvi.
In certain aspects, engineered cells as described herein are derived from bacteria of the genus Actinobacteria. In an embodiment, an engineered cell as described herein can be a derivative of the species Bifidobacterium asteroides or B. coryneforme.
In certain aspects, engineered cells as described herein are derived from bacteria of the genus Parasaccharibacter. In an embodiment, an engineered cell as described herein can be a derivative of the species Parasaccharibacter apium.
In certain aspects, engineered cells as described herein are derived from bacteria of the genus Bacillus. In an embodiment, an engineered cell as described herein can be a derivative of the Bacillus subtilis group (Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis), Bacillus megaterium, or Bacillus licheniformis. In an embodiment expression of the aforementioned enzymes is intracellular, in another embodiment expression can be extracellular, in a further embodiment, the gene(s) of interest is expressed in the spores or on their surface.
In certain aspects, engineered cells as described herein are derived from bacteria of the genus Pseudomonas, Stenotrophomonas, and/or Sphigobacterium.
Also described herein are modified bee breads. Modified bee breads can comprise bee bread, pollen and carbohydrates, or pollen and carbohydrates; and one or more purified recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes according to the present disclosure, one or more engineered cells according to the present disclosure, or both. The bee bread can be wild-type bee bread. The bee bread can be synthetic bee bread. The one or more purified recombinant carboxlyesterases can be Methyl Parathion Hydrolase (MPH), Pyrethroid Hydrolase (PytH), E3 carboxylesterase, E3Trp251Leu carboxylesterase, Deinococcus radiodurans phosphotriesterase, or Sulpholobus acidocaldarius phosphotriesterase. The modified bee bread can contain one or more purified recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The modified bee bread can comprise one or more engineered cells present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The one or more insecticides are one or more insecticides from the pyrethroid, synthetic pyrethroid, organophosphate, or neonicotinoid class of insecticides.
Described herein are methods of conferring protection from insecticides to one or more subjects. Methods as described herein can comprise providing a bee bread; providing one or more purified recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes according to the present disclosure, one or more engineered cells according to the present disclosure, or both; and introducing the one or more purified recombinant carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes according to the present disclosure, one or more engineered cells according to the present disclosure, or both into the bee bread. The bee bread according to methods of the present disclosure can be wild-type bee bread. The bee bread according to methods of the present disclosure can be synthetic bee bread (comprising about 5% to about 55% pollen and about 5% to about 25% sugar, such as fructose). One or more purified recombinant carboxlyesterases according to the present disclosure can be Methyl Parathion Hydrolase (MPH), Pyrethroid Hydrolase (PytH), E3 carboxylesterase, E3Trp251Leu carboxylesterase, Deinococcus radiodurans phosphotriesterase, or Sulpholobus acidocaldarius phosphotriesterase. One or more carboxylesterases or neonicotinoid (or neonicotinoid metabolite) degrading enzymes according to methods of the present disclosure can be present in an amount effective to reduce the amount of active ingredient of one or more insecticides from a first level to a second level, wherein the second level is less than the first level. One or more engineered cells according to methods of the present disclosure can be present in an amount effective to reduce the amount of active ingredient one or more insecticides from a first level to a second level, wherein the second level is less than the first level.
Also described herein are methods of creating an engineered cell. Methods of creating an engineered cell as described herein can comprise providing wild-type bee bread; isolating one or more bacteria from the wild-type bee bread; introducing one or more expression vectors comprising one or more recombinant carboxylesterase or neonicotinoid (or neonicotinoid metabolite) degrading enzyme coding sequences into the one or more bacteria; selecting one or more stably-expressing recombinant carboxylesterase or neonicotinoid (or neonicotinoid metabolite) degrading enzyme bacteria after the one or more expression vectors are introduced; and isolating the stably-expressing recombinant carboxylesterase or neonicotinoid (or neonicotinoid metabolite) degrading enzyme bacteria.
Further described herein are engineered cells created by any of the methods as described herein.
Also described herein are methods of treating a subject for pesticide exposure. Methods of treating a subject for pesticide exposure can comprise delivering a modified bee bread according to the present disclosure to a subject in need thereof. The modified bee bread can be delivered to the subject in an amount effective to reduce the active amount of one or more insecticides from a first level to a second level, wherein the second level is less than the first. The one or more insecticides according to methods of treatment as described herein can be one or more insecticides from the pyrethroid, synthetic pyrethroid, organophosphate class of insecticides, or neonicotinoid class of insecticides. A “subject in need thereof” according to methods of treatment of the present disclosure can be a subject as described herein (a bee of the Apis or Bombus genus) that is subjected to, or at risk for, pesticide exposure. In certain embodiments, a “subject in need thereof” is a honey bee that is subjected to, or at risk for, pesticide exposure.
Kits
Also provided herein are kits containing a cell or population thereof (homogeneous or heterogeneous) which are to express one or more recombinant carboxylases, oxalate decarboxylases, or neonicotinoid (or neonicotinoid metabolite) degrading enzymes as described herein, and/or one or more vectors configured to express one or more recombinant carboxylases, oxalate decarboxylases, or neonicotinoid (or neonicotinoid metabolite) degrading enzymes as described herein. In an embodiment, one or more vectors configured to express one or more recombinant carboxylases oxalate decarboxylases, or neonicotinoid (or neonicotinoid metabolite) degrading enzymes as described herein can comprise one or more of SEQ ID NOs: 1-24 (or, if the SEQ ID. referred to is a peptide sequence, a recombinant nucleotide sequence encoding the referenced peptide sequence). The kit can contain one or more substrates suitable for use with the engineered cells as described herein. The kit can further contain additional reagents, diluents, cell culture media, cell culture plates or other container, syringes, and other components (cells, vectors, transfection regents, etc.) that can be used with the a cell or population thereof and/or one or more vectors as described herein.
While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit the disclosure to the embodiments in these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.
Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.
Insect pollination is vital to terrestrial ecosystems and to crop production. First-line stakeholders include the farming industry, agri-food processors, and ultimately, the society at large. Although most calories humans gain from food are derived from wind-pollinated crop plants, it is estimated that about ¾ of cultivated plant species benefit from insect pollinators (77). The added value of pollination by honey bees alone is in the tens of billions of dollars worldwide (50, 23, 147). According to the 2017 USDA Annual Report, honey bees “add at least $15 billion to the value of U.S. agriculture annually through increased yields and superior-quality harvests” (1, 2). Wild pollinators (insects, birds, and some mammals), also substantially contribute to pollination, and in some cases, these may outperform honey bees (118, 113). Wild insect pollinators and honey bee colonies, both managed and feral, are currently experiencing high gross loss rates in North America and Europe (58). These losses depend on the type of operation, location, and year, and average between 20-30% annually (146, 3, 4). According to The Bee Informed Partnership, a non-profit organization connected to academic institutions, winter and summer combined losses currently range between 35-45% (6). The total cost of colony losses, the cost for their replacement and the loss of income combined, are likely to exceed hundreds of million annually (5).
The consensus is that honey bee colony losses are not linked to any single factor. Rather, honey bee decline can be due to the combined effect of several stressors that can be grouped into multiple categories (58): 1) malnutrition; 2) parasites and pathogens, especially the parasitic mite Varroa destructor and its associated pathogens; 3) queen quality and 4) sublethal exposure to pesticides, the focus of this proposal. Described herein are compositions, methods, and strategies to mitigate or overcome insecticide poisoning by exploiting a novel application of paratransgenesis.
As described herein, pollen contaminated by insecticides can be assimilated to a sludge in need of bioremediation, thus allowing the application of strategies and technologies developed over decades of environmental biotechnology. More specifically, by genetically engineering bee bread-adapted bacteria capable of degrading at least two classes of insecticides, organophosphates (OP) and synthetic pyrethroids (SP), individually or in combination, a safer environment for honey bees can be created. Furthermore applying the same genetic modifications to bacteria which are considered honey bee symbionts sensu stricto, a synergistic layer of protection can be bestowed upon honey bees. Compositions, methods, and strategies as described herein can entail the genetic manipulation of honey bee commensals to degrade OP and SP insecticides using insecticide-degrading carboxylesterases derived from soil and water-borne bacteria, (34, 71, 139). Compositions and methods as described herein can provide protection to honey bees against OP and SP insecticides.
Compositions and methods as described herein can involve the expression of a panel of pyrethroid and organophosphate degrading enzymes with broad-range activities in honey bees commensals and sensu stricto symbionts. In certain aspects, a single degradation enzyme can be expressed, in other aspects, more than one (ie a plurality) may be expressed.
Further described herein are bacteria, and their combinations, that can confer to honey bees protection against pesticides.
Last, described herein is the engineering of antibiotic resistance-free, food-grade genetically modified commensals that can stably express one or more enzymes needed for pesticide remediation.
Wild and managed pollinator declines are, in part, due to nutrition stress, pests, pathogens, queen quality, and exposure to agrochemicals. The occurrence and combination of these stressors vary in time and location (134, 51, 167), thus making it difficult to quantify the relative contribution each has to honey bee losses. While each of these stressors alone poses a significant economic impact, their combination is even more problematic (135, 84, 167).
Honey bees are intimately connected to their macro- and microbiota, similar to the relationship between mammals and their microbiomes. Upon closer examination, the honey bee colony's microbiome revealed similarities to those of mammals. Namely, the hive's microbiome provides both micro- and macro-nutrients; it stabilizes the mixture of pollen and nectar by converting these into bee bread (86, 110, 124, 52, 13, 85); and it maintains optimal insect health (14, 9, 49, 30, 15, 83). Similar to mammals, variations in the microbiome composition contribute to pathological conditions, although it is unclear if such changes are considered as the cause or an effect of Colony Collapse Disorder (CCD) (169). The relevance of the bee microbiome in maintaining adequate hive health has become a focus of intense research (7, 82).
A paratransgenic perspective of the honey bee microbiome. Paratransgenesis is an approach whereby members of the commensal community are genetically modified in lieu of the host. Currently, the honey bee microbiota is described in terms of a “core-microbiome,” which, according to studies based upon 16S rDNA sequencing, is composed by a set of nine niche-adapted bacterial species and clades of very closely related bacteria. (82). Several members of the proposed core microbiome are shared across honey bee subspecies and across continents, with some members being shared between corbiculate bees of the genera Apis and Bombus (149, 97, 12). Some investigators found only partial support for a “core microbiome” model (101, 12, 66, 90). An investigation of the metabolically active honey bee microbial community through the analysis of 16S rRNA proposes a more complex microbiome (99), but there is debate as to what approach best reflects the microbiome (127). The honey stomach harbors a different microflora than does the rest of the intestine (29); therefore, considering it a part of the gut or an external appendix influences the overall composition of the microbiome. For the purpose of the present disclosure, an exact distinction between the honey bees' commensals, straddling the line between the honey bee stomach and the hive food stores, and the symbionts sensu stricto is not needed.
The selection of paratransgenesis candidates. Regardless of the exact composition of the core microbiome, it may be impractical to propose a paratransgenesis strategy targeting all of its members. The modification of the entire microflora is also not necessary, in fact, the addition of one or very few microorganisms ameliorated Irritable Bowel Syndrome (IBS), diabetes, rheumatoid arthritis, and atopic dermatitis (100, 94, 128, 143, 160, 56). Disclosed herein are compositions and methods to enrich the honey bee microflora stratified with insecticide-degrading commensal bacteria to provide protection against insecticide-mediated poisoning. Honey bees can then be challenged with insecticides, and their tolerance can be compared to that of honey bees carrying unmodified microflora in order to evaluate the efficacy of compositions and methods as disclosed herein. It is believed that the honey bee stomach, also known as crop or foregut, and its associated microflora, can be relevant to the scope of this proposal. The current knowledge of the bee bread microbiology is based on a 50 year-old seminal work (110) that has recently been refined (12). Pollen contains its own microflora including lactic acid bacteria (LAB). Upon collection by honey bees, pollen is mixed with the regurgitated contents of the honey stomach, and begins the fermentation process that ultimately can contribute to the microbiological stability of the bee bread. Although the relative contribution of different bacteria is open to investigation, the microbiological transformation from pollen to bee bread needs a bacterial consortium to be fully palatable to honey bees (110). During this conversion process, the varied microflora of the pollen can progressively disappear while LAB rise in numbers and then fade away (part of this process is illustrated by the data of
The gut deep compartments, e.g., midgut, ileum, and rectum, are colonized by different bacteria than those that colonize the honey stomach and because microaerophilic, O2-tolerant bacteria become predominant. The Gram-positive LAB, which inhabit the O2-limited portions of the gut, are often described as two Operational Taxonomic Units (OTUs), named Firm-4 and Firm-5. Some assign species names to individual members of these two clades (107). For the very nature of this investigation, bacteria can be dealt with as single species as opposed to metadata categories. Accordingly, a binomial nomenclature is adopted herein, as detailed below. Two Gram-negative bacteria, Gilliamella apicola and Snodgrassella alvi, as well as a few species of Actinobacteria, mainly Bifidobacterium asteroides and B. coryneforme, are consistently found to dwell in the gut deep compartments. Members of each of the aforementioned groups have been isolated. While all the members of the honey bees' microflora may not have been isolated, a panel of honey bee commensals which are known to inhabit the gut have been isolated and investigated herein, (98, 150, 12, 114).
A novel approach to paratransgenesis. Paratransgenesis is a “Trojan horse” approach that was originally designed to control insect-borne diseases. The overall strategy entails the isolation of symbiotic microorganisms that are indigenous to the disease-transmitting insects, and subsequently genetically manipulating these to produce molecules that would hinder disease transmission. Genetically modified symbionts are then reintroduced into their host-vector, where they can deliver effector proteins or peptides. Such studies have shown promise (43, 46, 67). Genetically engineered Enterobacter cloacae expressing an insecticidal toxin from Photorhabdus luminescens has been shown to kill termites in lab experiments (165). More recently, genetically engineered yeast (Kluyveromyces lactis) expressing a ligand-Hecate fusion peptide was used to kill termites by eliminating their gut protozoa which assist the termite workers with the digestion of cellulose (137). It has been postulated, however, that paratransgenesis, when deployed to control insects, may negatively impact the fitness of the host, thus limiting or preventing the dissemination of the transformed symbionts into the targeted population (32). Instead, the concept of paratransgenesis as described herein is expanded as a preventative and intervention therapy to improve honey bees fitness by abating the amount of some insecticides that pollute the hive super-organism. This novel application for paratransgenesis has been speculated as a possible method to improve honey bee health (119, 93, 120, 17), but its application has yet to be tested.
The target insecticides. Described herein are compositions and methods to increase honey bee tolerance to organophosphate (OP) and synthetic pyrethroid (SP) insecticides by enabling selected members of the honey bee microflora to degrade these toxic xenobiotics enzymatically. OPs are esters of phosphoric acid, and they negatively impact almost every biological system they contact. They constitute the basis for some nerve agents developed for chemical warfare, as well as for some widely used insecticides (139). The lethality of OPs ultimately results from their interaction with the active site of acetylcholinesterase (AChE), which prevents breakdown of the neurotransmitter acetylcholine, thus causing continuous nerve impulses at these synapses. Because of their poisonous effects, OPs tend to be strictly regulated. SPs are neurotoxins that are chemical analogues of pyrethrins, originally detected in Chrysanthemum spp. SPs target the voltage-gated sodium channels and impede their inactivation, resulting in persistent sodium inflow and leaving the axonal membrane permanently depolarized, causing paralysis (183). Although, SPs are considered to be safer than other insecticides, the common and pervasive use of these compounds in a wide variety of fields, including in honey bee colonies for Varroa control, has resulted in widespread contamination of the environment posing an ecological concern. The results of many studies have revealed that SPs negatively affect non-target organisms such as fish and arthropods, including honey bees (182, 186,). Moreover, chronic exposure to SPs appears to have long-term effects on human health that are far more serious than previously thought (34). Pyrethroids differ from many other pesticides in that they contain one to three chiral centres. As a result, pyrethroids can have two to eight enantiomers. The chiral isomers of SP differ from each other in their biological properties, including insecticidal activity and their sensitivity to enzymatic hydrolysis (183). Such characteristics can be taken into consideration in the choice of the enzyme(s) responsible for their degradation, as detailed below. Two compounds, one from both insecticide classes, are used by beekeepers for the control of Varroa in honey bee colonies, coumaphos (OP) and fluvalinate (SP). Widespread resistance of Varroa to these compounds have been reported consistently (175, 178, and references therein) rising concerns about the sustainability of these approach. Consequently, high residues of both compounds have been found in honey bee colonies (102). A third class of insecticides, the neonicotinoids, is capable of severely impacting pollinator health (69), but to what extent is debated. However, the knowledge regarding their degradation pathways remains incomplete, even though it is known that neonicotinoids can undergo microbial bioremediation in soil and water (68). The lack of such knowledge is relevant because several neonicotinoid intermediate metabolites are more toxic and persistent than the original pesticide (103, 104 140).
Three groups have established the technology to perform honey bee paratransgenesis (93, 120,177). The genetic transformation of L. kunkeei and F. fructosus, has been established using both theta and rolling-circle replicons. Green fluorescent protein and m-Cherry were successfully expressed into both species. A 7.3 Kb operon from Lactococcus lactis, encoding the 5 genes of the Leloir pathway for lactose utilization, in F. fructosus (93) has been reconstructed and functionally expressed. In order to expand the microbial toolbox herein, commensal LAB encompassing the OTUs Firm4 and Firm5 have been isolated. (L. kullabergensis, L. melliventris, L. kimbladii, L. helsinborgensis, L. mellis, L. sp. AcjLac18. Parasaccharibacter apium (Genbank MH205731) and S. alvi pAJ198 (Genbank MH744154 have also been isolated.
The expression of carboxylesterases in Gram− (
Furthermore the capability to chromosomally engineer S. alvi, arguably, the most fastidious among honey bee commensals is shown in
Confocal microscopy detected the insertion of bacteria into the honey bee gut and will for the evaluation of the gene-expression achieved with different constructs in vivo (
In addition, it is shown that recombinant LABs expressing m-Cherry can grow 3 logs in high-pollen simulated bee bread (50% pollen/20% fructose) to very high-density, 109/ml, (
In order to prove the feasibility of an embodiment of a whole cell bioremediation strategy, a simulated bee bread was fermented with 1) no bacterial inoculum, 2) an inoculum of non-engineered bacteria, and 3) the corresponding mix of engineered bacteria. Mortality was then recorded and plotted (
In order to demonstrate the feasibility of the cell-free enzymatic bioremediation, the carboxylesterase was recombinantly expressed in E. coli BL21 and purified (
An aspect of novelty resides in considering the hive stores as a substrate for whole cell biocatalysis and enzyme bioremediation. As an added layer of protection honey bee symbionts sensu stricto will also be targeted to increase protection.
The metabolic capabilities of microorganisms to degrade OPs and SPs are widespread in the environments where these compounds are present, and the horizontal transmission of DNA fragments encoding degrading enzymes is an occurrence in nature, with carboxylesterase resistance genes apparently travelling even across continents (125). While the possibility that insecticide-degrading resistance genes may spread from engineered honey bees' commensals exists, such horizontal gene transfers already do occur without consequences. An example of horizontal gene transfer is the spread of resistance genes to the antibiotics kanamycin and hygromycin, and herbicide-resistance genes transmitted between transgenic crops and soil microorganisms, both have been occurring for more than 20 years upon hundreds of million acres with no apparent detrimental effects (137, 126, 144). The requirement for antibiotic-free GMOs is not universally enforced because a substantial fraction of transgenic crops carry either kanamycin or hygromycin resistance genes. Nonetheless, modifications are disclosed herein that will minimally impact the overall gene-flow, because i) the end-product of this disclosure will be antibiotic-free and food-grade genetic modifications; ii) the genetic elements used for engineering will not bear transposable-like sequences nor conjugational functions, which are instrumental to gene-flow in nature; and iii) the honey bee commensals are niche-specific, therefore their interaction with other microorganisms is limited. Not only can the aspects of the present disclosure limit or prevent the crossing of toxic xenobiotics through honey bee gut epithelium, but it can also remove insecticides from bee bread and decrease the honey bee metabolic burden associated with detoxification.
As described herein, the beneficial effects provided by the commensals to the host super-organism, coupled with the selective pressure caused by the presence of insecticides in the environment, can lead to a stable interaction between host and its modified commensals. Should this not be the case, the recombinant probiotics can be reapplied to colonies.
Honey bees emerge from larval stage as virtually germ-free organisms (53, 54), and their microflora is reconstituted mostly by nurse bees though trophallaxis and coprophagy (114), thus providing a plausible mechanism for the vertical transmission of recombinant probiotics (109). The present example focuses on two major classes of insecticides, OPs and SPs, and enzymes for the degradation and inactivation of these two classes of compounds (although additional aspects of the present disclosure describe degradation of other classes, for examples neonicotinoids and metabolites thereof). Moreover their capability to confer protection against insecticides has been extensively investigated as referenced hereafter. As described herein, cytochrome P450 or glutathione-S-transferase detoxifying enzymes are not exploited (44, 125, 157, 57, 133). The rationale behind this choice was to avoid any interference with the RedOx balance of the bacterial intracellular milieu, which may diminish fitness. Also, it is unclear if the P450 heme prosthetic group is present in all honey bee commensals. Instead, the emphasis as described herein will be on carboxylesterases, a class of enzymes which hydrolyze the ester bonds present in OPs and virtually all SPs. While not all carboxyesterases can hydrolyze OPs and/or SPs (125), the enzymes as described herein were selected for this capacity.
Aspects of the present disclosure has additional advantages. First, it is designed to keep the metabolic burden of the recombinant bacteria to a minimum, because enzymatic hydrolysis of esters does not require energy. Secondly, the activity of carboxylesterases can be monitored in vitro by colorimetric assays using a wide variety of substrates, thus providing a convenient screening tool for the preliminary evaluation of engineered bacteria. Thirdly, carboxylesterases do not require co-factors other than divalent metal ions (especially Zn++ and Mg++), which are naturally abundant in bee pollen and available to bacteria in the honey bee gut as well as in bee bread. This consideration is particularly relevant given that the need for expensive co-factors is a major hurdle in the field deployment of whole-cell biocatalysts. There is a compelling body of indirect and direct evidence that certain carboxylesterases can impart resistance to OPs and SPs insecticides (121, 154, 142, 125, 159, 18, 152, 47). Also, resistance to some OPs can be achieved by transforming the embryo with carboxylesterases (112, 16).
It has also been shown that the carboxylesterases have a relaxed specificity for their substrates and some carboxylesterases can even cross-hydrolyze both OP and SP (63, 158, 37, 71, 162, 168, 88). The use of promiscuous enzymes with broad-range substrate specificities is an aspect of the present disclosure because it can enhance the metabolic versatility of each engineered probiotic. In this framework, the expression of three enzymes in honey bee commensals is proposed. The first enzyme is the Methyl Parathion Hydrolase (MPH), an OP carboxylesterase, originally isolated from Plesiomonas sp. M6 (33), GenBank accession No DQ677027). This enzyme was expressed in Pseudomonas putida and it proved capable of hydrolysing a wide range of OP including methyl parathion, fenitrothion and chlorpyriphos (168). Intriguingly, MPH from Plesiomonas sp. M6 shares over 98% amino acid identity with proteins identified in several phylogenetically unrelated bacteria (71). The OP-hydrolytic activity of Pseudomonas sp.Wbc-3 carboxylesterase (which is 100% identical to MPH isolated from Plesiomonas sp. M6) was independently confirmed (117). Another major advantage of MPHs is that they appear to have a tremendous potential of improvement by in vitro mutagenesis (162, 105). The second enzyme, an SP carboxylesterase (GenBank accession No FJ688006), is encoded by the PytH gene from Sphingobium sp. JZ-1. This SP esterase was shown to be active against a panel of SPs including permethrin, deltamethrin and cypermethrin. Moreover, it did not show isomer selectivity, and it was active only on p-nitrophenyl esters of short-chain fatty acids, strongly suggesting that it does not have lipase activity (151, 168). The third enzyme is a naturally-occurring variant (Trp251Leu) of the E3 carboxylesterase (wild-type allele GenBank: AAB67728) from the blowfly Lucilia cuprina. This variant is believed to be the basis of the well documented cross-resistance between permethrin (SP) and malathion (OP) in various species (62,63, 125). Interestingly, this E3Trp251Leu variant showed a much higher activity than the wild-type enzyme for both trans permethrin (10-fold) and the more insecticidal cis permethrin (135-fold), so reducing the trans/cis preference from 27:1 to only 2:1. This mutation also shows a 1000-fold increased activity in the hydrolysis of malathion. Interestingly, the same Trp251Leu mutation was found in a malathion-resistant population of Drosophila melanogaster (63) and a closely similar variant, Trp to Gly was found in an orthologue protein present in a field population of malathion resistant hymenopterous parasitoid (166).
Modified bacteria can be fed to emerging honey bees which will be subsequently challenged with insecticides. Modified bacteria can then confer honey bees potent and potentially complete protection against orally-ingested OPs and SPs insecticides. All the insecticides according to the present disclosure can be relevant to pollinator health because they constitute the active principle in currently used insecticide preparations. Namely, the organophosphates methyl parathion, malathion, and fenitrothion; as well as the synthetic pyrethroids permethrin, deltamethrin, and cypermethrin are employed in the present disclosure.
A novel aspect of the present disclosure is that it adapts paratransgenesis from an insect pest-control strategy to a protection tool for pollinators, thus extending the concept of “probiotic” to beneficial insects. Another novel aspect is the translation to the hive stores of established technologies for the bioremediation of environmental pollutants. The strategies discussed herein are safe for many reasons. First, the engineered probiotics can be devoid of any antibiotic resistance genes. Concerns about horizontal gene transmission are misplaced because the genes that we will introduce into honey bee commensals are already widely present in the environment, especially where insecticides accumulate in the soil or water. The natural gene-flow related to insecticide-degradation pathways is substantial among these microbial populations. The proposed work will extend the metabolic capabilities, from the soil and water microorganisms into honey bees commensals. Second, as an added layer of safety, none of the genes, nor the vectors, will bear sequences related to transposable elements or to conjugational episomes, both of which facilitate horizontal gene transfer observed in nature for MPH genes (71). Third, most honey bee commensals are niche-restricted which contains interactions with other microorganisms. The proposed research is long-term and self-sustainable because it establishes a technology that is readily adaptable for problems other than those pertaining to this proposal (e.g., other pesticides and fungicides classes), and it also bears an intrinsic stability. In fact, the host may need to retain the engineered probiotics to protect against insecticides forcing the bacteria to retain the genetic modifications to preserve their environmental niche.
Approach
Embodiments of aspects of the present disclosure are disclosed below
Expression of a Pyrethroid and Organophosphate Degrading Enzymes with Broad Range Activities in Honey Bees Commensals.
As described herein, the tools for genetic manipulate of a variety of honey bee commensals have been established. In addition, a panel of honey bee commensal LABs which inhabit the mid- and hind-gut and are microaerophilic/O2-tolerant have been collected. As described herein, genetic transformations can be extended to L. kullabergensis, L melliventris, L. kimbladii, L. mellis, L. sp. AcjLac18. All these LAB are sensitive to erythromycin and kanamycin. LABs can be grown in MRS-2% fructose medium under CO2-enriched atmosphere at all times except during harvest, wash and electroporation. Transitory exposure to regular O2 atmosphere inhibits replication, but does not impair cell viability for at least two days. Prior to transformation, two aliquots will be inoculated in MRS-2% fructose supplemented with 2M glycine, grown until OD600=1, harvested, washed with sterile distilled H2O, and electroporated at high-voltage, 2-2.5 Kv (93). The DNA vector can be pIB184 or a derivative thereof. This vector carries an erythromycin resistance gene, and works flawlessly in a wide variety of LABs (93, 94). As described herein, Gram-negative Gilliamella apicola have been isolated and engineered by electroporation and Snodgrassella alvi by natural transformation (preliminary data). The latter was transformed chromosomally. A suitable vector for G. apicola and S. alvi is pBBR122, an improved commercially available (MoBiTec) non-mobilizable, non-conjugational version of pBBR1MCS2 (78). This vector has a broad host range carrying different antibiotic resistance cassettes.
Expression of Insecticide-Degrading Enzymes.
Recombinant genes and genetic elements as described herein can be synthesized by GenScript (Piscathaway, N.J.). This approach can be relatively inexpensive with maximal success. The recombinant genes can be designed for host-tailored expression and to feature all necessary regulatory sequences such as promoters, and transcription termination signals. All the recombinant genes can be synthesized with a suitable promoter and termination sequence, to readily allow for subcloning into the expression vector. The constructs can be transformed into their matching hosts, and the recombinant colonies can be tested in vitro for their capability to degrade in OP and/or SP using chromogenic substrates as a model system as detailed in Objective 1.3.2. The recombinant genes encoding for 1) Mph (GenBank: DQ677027), 2) PytH (GenBank: FJ688006), and 3) the mutated version of the E3 carboxylesterase E3Trp251Leu (wild-type allele GenBank: AAB67728) can be codon-optimized according to the final expression host. From our experience as evidenced data herein, overexpression of carboxylesterases is better achieved by secreting the enzyme outside the cytosol. The signal peptides for secretion can be selected by interrogating available resources as detailed above. Each signal peptide can be extended by a few amino acids beyond the cleavage site in order to preserve the signal peptidase requirements, as defined by a Y-score, a parameter that takes into account amino acid sequences and the geometry of the surroundings, and can be customized according to the final host. While suitable promoters for all the target bacteria have been identified, tools for expression can be optimized by defining the level of expression of the in vivo transcriptome. Transcriptomic data relative to honey bee commensals retrieved from honey bee guts can be generated at the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) and analyzed in house. The level of gene expression can be used as a proxy for promoter activity. The ideal promoter(s) can have a steady, robust but not excessive activity in vivo. The newly selected promoter can be confirmed by using a fluorescent marker and then used to express the carboxylesterases. These data will allow for a precise tuning of foreign gene expression in vivo in honey bee commensals. The vectors can be matched to the host. The synthetic DNA can be excised from the GenScript vector and ligated into the final vector according to standard molecular biology techniques. Sanger sequencing can be used to confirm DNA sequences. The expression of recombinant carboxylesterases can be preliminarily detected by standard immunological methods, e.g., Western blot. To detect the recombinant proteins, polyclonal antisera against the three target carboxylesterases can be produced in rabbits. The proteins can be expressed in E. coli as MBP fusions using the pMALp2 vector (New England Biolabs). Recombinant fusion proteins can be purified using a maltose affinity-column.
Chromogenic Detection of Insecticide-Degrading Activity.
The enzymatic activity of carboxylesterases can allow for the use of chromogenic substrates as a surrogate test to measure OP or/and SP degradation by the engineered bacteria. Bacterial colonies overlaid with 100 mM 5-bromo-4-chloro-3-indolyl caprylate (X-caprylate) turn blue if carboxylesterase activity is present (158, 89). This can provide a quick method to screen bacteria. The use of this approach is demonstrated herein. In another aspect, the colonies' carboxylesterase activity can be tested using a quantitative colorimetric assay which can rely on a series of chromogenic substrates, p-nitrophenyl esters, the hydrolysis of which can be quantitated by timing the formation of p-nitrophenol at 405 nm (62, 63, 158, 89). The esterase activity of the recombinant strains can be compared to their wild-type counterparts to assess how effectively the esterase activity is expressed. An alternative method is plating bacteria on an appropriate medium containing 100 mg/ml methyl parathion or 0.4 mM cypermethrin. Both of these compounds have poor solubility producing an opaque medium. Halos of degradation can become visible around colonies capable of hydrolyzing the esters into their alcohol and acid moieties (164, 151). The cumulative data obtained herein can be used to phase out strains with sub-par performance.
Analytical Detection of Insecticide-Degrading Activity.
A quantitative pesticides degradation test by Liquid chromatography tandem Mass Spectrometry (LC MS/MS) and Gas Chromatography tandem Mass Spectrometry (GCMS/MS) can be performed. The strains can be grown in 50 ml liquid medium batches supplemented with 0.2 mM of the insecticides (OP: parathion, malathion, fenitrothion, and SP: permethrin, deltamethrin, cypermethrin). A 50 ml sample of the uninoculated media can be set aside as a base-line control. At days 1 post-inoculation, the bacteria can be removed from the spent media and the supernatants will be titrated. Percentages of degradation can be plotted before and after the bacterial growth. To extract the pesticides from media, citrate QuEChERS following the method of (155) can be employed. QuEChERS stands for quick, easy, cheap, efficient, rugged and safe and was first used by (11). Since then, the method has been optimized for pesticides in honey and honey bees (155, 102, 65). Standards for pyrethroids and organophosphates can be obtained from Chem Service (West Chester, Pa.). Deuterated internal standards for each class of pesticides can be obtained from Cambridge Isotope Laboratories (Andover, Mass.). For the GC MS/MS, trans-permethrin d5 can be used and for LC MS/MS, malathion d9 can be used. Pesticides can be extracted from the media supernatants using 10 ml of media and an equal volume of acetonitrile. Citrate QuEChERS salts (Agilent Technologies, anhydrous MgSO4, NaCl, NaCitrate dehydrate, and disodium citrate sesquihydrate) and 200 μl of isotopically labeled standards at 1 μg/ml can be. The extract can be cleaned through 15 ml dispersive PSA/C18 tubes (containing anhydrous MgSO4, PSA bonded silica and C18 bonded silica). The extract can be evaporated to 100 μl and then analyzed by LC MS/MS or GC MS/MS using methods developed by (155, 48).
Identification of the Best Protecting Probiotics.
From a biotechnological perspective, the decontamination of honey bee food-sources can be assimilated into a bioremediation process to remove pollutants from the environment. Such an approach can adapt knowledge already gained from the bioremediation of contaminated soils and sludge. Data as shown herein demonstrate such, and the whole-cell biocatalyst strategy has proven extremely effective for a number of cases (179, 180, 181). Six insecticides will be alternatively tested (organophosphates methyl parathion, malathion, fenitrothion; and the synthetic pyrethroids permethrin, deltamethrin, cypermethrin).
At this point, the bacterial strains to be used can depend on the results above. Once completed, different strain-promoter combinations can offer a range of protection. Minimally, F. fructosus and L. kunkeei, each one expressing the three selected recombinant genes, can be used to ferment simulated bee bread (10% pollen+20% fructose). Other fructophilic bacteria have been collected, which may also be useful in the decontamination process. Their deployment can be evaluated.
The challenges can be delivered in simulated bee bread. The amount of simulated bee bread used daily by each honey bee is approximately 25 ml, therefore the dilutions of each insecticide in acetone can be calculated so that a 25 ml aliquot delivers the desired xLC50, daily. The insecticide-spiked bee bread can then be inoculated with the native bacteria, as a negative control, or with their engineered counterpart. The difference in survival can then be measured and statistically analyzed.
Capped combs containing emerging adult honey bees can be obtained from several colonies at the University of Florida apiary (Gainesville, Fla., USA). Combs with emerging bees can be placed in an incubator under simulated hive conditions (33° C., 65% RH) to allow bees to emerge. Twenty-four hours later, newly emerged bees, which cannot fly and have minimal stinging capability, can be brushed from the frames into holding tubs and allowed to intermix to minimize variation in the experimental population. The bees then can be distributed into clean cages (10 or 20 bees/cage), returned to the incubator and fasted for three hours.
Two doses of bacteria, (106 and 104 CFUs ml-1) can be mixed with simulated pollen, which can be fed to honey bees. Survival rates between the control and the treatment can be measured. Oral LC50 values for each insecticide can be extrapolated from (182, 187) or determined in house.
Based upon data as described herein, honey bees can be challenged daily with 10xLC50 of insecticide, and can be scaled-up or -down as needed. Each insecticide can be tested individually. The Kaplan-Meier method can be applied to obtain the survival fractions following pesticide challenge in honey bees. Using the Mantel-Haenszel log rank test, the P-value for statistical differences between surviving pesticide challenges among treated groups compared to those dosed with the corresponding strains lacking the pesticide-degrading enzymes can be discerned at the 95% confidence interval. The strains in appropriate combinations can also be tested for their ability to protect against more than one insecticide and belonging to more than one class. Data suggest that relatively low numbers of honey bees may be needed to test the efficacy of the treatments. In fact, 50 honey bees in the treatment and control groups can yield desired results. The capability to incorporate hundreds of bees daily can be utilized in experiments even up to 9-10 months a year.
Cell-Free Enzymatic Bioremediation of the Food Sources.
The use of formulated enzymes rather than whole cell biocatalysts is a process known as enzymatic bioremediation, the feasibility of which has been shown in different settings (reviewed in 174, 185) and references therein). As described herein, bee pollen can be a suitable target for this kind of intervention as an alternative or complement to whole cell catalysis described above. As shown herein, the recombinant carboxylesterase, when expressed in E. coli and purified, can reduce the toxicity of 40xLC50 cypermethrin to honey bees when tested in a 20% fructose syrup model system (
Symbiont-Mediated Bioremediation.
A maximum of 16 species/gene combination (6 LAB, G. apicola and S. alvi), each one expressing an OP recombinant gene, a SP recombinant gene or, the synthetic/purified E3 Trp251Leu carboxylesterase, which can degrade both OP and SP, can be tested. The LC50 delivered in 25 ml of feed for each insecticide can serve as a benchmark to test the efficacy of each recombinant commensal which can be fed to emerging honey bees at 105 CFUs ml-1 in 125 g/I of sucrose syrup. This dose is derived from available data showing that probiotics administered at 104-105 CFUs ml-1 in feed have a remarkable benefit (15, 30, 150,171, 172, 173, 181). Data demonstrating the beneficial effects of selected commensals for honey bees can allow dosing the syrup with the recombinant strains, and compare results obtained with syrup containing the isogenic, negative control strains lacking the enzymatic activity. Bacteria can be grown according to their required selective media and antibiotic, and then can be added to the syrup at the appropriate concentrations when at mid-to-late log phase is reached in order to preserve their metabolic activity. Next, the capacity of each strain can be tested for conferring protection against 1xLC50 of the cognate insecticide. It is worth at this point to recapitulate that eight different strains, each carrying either two (possibly, 1) recombinant genes (1 OP degrading, 1 SP degrading or, alternative, the E3Trp251Leu only) can be assessed in this bioassay. At this stage, only four insecticides (2 OP methyl parathion, malathion; and 2 SP permethrin, deltamethrin) can be evaluated singularly. Strains expressing Methyl Parathion Hydrolase (MPH) can be tested against OPs, and the strains expressing PytH can be tested against SPs. Strains expressing the naturally-occurring variant E3 (Trp251Leu) can be tested against combinations of OPs and SPs. For controls, insecticide-laced bee bread can contain equal CFUs of the naked vector strains. The mortality of bees fed protective (modified) commensals can be plotted against the mortality of bees fed bee bread containing vector controls, and the results can be evaluated over the course of 10 days. Survival rates can be determined using Kaplan-Meier test. All the following numbers may need +/− adjustments depending on the differences between the treatments and the controls. This approach can entail 64 batches (8 strains×1-2 genes×for 4 insecticides) of 3×20 honey bees each, total 3480 honey bees. The 8 control batches (each control strain carrying the naked DNA vector) on 4 insecticides will add 2000 honey bees, totaling 5480 bees. Should robust protection against 1xLC50 be achieved, then subsequent challenges can be performed using higher challenge doses, e.g., 5xLC50-20xLC50. It may not be necessary to test doses >20xLC50 since these levels of exposure would be expected to be rare catastrophic events and thus not worth addressing. In contrast to previous test, the challenge against 5-20xLC50 can be performed against four insecticides (2 OPs+2 SPs) mixed together. This approach can be performed by mixing together 106 CFUs ml-1 of bee bread and each of the strains which conferred protection against at least 10xLC50. The test can have six replica (6×30 bees each). The controls can be the same strains carrying the naked vector. If protection is achieved, the experiment can be repeated with 1/10 of CFUs.
Engineering Antibiotic Resistance-Free, Food-Grade Genetically Modified Commensals that Stably Express the Enzymes Needed for Pesticide Catabolism and Confer Protection for Pesticide-Exposed Honey Bees.
The generation of this kind of strains is an established, two-step process which is summarized in (55, 79, 108). As described herein, this strategy is adapted by inserting the carboxylesterase(s) and the m-Cherry (optionally) as a bi-cistronic minioperon.
At this stage, there can be around 5-7 strains. The suicide vector pCVD442 and its permissive host E. coli strain S17-1, which have an over 20 years record of successful chromosomal insertions in a very broad range of hosts (40, 111, 170), can be used in this project. The overall scheme of the recombinant suicide vectors is shown in (
Plasm id-engineered bacteria can be used to test if the engineered microbiota can be passed down to newly emerging honey bees. Optional fluorescent markers (for example, GFP or m-Cherry) can be used for tracking the bacterial flow between honey bees and for checking the stability of the genetic modifications over time in vivo. A procedure as described above can be adapted for this purpose. Color-marked honey bees reconstituted with recombinant microflora, and their fecal matter, can be intermixed with unmarked newly emerged honey bees not having the recombinant microflora, thus allowing for the determination of if those with the recombinant microflora trophallactically pass the microflora to other bees. Nurse bees containing the recombinant microflora can be color-labelled to distinguish them from emerging honey bees not having the microflora. The presence of recombinant bacteria in the newly emerged honey bees can be detected by flow-cytometry analysis. Should it be determined that the recombinant microflora can be vertically transmitted, depending on the resources and time available at the time, experiments can be repeated with insecticide-degrading plasmid-engineered microbiota and the newly emerged honey bees can be challenged with 1xLC50 of the suitable insecticide.
Honey bees (Apis mellifera) are key pollinators for a vast array of crops, with a global added value in the tens of billions of dollars for the US economy alone. One of the major stressors leading to the current honey bee colony losses is the sub-lethal exposure to pesticides. To address this problem, compositions and methods are described herein to mitigate and/or overcome insecticide poisoning by exploiting a novel application of paratransgenesis. As described herein, hive-related bacteria, engineered to degrade OPs and SPs insecticides efficiently, can confer potent protection against insecticide poisoning, thus complementing and synergizing with the honey bees' own defensive mechanisms. Pollen contaminated with insecticides can be assimilated to a sludge in need of bioremediation, thus allowing the potential to tap into strategies and technologies developed over decades of environmental biotechnology. This green technology is sometimes referred to as “bioaugmentation”. More specifically, as described herein, genetically engineering bee bread-adapted bacteria, as well as honey bee symbionts sensu stricto, enabling them to degrade two classes of insecticides, organophosphates (OP) and synthetic pyrethroids (SP). Compositions, methods, and strategies as described herein can lead to a safer environment for honey bees. This approach to bioremediation is referred to as “whole cell biocatalysis,” whereby the enzymatic reaction is catalyzed/operated by cells as opposed to purified enzymes. Furthermore, the purified carboxylestrases, recombinantly produced and appropriately applied to the hive food stores, can enhance the protection. This latter approach to bioremediation is referred to as “enzymatic bioremediation”.
Methods and strategies as described herein can entail the genetic manipulation of honey bee commensals to degrade OP and SP insecticides using insecticide-degrading carboxylesterases derived from soil and water-borne bacteria (34, 71, 139). Last, bacteria isolated from fresh bee bread can be molecularly bred to efficiently degrade insecticides adding an even deeper layer of protection. The long-term goal of this proposal is to protect honey bees against OP and SP insecticides.
Compositions and methods as described herein can utilize four materials:
1) The honey bee Lactic Acid Bacteria (LAB) engineered to express the carboxylesterase.
2) An E. coli strain engineered to express the carboxylesterase.
3) The purified carboxylesterase.
4) A consortium of bacteria derived from bee bread, subjected to in vitro evolution by genome shuffling and selected for over one year for being capable of using various insecticides as a sole carbon source. In actuality, 4 consortia are described herein, each one capable of growing on a different insecticide (the neonicotinoid, imidacloprid; the neonicotinoid, clothianidin; the synthetic pyrethroid, cypermethrin; and the orhanophosphate, methylparathion) as a sole carbon source.
The presented data were generated by challenging honey bees with the synthetic pyrethroid cypermethrin (which is highly toxic to honey bees), bioremediated with different combinations of the bacteria described above.
Whole-Cell Remediation:
Enzymatic Biomediation:
Conclusions
A) The data provide strong support to the overall strategy. There remains considerable debate about what concentration of insecticide should be considered “field concentration.” It is estimated that the challenge delivered herein in an amount of cypermethrin that is logarithmically (at least 10-fold, but likely, 100- to 1000-fold) higher than the average exposure in the field. It is reasonable to surmise that, the honey bees can be conferred with strong protection against any reasonable intoxication that they may be subjected to in the field according to compositions and methods as described herein.
B) The experimental approach involving the simulated bee pollen bioremediated with “whole cell biocatalysts”, (whereby the chemical reaction is catalyzed by whole cells) is feasible. Further embodiments of this approach can be undertaken, including genetic manipulations for enzyme secretion (to facilitate improved contact between the enzymes and the insecticides), and the selection of engineered bacteria.
C) Suitable results were also obtained when the challenge was delivered via a homogeneous fructose solution (syrup). This medium can easily be fed to honey bees, which use their ligula (tongue) to neatly retrieve it from the feeding device without causing spillage into the cups nor onto their own heads. As a result, the difference between the treatments and the controls can more readily be detectable and reproducible (
D) The bacterial consortium selected on cypermethrin as a sole carbon source (described hereafter with detail) is effective in bioremediation (
E) An aspect of this proposal resides in considering the hive stores as a substrate for whole cell biocatalysis and enzymatic bioremediation, which was defined above. This approach can enable the removal of pollutants from the environment. This enzymatic bioremediation can provide support for the overall idea that carboxylesterases can bioremediate food stores as a result of the enzyme's remarkable capacity for decontamination. On the other hand, the whole cell biocatalysis platform may provide a less labor-intensive, less expensive, and more sustainable protection in that engineered wild-type bacteria can self-support once they have been introduced in the system.
Described herein is an approach that can generate microbial communities and/or single microbial species that, while being adapted to the hive food stores, can degrade, besides the insecticides described above, recalcitrant insecticides including, but not limited to, neonocotinoids. Compositions, methods, and strategies as described herein can enable novel applications of the knowledge developed for the removal of pollutants from the environment. As described herein, pollen/bee bread-adapted bacteria can remove the same compounds from the honey bee food stores.
As described in the present example, native bacteria can be employed, naturally present in bee bread. From freshly isolated bee bread collected at the University of Florida apiary, bacteria can be grown on, alternatively, rich media and mineral media where the sole carbon source was the insecticide. Nitroguanidin was also used because that this compound may improve the capability of bacteria to use the nitrogen present within the neonicotinoid molecules as a nitrogen source. Incidentally, attempts to use the insecticides as a sole nitrogen source (instead of carbon) failed because diazotrophs were selected, bacteria that can fix atmospheric nitrogen. The bacteria underwent cycles of bentonic and planctonic growth to foster the exchange genetic materials amongst the pan-genome, while minimizing the selection of bacterial mats, where the degradation is operated by groups of bacterial species living in tight contact mutually assisting in the degradation process. The overall scheme of the selection process is presented in (
The consortia have partially been characterized by plating the bacteria to single colony on different media. The consortia as described herein in certain aspects may be composed also by species that cannot be cultured. The rationale for focusing on using culturable bacteria is that from a biotechnological perspective, bacteria that cannot be grown to a pure culture have a reduced practical utility. Colonies with different morphology were selected, and further analyzed. The carboxylesterase activity of 50 clones has been crudely assessed by overlaying X-caproylate (see Example 1 above and
Embodiments of coding sequences of recombinant carboxylesterases as described herein are listed below.
Described in the present example is the decontamination of the wax for recycling in the production of foundation (the sheets of wax which are inserted into the frames to invite honeybees to draw new honeycomb). Pesticides accumulate in beeswax of the honeycombs, which have to be discarded after, usually, three years. While most impurities such as propolis, feces and exuviae form larvae, can be removed (by melting the spent honeycombs in presence of water), pesticides contamination cannot. Accordingly, the honeycomb wax has to be discarded and it cannot be recycled to produce new. It is not desirable for beekeepers to trash old combs not only because the new foundation has a substantial costs, but also because it can contain pathogens (some of which are sporigenic Paenibacilli that cannot be eliminated unless the wax is autoclaved). Moreover commercial foundation, unless the fabled very, very expensive organic batches, is known to carry pesticides of its own. Beeswax melts between 62-64° C., therefore “regular” carboxylesterases (such as those described above) may not be suitable. On the contrary, carboxylesterases from thermophilic bacteria (for example SEQ. ID NOs. 4 and 5 below) can withstand and have optimum catalytic activities at higher temperatures (around 65-75° C. and remain active up to 90° C.) than carboxylesterases derived from other sources, which would make them ideal for this purposes. In embodiments, these enzymes can be immobilized on nonwoven textile-like matrices which can act as sieves to retain impurities (as mentioned above) and decontaminate the beeswax at the same time. Organophosphate carboxylesterases (phospotriesterases) received great attention because organophoshates, besides being insecticides, are warfare nerve agents.
Deinococcus radiodurans phosphotriesterase,
Sulpholobus acidocaldarius phosphotriesterase,
Neonicotinoids are recalcitrant to biodegradation and, among them, imidacloprid and clothianidin are particularly persistent. The degradation pathways of neonicotinoids are not completely understood. At least some of them eventually are transformed into 6-chloronicotinic acid (6-CNA) which can be transformed into 6-hydroxinicotinic acid (6-HNA) by the cch2 gene product from Bradyrhizobiaceae 6G-6C strain (Ref. Shettigar 2012). In turn, 6-HNA can be funneled into the Kreb's cycle through different routes (Jimenez 2008, Shettigar 2012). In Pseudomonas putida KT2440 the pathway of 6-HNA degradation has been thoroughly dissected (Jimenez 2008).
Described herein is a neonicotinoid detoxifying strategy of assembling several parts into devices and combining them into a cassette-in, cassette-out flexible genetic system where the catabolic genes converting 6-CNA into 6-HNA and then into fumaric acid, which is an intermediate of the Kreb's cycle, can be paired into the same plasmid with appropriate transporters (
Described herein are genetic systems which, when introduced into a novel host, have the potential to confer the capability of degrading 6-CNA, thus creating a metabolically advantageous outlet for the mineralization of some intermediates of neonicotinoids catabolism. The P. putida 6-HNA operon was cloned by PCR using the genomic DNA as a template. The cch2 gene and its associated transporter were derived from a synthetic gene (
Several constructs were made bearing different configurations of the above described genetic parts and transformed into E. coli BL-21 testing for the ability to grow on 6-HNA as a sole carbon source. Despite the extremely slow growth which cannot be measured by the increase in the OD600, the E. coli BL-21 cells, transformed with system were still alive, and capable of generating CFUs after 6 weeks incubation, whereas the control cells were not. This observation suggests that these constructs provide E. coli BL-21 limited, but sizeable, capability to use 6-HN to support metabolism.
The constructs and aspects thereof are shown in (
Oxalic acid, legally or brevi manu, is widely used to control phoretic Varroa destructor mites, a major drive of current honey bees' colony losses. Previous data suggests that varroa may become at least partially resistant to oxalic acid by associating with oxalotrophic bacteria, which use oxalates as a sole carbon source. Despite the efficacy against the varroa mites, oxalic acid is somewhat detrimental also to honey bees. In this composite scenario it is possible that honey bee commensals could be engineered to degrade oxalic acid. From a human probiotic strain of Bacillus subtilis, a gene encoding for oxalate decarboxylase was cloned, an enzyme that degrades both oxalates and oxalic acid to formate and CO2, and was expressed in Lactobacillus kunkeei and Fructobacillus fructosus. Data show that these recombinant strains robustly degrade calcium oxalate. These strains will assist for a better understanding of the complex dynamics of oxalic acid inside the hive, and they may allow for precision interventions to shelter honey bees from the side-effects of miticides treatments. More importantly, this investigation provides a proof-of concept for the bioremediation of more toxic organic compounds inside the hive.
The current economic added-value of honey bees as crops pollinators, it is estimated in the tens of billions of dollars worldwide, thus exceeding by two logs (Gallai et al. 2009; Calderone 2012) the value of honey, wax, propolis, and bee-venom combined (van Engelsdorp and Meixner 2010). In the past 20 years a steady, dramatic decline of honey bees in has occurred. These losses depend on the type of operation, location, and year, and average 20-30% annually (van Engelsdorp D, 2008). According to (The Bee Informed Partnership), a non-profit organization connected to academic institutions, winter and summer combined losses currently range between 35-45%. The total cost of colony losses, the cost for their replacement and the loss of income combined, are likely to exceed hundreds of million annually. Scientific and regulatory institutions agree on these estimates (USDA, European Parliament).
Losses are in part attributed to the so-called colony collapse disorder (CCD), an umbrella name which defines a syndrome more than a specific disease, as well as to other ‘disappearing diseases’ (Cornman et al. 2012; van Engelsdorp et al. 2013). The causes of CCD and related diseases are poorly understood. Three stressors play major role in colony losses. 1) lack of a steady food supply throughout the entire year, 2) transfer of pathogens and parasites across the globe, 3) widespread use of pesticides. (Genersh 2010; Genersh and Aubert 2010; Evans and Schwartz 2011; Cornman et al. 2012; Becher 2013; Derecka et al. 2013). (Goulson et al. 2015 and references therein).
There is also a substantial agreement that the hemophagous honey bee mite Varroa destructor is a major drive of colony losses (Rosenkranz et al. 2009) and, while breeding aimed at improving honey bee resistance to varroasis is showing good potential and promising result (Moritz 1994; DeGuzman et al. 2005; Rinderer et al. 2014; Lattorf et al. 2015, Sabahi et al. 2017), mite control by beekeepers mostly relies on the use of chemicals, whose regulation is subjected to substantial differences worldwide and over the course of time. Over the last decades, the most noted synthetic acaricides against V. destructor are the organophosphate coumaphos, the pyrethroids tau-fluvalinate, as well as the formamidine amitraz against which V. destructor eventually developed resistance (Rosenkranz et al. 2009; Kanga et al. 2010). Some essential oils are reported to be effective against V. destructor (DeGrandi-Hoffman et al. 2012; Umpierrez et al. 2013; Damiani et al. 2014). A third category of miticides is represented by organic acids, lactic, formic and oxalic, which are empirically believed to carry minimal danger of resistance (Rosenkranz et al. 2009. and references therein). The use of organic acids against varroa dawned in the mid-80s (Klepsch et al. 1984,) and their use preceded, and in some cases subsided and/or disregarded regulations by government agencies. In certain regions, including Europe, when the honey supers, the combs where the honey is stored, separated from the nest, are not deployed, oxalic acid is legal to use and it is remarkably effective against varroa. Despite the efficacy against the varroa mites, oxalic acid is also detrimental to honey bees (Aliano et al. 2006; Aliano and Ellis 2009) possibly by interfering with the microbiome composition (Diaz et al. 2019). Accordingly, it is recommended that no more than one treatment is applied per generation, which in the active season averages about 45 days. It has been previously shown that varroa associates with oxalotrophic bacteria (Maddaloni and Pascual 2015), an observation that may explain why beekeepers lament that in certain instances they need to increase the frequency of oxalic acid treatments. In this complex scenario the ability to modulate the fate of oxalic acid in the hive through the microbiota offers novel approaches to refine its use for the control of phoretic varroa. Oxalic acid is a highly oxidized molecule that combines with various cations to form an array of salts. Among them, calcium oxalate has received much attention as 75% of all kidney stones are composed primarily of calcium oxalate (Robijn et al. 2011). A probiotic Lactobacillus plantarum has been engineered to degrade oxalate (Anbazhagan et al 2013,) and successfully used in a rat model of hyperoxaluria (Sasikumar et al 2014). In the present example the degradation of oxalate can be mediated by oxalate decarboxylase (OXDC, EC 4.1.1.2), an enzyme that catalyzes the oxidization of both oxalate and oxalic acid to formate and CO2, both being volatile compounds (Tanner et al. 2001, Makela et al. 2010). It is reasoned that the cupin domain-containing oxalate decarboxylase from Bacillus subtilis can be functionally expressed intracellularly in honeybee commensals Fructobacillus fructosus and Lactobacillus kunkeei and that the recombinant enzyme would ultimately interact with the substrate, without the need for providing a specific ectopic transporter. The present example highlights the ample potential of enhanced honey bee commensals for the bioremediation of organic molecules in the hive.
Microbiological techniques, vectors and deoxyribonucleic acid (DNA) manipulation procedure are detailed in (Maddaloni et al. 2014). Restriction enzymes, alkaline phosphatase, Phusion® Taq polymerase, and DNA ligase were purchased from New England Biolabs (NEB, Ipswich, Mass.) and used according to manufacturer's instructions. DNA sequencing was performed at the Sanger Sequencing Core Laboratory of the Interdisciplinary Center for Biotechnology Research, University of Florida. Synthetic DNA sequences were purchase from Genscript, Piscataway, N.J. To clone the oxalate decarboxylase, B. subtilis genomic DNA from a human probiotic supplement served as a template. The genomic DNA was amplified by a pair of primers, LAB258 GGATCCGTAAGGAGGAAACATTTCATG and LAB259 GAATTCGCAAGTCTTTTATTTACTGC, containing 5′ BamHI and 3′ EcoRI restriction sites. Amplification was performed for 35 cycles, extension time 20 seconds, annealing temperature 48° C. After cloning into the pMiniT vector, provided with the PCR cloning kit, the fragment was sequenced and they and 100% matched the reference sequence WP_042974741. Subsequently this insert was excised by BamHI and EcoRI restriction enzymes and inserted into expression vectors pGnMM37 and pGnMM38 (
GGGCCCTGCATTAATTTTCTTTTTTGGTTAAAATAAGCGAAAGAACCTGG
Transformation of Lactobacillus kunkeei and Fructobacillus fructosus
All cultures were grown static. Both F. fructosus and L. kunkeei were stocked at −80° C. in 200 μl 15% glycerol end-use aliquots and pre-cultured in 50 ml of MRS, fructose 1% overnight at 30° C. before transformation. The entire 50 ml pre-cultures were diluted in 250 ml of MRS, fructose 1%, 2 moles l−1 glycine and grown at 30° C. for 4 hours. Cells were harvested and further washed 3 times with 50 ml of ice-cold distilled water and one time with 20 ml of ice-cold glycerol 10%, sucrose 0.5 moles l−1. Cells were resuspended for electroporation in 0.5-0.8 ml of ice-cold glycerol 10%, sucrose 0.5 moles l−1, and 70 μl aliquots were moved into 1.5 ml pre-chilled tubes, and 1-5 μg of plasmid DNA were added. After 5 min. incubation, bacterial cells were electroporated with a BTX ECM630 in 1 mm gap at 1.7 Kv, 125 Ohm, 50 μF. Cells were rested for 5 min. on ice, resuspended in 1 ml of MRS, 1% fructose, 0.5 moles l−1 sucrose and recovered 3 hours at 30° C. Cells were then plated onto MRS Petri dishes containing 1% fructose containing 5 μg ml−1 erythromycin and grown at 30° C. for 2-3 days, until colonies were clearly visible.
Depletion of oxalate in solid medium was detected as in (Kost et al. 2014) with minor modifications. A MRS base agar was enriched with fructose 1% and 5 mmoles l−1 of MnCl2. Bacteria were appropriately diluted and spread with a sterile loop. Petri dishes were briefly dried under laminar flow. A second layer, which contained 0.7% calcium oxalate and 0.7% agarose was freshly prepared, melted in a microwave and cooled to 47° C. in a water bath. The top agar was then quickly overlaid. Plates were incubated for at least five days before the halos became visible. Plates were then photographed using natural light and a black background. For the detection of oxalate in liquid medium, potassium oxalate was used due to its higher solubility. Bacteria were inoculated at 107 CFUs/ml in 6 ml of MRS+fructose+Mn++ in a 14 ml tube, and incubated static at 34° C. After 7 days growth, oxalate in the supernatants was titrated with an Oxalate Assay Kit (Sigma-Aldrich).
With the aim of increasing the molecular toolbox for fructophilic bacteria, the annotated F. fructosus genome (Joint Genome Institute) was analyzed. Two non-coding regions upstream the operons were arbitrarily selected hypothesizing these might act as robust promoters. The first region (
Cloning of B. subtilis Oxalate Decarboxylase.
Bacillus subtilis was isolated from a commercially available probiotic and reportedly containing 15 species of bacteria (Garden of Life, Palm Beach Gardens, USA). Dehydrated bacteria were resuspended in phosphate-saline buffer (PBS) and heated at 90° C. for 15 minutes. The heated bacterial suspension was serially diluted and plated on LB medium and incubated at 37° C. As expected, the heat treatment killed all bacteria except B. subtilis spores. In fact, the colonies retrieved on LB looked morphologically homogeneous displaying the typical swarming habit of B. subtilis. One colony was chosen and confirmed as B. subtilis, by ribosomal DNA genotypying. B subtilis genomic DNA was purified and used as template for PCR amplification of oxalate decarboxylase using the primers Fwd LAB258 and Rev LAB259. The amplified fragment migrated at the expected MW and it was cloned into pMiniT (NEB). The sequence was confirmed to be 100% identical to the original, including the Shine-Dalgarno (SD) sequence. This strategy generates expression vectors with two Shine-Dalgarno sequences upstream the ATG initiation codon. The BamHI-EcoRI insert was excised, purified, and cloned into pGNMM15 (Maddaloni et al. 2014) a construct based on pIB184Term devoid of GFP. The resulting construct, named pGnMM56 (
The constructs pGnMM56 and pGnMM64 were introduced into both F. fructosus and L. kunkeei with similar efficiency of transformation. Expression was evaluated both by calcium oxalate depletion halos in Petri dishes (Allison et al 1985, Kost et al 2014) and by quantitative potassium oxalate bioremediation in culture supernatant. Wild type F. fructosus and L. kunkeei, cannot degrade calcium oxalate in Petri dishes, whereas their engineered counterparts produced clear halos.
With the rapid and hotly debated global decline of honey bee colonies, it is becoming increasingly evident that translational research, aimed at providing beekeepers with tools to improve colony health, needs to become a priority. There is a substantial hiatus between researchers and beekeepers with the latter at times, being ahead of sanctioned science, a statement that can be verified by matching the existing scientific literature and the beekeepers' chat in online fora all around the world. As an example, studies on optimization of cattle, horse and poultry nutrition initiated well over a hundred years ago. Conversely, honey bees have been, and still are, the only livestock that is kept for extended periods of times on an artificial diet composed of 100% sucrose. The practice of feeding sucrose is adopted to help bees during dearth and to build new comb. It also allows beekeepers to skim more honey. Only recently has honeybees' nutrition come under scrutiny of science and, according with what beekeepers discovered decades ago, poor diets correlated to diminished immune competence and increased overall hive weakness (Alaux et al. 2010; DeGrandi-Hoffman et al. 2010; DiPasquale et al. 2013; Morais et al. 2013). More poignant to the present investigation is the research on oxalic acid which received an inexplicable low attention by the scientific community despite the paramount relevance that this compound currently has in managing honey bee health.
The hematophagous Varroa destructor (varroa) mites are commonly believed to be a major factor driving colony losses by feeding on hemolymph, thus weakening the bees, and ferrying pathogens (reviewed in Rosenkranz et al. 2009). Despite the current relevance of oxalic acid in the fight against varroa, and the fact that first scientific reports on its use date back to the mid 90's (Radetzki 1994; Nanetti et al. 1995; reviewed in Gregorc and Planinc 2002) the recent scientific literature shows only a handful of investigations on this topic. Oxalic acid as a miticide is not uniformly regulated. However, it is safe to say that, legally or brevi manu, it is being used worldwide on a regular basis. While oxalic acid is acutely toxic to varroa, it is also toxic to honey bees (Aliano et al. 2006; Aliano et al. 2009) and it is recommended that no more than one treatment per generation of honeybees, averaging 4-6 weeks during the active season, is applied. Due to their extended lifespan, honeybee queens receive multiple treatments, a factor that may contribute to their overall decreased overall longevity. In this scenario, the capability of fine tuning the fate of oxalic acid in the hive may lead to interesting applications. Oxalic acid is a highly oxidized organic compound that is toxic to almost all organisms and cells. Oxalate anions are major chelators of metal cations, and calcium oxalate has received a great deal of attention in the medical field because it is one of the leading causes in the formation of kidney stones (Cochat and Rumsby 2013). The ability of microflora to metabolize and scavenge oxalates form the body is believed to be relevant in preventing the formation of kidney stones. In this respect, the idea of harnessing the commensal microflora, be it naturally capable of degrading oxalate or engineered, has received much attention over the last 30 years (Allison et al. 1985; Lung et al. 1991; Duong et al. 2010; Anbazhagan et al. 2013). Recently, an aggressive treatment with two weeks of daily doses of 5×1010 Lactobacillus plantarum cells, engineered with oxalate decarboxylase, proved effective in the reduction of oxalate in the kidney tissues in a rat model (Sasikumar 2014).
As a tool for fine-tuning the persistence of oxalic acid in the control of the varroa mites, oxalate decarboxylases (EC 4.1.1.2) are uniquely posed for the task, because it oxidizes both oxalates and oxalic acid generating formate and CO2 (reviewed in Makela et al. 2010) which are both volatile and do not leave residues in the comb (Calderone 2010). In the process, energy is stored because formate can be potentially further oxidized with concomitant reduction of NAD to NADH, which is a substrate for anabolic reactions, thus decreasing the metabolic burden to engineered bacteria. Here we engineered two honey bee commensals, F. fructosus and L. kunkeei, to express a gene encoding for an oxalate decarboxylase cloned from a strain of B. subtilis that we isolated from a commercially available probiotic, advertised to be effective against kidney stones. Because oxalate decarboxylase can catabolize both oxalic acid and oxalates, calcium oxalate (in qualitative Petri dish test) and potassium oxalate (in a quantitative liquid culture test) were used in lieu of oxalic acid, under the constraint to minimize the impact of low pH on the bacterial growth. This approach, besides determining the bioremediation capability of the bacteria, is also functional in assessing the potential need to provide an oxalate transporter (Anantharam et al 1989; lyalomhe et al. 2014). As it can be seen from
Paratransgenesis, a “Trojan horse” approach originally designed to control insect-borne diseases, is being re-thought in broader terms to improve the health of beneficial arthropods, particularly honey bees (Rangberg et al. 2012, Maddaloni et al. 2014, Rangberg et al. 2015). Extensive applications of this technology can be envisioned. For example the expression of enzymes, such as some carboxylesterases capable of degrading extremely toxic insecticides, is expected to mitigate their negative effects. The present example focuses on oxalate, a molecule which, while having a moderate toxicity to honey bees, is used as a medication against the devastating varroa mites. The data presented here show that engineered honey bee commensals have the potential to remove organic molecules, oxalates in this case, in solutions at concentrations in the mg/ml range. Such a range of concentrations exceeds by several orders of magnitude the insecticide LD50 that a single honey bee can absorb even from highly contaminated food. This can be projected by combining any of the honey bee LD50 (ng- to μg/bee) (Sanchez-Bayo and Goka 2014) for a wide panel of insecticides, the full capacity of the honey stomach of individual honey bees (40 μl/bee) (Nicolson et al. 2013) and the expected number of bacteria contained in the whole honey bee intestine, including the honey stomach, which varies greatly depending on the study (Olofsson and Vasquez 2008, Martinson et al 2012, Corby-Harris et al. 2014). In line with this expectation the expression of a Lucilia cuprina carboxylesterase (Jackson et al 2014) in honey bee commensals can confer honey bees protection against a 40 LD50 of cypermethrin (Maddaloni et al. in preparation). In conclusion the present example provides for the bioremediation of problematic organic compounds in the honey bee hive.
Pseudomonas putida KT2440 engineered with a construct bearing the cassette borne on pGnMM83 (
Additional examples of oxalate decarboxylases, with sequences, are described below:
It is noted that the above cassette is optimized for expression in Lactococci with adaptability to E. coli and Pseudomonas putida.
The cch2 cassette described above in the examples, and examples of sequences of which are shown in Example 10, can be expressed in a host with an endogenous 6-HNA degradation operon, for example that of P. putida KT2440. Such expression of the cch2 cassette in an organism with an operable 6-HNA degradation operon allows for the utilization of 6-CNA as a carbon source, as neonicotinoids can be degraded into 6-CNA, and the resultant 6-CNA funneled into the 6-HNA degration operon pathway (
Further information regarding the P. putida 6-HNA degradation operon is described in Jimenez J I, Canales A, Jimenez-Barbero J, Ginalski K, Rychlewski L, Garcia J L, Diaz E. “Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440. Proc Natl Acad Sci USA. 2008 Aug. 12; 105(32):11329-34. doi: 10.1073/pnas.0802273105. Epub 2008 Aug. 4, which is incorporated by reference as if fully set forth herein.
Briefly, an example of a fully-functional 6-HNA degradation operon (top of
If not expressed endogenously, as illustrated in
It is noted that recombinant codon-optimized coding sequences of gene products as listed above can be derived from the 13 kb region of the Pseudomonas putida KT2440 chromosome, complete genome, from base 4441000 to 4454000 (NCBI reference sequence NC_002947.4) using the full peptide coding sequences above and cloned into expression backbones as fully functional cassettes or individual coding sequences for one or more of the gene products above.
Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of separating, testing, and constructing materials, which are within the skill of the art. Such techniques are explained fully in the literature.
It should be emphasized that the above-described embodiments are merely examples of possible implementations. Many variations and modifications may be made to the above-described embodiments without departing from the principles of the present disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.
This application claims priority to U.S. Provisional application entitled “BIOREMEDIATION OF XENOBIOTICS IN THE HONEY BEE HIVE,” having Ser. No. 62/750,396, filed on Oct. 25, 2018, which is entirely incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/058146 | 10/25/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62750396 | Oct 2018 | US |
