Bacteria expressing lysins for inhibition of methanogenesis

Abstract

The disclosure relates to compositions and methods of bacterial species expressing endolysins for the prophylaxis and treatment of methanogen growth in the digestive tract of an animal. More specifically, the disclosure relates to agents, compositions, and methods for preventing and / or inhibiting growth of methanogens.

Description

BACTERIA EXPRESSING LYSINS FOR INHIBITION OF METHANOGENESISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing dates of U.S. Provisional Application No. 63 / 569,351, filed March 25, 2024, the entire contents of which is incorporated by reference herein.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 24, 2025, is named 064674-501001WO.xml and is 150,993 bytes in size.BACKGROUND

[0003] Silage is a type of livestock feed which is produced from green plant material preserved via fermentation. The process of ensiling is one of the largest scale fermentations carried out by humans, with —130 million tonnes produced in the US for com silage alone in 2023. In general, silage begins with green forage material or crops which would otherwise be spoiled by aerobic organisms, being stored in an anaerobic environment (often a silo). In the anaerobic environment, the green material is acidified by fermentation, for example by lactic acid bacteria (LABs), which preserves it from spoilage. This process can be enhanced by deliberate introduction of selected strains of LAB into the ensiling process to promote desirable fermentation characteristics. By enhancing fermentation efficiency and reducing nutrient losses, LAB inoculants improve silage stability, palatability, and overall feed value, contributing to more sustainable ruminant production systems.

[0004] Almost 30% of all green-house gas warming in agriculture in the United States comes from the methane produced by enteric fermentation in cows, sheep, and goats. Methane production in the cow rumen is a significant contributor to greenhouse gas emissions, occurring as a natural byproduct of microbial fermentation during the breakdown of feed by anaerobic microorganisms. The primary methane-producing microbes in the rumen are methanogenic archaea, which utilize hydrogen and carbon dioxide produced during fermentation to generate methane. Strategies to mitigate methane emissions from cow rumen include dietary interventions, such as altering feed composition or using feed additives, as well as microbial interventions aimed at modifying the rumen microbiome to reduce methanogen populations or enhance hydrogen utilization pathways.SUMMARY

[0005] The present disclosure provides for microbial species capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when consumed by an animal.

[0006] In one aspect, a lactic acid bacteria genetically engineered to express a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when consumed by an animal is provided.

[0007] In one aspect, a lactic acid bacteria comprising a nucleic acid encoding a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when consumed by an animal is provided.

[0008] In some embodiments, the lysin is a lysin from a bacteriophage. In some embodiments, the lysin is selected from the lysins in Table 2 A or Table 2B. In some embodiments, the lysin is selected from SEQ ID NOs: 1-12 or SEQ ID NOs: 13-124. In some embodiments, the lysin inhibits the growth of the methanogen species. In some embodiments, the lysin kills the methanogen species. In some embodiments, the lysin reduces the growth of the methanogen species. In some embodiments, the lysin is encoded on a plasmid. In some embodiments, the lysin is encoded on the bacterial chromosome. In some embodiments, the lysin comprises a signal sequence. In some embodiments, the lysin comprises a protein tag. In some embodiments, the methanogen species is selected from the group of Methanobrevibacter, Methanothermobacter, Methanomicrobium, Methanosphaera, Methanothrix, Methanobacterium, and Methanosarcina. In some embodiments, the methanogen species is located in the digestive tract of the animal. In some embodiments, the lactic acid bacteria are present in the digestive tract of the animal. In some embodiments, the animal is a ruminant, optionally wherein the ruminant is a bovine. In some embodiments, the lactic acid bacteria are selected from Table 1. In some embodiments, the lactic acid bacteria are present in silage. In some embodiments, silage is consumed by the animal.

[0009] In one aspect, a composition comprising a microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the silage is consumed by an animal is provided.

[0010] In some embodiments, the composition further comprises a second microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the silage is consumed by an animal. In some embodiments, a silage comprising the microbial species ofthe disclosure is provided. In some embodiments, the lysin is selected from the lysins in Table 2A or Table 2B. In some embodiments, the lysin is selected from SEQ ID NOs: 1-12 or SEQ ID NOs: 13-124. In some embodiments, the microbial species is selected from Table 1.

[0011] In one aspect, a method of producing or treating silage, the method comprising contacting the silage with a composition comprising on or more microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the silage is consumed by an animal is provided.

[0012] In some embodiments, the microbial species is selected from the microbial species in Table 1. In some embodiments, the lysin is selected from the lysins in Table 2 A or Table 2B.

[0013] In a further aspect, the lysin is encoded by a nucleic acid comprising a polynucleotide sequence operatively linked to a promoter active in lactic acid bacteria.

[0014] In some embodiments, a vector comprises the polynucleotide sequence of a lysin.

[0015] In a further aspect, an additive composition for use in feed, comprising the lactic acid bacteria is disclosed. In embodiments, a feedstock for a ruminant, comprising the lactic acid bacteria is disclosed.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows a schematic for the cloning strategy for constructing the plasmid pPG 1 to deliver the lysin gene in a lactic acid bacteria. The gene cassette CPl-6x-His-Lysin was synthesized and cloned resulting in the pMET-Lysin plasmid. pMET-Lysin and a broad host range vector BHR1H was linearized by Xhol and PstI restriction enzyme digestion and subsequently ligated in a sticky-end ligation reaction resulting in the plasmid pPG 1.

[0017] FIGs. 2A and 2B show RT-PCR analysis of a lysin-expressing E.faecium strain. FIG. 2A shows a representative agarose gel following RT-PCR of transformed E.faecium strains compared to the control. Arrow represents the PCR product of the expected size. FIG. 2B provides sample identification corresponding to lanes of the agarose gel shown in FIG. 2A.

[0018] FIG. 3 is a graphical illustration of the lab-scale silage setup.

[0019] FIGs. 4A and 4B show RT-PCR analysis of a lysin-expressing E.faecium strain grown on silage. FIG. 4A shows a representative agarose gel following RT-PCR of transformed E. f aecium strains compared to the control. Arrows represent PCR products ofthe expected size. FIG. 4B provides sample identification corresponding to lanes of the agarose gel shown in FIG. 4A.

[0020] FIG. 5 is a boxplot showing growth of Methanobrevibacter ruminatium measured as OD(600nm) in the presence of purified, exemplary methanogen lysin set forth by SEQ ID NO: 13 compared to the control.

[0021] FIG. 6 is a boxplot showing the percentage (%) of endpoint methane (CH4) in the laboratory culture of Methanobrevibacter ruminatium in the presence of purified, exemplary methanogen lysin set forth by SEQ ID NO: 13 compared to the controls.

[0022] FIG. 7 is a graphical illustration of the composition and method of the disclosure. (1): on harvest, fresh silage is inoculated with genetically engineered lactic acid bacteria, (2): during ensiling and fermentation engineered lactic acid bacteria multiply and also express a lysin protein of interest, and (3): when ruminants ingest silage produced in this manner the lysin protein is active in the rumen and kills methanogenic archaea, reducing the production of methane in the rumen and thus methane emissions.DETAILED DESCRIPTION

[0023] The present disclosure relates, in part, to compositions and methods of bacterial species expressing a phage derived lysin specific for a methanogen species. The bacterial compositions can be used to inhibit the growth, reduce the growth, and / or kill a methanogen species that resides in the digestive tract of an animal, for example a bovine. The bacterial compositions of the disclosure can be used to prepare or treat silage that can be fed to the animal to reduce methanogens in the animal digestive tract.Definitions

[0024] The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0025] The terms “a” or “an,” as used in herein means one or more.

[0026] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about,” when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.

[0027] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to havespecifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96- 99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.[0028J “Gram-negative bacteria” generally refers to bacteria which produce a crystal violet stain that is decolorized in Gram staining, i.e., they do not retain crystal violet dye in the Gram staining protocol. Gram-negative bacteria can be characterized by an outer membrane composed of lipopolysaccharide (LPS) molecules with a highly variable O-polysaccharide (O-antigen).

[0029] “Gram-positive bacteria” generally refers to bacteria which produce a crystal violet stain that is colorized in Gram staining, i.e., they do retain crystal violet dye in the Gram staining protocol. Gram-positive bacteria can have essential components such as (lipo)teichoic acids embedded in peptidoglycan (PG). Gram-positive bacteria include but are not limited to the genera Actinomyces, Bacillus, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, and Clostridium. Medically relevant species include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. Bacillus species, which are spore-forming, cause anthrax and gastroenteritis. Spore-forming Clostridium species are responsible for botulism, tetanus, gas gangrene and pseudomembranous colitis. Corynebacterium species cause diphtheria, Listeria species cause meningitis. Enterococcus species can cause urinary tract infections (UTI), bacteremia, and infective endocarditis and rarely cause intra-abdominal infections and meningitis. Staphylococcus species can cause skin infections, bacteremia, bone infections, endocarditis, food poisoning, pneumonia, and toxic shock syndrome. Streptococcus species can cause pharyngitis, pneumonia, wound and skin infections, sepsis, and endocarditis.

[0030] The term “bactericidal” in the context of an agent or composition conventionally means having the property of causing the death of bacteria or capable of killing bacteria to an extent of at least a 3-log (99.9%) or better reduction among an initial population of bacteria.

[0031] The term “bacteriostatic” in the context of an agent or composition conventionally means having the property of inhibiting bacterial growth, including inhibiting growingbacterial cells, thus causing a 2-log (99%) or better and up to just under a 3 -log reduction among an initial population of bacteria.

[0032] The term “antibacterial” in the context of an agent or composition is used generically to include both bacteriostatic and bactericidal agents.

[0033] The term “drug resistant” in a context of a pathogen and more specifically a bacterium, generally refers to a bacterium that is resistant to the antimicrobial activity of a drug. When used in a more particular way, drug resistance specifically refers to antibiotic resistance. In some cases, a bacterium that is generally susceptible to a particular antibiotic can develop resistance to the antibiotic, thereby becoming a drug resistant microbe or strain. A “multi-drug resistant” pathogen is one that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, certain strains of Pseudomonas aeruginosa have been found to be resistant to nearly all or all antibiotics including aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems (Antibiotic Resistant Threats in the United States, 2013, U.S. Department of Health and Services, Centers for Disease Control and Prevention). One skilled in the art can readily determine if a bacterium is drug resistant using routine laboratory techniques that determine the susceptibility or resistance of a bacterium to a drug or antibiotic.

[0034] The term “fusion polypeptide” refers to an expression product resulting from the fusion of two or more nucleic acid segments, resulting in a fused expression product typically having two domains or segments with different properties or functionality. In a more particular sense, the term “fusion polypeptide” also refers to a polypeptide or peptide comprising two or more heterologous polypeptides or peptides covalently linked, either directly or via an amino acid or peptide linker. The polypeptides forming the fusion polypeptide are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The term “fusion polypeptide” can be used interchangeably with the term “fusion protein.” Thus the open-ended expression “a polypeptide comprising” a certain structure includes larger molecules than the recited structure such as fusion polypeptides.

[0035] The term “active fragment” refers to a portion of a full-length polypeptide disclosed herein which retains one or more functions or biological activities of the isolated original polypeptide.

[0036] The term “derivative” in the context of a peptide or polypeptide (which as stated herein includes an active fragment) is intended to encompass for example, a polypeptide modified to contain one or more-chemical moieties other than an amino acid that do notsubstantially adversely impact or destroy the endolysin activity. The chemical moiety can be linked covalently to the peptide, e.g., via an amino terminal amino acid residue, a carboxy terminal amino acid residue, or at an internal amino acid residue. Such modifications include the addition of a protective or capping group on a reactive moiety, addition of a detectable label, such as antibody and / or fluorescent label, addition or modification of glycosylation, or addition of a bulking group such as PEG (pegylation) and other changes that do not substantially adversely impact or destroy the activity of the endolysin polypeptide. Polyethylene glycol (PEG) conjugation to proteins has been used as a method for extending the circulating half-life of many pharmaceutical proteins. Thus, in the context of endolysin polypeptide derivatives, the term “derivative” encompasses endolysin polypeptides chemically modified by covalent attachment of one or more PEG molecules. It is anticipated that pegylated endolysin polypeptides may exhibit prolonged circulation half-life compared to the unpegylated endolysin polypeptides, while retaining biological and therapeutic activity.

[0037] The terms “bind” and “bound” as used herein is used in accordance with its plain and ordinary meaning and refers to the association between atoms or molecules. The association can be direct or indirect. For example, bound atoms or molecules may be bound, e.g., by covalent bond, linker (e.g., a first linker or second linker), or non-covalent bond (e.g. electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).

[0038] As used herein, the term “conjugated” when referring to two moieties means the two moieties are bonded, wherein the bond or bonds connecting the two moieties may be covalent or non-covalent. In embodiments, the two moieties are covalently bonded to each other (e.g., directly or through a covalently bonded intermediary). In embodiments, the two moieties are non-covalently bonded (e.g., through ionic bond(s), van der Waal’s bond(s) / interactions, hydrogen bond(s), polar bond(s), or combinations or mixtures thereof).

[0039] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modifiedR groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0040] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0041] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers.

[0042] A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0043] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0044] The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0045] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0046] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0047] An amino acid or nucleotide base “position” is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N- terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to aposition in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0048] The terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0049] An amino acid residue in a protein “corresponds” to a given residue when it occupies the same essential structural position within the protein as the given residue.

[0050] The term “isolated”, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0051] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non- limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may include natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0052] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T)when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0053] As used herein, a “subsequence” refers to a length of contiguous amino acids or nucleotides that form a part of a sequence described herein. A subsequence may be identical to a part of a full length sequence when aligned to the full length sequence, or less than 100% identical to the part of the full length sequence to which it aligns (e.g., 90% identical to 50% of the full sequence, or the like).

[0054] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0055] A polynucleotide is “operably linked” to another polynucleotide when it is placed into a functional relationship with the other polynucleotide. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. A peptide is “operably linked” to another peptide when the polynucleotides encoding them are operably linked, preferably they are in the same open reading frame.

[0056] A “promoter” is a sequence of DNA needed to turn a gene on or off. Promoters are located immediately upstream and / or overlapping the transcription start site, and are usually between about one hundred to several hundred base pairs in length.

[0057] A “cell” as used herein, refers to a cell carrying out metabolic or other functions sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria or archae. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

[0058] The term “plasmid,” “expression vector,” or “viral vector” refers to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids. Suitable viral vectors contemplated herein include, for example, lentiviral vectors and onco-retroviral vectors.

[0059] As used herein, the term “expression” is used in accordance with its plain ordinary meaning and refers to a step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression may be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0060] A “methanogen,” as used herein, refers to microbes that produce methane gas. Methanogens may reside in an animal, for example a ruminant. Methanogens include Methanobrevibacter, Methanothermobacter, Methanomicrobium, Methanosphaera, Methanothrix, Methanobacterium, and Methanosarcina. Specific methanogens include, but are not limited to, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter acididurans, Methanobrevibacter thaueri, Methanobacterium bryantii, Methanobacterium formicicum, Methanothermobacter marburgensis, Methanothermobacter wolfeii, Methanosphaera stadtmanae, Methanomicrobium mobile, Methanosarcina barkeri, Methanosarcina mazei, Methanococcoides burtonii, and Methanolobus taylorii.

[0061] As used herein, the terms “silage” to any plant material suitable for consumption by a ruminant. Examples of silage or forage include, but are not limited to, com silage, sorghum silage, wheat silage, oat silage, grass silage, or alfalfa silage.

[0062] As used herein, “bacteriophage” and “phage” refer to viruses which infect bacteria and / or archaea.Bacteria Expressing Lysins

[0063] Provided herein are microbial species expressing lysin polypeptides (including active fragments thereof). In particular, the present disclosure relates to microbial species expressing lysin polypeptides (including active fragments thereof) active against methanogens residing in animals, such as Methanobrevibacter, Methanothermobacter, Methanomicrobium, Methanobacterium, and Methanosarcina.

[0064] Lysins (for example endolysins) are bacteriophage-encoded enzymes. Bacterial and archaea cells can be lysed by lysins specific for the bacterial or archaea species by targeting peptidoglycans or similar in structure to peptidoglycans in the cell wall. Gram-positive bacteria can be targeted with endolysins due to their naturally exposed peptidoglycan layer. Endolysins can have a modular architecture composed of one or more catalytic or enzymatically active domains (EAD) that may be connected by flexible linkers to one or more cell wall-binding domains (CBD). The EADs can be broadly distributed into three classes (glycosidases, amidases, and peptidases) depending on the type of bond in the murein or pseudomurein cell wall that they can act upon. Differences in CBD presence and composition can be largely dependent upon the microbial target. Endolysins targeting Gram positive bacteria can contain one or more N-terminal EAD and one or more C-terminal CBD, but endolysins acting on Gram-negative bacteria typically only have one EAD. In both cases, a successful phage lytic cycle relies on endolysin access to the peptidoglycan cell wall, meaning the enzyme must cross the inner or cytoplasmic membrane. This access is typically achieved through holins, proteins that form channels in the inner membrane which allow the enzyme access to the periplasmic space. The mode of action for endolysins can have implications for their application as antimicrobials. For Gram-positive bacteria, externally applied endolysins can directly access and degrade the cell wall, but for Gram negative bacteria the outer membrane poses a barrier that requires further consideration when selecting and screening candidate endolysins. For archaea, pseudomurein endopeptidases have been described that can bind the archaea cell wall and cleave the peptide links that connect adjacent pseudomurein glycan strands. The activity of an endolysin can include degrading the cell wall / membrane, binding of the cell wall / membrane, or cell surface receptors, penetrating the cell wall / incinbranc, and / or outer membrane destabilization.

[0065] In one aspect, a microbial species genetically engineered to express a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when consumed by an animal is provided.

[0066] In one aspect, a microbial species comprising a nucleic acid encoding a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when consumed by an animal is provided.

[0067] In one aspect, a composition comprising one or more microbial species genetically engineered to express one or more lysins that specifically inhibit the growth, reduce the growth, and / or kills a methanogen species when consumed by an animal is provided.

[0068] In one aspect, a composition comprising one or more microbial species comprising a nucleic acid encoding one or more lysins that specifically inhibit the growth, reduce the growth, and / or kill a methanogen species when consumed by an animal is provided.

[0069] In some embodiments, the microbial species expressing the lysin polypeptide is a lactic acid bacteria (LAB). In some embodiments, the microbial species can be a strain of Enterococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Lactococcus, Lentilactobacillus, Levilactobacillus, Pediococcus, Weisella, or Bacillus, or other probiotic bacteria. In some embodiments, the microbial species is selected from a microbial species of Table 1.

[0070] In some embodiments, the microbial species can be a strain of Enterococcus. In some embodiments, the microbial species can be a strain of Lactobacillus. In some embodiments, the microbial species can be a strain of Lactobacillus. In some embodiments, the microbial species can be a strain of Lacticaseibacillus. In some embodiments, the microbial species can be a strain of Lactiplantibacillus. In some embodiments, the microbial species can be a strain of Lactococcus. In some embodiments, the microbial species can be a strain of Lentilactobacillus. In some embodiments, the microbial species can be a strain of Levilactobacillus. In some embodiments, the microbial species can be a strain of Pediococcus. In some embodiments, the microbial species can be a strain of Weisella. In some embodiments, the microbial species can be a strain of Bacillus.

[0071] Exemplary bacterial strains for Endo lysin expression are listed in Table 1.Table 1: Exemplary Bacterial Strains for Endolysin Expression

[0072] In some embodiments, the microbial species genetically engineered to express one or more lysins that specifically inhibit the growth, reduce the growth, and / or kills a methanogen species when consumed by an animal is present in the digestive tract of the animal.

[0073] In some embodiments, the microbial species genetically engineered to express one or more lysins that specifically inhibit the growth, reduce the growth, and / or kills a methanogen species when consumed by an animal is provided to an animal.

[0074] In some embodiments, the microbial species genetically engineered to express one or more lysins that specifically inhibit the growth, reduce the growth, and / or kills a methanogen species when consumed by an animal is provided to an animal in silage.Lysins

[0075] Provided herein are lysins and compositions comprising the same.

[0076] In one embodiment, the lysin is a lysin from a bacteriophage. In aspects, lysins are from phage that infect archaea. In embodiments, the major phyla in archaea comprise Crenarchaeota, Euryarchaeota, Korarchaeota, Nanoarchaeota, and Thaumarchaeota. In aspects, the Euryarchaeota phylum comprises Methanobrevibacterium.

[0077] In embodiments, the Methanobrevibacterium comprise species Methanothermobacter marburgensis, Methanothermobacter wolfeii, and Methanobrevibacter ruminantium Ml . In embodiments, PeiP is derived from phage M2 infecting Methanothermobactermarburgensis, PeiW is derived from phage M 100 infecting Methanothermobacter wolfeii and PeiR is derived from phage cp-mru infecting Methanobrevibacter ruminantium Ml. In embodiments, the Methanobrevibacter ruminantium lysin is PeiR.

[0078] In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In one embodiment, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to ane one of SEQ ID NOs: 14-124.

[0079] In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuoussequence within SEQ ID NO: 1. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 2. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 3. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 4. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 5. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 6. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 7. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 8. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 9. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 10. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 11. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 12. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within SEQ ID NO: 13. In one aspect, the lysin is a lysin having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a 200 amino acid continuous sequence within any one of SEQ ID NOs: 14-124.Table 2 A: Illustrative Lysin sequences and Targeted Microbial GenusTable 2B: Illustrative Lysin sequences and Targeted Microbial Genus

[0080] In one embodiment, the endolysin targets a microbial species. In one embodiment, the lysin targets more than one microbial species. In one embodiment, the microbial species is a Gram-positive bacteria. In one embodiment, the microbial species is a Gram-negative bacteria. In one embodiment, the microbial species is a archaea. In one embodiment, the microbial species is a methanogen species. In one embodiment, the lysin targets a methanogen species. In one embodiment, the lysin targets a methanogen species from Table 2A or Table 2B. In one embodiment, the lysin targets a Methanobrevibacter, Methanothermobacter, Methanomicrobium, Methanobacterium, Methanosphaera, Methanothrix, or Methanosarcina species or a combination thereof. In one embodiment, the lysin targets a Methanobrevibacter. In one embodiment, the lysin targets a Mathanosphaera. In one embodiment, the lysin targets a Methanothrix. In one embodiment, the lysin targets a Methanothermobacter. In one embodiment, the lysin targets a Methanomicrobium. In one embodiment, the lysin targets a Methanobacterium. In one embodiment, the lysin targets a Methanosarcina. In one embodiment, the methanogen species targeted by the lysin is Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter acididurans, Methanobrevibacter thaueri, Methanobacterium bryantii, Methanobacterium formicicum, Methanothermobacter marburgensis, Methanothermobacter wolfeii, Methanosphaera stadtmanae, Methanomicrobium mobile, Methanosarcina barkeri, Methanosarcina mazei, Methanococcoides burtonii, or Methanolobus taylorii.

[0081] In one embodiment, the methanogen species is located in the digestive tract of the animal. In one embodiment, the animal is a ruminant, for example a bovine. Other ruminants comprise but are not limited to goats, sheep, giraffes, yaks, deer, and antelope.

[0082] In one embodiment, the targeting comprises a lytic activity against a microbial species. In one embodiment, the targeting comprises a hydrolase activity against a microbial species. In one embodiment, the targeting comprises a glycosidase activity against a microbial species. In one embodiment, the targeting comprises an amidase activity against a microbial species. In one embodiment, the targeting comprises a peptidase activity against a microbial species.

[0083] In one embodiment, the lysin has activity against a microbial species. In one embodiment, the activity comprises inhibiting the growth of a microbial species. In one embodiment, the activity comprises killing a microbial species. In one embodiment, the activity comprises reducing the growth of a microbial species.Engineered Lysins

[0084] In one embodiment, the lysin is engineered to reduce protease degradation and increase stability of the lysin. Proteases that degrade lysins include but are not limited to trypsin, chymotrypsin, pepsin, and peptidase. Protease degradation can be reduced, for example by removing or mutating protease cleavage sites. In one embodiment, the lysin is protease resistant.

[0085] In one embodiment, the lysin engineered to reduce immunogenicity.In one embodiment, the lysin is a fusion protein. In one embodiment, the lysin comprises an N or C terminal fusion protein. In one embodiment, the lysin comprises a signal sequnence, for example a secretion signal.

[0086] In one embodiment, the lysin is a chimeric lysin. In one embodiment, the chimeric lysin comprises domains from one or more lysins. In one embodiment, the chimeric lysin comprises domains from one or more lysins selected from Table 2 A or Table 2B. In one embodiment, the chimeric lysin comprises catalytic N-terminal domains and cell wall-binding domains from one or more lysins. In one embodiment, the chimeric lysin comprises rearranged N- and C-terminal domains of the same lysin. In one embodiment, the chimeric lysin comprises rearranged N- and C-terminal domains of the different lysins.

[0087] In embodiments, a chimeric lysin of the disclosure, such as any of the sequences of Table 2 A or Table 2B can be modified.

[0088] In one embodiment, the endolysin is a circular permuted lysin. A circular permutation is a relationship between proteins whereby the proteins have a changed order of amino acids in their peptide sequence. The result is a protein structure with different connectivity, but overall similar three-dimensional (3D) shape.

[0089] In one aspect, the lysin comprises an N or C terminal fusion protein. In embodiments, the fusion protein is a tag. In one embodiment, the fusion protein is a signal sequence. In one embodiment, the fusion protein is a SPK1 signal peptide. In one embodiment, the fusion protein is a protein tag. In one embodiment, the fusion protein is a fluorescent protein. In one embodiment, the fluorescent protein is a GFP protein. In one embodiment, the fluorescent protein is a GFP, an eGFP, a RFP, a YFP, a BFP, or a CFP.

[0090] Provided herein are, inter alia, nucleic acids encoding a lysin described herein. In some embodiments, the nucleic acid is a DNA or RNA. In some embodiments, the DNA is circular plasmid DNA, linear double-strand DNA, single strand DNA, or chimeric RNA and DNA. In some embodiments, the bacterial chromosome is engineered to encode the lysin. In some embodimnets the lysin is encoded on a plasmid.

[0091] Codons may be selected to increase the rate at which expression of the polypeptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. For example, codons can be optimized for expression in a lactic acid bacteria in accordance with known methods. Other reasons for substantially altering the nucleotide sequence encoding polypeptides and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.Methods for Producing Bacteria

[0092] In an aspect, the disclosure includes methods for producing the microbial species expressing the lysin polypeptides of the present disclosure.

[0093] In some embodiments, the method comprises contacting the microbial species with a nucleic acid encoding the lysin polypeptides of the present disclosure. In some embodiments, the method comprises contacting the microbial species with a plasmid. In embodiments, the microbial species is a E. coli strain. In embodiments, the microbial species is a Lactic acid bacteria. In embodiments, the microbial species is selected from Lactobacillus, Lactococcus, Enterococcus, Pediococcus, Leuconostoc, Streptococcus, Camobacterium, Fructobacillus, Oenococcus, and Weissella or any of the species listed in Table 1. In embodiments, the Lactic acid bacteria are an Enterococcus strain. In embodiments, the Enterococcus strain is E.faecium. In some embodiments, the microbial species is Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus acidilactici, or Pediococcus pentacaceus, Lactobacillus buchneri, Lactobacillus casei, Enterococcus faecalis, Enterococcus hirae, Enterococcus casseliflavus, Enterococcus mundtii, or Bacillus subtilis. In some embodiments, the method comprises genetically engineering the chromosome of the microial species.

[0094] To obtain a level of lysin polypeptide expression, lysin polynucleotide sequences are typically expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate cellular host. Such operative linking of a polynucleotide sequences encoding lysin polypeptides of the present disclosure to an expression control sequence, includes, the provision of an initiation codon, ATG, in the correct reading frame upstream of the polynucleotide (DNA) sequence. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and / or to express a polypeptide in a host may be used for expression of lysin polypeptides. The appropriate DNA / polynucleotide sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning, A Laboratory Manual. Additionally, tags can also be added to endolysin polypeptides to provide convenient methods of isolation, e.g., c-myc, biotin, poly-His, etc. Kits for such expression systems are commercially available.Vectors

[0095] In a further aspect, the lysin is encoded by a nucleic acid comprising a polynucleotide sequence operatively linked to a promoter active in lactic acid bacteria. In some emdodiments, a vector comprises the polynucleotide sequence of a lysin.

[0096] A wide variety of host / expression vector combinations may be employed in expressing the polynucleotide sequences encoding lysin polypeptides of the present disclosure. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Examples of suitable vectors are provided in Sambrook et al, eds., Molecular Cloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (2001). Such vectors include, among others, chromosomal, episomal and virus- derived vectors, e.g. , vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derivedfrom combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Furthermore, said vectors may provide for the constitutive or inducible expression of endolysin polypeptides of the present disclosure. More specifically, suitable vectors include but are not limited to derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids colEl, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4, pBAD24 and pBAD-TOPO; phage DNAS, e.g., the numerous derivatives of phage Z, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 D plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like. Many of the vectors mentioned above are commercially available from vendors such as New England Biolabs, Addgene, Clontech, Life Technologies etc many of which also provide suitable host cells).

[0097] Additionally, vectors may comprise various regulatory elements (including promoter, ribosome binding site, terminator, enhancer, various cis-elements for controlling the expression level) wherein the vector is constructed in accordance with the host cell. Any of a wide variety of expression control sequences (sequences that control the expression of a polynucleotide sequence operatively linked to it) may be used in these vectors to express the polynucleotide sequences encoding endolysin polypeptides. Useful control sequences include, but are not limited to: the early or late promoters of the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage Z, the control regions of fd coat protein, the promoter for 3 -phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast-mating factors, E. coli promoter for expression in bacteria, and other promoter sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0098] In embodiments, the vector to express a lysin in lactic acid bacteria is pAF 100, pAK80, pDK6, pDL, pFUN, pGEM, pLB141, pLB85, pNZ273, pNZ8048, pNZ8148, pOTHY12, pRV300, pSEC, pSH71, pSIP, pTINX, or pValac. In embodiments, the vector is pPGl. In embodiments, the vector is pET-29b(+).

[0099] In embodiments, the lysin is operatively linked to a purification tag. In embodiments, the purification tag is a histidine tag or a His-tag. In embodiments, the purification tag may be selected from the list, but not limited to, a HaloTag, HiBiT, Maltose binding protein (MBP),Calmodulin binding peptide (CBP), Strep-tag, Chitin binding domain (CBD), Epitope tags, GST-tagged proteins, myc-tag, FLAG tag, or Streptavidin-binding peptide (SBP).

[0100] In embodiments, the vector to express a lysin in a lactic acid bacteria is a broad host range vector. Such vectors include, without limitation, those derived from major broad-host- range incompatibility groups IncO (<?.g.,RSF1010, R300B, R1162), IncW (e.g., pSa, pR388), IncP (e.g., R18, R68, RK2, RP1, RP4), IncN and IncU (e.g, RA3), pBBRl, pDSK509, pDSK519, pRK415, pLAFR3, pLAFR5, pUC128, pUC129. In embodiments, the broad host range vector is pTRKH2. In embodiments, the broad host range vector is based on pTRKH2. In embodiments, the broad host range vector is BHR1H.

[0101] In embodiments, the vector to express a lysin in a lactic acid bacteria further comprises an E. coli origin of replication sequence. In embodiments, the vector to express a lysin in a lactic acid bacteria further comprises an Enterococcus origin of replication sequence.

[0102] In embodiments, the vector further comprises a native Lactic acid bacteria promoter. In embodiments, the vector comprises a native Lactic acid bacteria promoter CPI. In aspects, the vector comprises a native Lactic acid bacteria promoter Ptuf.

[0103] In embodiments, the lactic acid promoter is the promoter of the L-lactate dehydrogenase (IdhL) gene from Lactobacillus plantarum, Ppgm, ery, Pl, P8, P5, P6, P170, P45 series, P170, lacA promoter from Lactococcus lactis, promoters from FOS (Pfos), lac (Plac), and tre (Ptre) operons, PsrfA, PnisA, nisA, PsapA, Papf, PczcD, PgroESL, Pnis, PSPL, PthyA, PxylT, Pzn, pBacA, or PslpA.

[0104] In aspects, a vector comprises a nucleic acid sequence encoding for lysin. In aspects, the nucleic acid sequence comprises at least 80%, 85%, 90%, 95%, 98% or 99% identity to a corresponding nucleic acid sequence of lysins from Table 2A or Table 2B.

[0105] Efficient expression of endolysin polypeptides and vectors thereof depends on a variety of factors such as optimal expression signals (both at the level of transcription and translation), correct protein folding, and cell growth characteristics. Regarding methods for constructing the vector and methods for transducing the constructed recombinant vector into the host cell, conventional methods known in the art can be utilized. While it is understood that not all vectors, expression control sequences, and hosts will function equally well to express the polynucleotide sequences encoding endolysin peptides of the present disclosure, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this disclosure.

[0106] The engineered microbial species can then be produced in large scale. Large scale production of microbial species is known in the art, for example in fermenters.Silage and Silage Additives

[0107] In an aspect, the disclosure provides silage treated with lactic acid bacteria, as disclosed herein, for use, e.g., as a feedstock for cattle. In a further aspect, an additive composition for use in feed, comprising the lactic acid bacteria is disclosed. In embodiments, a feedstock for a ruminant, comprising the lactic acid bacteria is disclosed.

[0108] In some embodiments, a composition comprising the microbial species and a water supplement, e.g. , drenching composition, or food supplement, e.g. , ruminant feed component, is provided. In some embodiments, the feed component component comprises a forage. In some embodiments, the forage comprises hay, grass, grain, or meal, for example, legume hay, grass hay, com silage, grass silage, legume silage, com grain, oats, barley, distillers grain, brewers grain, soy bean meal, and cotton seed meal. In some embodiments, the forage is silage.

[0109] In an aspect, a composition comprising a microbial species capable of being cultured on forage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the forage is consumed by an animal is provided.

[0110] In some embodiments, the composition further comprises a second microbial species capable of being cultured on forage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the forage is consumed by an animal.

[0111] In some embodiments, a forage comprising a microbial species of the disclosure is provided.

[0112] In embodiments, the siliage comprises lactic acid bacteria comprising a polynucleotide encoding and / or expressing a lysin selected from Table 2 A or Table 2B.Methods of Use

[0113] Provided herein are methods of using the microbial species expressing a lysin of the disclosure.

[0114] In one aspect, a method of producing silage is provided, the method comprising contacting the silage with a composition comprising on or more microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the silage is consumed by an animal.

[0115] In one aspect, a method of treating silage is provided, the method comprising contacting the silage with a composition comprising on or more microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the silage is consumed by an animal.

[0116] In one aspect, a method of producing forage is provided, the method comprising contacting the silage with a composition comprising on or more microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the silage is consumed by an animal, and culturing the microbial species on the silage.

[0117] In one aspect, a method of treating forage is provided, the method comprising contacting the silage with a composition comprising on or more microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the silage is consumed by an animal, and culturing the microbial species on the silage.

[0118] In one aspect, a method of inhibiting the growth, or reducing the population, or killing of at least one species of methanogen in an animal is provided, the method comprising delivering to animal with a composition comprising a lactic acid bacteria genetically engineered to express a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when consumed by an animal.

[0119] In one aspect, a method of inhibiting the growth, or reducing the population, or killing of at least one species of methanogen in an animal is provided, the method comprising delivering to the animal with a composition comprising a lactic acid bacteria comprising a nucleic acid encoding a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when consumed by an animal.

[0120] In some embodiments, the microbial species is selected from the microbial species in Table 1.

[0121] In some embodiments, the lysin is selected from the lysins in Table 2A or Table 2B.

[0122] In some embodiments, the animal is a ruminant.

[0123] In some embodiments, the ruminant is a bovine.

[0124] In some embodiments, the delivery comprises oral delivery.EXAMPLES

[0125] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.Example 1: Nucleic Acids and Vectors Encoding a Lysin.

[0126] This example describes construction of a plasmid vector containing the polynucleotide sequence encoding a lysin gene construct to be used for transformation a lactic acid bacteria strain.

[0127] The cloning strategy used to generate a vector containing the nucleotide sequence of a lysin of interest and capable of being expressed in a lactic acid bacterial strain is illustrated in FIG. 1. Briefly, a gene cassette CPl-6xHis-Lysin was designed wherein a lysin sequence tagged with 6xHis at the N-terminal was downstream of a lactic acid bacteria specific constitutive promoter CPI. Exemplary amino acid lysin sequences are provided in Tables 2 A and 2B. The gene cassette flanked with Xhol and PstI restriction enzyme sites was subsequently cloned to a pMET plasmid to generate a pMET-Lysin vector. A broad host range shuttle vector BHR1H and the pMET-Lysin plasmids were linearized by restriction enzyme digestion with Xhol and PstI enzymes. The restriction digests were then run on an agarose gel and the resulting fragments, harboring plasmid backbones and the gene cassette of interest, were purified. The digested plasmid backbone from BHR1H and the fragment containing the gene cassette CPl-6xHis-Lysin were then ligated by sticky-end ligation to generate plasmid pPG 1.Example 2: Lactic acid bacteria Genetically Engineered to Express a Lysin

[0128] This example describes a method of genetically engineering a lactic acid bacteria to express a lysin. This example describes growth conditions and transformation of the representative lactic acid bacteria strain Enterococcus faecium (E. faecium) strain NCTC 7171 to express an exemplary lysin of interest generated as described in Example 1.

[0129] E. faecium strain NCTC 7171 was cultured at 37°C in Brain Heart Infusion (BHI) broth without shaking or on BHI-agar (1.5% agar) plates containing erythromycin. The antibiotic erythromycin stock (Millipore Sigma) was prepared as 150 mg / ml in 70% ethanol.

[0130] Electrocompetent E. faecium strains were generated using a modification of a protocol as previously described. Bhardwaj P et al, PLoS One, 16(4):e0249631 (2021). Briefly, overnight E. faecium culture was diluted (1 :10) in 50 ml pre-warmed BHI broth and cultured at 37°C without shaking till the OD600 reached 0.7. The cell pellet was collected by centrifugation at 6,000 x g at 4°C for 10 minutes. The pellet was resuspended in 10 ml lysozyme buffer (10 mM Tris-HCL pH 8.0, 10 mM EDTA pH 8.0, 50 mM NaCl)supplemented with 25 pg / ml lysozyme and incubated at 37°C for 30 mins without shaking. The cells were then pelleted by centrifugation at 10,000 x g at 4 °C for 10 min and washed three times with ice-cold electroporation buffer (0.5M sucrose and 10% glycerol). The cells were resuspended in 500 pl ice-cold electroporation buffer, aliquoted into 150 pl in individual Eppendorf tubes and immediately transformed with 1 pg pPG 1 plasmid containing an exemplary lysin set forth by SEQ ID NO: 13 generated as previously described (see Example 1) by electroporation at 1.25 kV in a cuvette with 0.1 cm gap. The electroporated cells were immediately mixed with 1 ml ice-cold BHI broth, transferred to an Eppendorf tube and incubated at 30°C for 2.5 hours without shaking. The cells were then spread on BHI-agar plate supplemented with 5 pg / ml or 50 pg / ml erythromycin and incubated at 37°C for 24-48h or until transformed colonies appear.

[0131] Transformed colonies of E. faecium strain were confirmed to harbor the pPG 1 plasmid by colony PCR using with M13 primers (Table 3). To confirm the presence of lysin construct, total RNA was extracted from both transformed and un-transformed E. faecium strains and was reverse transcribed into cDNA. The presence of lysin specific cDNA was confirmed by PCR using the Lysin primers (Table 3). RT-PCR confirmed the presence of the exemplary methanogen lysin when expressed in the E.Faecium (FIGs. 2A and 2B).Table 3: List of Primers for Screening Successful TransformantsExample 3: Silage Comprising Genetically Engineered Lactic acid bacteria

[0132] This example describes lab-scale silage setup, recovery, and analysis of transformed E. faecium bacterial colonies when grown on silage.

[0133] Fresh whole com stalks were cut into 2 foot long pieces for ease of handling. The pieces were cleaned by spraying 70% ethanol, soaking briefly in 200ppm bleach solution and water, dried under UVC, and cut into a 0.5 inch pieces. These com pieces were dehydrated to 35% dry matter (DM) content using a food dehydrator. A total of 175 g of the dehydrated com was packed into a 250 mL glass jar using an acrylic rod to achieve an approximately 0.7 g / cm3 pack density (FIG. 3). During packing, the com was inoculated with ImL of transformed E. faecium culture grown overnight generated as described in Example 2. A rubber stopper was modified by drilling holes to hold both a fermentation lock filled withglycerol and a self-healing port held in place with 100% silicone caulk. The packed jar was sealed with the modified rubber stopper and held in place with a tape brace. The jar was then placed in a fermentation chamber consisting of a holding rack within a plastic covering containing an automated temperature control system.

[0134] The system consisted of a small fan and space heater connected to an InkBird™ ITC- 308 Wifi Smart Thermostat. Internal temperature was set to maintain 32 °C, and conditions were monitored with the InkBird™ system and a Govee™ Smart Thermo-Hygrometer. Ensiling took place for 14-21 days.

[0135] Upon opening the silage jar after 14-21 days, approximately 10g of ensiled com material was placed into a 20mL syringe containing a filter paper at the bottom and placed inside of a 50 mL conical vial. The setup was centrifuged at 500 x g for 5 minutes to collect the effluent in the conical. The pH of the effluent was measured using a pH strip. The rest of the contents of the silage jar were then emptied into a IL glass beaker and PBS (pH 7.0) was added until the contents were sufficiently submerged, and the top of the beaker was covered with aluminum foil. The beaker was vortexed aggressively twice for five minute intervals. The PBS was then collected into 50 mL conicals and subsequently centrifuged at 500 x g for 5 minutes to pellet any plant material. The resulting supernatant was then transferred to new vials and centrifuged at 3000 x g for 10 minutes to pellet bacterial cells. Harvested bacterial cells were analyzed for the presence of lysin as described previously in Example 2 using primers listed in Table 3.

[0136] FIGs. 4A and 4B shows that the genetically engineered Enterococcus faecium strain expressing an exemplary lysin sequence under constitutive promoter when grown on silage expresses the lysin. In sum, data from this example successfully demonstrates growth and recovery of a genetically engineered lactic bacteria strain expressing a lysin grown on silage. Example 4: Growth Inhibition of Methanogens by a Lysin

[0137] This example demonstrates inhibition of a representative methanogenic species in the presence of an exemplary lysin.

[0138] The nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 13 with the addition of a 6X-His N-terminal tag was synthesized in a pET-29b(+) expression vector under kanamycin selection. This expression vector was transformed into E. coli BL21 cells (ATCC C2527H). Overnight cultures of the transformed cells were prepared by inoculating single colony in 5 ml LB supplemented with 50 ug / ml kanamycin and incubating at 37°C with 250 rpm shaking for 18-20 hrs. The overnight culture was then diluted 50 fold in LB supplemented with 50 ug / ml kanamycin and grown till the OD600 reached 0.6-0.7. Theprotein expression in the cells were then induced with 1 mM isopropyl-d- thiogalactopyranoside (IPTG, Zymo Research) for 18 hrs at 4°C while shaking at 150 rpm. Cells were harvested by centrifugation (2300 x g, 15 min, room temperature) and lysed with B-PER lysis buffer (Thermo Scientific). The soluble 6xHis-tagged lysin was then isolated from the crude lysate with HisPur™ Ni-NTA Spin Columns (Thermo Scientific). The purified lysin was then dialyzed with Slide-A-Lyzer™ MINI Dialysis Devices, 10K MWCO (Thermo Scientific, catalog 88404) against PBS to exclude the 250 mM imidazole of the protein elution buffer. A final concentration of 2 mM dithiothreitol (DTT, Thermo Scientific) was added to the dialysed protein to maintain reducing condition.

[0139] The purified lysin thus produced was subsequently incubated after sparging with N2 / CO2 with a laboratory culture of Methanobrevibacter ruminatium (DSM1093), a well- known rumen methanogenic archaea for 30h at 30°C in DSM medium 119 under anaerobic conditions at 6PSI with a 1 :4 mixture of CO2+H2. Inhibition of methanogen growth was evaluated by measuring OD600 at the experimental end-point. In addition, reduction of methane was evaluated by gas chromatography using standard methods.

[0140] As shown in FIG. 5, purified lysin reduced the growth of methanogenic bacteria at least 2-fold. Consistent with these results, a significant reduction in endpoint methane emissions were also noted in the laboratory culture of the methanogen in the presence of the purified lysin (FIG. 6).

[0141] In sum, purified lysin significantly inhibited growh of the methanogen and reduced endpoint methane compared to the controls.EQUIVALENTS

[0142] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is claimed is:

1. A lactic acid bacteria genetically engineered to express a lysin.

2. The lactic acid bacteria of claim 1, wherein the lysin specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the bacteria are consumed by an animal.

3. A lactic acid bacteria comprising a nucleic acid encoding a lysin.

4. The lactic acid bacteria of claim 3, wherein the lysin specifically inhibits the growth, reduces the growth, and / or kills a methanogen species when the bacteria are consumed by an animal.

5. The lactic acid bacteria of any one of claims 1 to 4, wherein the lysin is a lysin from a bacteriophage.

6. The lactic acid bacteria of any one of claims 1 to 5, wherein the lysin is selected from the lysins in Table 2 A or Table 2B.

7. The lactic acid bacteria of any one of claims 1 to 6, wherein the lysin is selected from SEQ ID NOs: 1-12 or SEQ ID NOs: 13-124.

8. The lactic acid bacteria of any one of claims 1 to 7, wherein the lysin inhibits the growth of the methanogen species.

9. The lactic acid bacteria of any one of claims 1 to 8, wherein the lysin kills the methanogen species.

10. The lactic acid bacteria of any one of claims 1 to 9, wherein the lysin reduces the growth of the methanogen species.

11. The lactic acid bacteria of any one of claims 1 to 10, wherein the lysin is encoded on a plasmid.

12. The lactic acid bacteria of any one of claims 1 to 11, wherein the lysin is encoded on the bacterial chromosome.

13. The lactic acid bacteria of any one of claims 1 to 12, wherein the lysin comprises a signal sequence.

14. The lactic acid bacteria of any one of claims 1 to 13, wherein the lysin comprises a protein tag.

15. The lactic acid bacteria of any one of claims 1 to 14, wherein the methanogen species is selected from the group of Methanobrevibacter, Methanothermobacter, Methanomicrobium, Methanobacterium, and Methanosarcina.

16. The lactic acid bacteria of any one of claims 1 to 15, wherein the methanogen species is located in the digestive tract of the animal.

17. The lactic acid bacteria of any one of claims 1 to 16, wherein the lactic acid bacteria are present in the digestive tract of the animal.

18. The lactic acid bacteria of any one of claims 1 to 17, wherein the animal is a ruminant, optionally wherein the ruminant is a bovine.

19. The lactic acid bacteria of any one of claims 1 to 18, wherein the lactic acid bacteria are selected from Table 1.

20. The lactic acid bacteria of any one of claims 1 to 19, wherein the lactic acid bacteria are present in silage.

21. The lactic acid bacteria of claim 19, wherein the silage is consumed by the animal.

22. A composition comprising a microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species.

23. The composition of claim 22, wherein the silage is consumed by an animal.

24. The composition of claim 23, further comprising a second microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species.

25. The composition of any one of claims 22 to 24, wherein the silage is consumed by an animal.

26. A silage comprising the microbial species of claim 22 or claim 24.

27. The composition of any one of claims 22 to 26, wherein the lysin is selected from the lysins in Table 2 A or Table 2B.

28. The composition of any one of claims 22 to 27, wherein the lysin is selected from SEQ ID NOs: 1-12 or SEQ ID NOs: 13-124.

29. The composition any one of claims 22 to 28, wherein the microbial species is selected from Table 1.

30. A method of producing or treating silage, the method comprising contacting the silage with a composition comprising on or more microbial species capable of being cultured on silage and capable of expressing a lysin that specifically inhibits the growth, reduces the growth, and / or kills a methanogen species.

31. The method of claim 30, wherein the silage is consumed by an animal.

32. The method of claim 30 or claim 31, wherein the microbial species is selected from the microbial species in Table 1.

33. The method of claim 30 or claim 31, wherein the lysin is selected from the lysins inTable 2A or Table 2B.

34. A nucleic acid comprising a polynucleotide sequence encoding a lysin operatively linked to a promoter active in lactic acid bacterium.

35. A vector comprising the nucleic acid of claim 34.

36. An additive composition for use in feed, comprising the lactic acid bacteria of any one of claims 1-21.

37. A feedstock for a ruminant, comprising the lactic acid bacteria of any one of claims 1-21.

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