Genetically modified bacillus subtilis strains and their use as in vivo delivery and production systems

Abstract

The present invention relates to genetically modified Bacillus subtilis, compositions and their use in the production and delivery of biomolecules and heterologous proteins in animals, and related methods for improving the health of animals.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. application serial number 63 / 2247271 filed on September 22, 2021, U.S. application serial number 63 / 247273 filed on September 22, 2021, and U.S. application serial number 63 / 247400 filed on September 23, 2021, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted in XML format via EFS-Web and is incorporated herein by reference in its entirety. The XML copy was created on September 21, 2022, is named "2950-12_ST26.xml" and is 12483777 bytes in size. Technical Field

[0005] The present invention relates to genetically modified Bacillus subtilis, compositions and uses thereof for producing and delivering biomolecules and heterologous proteins in animals, and related methods for improving animal health. Background Art

[0006] Direct-feed microorganisms (DFMs), also commonly referred to as probiotics, are microorganisms that colonize the gastrointestinal tract of animals and provide some beneficial effect to the animal. The microorganisms can be bacteria, such as those from the genera Bacillus, Lactobacillus, Lactococcus, and Enterococcus. The microorganisms can be delivered to the animal orally or through the mucous membranes, or, in the case of birds, to the fertilized egg, i.e., in ovo. The beneficial activity provided by DFMs can be through the synthesis and secretion of vitamins or other nutrients required for healthy metabolism in the host animal. DFMs can also protect the host animal from diseases, conditions, or clinical symptoms caused by pathogenic microorganisms or other pathogens. For example, DFMs may naturally produce factors that have inhibitory or cytotoxic activity against certain pathogens, such as harmful or pathogenic bacteria. Probiotics and DFMs offer an attractive alternative or supplement to the use and application of antibiotics in animals. Antibiotics can promote resistant or non-susceptible bacteria, which may eventually appear in feed products or foods consumed by other animals or humans. DFMs are generally safe (even designated as Generally Recognized as Safe (GRAS)), and most do not harbor natural resistance to antibiotics.

[0007] However, DFMs may not produce sufficient quantities of such factors to reduce pathogen infection in the host, or these factors may only affect a limited set of pathogens, leaving the host vulnerable to other pathogens. Strains suitable for use as DFMs can provide attractive and useful starting points for applications in the production or generation of biomolecules and heterologous proteins, including as living delivery systems for the synthesis and delivery of molecules or proteins, with a wide range of applications, including in therapeutics and animal health.

[0008] The production of recombinant proteins in microbial cells is an important aspect of the modern biotechnology industry. Intracellular expression of heterologous proteins in host cells is widely exploited, and these proteins are isolated from the cultures in which the host cells were produced. Biomolecules or heterologous proteins can be expressed from plasmids transfected into bacterial cells or from coding sequences integrated into the host bacterial genome.

[0009] In addition, recent achievements in secretory expression of recombinant proteins have encouraged the scientific and industrial communities to apply and implement bacteria with secretion capabilities to produce proteins. Using secretory host cells, the synthesized target biomolecules and proteins are directly secreted and accumulated in the extracellular medium, which provides cost-effective downstream purification processes. In addition, this can allow the production and isolation of target biomolecules and proteins without the need or requirement to lyse the host cells. In addition, secretory expression of recombinant proteins prevents the accumulation of target biomolecule heterologous proteins in host cells, which can limit cell growth and production, lead to cytotoxicity and cause incorrect protein folding (Mergulhao, FJ; Summers, DK; Monteiro, GA (2005) Biotechol Adv 23 (3): 177-202; Song, Y.; Nikoloff, JM; Zhang, D. (2015) J Microbiol Biotechol 25 (7): 963-77).

[0010] Bacillus subtilis is a Gram-positive model bacterium that is widely used for the industrial production of recombinant proteins such as α-amylase, protease, lipase, and other industrial enzymes. Due to the ability of bacteria to produce and secrete large amounts of target proteins into the culture medium, as well as the availability of low-cost downstream production and purification processes, more than 60% of commercially available industrial enzymes are produced in Bacillus subtilis and related Bacillus species (Schallmey, M.; Singh, A.; Ward, OP (2004) 50(1): 1-17). Compared to the commonly used recombinant protein expression host Escherichia coli, Bacillus subtilis has no risk of endotoxin contamination and has been certified as a GRAS (generally recognized as safe) organism by the U.S. Food and Drug Administration, making it an option for food-grade and pharmaceutical protein production.

[0011] This paper provides a kind of bacillus subtilis expression system, which is modified and engineered to produce biomolecules or heterologous proteins.In some cases, modified bacillus subtilis can produce high levels of at least one or more biomolecules or heterologous proteins, including as surface display or secreted molecules in some cases.In other aspects, modified bacillus subtilis can produce or deliver therapeutic biomolecules or heterologous proteins when bacteria are introduced into host animals.Therefore, in this case, provided is a kind of required delivery system, which can continuously deliver useful therapeutic molecules and biomolecules, such as anti-infective molecules, directly to the host, such as delivered to the gastrointestinal tract, and pathogenic bacteria replicate in the host gastrointestinal tract.The gastrointestinal system is also usually the entrance for pathogens to enter the host.Preferably, the delivery system is a living genetically modified microorganism, such as bacteria, which can breed in the host, even colonize in some cases, and directly deliver therapeutic molecules and biomolecules, such as anti-infective, antipathogenic or antimicrobial agents, to reduce the quantity of pathogens or to block the entry of pathogens.

[0012] There is a need in the art for bacterial strains, compositions, and methods that provide improved production of beneficial molecules and / or delivery of beneficial molecules to the gastrointestinal tract of animals, thereby improving animal health. There is a need for improved delivery platforms and systems, including suitable vectors and nucleic acid-based systems, for rapid and efficient expression of target heterologous proteins or genes, and robust production of large quantities of vectors using a single platform. There is a need for strategies for intracellular and systemic delivery of therapeutic biomolecules, including antigens, antibodies, proteins, and other therapeutic biomolecules.

[0013] Citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. Summary of the Invention

[0014] The present invention provides compositions and methods for improving animal health and animal production and performance. The present invention provides recombinantly manipulated and genetically modified Bacillus subtilis, compositions, and their use in the production and / or direct delivery of biomolecules and heterologous proteins. The production or delivery of biomolecules and heterologous proteins provides materials, reagents, compounds, and related methods for improving animal health.

[0015] The present invention provides a Bacillus subtilis strain that is modified to promote the expression and / or production and / or delivery of a target biomolecule or heterologous protein. In embodiments, the Bacillus subtilis strain is modified to introduce a strong promoter or an inducible promoter that drives the expression and / or production of a target natural or heterologous biomolecule or protein. The strain can be modified to introduce a signal sequence that drives or promotes the secretion of a target natural or heterologous biomolecule or protein. In some embodiments, the Bacillus subtilis strain is modified to introduce a nucleic acid that encodes a heterologous protein or encodes one or more proteins required or utilized for the production of a heterologous protein. In embodiments, the introduced nucleic acid includes a strong promoter or an inducible promoter that drives the expression and / or production of a target heterologous protein. In embodiments, the introduced nucleic acid includes a signal sequence that drives the expression and secretion of a target heterologous protein.

[0016] The present invention provides a production and delivery system that can continuously produce and deliver useful therapeutic molecules and biomolecules, such as anti-infective molecules, with growth and production capacity, and / or directly deliver them to a host, such as the gastrointestinal tract, where pathogens replicate in the host's gastrointestinal tract. The gastrointestinal system is also typically a portal for pathogens to enter the host. Preferably, the delivery system is a living genetically modified microorganism, such as bacteria, which can reproduce in the host and even colonize in some cases, and directly deliver therapeutic molecules and biomolecules, such as anti-infective, anti-pathogenic, antibacterial, anti-inflammatory or immunomodulatory peptides, polypeptides or preparations to reduce the number of pathogens or block the entry of pathogens. For example, in-ovum delivery of a living delivery platform may prevent pathogens from colonizing the embryo in the early stages through competitive exclusion or direct or indirect anti-infective effects. In-ovum delivery has the further advantage of bypassing any limitations on colonization of genetically modified microorganisms due to maternal antibody interference. Preferably, the live bacterial delivery system synthesizes sufficient amounts of anti-infective factors to have the desired effect on the pathogens. The target pathogen can be but not limited to Salmonella, Clostridium, Campylobacter, Staphylococcus or Streptococcus bacteria, or Escherichia coli, or parasites such as Eimeria species. Preferably, the live bacterial system continues for a period of time in the host gastrointestinal tract. Preferably, the live bacterial delivery system produces a broad spectrum anti-infective factor or multiple anti-infective factors, thereby targeting multiple pathogens. Alternatively, the combination of live delivery systems can be applied to a single animal, wherein the genetically modified bacteria produces multiple anti-infective factors, immunomodulatory molecules or growth-promoting biomolecules, or any combination thereof. Therefore, more than one disease state can be prevented or reduced, or diseases and syndromes with multiple causes can be effectively treated.

[0017] The present invention relates to a protein production and intracellular delivery platform that utilizes genetically modified bacteria to produce or deliver preventive or therapeutic anti-infective active factors, immunomodulatory factors or growth-promoting biomolecules directly to the mucosa of animals in need.

[0018] The present invention provides modified Bacillus, particularly modified Bacillus subtilis strain 105 (ELA191105), as a bacterial strain for producing one or more biomolecules and heterologous proteins. In one embodiment, the modified Bacillus, particularly modified Bacillus subtilis strain 105 (ELA191105), is a bacterial strain for producing and secreting one or more biomolecules and heterologous proteins.

[0019] In embodiments, Bacillus subtilis strain 105 (ELA191105) is modified to include nucleic acids that directly encode one or more biomolecules or heterologous proteins. In embodiments, Bacillus subtilis strain 105 (ELA191105) is modified to include nucleic acids that encode one or more proteins that promote, induce, enhance, or otherwise enable the production of one or more biomolecules or heterologous proteins. In one such embodiment, Bacillus subtilis strain 105 (ELA191105) is modified to include nucleic acids that encode one or more proteins, enzymes, or substrates in a production pathway, synthetic pathway, or the like that results in the production or production of a target biomolecule or heterologous protein.

[0020] In one embodiment, a live delivery platform comprising a genetically modified Bacillus sp. is provided for producing one or more biomolecules and heterologous proteins in an animal, wherein the modified Bacillus sp. comprises Bacillus subtilis strain 105 (ELA191105), which is genetically modified to contain a nucleic acid encoding one or more biomolecules or heterologous proteins that are produced and delivered when the modified Bacillus sp. is administered to the animal.

[0021] In one embodiment, the modified Bacillus comprises Bacillus subtilis strain 105, or a Bacillus strain having a genome sequence that is at least 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein the Bacillus subtilis strain 105 comprises the nucleic acid sequence of SEQ ID NO: 1.

[0022] In one embodiment, the modified Bacillus comprises a Bacillus subtilis strain corresponding to ATCC deposit PTA-126786 or a Bacillus strain having at least 95% identity, 97% identity, 98% identity, and 99% identity in genomic sequence to the ELA191105 sequence corresponding to ATCC deposit PTA-26786.

[0023] Bacillus subtilis strain 105, also known as ELA191105, also known as Bs PTA-86, is an isolated Bacillus subtilis strain having probiotic properties and capabilities. In one embodiment, Bacillus subtilis strain 105 corresponds to ATCC deposit PTA-126786. In embodiments, the Bacillus subtilis strain corresponds to ATCC deposit PTA-126786 or a Bacillus strain having at least 90% identity, 95% identity, 97% identity, 98% identity, and 99% identity in genomic sequence to the sequence of ELA191105 corresponding to ATCC deposit PTA-26786.

[0024] In one embodiment, Bacillus subtilis strain 105 comprises the nucleic acid sequence set forth in SEQ ID NO: 1, or a Bacillus strain having at least 90%, 95%, 97%, 98%, or 99% identity in its genomic sequence to SEQ ID NO: 1. In one embodiment, Bacillus subtilis strain 105 comprises the nucleic acid sequence set forth in SEQ ID NO: 2, 3, 4, 5, and / or 6, or comprises a Bacillus strain having at least 90%, 95%, 97%, 98%, or 99% identity in its genomic sequence to SEQ ID NO: 2, 3, 4, 5, and / or 6. In one embodiment, Bacillus subtilis strain 105 comprises the nucleic acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, or 6, or comprises a Bacillus strain having at least 90%, 95%, 97%, 98%, or 99% identity in its genomic sequence to SEQ ID NO: 1, 2, 3, 4, 5, or 6. SEQ ID NO: 1 provides the complete genomic nucleic acid sequence of ELA191105, deposited as PTA-126786.

[0025] The present invention relates to a production and live delivery system based on Bacillus bacteria, wherein a genetically modified Bacillus subtilis, particularly a safe Bacillus subtilis strain with probiotic properties, is modified to encode and produce one or more biomolecules or heterologous proteins, or to increase the production of one or more biomolecules or heterologous proteins or to provide inducible production / expression of one or more biomolecules or heterologous proteins. The target biomolecule or protein can be a homologous Bacillus subtilis protein or a heterologous protein. There are one or more, two or more, three or more, or a complex or gene cassette-encoded group of proteins or biomolecules. In one embodiment, the biomolecule or heterologous protein is a therapeutic agent. In one embodiment, the biomolecule or heterologous protein is a compound, agent, or reagent important in a biological or chemical reaction. In one embodiment, the biomolecule or heterologous protein is an antigen or one or more antigens. In one embodiment, the biomolecule or heterologous protein is an antibody or fragment thereof, such as a domain antibody or nanobody. In one embodiment, the biomolecule or heterologous protein is an anti-infective, antibacterial, or antipathogen agent. In one embodiment, the biomolecule or heterologous protein is a lytic protein. In one embodiment, the biomolecule is a therapeutic biomolecule, particularly a molecule having prophylactic or therapeutic anti-infective activity, one or more immunomodulatory factors, or one or more growth-promoting biomolecules. Any of a variety of known or important biomolecules or proteins can be expressed by the system and modified strain 105 of the present invention.

[0026] In embodiments of Bacillus-based production and in vivo delivery systems, genetically modified Bacillus subtilis is used to simultaneously produce one or more biomolecules or heterologous proteins. In one embodiment, strain 105 is modified to produce a combination of biomolecules or heterologous proteins. In one embodiment, the combination can produce a target molecule. In one embodiment, the combination can be used as a combination formulation. In one embodiment, the combination can be a panel of antigens, such as for use in a vaccine or immunogenic composition.

[0027] In one embodiment, strain 105 is modified to increase capacity. In one embodiment, strain 105 is modified to increase its ability to absorb and internalize extracellular nucleic acids or DNA. In one embodiment, strain 105 is modified to express, overexpress or induce expression of genes encoding comK and comS. In one embodiment, strain 105 is modified to express, overexpress or induce expression of competent comK and comS proteins. In one embodiment, strain 105 is modified to induce expression or overexpression of genes encoding comK and comS. In an embodiment, overexpression refers to the expression of a gene or the production of a protein that is greater than, in particular significantly greater than, the natural expression of a gene or protein production. In an embodiment, overexpression refers to the expression of a gene or the production of a protein that is greater than, in particular significantly greater than, the expression of an unmodified or wild-type strain. In one embodiment, the promoter is a natural promoter of strain 105. In one embodiment, the promoter is a natural inducible promoter of strain 105. In one embodiment, the promoter is a non-natural promoter or a non-natural inducible promoter. Exemplary and suitable promoters are provided herein. Alternative promoters are known or can be selected by those skilled in the art.

[0028] In one embodiment, a nucleic acid encoding or promoting production of a biomolecule or homologous protein or heterologous protein is linked to a native strain 105 promoter. In one embodiment, a nucleic acid encoding or promoting production of a biomolecule or homologous protein or heterologous protein is linked to one or more promoters of the native strain 105. In one embodiment, a nucleic acid encoding or promoting production of a biomolecule or homologous protein or heterologous protein is linked in tandem to at least two promoters of the native strain 105. In some embodiments, these promoters promote expression and production in strain 105. Exemplary and suitable promoters are provided herein.

[0029] In one embodiment, bacterial strain 105 is modified to secrete or more effectively secrete biomolecules, homologous proteins or heterologous proteins. In one embodiment, bacterial strain 105 is modified to include a nucleic acid that encodes or otherwise can produce a biomolecule, homologous protein or heterologous protein, wherein the nucleic acid includes a signal sequence. In one embodiment, the signal sequence is the natural signal sequence of bacterial strain 105. In one embodiment, the signal sequence is a non-natural signal sequence. Exemplary and suitable signal sequences are provided herein. Alternative signal sequences are known or can be selected by those skilled in the art. The signal sequence for secretion can be at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 44 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids or at least 65 amino acids. The secretory signal sequence can also be 20-65 amino acids, 20-60 amino acids, 20-55 amino acids, 20-50 amino acids, 20-45 amino acids, 20-40 amino acids, 20-35 amino acids, 20-30 amino acids, 25-65 amino acids, 25-60 amino acids, 25-55 amino acids, 25-50 amino acids, 25-45 amino acids, 25-40 amino acids, 25-35 amino acids, 25-30 amino acids, 30-65 amino acids, 30-60 amino acids; 30-55 amino acids; 30-50 amino acids, 30-45 amino acids, 30-40 amino acids or 30-35 amino acids.

[0030] In one embodiment, strain 105 is modified to enhance maintenance metabolism and promote more efficient growth and growth cycles. In one embodiment, strain 105 is modified to produce a non-spore-forming bacterial strain. In one embodiment, strain 105 is modified to delete or otherwise inactivate one or more native sequences responsible for or conducive to spore formation. In one embodiment, one or more genes encoding SpoA and / or SoIVB proteins are deleted or otherwise inactivated.

[0031] In one embodiment, bacterial strain 105 is modified to block the production of one or more natural proteases, to delete or otherwise inactivate one or more natural proteases. In one embodiment, the proteases are one or more extracellular proteases. In one embodiment, the inactivation or deletion is used to stabilize or increase the half-life of one or more biomolecules, proteins, etc. discharged from the bacterial strain. In an embodiment, one or more of the natural extracellular proteases NprE, AprE, Epr (Epr1 and Epr2), Bpr, Mpr, NprB, Vpr, and WprA from Bacillus subtilis strain 105 are deleted or otherwise inactivated. In an embodiment, one or more of the natural extracellular proteases NprE, AprE, NprB, Vpr, and WprA from Bacillus subtilis strain 105 are deleted or otherwise inactivated. In one embodiment, one or more of the natural extracellular proteases NprE, AprE, and Epr (Epr1 and Epr2) from Bacillus subtilis strain 105 are deleted or otherwise inactivated. In one embodiment, the native extracellular proteases NprE and Vpr from Bacillus subtilis strain 105 are deleted or otherwise inactivated. In one embodiment, the native extracellular proteases AprE, NprB, and WprA from Bacillus subtilis strain 105 are deleted or otherwise inactivated.

[0032] In one embodiment, strain 105 is modified to block the production of, delete, or otherwise inactivate one or more native lytic enzymes or antimicrobial proteins. Exemplary strain 105 native lytic enzymes and antimicrobial proteins that can be deleted or otherwise inactivated are provided herein.

[0033] In an embodiment, strain 105 is modified to contain one or more self-amplifying nucleic acids encoding one or more biomolecules or proteins of interest. In some embodiments, the self-amplifying nucleic acids encode biomolecules with therapeutic effects, such as antibodies, anti-infective peptides, immunomodulatory proteins, and antigens.

[0034] The present invention provides an expression cassette within a genetically modified strain 105, comprising a heterologous coding region encoding a desired biomolecule or heterologous protein. The desired biomolecule may be a biomolecule with anti-infective activity, a probiotic factor, an immunomodulatory factor, a growth-promoting biomolecule, or the like. The biomolecule may have anti-infective activity against pathogenic bacteria or parasites. The expression cassette may be a plasmid or vector, including a vector for integration into the Bacillus strain genome. The expression cassette, plasmid, or vector may comprise a promoter sequence, a signal sequence, one or more biomolecule or protein coding sequences, or one or more selection sequences for selecting or determining the growth of the plasmid or vector and / or for selecting or determining the integration of the plasmid or vector. Suitable promoters and signal sequences are provided herein or are known and available to those skilled in the art.

[0035] The present invention provides the use of any genetically modified Bacillus subtilis disclosed herein in the preparation of a medicament. The present invention provides the use of any genetically modified Bacillus subtilis disclosed herein in the preparation of a feed additive or an animal feed component.

[0036] In one embodiment, the present invention provides a probiotic and therapeutic composition comprising a genetically modified Bacillus subtilis strain, particularly the genetically modified Bacillus subtilis species 105 as described and described in detail herein. In one embodiment, the present invention provides a probiotic and therapeutic composition comprising a genetically modified Bacillus subtilis strain, particularly the genetically modified Bacillus subtilis species 105 as described and described in detail herein, and a carrier suitable for administration to an animal; wherein when administered to an animal in an effective amount, the composition results in expression and production of one or more biomolecules or heterologous proteins in the animal as compared to an animal not administered the composition.

[0037] Provided herein are methods for treating or alleviating a condition, disorder, infection, or disease in an animal comprising administering to the animal a genetically modified Bacillus subtilis strain, particularly the genetically modified Bacillus subtilis species 105 as described and described in detail herein. In one embodiment, the strain is administered with a carrier suitable for administration to an animal. In one embodiment, the strain is administered orally as part of or as a component of feed.

[0038] In one embodiment, the present invention provides a feed additive comprising a genetically modified Bacillus subtilis strain, in particular the genetically modified Bacillus strain 105 as described and detailed herein.

[0039] The present invention provides a method for producing one or more target biomolecules or proteins, comprising:

[0040] (a) modifying the Bacillus subtilis strain 105 to introduce one or more nucleic acids encoding the one or more target biomolecules or proteins,

[0041] (b) cultivating the modified strain 105, and

[0042] (c) isolating the one or more target biomolecules or proteins.

[0043] In one embodiment, the Bacillus subtilis strain 105 is modified prior to step a to improve or otherwise increase expression and / or production of the target biomolecule. In one embodiment, the Bacillus subtilis strain 105 is modified prior to step a by altering its competence, deleting or inactivating one or more genes, such as one or more native proteases or lytic enzymes, or deleting or inactivating one or more genes or proteins responsible for spore formation.

[0044] The present invention relates to and provides a modified Bacillus for producing or delivering one or more biomolecules or heterologous proteins in vivo, wherein the bacterium comprises a Bacillus subtilis strain 105 (ELA191105) genetically modified in one or more aspects selected from the following:

[0045] (a) genetically modified to increase competence;

[0046] (b) genetically modified to reduce or block sporulation;

[0047] (c) genetically modified to delete or inactivate one or more native proteases; and

[0048] (d) genetically modified to include a nucleic acid encoding one or more biomolecules or heterologous proteins.

[0049] In one embodiment of the modified Bacillus, the Bacillus subtilis strain 105 comprises the nucleic acid sequence set forth in SEQ ID NO: 1, or comprises a nucleic acid sequence in its genomic sequence that is at least 95% identical, 97% identical, 98% identical, and 99% identical to SEQ ID NO: 1.

[0050] In one embodiment of the modified Bacillus, the Bacillus subtilis strain corresponds to ATCC deposit PTA-126786, or its genomic sequence has at least 95% identity, 97% identity, 98% identity, or 99% identity to the sequence of ELA191105 corresponding to ATCC deposit PTA-26786.

[0051] In one embodiment of the modified Bacillus, the Bacillus subtilis strain 105 comprises the nucleic acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5 or 6, or comprises in the genomic sequence a nucleic acid having at least 90% identity, 95% identity, 97% identity, 98% identity, or 99% identity to SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0052] Provided herein are embodiments wherein in (a), the bacteria are modified to overexpress comK, comS, or comK and comS to increase competence. In one embodiment, the gene cassette encoding comK and comS is integrated into the Bacillus subtilis genome.

[0053] In one embodiment, the competence is increased and the transformation efficiency of the strain increases by at least 20 times; 50 times; 50 times or more; 60 times; 80 times; 80 times or more; 90 times; 100 times; or 100 times or more. In one embodiment, the competence is increased and the transformation efficiency of the strain increases by about 80 times, 80 times or more, 90 times, 100 times. In one embodiment, the competence is increased and the transformation efficiency of the strain increases by about 100 times.

[0054] Provided herein are embodiments wherein in (b), the bacterium is modified to delete or inactivate one or more native genes encoding Spo0A, SpoIVB, or SpoA and SpoIVB.

[0055] Provided herein are embodiments wherein in (c), the bacteria are modified to delete or inactivate one or more native proteases or genes encoding one or more native proteases selected from the group consisting of NprE, AprE, Epr1, Epr2, Bpr, Mpr, NprB, Vpr, and WprA. In one embodiment, the bacteria are modified to delete or inactivate genes encoding the native proteases NprE and Vpr. In one embodiment, the bacteria are modified to delete or inactivate genes encoding the native proteases AprE, NprB, and WprA.

[0056] In other aspects, the modified Bacillus, particularly Bacillus subtilis strain 105, is further genetically modified to delete or inactivate one or more native lytic enzymes or antimicrobial peptides. In one embodiment, one or more native lytic enzymes or antimicrobial peptides selected from xpf, lytCl, lytC2, and sdpC are deleted or inactivated.

[0057] In other aspects, the modified Bacillus, particularly Bacillus subtilis strain 105, is further genetically modified to delete or inactivate one or more native genes encoding virulence factors, toxins, or antimicrobial resistance (AMR). In embodiments, the one or more virulence factors, toxins, or antimicrobial resistance (AMR) are selected from macrolide 2' phosphotransferase (mphK), ABC-F type ribosomal protection protein (vmlR), streptothricin-N-acetyltransferase (satA), tetracycline efflux protein (tet(L)), aminoglycoside 6-adenylyltransferase (aadK) (29), and rifamycin inactivated phosphotransferase (rPhC) as described in Table 16.

[0058] In some embodiments, the modified Bacillus comprises a Bacillus subtilis isolate having at least one gene knockout selected from the group consisting of spo0A, spoIIIE, spoIVB, NprE, AprE, NprB, Vpr, WprA; and one or more heterologous genes encoding one or more biomolecules or heterologous proteins operably linked to one or more promoters selected from the group consisting of a tuf promoter, a sigx promoter, a gros promoter, a ftsh promoter, a PxylA promoter, a mannose-inducible promoter, and a Physpank promoter.

[0059] In some embodiments, the modified Bacillus comprises a Bacillus subtilis strain 105 isolate modified to overexpress comK, comS, or both comK and comS to increase competence; having at least one gene knockout selected from at least one gene selected from the group consisting of spo0A, spoIIIE, spoIVB, NprE, AprE, NprB, Vpr, and WprA; and modified to comprise one or more heterologous genes encoding one or more biomolecules or heterologous proteins operably linked to one or more promoters selected from the group consisting of tuf promoter, sigx promoter, gros promoter, ftsh promoter, PxylA promoter, mannose-inducible promoter, and Physpank promoter. In one embodiment, the one or more promoters are selected from the group consisting of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 40, and SEQ ID NO: 41.

[0060] In other aspects, one or more heterologous genes encoding one or more biomolecules or heterologous proteins are integrated into the genome of the host Bacillus subtilis strain 105. In some embodiments, one or more heterologous genes encoding one or more biomolecules or heterologous proteins are integrated into the genome of the host Bacillus subtilis strain 105 at one or more gene locations selected from the group consisting of amyE, NprE, AprE, Epr1, Epr2, Bpr, Mpr, NprB, Vpr, and WprA.

[0061] In further embodiments, the one or more biomolecules or heterologous proteins are selected from anti-infective agents, antibacterial agents, antipathogenic agents, immunomodulatory factors or agents, antigens, antibodies, growth promoting biomolecules, probiotics, and bio-based chemicals.

[0062] In one aspect, the one or more biomolecules or heterologous proteins are antimicrobial agents. In another aspect, the one or more antimicrobial agents are one or more lysins or lytic peptides. In another aspect, the one or more lysins or lytic peptides are PlyCM, CP025C, lysostaphin, or native Bacillus subtilis 105 lytic enzyme.

[0063] In some embodiments, the one or more antimicrobial agents are one or more antimicrobial peptides (AMPs). In one embodiment, the one or more antimicrobial peptides (AMPs) are mersacidin or cathelicidin peptides. In one embodiment, the one or more antimicrobial peptides (AMPs) are CAP18 peptides. In embodiments thereof, the CAP18 peptide can be rabbit CAP18 or human CAP18LL37, or a CAP18 peptide from another animal, or a variant thereof. In embodiments thereof, the CAP18 peptide can be SEQ ID NO: 95 or SEQ ID NO: 96, or a variant thereof.

[0064] In some embodiments, the one or more biomolecules or heterologous proteins are one or more antibodies or fragments thereof. In one embodiment, the one or more antibodies or fragments thereof are one or more single-chain antibodies, domain antibodies, VHH antibodies, or nanobodies. In other embodiments, the one or more single-chain antibodies, domain antibodies, VHH antibodies, or nanobodies are one or more single-chain antibodies, domain antibodies, VHH antibodies, or nanobodies directed against pathogenic bacteria.

[0065] In another embodiment, the one or more antibodies are one or more VHH antibodies or Nanobodies against Clostridium perfringens. In one embodiment, the one or more antibodies are one or more VHH antibodies or Nanobodies against Clostridium perfringens alpha toxin and NetB. In some embodiments, the one or more VHH antibodies are selected from SEQ ID NO: 97, SEQ ID NO: 88, SEQ ID NO: 101 and SEQ ID NO: 102.

[0066] In other aspects, the one or more biomolecules or heterologous proteins are one or more antigens, and wherein the antigens are capable of stimulating an immune response against a parasite, bacterium, or virus. In one aspect, the one or more biomolecules or heterologous proteins are one or more antigens that can stimulate an immune response against an Eimeria parasite. In one aspect, the one or more antigens are selected from Eimeria tenella elongation factor-1α, EtAMA1, EtAMA2, Eimeria 5401, Eimeria acervuline lactate dehydrogenase antigen gene, Eimeria maxima surface antigen gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Eimeria common antigen 14-3-3. In a specific aspect, the one or more antigens are Eimeria antigens encoded by one or more of SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or SEQ ID NO:109.

[0067] In other embodiments, one or more heterologous genes encoding one or more biomolecules or heterologous proteins are provided on a biosynthetic gene cluster (BGC), and wherein the BGC or a portion thereof is integrated into the host Bacillus subtilis strain 105 genome.

[0068] In one embodiment, the biosynthetic gene cluster (BGC) is a PKS BGC or a mericidin BGC. In one embodiment, the PKS-BGC is capable of producing metabolites that activate AhR. In another embodiment, the mericidin BGC is capable of producing one or more mericidin polypeptides of SEQ ID NO: 22 or SEQ ID NO: 23, which are capable of inhibiting or killing one or more bacteria or viruses.

[0069] In some embodiments, the PKS BGC comprises the nucleic acid set forth in SEQ ID NO: 110 or comprises a nucleic acid encoding one or more polypeptides selected from SEQ ID NOs: 7 to 21. In some embodiments, the mesaccharin BGC comprises the nucleic acid set forth in SEQ ID NO: 24 or comprises a nucleic acid encoding one or more polypeptides selected from SEQ ID NOs: 25 to 32.

[0070] In other aspects, the one or more biomolecules or heterologous proteins are biobased chemicals. Chemicals or agents that are biobased, produced by animals, bacteria, or can be synthesized by fungal host cells, are well known to those skilled in the art. These chemicals or agents may include enzymes or intermediates in enzymatic reactions. These chemicals or agents may include additives to stabilize other reagents. These chemicals or agents may include molecules or proteins useful in the food, cosmetic, or pharmaceutical industries.

[0071] In one embodiment, the biobased chemical is gamma-polyglutamic acid (γ-PGA). In one embodiment, the γ-PGA is encoded by the CapABC locus, and the Bacillus subtilis strain 105 is modified to produce increased amounts of γ-PGA by integrating at least one additional copy of the CapABC locus into the genome of the Bacillus subtilis strain 105. In one such embodiment, the at least one additional copy of the CapABC locus is integrated into the genome of the Bacillus subtilis strain 105 at one or more loci selected from amyE, nprE, apr, and wprA.

[0072] In some embodiments of the present invention, one or more heterologous genes encoding one or more biomolecules or heterologous proteins include a native Bacillus subtilis 105 strain or other bacterial strain signal sequence for modifying the bacteria to secrete one or more biomolecules or heterologous proteins.

[0073] In embodiments, the native Bacillus subtilis 105 strain or other bacterial strain signal sequence used for secretion is selected from SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NOs: 50-64.

[0074] In another aspect, provided herein is a live delivery platform comprising a genetically modified Bacillus for producing one or more biomolecules or heterologous proteins in an animal, wherein the modified Bacillus comprises Bacillus subtilis strain 105 (ELA191105), which is genetically modified to contain a nucleic acid encoding one or more biomolecules or heterologous proteins, and the biomolecules or heterologous proteins are produced and delivered when the modified Bacillus is administered to the animal.

[0075] In some aspects, the bacteria include Bacillus subtilis strain 105 (ELA191105), which is genetically modified in one or more aspects selected from the group consisting of:

[0076] (a) genetically modified to increase competence;

[0077] (b) genetically modified to reduce or block sporulation;

[0078] (c) genetically modified to delete or inactivate one or more native proteases; and

[0079] (d) genetically modified to include a nucleic acid encoding one or more biomolecules or heterologous proteins.

[0080] In one aspect, the Bacillus subtilis strain 105 comprises the nucleic acid sequence of SEQ ID NO: 1, or the genomic sequence comprises at least 95% identity, 97% identity, 98% identity, and 99% identity to SEQ ID NO: 1.

[0081] In one aspect, the Bacillus subtilis strain corresponds to ATCC deposit PTA-126786, or its genomic sequence has at least 95% identity, 97% identity, 98% identity, or 99% identity to the ELA191105 sequence corresponding to ATCC deposit PTA-26786.

[0082] In other aspects, the Bacillus subtilis is genetically modified to include a nucleic acid encoding one or more biomolecules or heterologous proteins and includes an expression cassette;

[0083] The expression cassette comprises one or more of the following:

[0084] Promoter, for transcriptional expression,

[0085] A nucleic acid sequence encoding a signal sequence for secretion,

[0086] at least one heterologous coding region encoding a desired biomolecule or heterologous protein, and

[0087] Terminators are used to terminate translation and transcription.

[0088] In some embodiments, the promoter used for transcriptional expression is one or more promoters selected from the group consisting of tuf promoter, sigx promoter, gros promoter, ftsh promoter, PxylA promoter, mannose-inducible promoter, and Physpank promoter. In embodiments, the one or more promoters are selected from the group consisting of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 40, and SEQ ID NO: 41.

[0089] In embodiments, the nucleic acid sequence encoding a signal sequence for secretion encodes at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 44 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, or at least 65 amino acids.

[0090] In some embodiments, the nucleic acid sequence encoding a signal sequence for secretion encodes a signal sequence for secretion of a native Bacillus subtilis 105 strain or other strains, wherein the signal sequence comprises a sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NOs: 50-64.

[0091] In some embodiments, the expression cassette or at least one heterologous coding region is integrated into the genome of the host Bacillus subtilis strain 105, wherein the heterologous coding region encodes a desired biomolecule or a heterologous protein.

[0092] In an embodiment, the expression cassette or at least one heterologous coding region is integrated at one or more gene locations in the genome of the host Bacillus subtilis strain 105, wherein the at least one heterologous coding region encodes a desired biomolecule or heterologous protein, and the gene location is selected from the group consisting of amyE, NprE, AprE, Epr1, Epr2, Bpr, Mpr, NprB, Vpr and WprA.

[0093] In certain embodiments, the desired biomolecule or heterologous protein is selected from the group consisting of anti-infective agents, antibacterial agents, antipathogenic agents, immunomodulatory agents or agents, antigens, antibodies, growth promoting biomolecules, probiotics, and bio-based chemicals.

[0094] The present invention also relates to a method for reducing the colonization of an animal by pathogenic bacteria, parasites or viruses, comprising treating the animal with the modified Bacillus provided herein or the in vivo delivery platform provided herein.

[0095] In embodiments thereof, the animal is a bird, a human, or a non-human mammal.

[0096] In an embodiment thereof, the pathogenic bacteria is selected from the group consisting of Salmonella, Clostridium, Campylobacter, Staphylococcus, Streptococcus and Escherichia coli.

[0097] In embodiments, the pathogenic parasite is Eimeria.

[0098] In embodiments, the modified Bacillus or living delivery platform is administered orally, parenterally, nasally, or mucosally.

[0099] In some embodiments, the animal is a bird, and wherein the treatment is administered in ovo.

[0100] In aspects of the present invention, a modified Bacillus and a live delivery platform for use in therapy are provided. In some aspects, a modified Bacillus and a live delivery platform for reducing colonization of an animal by pathogenic bacteria, parasites, or viruses are provided.

[0101] In other aspects, modified Bacillus and live delivery platforms are provided for use in preparing drugs that reduce colonization of animals by pathogenic bacteria, parasites, or viruses. In other aspects, modified Bacillus and live delivery platforms are provided for use in preparing drugs for stimulating an immune response in an animal against pathogenic bacteria, parasites, or viruses. In other aspects, the modified Bacillus and live delivery platforms are provided for use in preparing drugs for passive immunization against pathogenic bacteria, parasites, or viruses in animals.

[0102] While there have been described what are presently considered to be preferred embodiments of the present invention, those skilled in the art will understand that other and further changes and modifications may be made thereto without departing from the spirit of the invention, and it is intended that all such changes and modifications come within the true scope of the invention.

[0103] Other objects and advantages will become apparent to those skilled in the art from a reading of the following detailed description, which proceeds with reference to the following illustrative drawings and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 The Spo0A and SpoIVB loci of Bacillus subtilis strain 105 are shown, where SpoIVB and SpoA are encoded by tandem mapping sequences.

[0105] Figure 2 The gene maps of amyE, nprE, apr, and wprA on the genome of Bacillus subtilis strain 105 are provided.

[0106] Figure 3AA and B show the pathway for poly-gamma-glutamic acid biosynthesis. B shows the native locus for PGA production in Bacillus subtilis strain 105. The native Bacillus subtilis locus includes capC, capB, and capA, which are encoded by a single promoter.

[0107] Figure 4 The engineering and design of the comKS cassette for integration and expression are shown.

[0108] Figure 5 Engineering of a PKS biosynthetic gene cluster (BGC) into Bacillus subtilis 105 is shown.

[0109] Figure 6 The Bacillus BGC expression vector with left and right amyE arms is shown for homologous integration into Bacillus subtilis 105. The genes shown to the left of the vector ring are required for single-copy replication in E. coli. The lacI and kan genes are required for homologous recombination into the Bacillus genome, selection, and expression. Figure 6 Also shown are the PxylA and Physpank promoter elements.

[0110] Figure 7 The engineering of the merapidine biosynthetic gene cluster (BGC) into Bacillus subtilis 105 is shown. (A) A merapidine cluster including a lagD coding sequence (lagD-CDS) is provided. (B) A merapidine cluster without a lagD coding sequence is provided. DETAILED DESCRIPTION

[0111] According to the present invention, conventional molecular biology, microbiology and recombinant DNA techniques may be employed within the skill of the art. Such techniques are fully explained in the literature.

[0112] As used herein, "isolated" means that a subject isolate has been separated from at least one material with which it is associated in a particular environment, such as its natural environment.

[0113] Thus, an "isolate" is not present in its naturally occurring environment; rather, it is a microorganism that has been removed from its natural environment and placed in a non-naturally occurring state by various techniques known in the art. Thus, an isolated strain or isolated microorganism can exist, for example, as a biologically pure culture associated with an acceptable carrier.

[0114] As used herein, "single isolate" should be considered to refer to a composition or culture that primarily comprises a single species or strain after separation from one or more other microorganisms. The phrase should not be considered to indicate the extent to which the microorganism has been isolated or purified. However, a "single isolate" may include essentially only one microorganism or strain.

[0115] In certain aspects of the present disclosure, the isolated Bacillus strain is present in the form of an isolated and biologically pure culture. It will be understood by those skilled in the art that an isolated and biologically pure culture of a particular Bacillus strain means that the culture is substantially free (within the scientific scope) of other living organisms and contains only the single Bacillus strain. The culture can contain varying concentrations of the isolated Bacillus strain. The present disclosure indicates that isolated and biologically pure microorganisms are often necessarily distinguished from less pure or impure materials.

[0116] As used herein, "spore" or "spores" refers to a structure produced by bacteria that is suitable for survival and propagation. Spores are typically characterized as dormant structures; however, spores are capable of differentiation through the process of germination. Germination is the differentiation of a spore into a vegetative cell capable of metabolic activity, growth, and reproduction. Germination of a single spore produces a single bacterial vegetative cell. Bacterial spores are structures that are used for survival conditions that would normally be unfavorable for the survival or growth of vegetative cells.

[0117] As used herein, the terms "colonize" and "colonization" include "temporary colonization" and "temporary colonization".

[0118] As used herein, "microbiome" refers to the collection of microorganisms that inhabit the gastrointestinal tract of an animal and the microbial physical environment (i.e., the microbiome has biological and physical components). The microbiome is fluid and may be regulated by many natural and artificial conditions (e.g., changes in diet, disease, antimicrobial agents, influx of additional microorganisms, etc.). Modulation of the gastrointestinal microbiome can be achieved by administering the compositions of the present disclosure in the form of: (a) increasing or decreasing microorganisms of a particular family, genus, species, or function (i.e., alteration of the biological component of the gastrointestinal microbiome) and / or (b) increasing or decreasing gastrointestinal pH, increasing or decreasing volatile fatty acids in the gastrointestinal tract, increasing or decreasing any other physical parameter important to gastrointestinal health (i.e., alteration of the non-biological component of the intestinal microbiome).

[0119] As used herein, "probiotics" refers to substantially pure microorganisms (i.e., single isolates) or mixtures of desired microorganisms, and may also include any additional ingredients (e.g., carriers) that can be administered to animals to provide a beneficial health effect. The probiotics or microbial compositions of the present invention can be administered with an agent or carrier to allow the microorganisms to survive in the environment of the gastrointestinal tract, i.e., to withstand the low pH and grow in the gastrointestinal environment.

[0120] As used herein, the term "growth medium" refers to any medium suitable for supporting the growth of microorganisms. For example, the medium can be natural or artificial, including gastrin-supplemented agar, minimal medium, complete medium, LB medium, serum, and tissue culture gel. It should be understood that the medium can be used alone or in combination with one or more other mediums. It can also be used with or without the addition of exogenous nutrients.

[0121] As used herein, "improvement" should be broadly understood to include improvement in the target characteristic compared to a control group or a known average value associated with the characteristic. In the present disclosure, "improvement" does not necessarily require that the data be statistically significant (i.e., p < 0.05); rather, any quantifiable difference indicating that one value (e.g., the mean treatment value) is different from another value (e.g., the mean control value) may rise to the level of "improvement."

[0122] As used herein, the term "metabolite" refers to an intermediate or product of metabolism. In some embodiments, metabolites include small molecules. Metabolites have a variety of functions, including energy, structure, signal transduction, stimulation and inhibition of enzymes, as cofactors of enzymes, defense, and interactions with other organisms (such as pigments, odors and pheromones). Primary metabolites are directly involved in normal growth, development and reproduction. Secondary metabolites are not directly involved in these processes, but often have important ecological functions. Examples of metabolites include, but are not limited to, antibiotics and pigments, such as resins and terpenes. Metabolites used herein include small hydrophilic carbohydrates; large hydrophobic lipids and complex natural compounds.

[0123] As used herein, "carrier," "acceptable carrier," or "pharmaceutical carrier" are used interchangeably and refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin; such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous saline solutions, as well as aqueous dextrose and glycerol solutions, are preferably used as carriers, in some embodiments as injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more of a binder (for compressed pills), a tackifier, an encapsulating agent, a flavoring, and a coloring agent. The choice of carrier can be selected based on the intended route of administration and standard pharmaceutical practice. See Handbook of Pharmaceutical Excipients, 8th ed. (Sheskey, Cook and Cable) 2017, Pharmaceutical Press; Remington's Parmaceutical Sciences, 18th ed. (Remington and Gennaro) 1990, Mack Publishing Company; Development and Formulation of Veterinary Dosage Forms (Hardee and Baggot) 2nd ed. (CRC Press) 1998.

[0124] As used herein, "delivery" or "administration" refers to the act of providing a beneficial activity to a host. Delivery can be direct or indirect. Administration can be by oral, nasal, or mucosal routes. For example, but not limited to, the oral route can be administration through drinking water, the nasal route can be administration by spray or steam, and the mucosal route can be administration by direct contact with mucosal tissue. Mucosal tissue is a membrane rich in mucous glands, such as the mucous glands that line the inner surface of the nose, mouth, esophagus, trachea, lungs, stomach, digestive tract, intestines, and anus. In the case of birds, administration can be in ovo, that is, administration to a fertilized egg. In ovo administration can be by liquid, sprayed on the surface of the eggshell, or injected through the eggshell.

[0125] As used herein, "animal" includes birds, poultry, humans, or non-human mammals. Specific examples include chickens, turkeys, dogs, cats, cows, salmon, fish, pigs, and horses. The chicken can be a broiler chicken, an egg-laying chicken, or an egg-producing chicken. As used herein, the term "poultry" includes domesticated fowl, such as chickens, turkeys, ducks, and geese.

[0126] As used herein, "digestive tract" refers to the gastrointestinal tract, including the stomach, small intestine, and large intestine. The term "digestive tract" can be used interchangeably with "gastrointestinal tract."

[0127] As used herein, "genetically modified microorganism" refers to any microorganism that has been altered from its natural state using molecular biology techniques. The genetic modification can be the deletion of a portion of a bacterial chromosome or a naturally occurring plasmid. Genetic modification can also be the introduction of artificial or exogenous nucleic acids into a portion of a chromosome. The introduction may or may not interfere with the expression of bacterial genes. Genetic modification can also be the introduction of artificial plasmids. The genetically modified microorganism can be a bacterium, a virus, a yeast, a mold, or a single-celled organism.

[0128] "Artificial nucleic acid" or "artificial plasmid" refers to any nucleic acid or plasmid that does not occur naturally, but is constructed using molecular biology techniques. Parts of the nucleic acid or plasmid may occur naturally, but these parts are artificially joined or organized.

[0129] As used herein, an "expression cassette" is an artificial nucleic acid constructed to express a desired biomolecule by a genetically modified microorganism. The expression cassette contains one or more promoters for transcriptional expression, a nucleic acid sequence encoding a signal sequence for secretion, a nucleic acid sequence encoding a cell wall anchor, at least one heterologous coding region encoding the desired biomolecule, a nucleic acid sequence encoding an expression peptide tag for detection, and a terminator for translation and transcription termination. The promoter directs the initiation of transcription of the coding region into messenger RNA and translation of the mRNA into a peptide. The signal sequence for secretion, or secretory signal sequence, directs the peptide to be located outside the cell membrane. The extracellular peptide can be a soluble secreted protein, or it can be cell-associated, particularly if the expression cassette contains a cell wall anchor sequence that connects the extracellular peptide to the bacterial cell wall. The expressed peptide tag is any amino acid sequence that can be recognized by an antibody or other binding protein. The expressed peptide tag can also be bound to an inorganic substance, such as a hexahistidine tag that binds to a nickel molecule. The terminator of translation can be a stop codon or an intervening open reading frame containing a stop codon.

[0130] As used herein, a "heterologous coding region" is a nucleic acid sequence comprising an open reading frame encoding a peptide. The coding region is heterologous to the associated promoter, meaning that the coding region and promoter are not linked in their native state.

[0131] A "heterologous" region of a nucleic acid, RNA, or DNA construct is a recognizable segment of RNA or DNA within a larger RNA or DNA molecule that is not found associated with the larger molecule in nature. Thus, when the heterologous region encodes a gene, the gene is typically flanked by RNA or DNA that is not the genomic RNA or DNA located in the genome of the source organism.

[0132] As used herein, a "protein" is a sequence of amino acids that exhibits a three-dimensional structure. A "peptide" is used interchangeably with protein, but may also refer to a short linear sequence of amino acids that does not have a well-defined three-dimensional structure.

[0133] As used herein, a "desirable biomolecule" is any molecule or peptide that, when administered via a live delivery platform, has a beneficial effect on the host. The desired biomolecule can be a peptide with anti-infective activity, a probiotic factor, an immunomodulatory factor, an anti-antinutritional factor, or a growth-promoting biomolecule. The desired biomolecule can also be an enzyme that produces a substance with anti-infective activity or a probiotic factor, such as a vitamin.

[0134] As used herein, "anti-infective activity" includes any activity that prevents a host from being infected with a pathogenic organism. The following molecules are examples of biomolecules with anti-infective activity: antimicrobial peptides; hemolysins or lytic enzymes; prophages, phages or viruses; enzymes, such as enzymes that cut or disable proteins produced by pathogens; and antibodies that block, inhibit or clear pathogenic molecules. Anti-infective drugs may have bactericidal activity, thereby slowing, reducing or preventing the growth of pathogenic species. Non-limiting examples of antimicrobial peptides are members of the mesicidin family or mesicidin-like molecules, such as those described in EP0700998. Non-limiting examples of hemolysins are lytic molecules produced by bacteriophages. Lysine may be specific to certain pathogenic bacteria and has been suggested as an alternative to traditional antibiotics. VA Fischetti, Viruses, vol. 10, no. 310 (2018); and R. Vazquez et al. Frontiers in Immunology, vol. 9, article 2252 (2018).

[0135] As used herein, a "probiotic factor" is a substance that, when produced by a genetically modified microorganism, demonstrates beneficial effects on the host. A probiotic factor can be an attachment molecule or agglutination molecule that promotes colonization of the host with the genetically modified microorganism and / or prolongs the time that the genetically modified microorganism colonizes the host. The longer the genetically modified microorganism persists in the host, the longer the beneficial effects provided.

[0136] As used herein, an "immunomodulatory factor" can be a cytokine, lymphokine, chemokine, interleukin, interferon, colony stimulating factor, or growth factor. The immunomodulatory factor can provide a nonspecific enhancement of the immune response, or the immunomodulatory factor can increase the number or tissue distribution of immune cells present in the host. Immunomodulatory factors can also reduce inappropriate immune responses, such as, but not limited to, autoimmune responses.

[0137] As used herein, a "growth promoting biomolecule" can be a growth factor, a transfer factor (such as an iron chelating molecule), a hormone, or any other factor that promotes healthy metabolic activity.

[0138] As used herein, "anti-nutritional factors" may include protease inhibitors, such as trypsin inhibitors.

[0139] As used herein, "delivery" or "administration" refers to the act of providing a beneficial activity to a host. Delivery can be direct or indirect. Administration can be by oral, nasal, or mucosal routes. For example, but not limited to, the oral route can be administration through drinking water, the nasal route can be administration by spray or steam, and the mucosal route can be administration by direct contact with mucosal tissue. Mucosal tissue is a membrane rich in mucous glands, such as the mucous glands that line the inner surface of the nose, mouth, esophagus, trachea, lungs, stomach, digestive tract, intestines, and anus. In the case of birds, administration can be in ovo, that is, administration to a fertilized egg. In ovo administration can be by liquid, sprayed on the surface of the eggshell, or injected through the eggshell.

[0140] As used herein, the terms "treating," "to treat," or "treatment" include inhibiting, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Treatment can also be applied prophylactically to prevent or reduce the incidence, rate, risk, or severity of a clinical symptom, disorder, condition, or disease.

[0141] As used herein, "subject" includes birds, poultry, fish, humans, or non-human animals. Specific examples include chickens, turkeys, dogs, cats, cows, and pigs. The chicken can be a broiler chicken, an egg-laying chicken, or an egg-producing chicken. As used herein, the term "poultry" includes domesticated birds such as chickens, turkeys, ducks, quail, and geese.

[0142] A "heterologous" region of a nucleic acid, RNA, or DNA construct is a recognizable segment of RNA or DNA within a larger RNA or DNA molecule that is not found associated with the larger molecule in nature. Thus, when the heterologous region encodes a gene, the gene is typically flanked by RNA or DNA that is not the genomic RNA or DNA located in the genome of the source organism.

[0143] The term "primer" as used herein refers to an oligonucleotide, whether naturally occurring or synthetically produced in a purified restriction digest, which can serve as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain, i.e., in the presence of nucleotides and an inducing agent (such as DNA polymerase), and at a suitable temperature and pH. A primer can be single-stranded or double-stranded and must be long enough to initiate the synthesis of the desired extension product in the presence of an inducing agent. The exact length of a primer depends on many factors, including the use of temperature, primer source, and method. For example, for diagnostic applications, an oligonucleotide primer typically contains 15-25 or more nucleotides, depending on the complexity of the target sequence, although it may contain fewer nucleotides.

[0144] The primers herein are selected to be "substantially" complementary to the different chains of a specific target DNA sequence. This means that primers must have enough complementarity to hybridize with their respective chains. Therefore, the primer sequence does not need to reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be connected to the 5' end of the primer, and the remainder of the primer sequence is complementary to the chain. Alternatively, non-complementary bases or longer sequences can be inserted into the primer, provided that the primer sequence has enough complementarity to hybridize with it with the sequence of the chain, thereby forming a template for the synthesis of extension products.

[0145] A "chimeric protein" or "fusion protein" comprises all or (preferably biologically active) a portion of a first polypeptide operably linked to a heterologous polypeptide. A chimeric protein or peptide is produced, for example, by combining two or more proteins having two or more active sites. In a chimeric or fusion protein, the first polypeptide may be covalently linked to an entity that can provide additional functionality or enhance the use or application of the first polypeptide, including, for example, a tag, a label, a targeting moiety or ligand, a cell binding or cell recognition motif or agent, an antimicrobial agent, an antibody, an antibiotic. Exemplary labels include radiolabels, such as isotopes 3 H. 14 C. 32 P. 35 S. 36 Cl, 51 Cr, 57 Co、 58 Co、 59 Fe, 90 Y. 125 I. 131 I and 186Re. The label can be an enzyme, and detection of the labeled lysine polypeptide can be accomplished by any currently used or accepted colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric technique known in the art. Chimeric proteins and peptides can act independently on the same or different molecules or targets, and thus have the potential to provide multiple activities, such as simultaneously treating or stimulating immune responses against two or more different bacterial infections or infectious agents.

[0146] As used herein, the term "mutant" refers to a variant of a nucleic acid, DNA or RNA sequence or chromosome structure that is different from a normal or wild-type sequence or a non-defective chromosome. In the case of nucleic acid, DNA or RNA sequences, examples of mutations include point mutations, insertions and deletions. Deletions include deletions of part or all of a gene. Such mutations can have functional effects, such as reduced function of a gene product, loss of function in a gene product, and / or new or altered functions in a gene product.

[0147] As used herein, "mutation" includes any change in one or more nucleic acids in a genomic sequence, including one or more base changes, deletions and / or insertions, which result in silent mutations, nonsense mutations, missense mutations, or any other such mutations that result in reduced gene function or result in an inactivated protein encoded by the gene or result in an otherwise non-functional protein encoded by the gene. Mutations include, but are not limited to, mutations that result in premature stop codons, abnormal splicing, transcriptional changes or failures, translational changes or failures. A gene comprising a mutation may have more than one mutation. Mutations include deletions of a gene or an important part of a gene, particularly such that the protein of the gene is not produced or expressed and / or inactivated. Mutations include insertions, for example, in which exogenous or heterologous sequences or nucleic acids are introduced or otherwise inserted into a gene. Such insertions can block or eliminate translation to an active protein or full-length protein, or can produce significantly altered and different proteins that are not as active as the wild type. Insertions can facilitate the separation, detection and selection of gene mutants, for example by introducing or inserting antibiotic resistance genes or detectable markers or proteins. In specific embodiments of the invention, as described herein, the mutation is a non-natural mutation, which includes one or more mutations and is genetically engineered or recombinantly produced. In some embodiments, the mutation is genetically engineered or recombinantly produced in vitro. In some embodiments, the mutation is genetically engineered or recombinantly produced in cells.

[0148] In some embodiments, when a gene or a gene encoding nucleic acid or a major part or important part of a protein is missing, a sudden change occurs. In embodiments, for example, one or more genes or genes encoding nucleic acid or a major part or important part of a protein are deleted by a recombination method. The recombination method of the targeted deletion gene is known and available to those skilled in the art. This method includes homologous recombination, for example, by the plasmid, phage or nucleic acid (such as DNA or linear DNA fragments) introduced, recombinase or recombinase-mediated recombinase, for example, by recombinase recognition or target sequence sequence, transposon-mediated compound and gene replacement.

[0149] According to some embodiments of the present invention, deletion or inactivation mutations are generated, thereby deleting or inactivating one or more genes in the genome of Bacillus subtilis bacteria. Thus, deletion mutants have been generated and utilized, or deletion mutations have been utilized to construct deletions in each gene, thereby providing new Bacillus subtilis mutant strains. In some embodiments, the growth of these mutant bacteria is altered.

[0150] In an embodiment of the present invention, the gene mutation is a gene deletion mutation. In an embodiment, the gene mutation is a deletion produced by recombination, including a substantial portion of the coding region of a gene being deleted. In some embodiments, a substantial portion of the coding gene is deleted and replaced by inserting a tag or marker (e.g., a detectable tag or selectable marker).

[0151] Therapeutic or biologically active molecules can be any molecules with useful or desired activity, including polypeptides or nucleic acids. Therapeutic biomolecules include biomolecules with therapeutic effects. Examples of therapeutic biomolecules include antibodies, ribonucleic acids (RNA), and antigens. Antibodies include antibody fragments, such as VHH. RNA includes inactivated RNA, such as shRNA and siRNA. Antigens include biomolecules that stimulate an immune response. Examples of antigens include peptides, polypeptides, proteins, nucleic acid molecules, and carbohydrate molecules. In some embodiments, the molecule can be selected from antibodies, ribonucleic acids (RNA), peptides or proteins, and antigens.

[0152] Antibodies according to the present disclosure include immunoglobulins, in particular any polypeptide or protein having a binding domain that is homologous to an antibody binding domain or to an antibody binding domain. The term also includes CDR-grafted antibodies. "Antibody" refers to any immunoglobulin that binds to a specific epitope, including antibodies and fragments thereof. The term includes polyclonal antibodies, monoclonal antibodies and chimeric antibodies. The term "antibody" includes wild-type immunoglobulin (Ig) molecules, which typically comprise four full-length polypeptide chains, two heavy (H) chains and two light (L) chains, or their equivalent Ig homologs (e.g., camelid nanobodies comprising only heavy chains; including full-length functional mutants, variants or derivatives thereof that retain the basic epitope binding characteristics of Ig molecules, and including dual specific, bispecific, multispecific and dual variable domain antibodies. The term "antibody" also includes any "antibody fragment" in its meaning."Antibody fragments" refer to molecules containing at least one non-full-length polypeptide chain, including (i) Fab fragments, which are monovalent fragments consisting of the variable light (VL), variable heavy (VH), constant light (CL), and constant heavy 1 (CH1) domains; (ii) F(ab')2 fragments, which are bivalent fragments consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) the heavy chain portion of a Fab (Fd) fragment, which consists of the VH and CH1 domains; (iv) variable fragments (Fv), which consist of the VL and VH domains of a single arm of an antibody; and (v) domain antibody (dAb) fragments, which contain a single variable domain (Ward, ES et al., Nature 341, 544-546 (1989)); (vi) camelid antibodies; (vii) isolated complementarity determining regions (CDRs); (viii) single-chain Fv fragments in which the VH and VL domains are connected by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); (ix) diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (WO 94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) linear antibodies, which comprise a pair of tandem Fv fragments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and / or tetramers (Power and Hudson, J Immunol. Methods 242: 193-2049 (2000)); (xii) minibodies, which are bivalent molecules consisting of scFv fused to constant immunoglobulin domains CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al. (2004) Prot Eng Des Sell 7(4): 315-323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9):1126-1136)); and (xiii) other non-full-length portions of the heavy and / or light chains, or mutants, variants or derivatives thereof, alone or in any combination. Also included are chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide.

[0153] An "antibody combining site" is a structural portion of an antibody molecule consisting of a light chain or heavy chain and light chain variable and hypervariable regions that specifically binds to an antigen. Exemplary antibody molecules are complete immunoglobulin molecules, substantially complete immunoglobulin molecules, and those portions of immunoglobulins that comprise an antibody combining site, including those portions of Fab, Fab', F(ab')2, and F(v) as known in the art. Antibodies can also be bispecific, in which one binding domain of the antibody has a first binding specificity and the other binding domain has a different specificity, such as to supplement effector functions. The other binding domain can be an antibody that recognizes or targets a specific cell type or recognizes a specific cell receptor and / or modulates the cell in a specific manner, such as an immunomodulator (e.g., an interleukin), a growth regulator, or a cytokine or toxin (e.g., ricin), or an anti-mitotic or apoptotic agent or factor.

[0154] The term "antigen binding domain" describes a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. In the case where the antigen is large, the antibody may only bind to a specific portion of the antigen, which is referred to as an epitope. The antigen binding domain can be provided by one or more antibody variable domains. The antigen binding domain may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), or may include only an antibody heavy chain variable region (VH).

[0155] Also contemplated are immunoconjugates or antibody fusion proteins in which antibodies, antibody molecules, or fragments thereof of the present invention are coupled or connected to other molecules or formulations. Such immunoconjugates or antibody fusion proteins may further include, but are not limited to, antibodies, molecules, or fragments coupled to chemoablators, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutics, antimicrobials, or peptides, cell wall and / or cell membrane disruptors, or drugs.

[0156] Single domain antibodies are included as specific embodiments of therapeutic or biologically active molecules delivered according to the intracellular delivery platform provided herein and expressed by self-amplifying nucleic acids. Single domain antibodies were originally isolated from camelids and are interchangeably designated as camelid antibodies, nanobodies or VHHs. VHH antibodies correspond to the variable region of the heavy chain of antibodies and are very small in size, about 15 kDa, hence the name "nanoantibodies". The advantage of these antibody-derived molecules is their small size, which allows them to bind to hidden epitopes that are inaccessible to whole antibodies. In the context of therapeutic applications, small molecular weight also means effective penetration and rapid clearance. Both scFv and VHH nanoantibodies can be connected to the Fc fragment of the desired species and retain their specificity and binding properties, and are then called miniantibodies.

[0157] Delivery of Nanobodies: Nanobodies are small, low-molecular-weight, single-domain, heavy-chain-only antibodies found in camels. Due to their small size, the genes for these proteins are easily cloned in plasmids. Therefore, by using molecular cloning techniques, Nanobodies against various antigens can be presented in the systemic circulation. This invention and intracellular delivery platform has been used to deliver and express antibody fragments, particularly VHHs or Nanobodies.

[0158] Antigen is a substance, such as a protein or peptide, that can induce an immune response, especially the production of antibodies. In immunology, an antigen is a molecule or molecular structure that can be present in the outside of a pathogen, which can be bound by antigen-specific antibodies or B cell antigen receptors. The presence of antigens in the body will usually trigger an immune response. In many systems, antigens or peptides or protein sequences that can trigger an immune response, particularly a protective or neutralizing immune response, have been defined. The basis of a vaccine is to present one or more antigens to an animal or host from a pathogenic factor so that the animal or host has an immune response and produces antibodies against the pathogenic factor. This immune response and these recovered antibodies are used to protect the host or animal from further infection, disease or ailment caused by the pathogenic factor. In embodiments of the present invention, the vaccines provided herein and intended can and are used to produce mucosal, systemic and cellular immunity against one or more pathogens.

[0159] Antigens can include all or part of a protein. In particular, an antigen can be an antigenic portion or fragment of a full-length protein. An antigen can be a non-natural fragment of a protein. The delivery platform can be used to express one or more antigens of a specific pathogen. Multiple antigens can be expressed from, for example, a single self-amplifying RNA. Multiple antigens of a pathogenic agent or pathogen can be expressed from a single Bacillus subtilis 105 strain.

[0160] There are various peptides or proteins that are independently used as therapeutic biomolecules. Among them are anti-infective or antimicrobial peptides, which can be used to block or treat infection by pathogenic factors or bacteria.

[0161] Plants and animals secrete a variety of antimicrobial peptides to combat foreign viral, bacterial, or fungal attacks (Boman, HG (2003) J. Intern. Med. 254 (3): 197-215). These form part of the innate immune response to infection, which is short-lived and rapid compared to humoral immunity. These peptides are heterogeneous in length, sequence, and structure, but most are small, cationic, and amphipathic (Zasloff, M. (2002) Nature 415 (6870): 389-395). Exemplary known antimicrobial peptides of this type are listed in the Antimicrobial Database (aps.unmc.edu / AP / main.php; Wang Z and Wang G (2004) NAR 32: D590-D592), and the contents and disclosures of this site are incorporated herein by reference in their entirety. Although the cell wall may be the primary target, several lines of evidence suggest that antimicrobial peptides act by lysing bacterial membranes. Cells become permeable after exposure to the peptides, and their membrane potential decreases accordingly. Protamine or polycationic amino acid peptides containing combinations of one or more repeating units of cationic amino acids such as arginine (R), tryptophan (W), lysine (K), or even synthetic polyarginine, polytryptophan, and polylysine have been shown to kill microbial cells.

[0162] A cell wall degrading enzyme is an enzyme that degrades cell wall components, including peptidoglycans, such as murein and pseudomurein, chitin, and teichoic acid. Cell wall degrading enzymes may include, but are not limited to, amidases, muramamidases, endopeptidases, and glucosaminidase. Phage lytic enzymes are cell wall degrading antimicrobial enzymes encoded by bacteriophages in bacteria. Lytic enzymes are peptidoglycan hydrolases that break bonds in the bacterial wall, rapidly hydrolyzing covalent bonds necessary for the integrity of the peptidoglycan, leading to bacterial lysis and the concomitant release of progeny phage. Bacterial phage lytic enzymes have been shown to be useful for the assessment and specific treatment of various types of infections in subjects via various routes of administration. Phage-associated lytic enzymes have been identified and cloned from various bacterial phages, each of which has been shown to be effective in killing specific bacterial strains.

[0163] The use of bacteria such as Bacillus as vectors to express, produce or deliver immune, prophylactic or any other therapeutic biomolecules provides a wide range of applicable products and therapies targeting a variety of disease conditions across a range of host species. There are various ways to modify live bacterial vectors and expression systems to deliver heterologous antigens, such as as payloads for chromosomal or plasmid-integrated genes, or eukaryotic antigen expression plasmids (so-called DNA vaccines), but these systems have limitations, including their means of expressing heterologous antigens. Recently, RNA-based vaccines such as messenger RNA (mRNA) and self-amplifying replicons (SAMs) are becoming an increasingly promising alternative to traditional plasmid DNA gene vaccines (DNA vaccines). RNA vaccines have been shown to elicit antigen-specific antibody and cellular immune responses against several viral pathogens and have several distinct advantages over DNA. The present invention provides a new delivery platform for delivering antigens, immunogens, antibodies, bioactive peptides, RNA and other biotherapeutics and therapeutic biomolecules. The present invention provides a new delivery platform for delivering immunogens, antibodies and therapeutic biomolecules as vaccines, including prophylactic and therapeutic vaccines.

[0164] The intracellular delivery platforms and production systems of the present disclosure include genetically modified bacteria with self-amplifying or integrated nucleic acids capable of encoding biomolecules or heterologous proteins.

[0165] In one embodiment, a probiotic composition is provided that comprises the genetically modified Bacillus subtilis strain 105 described herein, wherein the Bacillus subtilis strain 105 comprises a nucleic acid encoding a biomolecule or heterologous protein for production, delivery, targeting, or therapeutic importance.

[0166] In some embodiments, the composition comprises a genetically modified Bacillus subtilis strain 105, wherein ELA191105 or an active, effective variant thereof has been modified. In some embodiments, the composition comprises a genetically modified Bacillus subtilis strain 105 and further comprises a combination with another isolated Bacillus strain, particularly a different Bacillus species having probiotic properties or activity, particularly including a combination with strain 105. In some embodiments, Bacillus subtilis strain 105 can be combined with one or more isolated Bacillus amyloliquefaciens strains, particularly selected from ELA191024 (corresponding to ATCC deposit PTA-126784), ELA191036 (corresponding to ATCC deposit PTA-126785), ELA191006 (corresponding to ATCC deposit PTA-127065), and ELA202071 (corresponding to ATCC deposit PTA-127064).

[0167] These probiotic strain combinations, compositions, and methods thereof are described and provided in PCT / US2021 / 051973, published as WO2022 / 067052 on March 31, 2022, which is incorporated herein by reference.

[0168] In some embodiments, the composition does not include Lactobacillus. Examples of Lactobacillus species include Lactobacillus reuteri and Lactobacillus crispatus, Lactobacillus vaginalis, Lactobacillus helviticus, and Lactobacillus johnsonii.

[0169] In some embodiments, the composition does not include a non-Bacillus strain. Examples of non-Bacillus strains include Lactobacillus, Leuconostoc (eg, Leuconostoc mesenterioides).

[0170] The composition may include or comprise live bacteria or bacterial spores, or a combination thereof.

[0171] In some embodiments, the composition does not include an antibiotic. Exemplary antibiotics include tetracycline, bacitracin, tylosin, salinomycin, virginiamycin, and bambemycin.

[0172] The above-mentioned composition can include a carrier suitable for animal consumption or use. Examples of suitable carriers include edible food-grade materials, mineral mixtures, gelatin, cellulose, carbohydrates, starch, glycerol, water, ethylene glycol, molasses, corn oil, animal feed, such as cereals (barley, corn, oats, etc.), starch (cassava, etc.), oilseed cakes, and vegetable waste. In some embodiments, the composition includes vitamins, minerals, trace elements, emulsifiers, aroma products, adhesives, colorants, flavor enhancers, thickeners, etc.

[0173] In some embodiments, the composition includes one or more biologically active molecules or therapeutic molecules. Examples of such molecules include ionophores, vaccines, antibiotics, antihistamines, virucides, nematicides, amino acids (such as methionine, glycine, and arginine), fish oils, krill oils, and enzymes.

[0174] In some embodiments, the composition or combination can include one or more prebiotics in addition. In some embodiments, the composition can be used together with one or more prebiotics, or can be used altogether with it. Prebiotics may include organic acid or indigestible feed ingredients, which ferment in the lower digestive tract and can be used for selecting beneficial bacteria. Prebiotics can include manno-oligosaccharides, oligofructose, oligogalactose, chito-oligosaccharides (chito-oligosaccharides), isomaltooligosaccharides, pectin oligosaccharides, oligoxylose and lactose oligosaccharides.

[0175] The composition can be formulated as an animal feed, a feed additive, an animal food, a food ingredient, a water additive, a water mixing additive, a consumable solution, a consumable spray additive, a consumable solid, a consumable gel, an injection, or a combination thereof. The composition can be formulated and suitable for use as one or more of an animal feed, a feed additive, a food ingredient, a water additive, a water mixing additive, a consumable solution, a consumable spray additive, a consumable solid, a consumable gel, an injection, or a combination thereof. The composition can be suitable for use as an animal feed, a feed additive, an animal food, a food ingredient, a water additive, a water mixing additive, a consumable solution, a consumable spray additive, a consumable solid, a consumable gel, an injection, or a combination thereof.

[0176] In some embodiments, the present disclosure provides for use of any of the compositions described above in therapy or treatment, or to improve a phenotypic characteristic of an animal.

[0177] In an embodiment of the present invention, animals may include farmed animals or livestock or domesticated animals. Livestock or farmed animals may include cattle (such as dairy cows or bulls (including calves)), poultry (including broilers, chickens and turkeys), pigs (including piglets), birds, aquatic animals (such as fish, agastric fish, gastric fish, freshwater fish (such as salmon, cod, trout and carp (such as koi)), marine fish (such as sea bass) and crustaceans (such as shrimp, mussels and scallops), horses (including racehorses), sheep (including lambs). Domesticated animals may be pets or animals raised in a zoological environment, and may also include any related animals, including canines (such as dogs), felines (such as cats), rodents (such as guinea pigs, rats, mice), birds, fish (including freshwater fish and marine fish) and horses. The animal may be a human.

[0178] The animal may be a pregnant or breeding animal, such as a pregnant sow or a pregnant pig.

[0179] Examples of improved phenotypic traits include reducing the formation of pathogen-associated lesions in the gastrointestinal tract or within the animal, reducing pathogen colonization, reducing the spread of one or more pathogens, promoting an immune response or antibody production against a pathogen, and increasing intestinal health or properties (reduced permeability and inflammation).

[0180] Examples of pathogens include Eimeria spp., Salmonella Typhimurium, Salmonella Infantis, Salmonella Hadar, Salmonella Enteritidis, Salmonella Newport, Salmonella Kentucky, Clostridium perfringens, Staphylococcus aureus, Streptococcus uberis, Streptococcus suis, Streptococcus pneumoniae, Escherichia coli, Campylobacter jejuni, Campylobacter perfringens, Fusobacterium necroticus, avian pathogenic Escherichia coli (APEC), Pisciricketsia salmonis, Flavobacterium spp., Salmonella Lubbock, Cryptococcus pyogenes, Shiga toxin-producing Escherichia coli, enterotoxigenic Escherichia coli, Campylobacter coli, and Lawsonia intellularis.

[0181] The pathogen may be a bacterium, a parasite or a virus. The virus may include a pathogenic virus that infects animals (including humans, livestock or domesticated animals) and may be specific to a particular animal (such as a poultry virus or a pig virus).

[0182] The composition can be used for treating infection, particularly bacterial infection. In some aspects, the above composition is used for treating an infection of at least one of Eimeria, Salmonella typhimurium, Salmonella infantis, Salmonella Hadar, Salmonella Enteritidis, Salmonella Newport, Salmonella Kentucky, Clostridium perfringens, Staphylococcus aureus, Streptococcus tuberculosis, Streptococcus suis, Escherichia coli, Campylobacter jejuni, Fusobacterium necrotizing, avian pathogenic Escherichia coli (APEC), Salmonella Lubbock, Cryptobacterium pyogenes, Shiga toxin-producing Escherichia coli, enterotoxigenic Escherichia coli, Campylobacter coli and Lawsonia intracellularis. The composition can be used for suppressing infection, particularly bacterial infection. Infection may be caused by one or more of the following: Eimeria spp., Salmonella Typhimurium, Salmonella Infantis, Salmonella Hadar, Salmonella Enteritidis, Salmonella Newport, Salmonella Kentucky, Clostridium perfringens, Staphylococcus aureus, Streptococcus uberis, Streptococcus suis, Escherichia coli, Campylobacter jejuni, Fusobacterium necroticus, avian pathogenic Escherichia coli (APEC), Salmonella Lubbock, Cryptococcus pyogenes, Shiga toxin-producing Escherichia coli, enterotoxigenic Escherichia coli, Campylobacter coli, and Lawsonia intracellularis.

[0183] In some aspects, the compositions are used to reduce or inhibit the colonization of bacteria in animals or in herds of animals, particularly pathogenic bacteria. In some aspects, the compositions are used to reduce or inhibit the colonization of Eimeria, Salmonella Typhimurium, Salmonella Infantis, Salmonella Hadar, Salmonella Enteritidis, Salmonella Newport, Salmonella Kentucky, Clostridium perfringens, Staphylococcus aureus, Streptococcus uberis, Streptococcus suis, Escherichia coli, Campylobacter jejuni, Fusobacterium necroticum, avian pathogenic Escherichia coli (APEC), Salmonella Lubbock, Cryptococcus pyogenes, Shiga toxin-producing Escherichia coli, enterotoxigenic Escherichia coli, Campylobacter coli, and Lawsonia intracellularis.

[0184] In some aspects, the compositions are used to reduce the spread of bacteria, particularly pathogenic bacteria, within an animal enclosure or within a group or herd of animals. In some aspects, the compositions are used to reduce the spread of at least one of Eimeria spp., Salmonella Typhimurium, Salmonella Infantis, Salmonella Hadar, Salmonella Enteritidis, Salmonella Newport, Salmonella Kentucky, Clostridium perfringens, Staphylococcus aureus, Streptococcus uberis, Streptococcus suis, Escherichia coli, Campylobacter jejuni, Fusobacterium necroticum, avian pathogenic Escherichia coli (APEC), Salmonella Lubbock, Cryptococcus pyogenes, Shiga toxin-producing Escherichia coli, Enterotoxigenic Escherichia coli, Campylobacter coli, and Lawsonia intracellularis within an animal enclosure or within a group or herd of animals.

[0185] In some aspects, the above composition is used to reduce bacterial load, particularly pathogenic or clinically important bacteria, including the number or amount of bacteria in the digestive tract or gastrointestinal tract of an animal. The bacteria can be selected from at least one of Eimeria, Salmonella typhimurium, Salmonella infantis, Salmonella hadal, Salmonella enteritidis, Salmonella Newport, Salmonella kentucky, Clostridium perfringens, Staphylococcus aureus, Streptococcus uberis, Streptococcus suis, Escherichia coli, Campylobacter jejuni, Fusobacterium necroticum, avian pathogenic Escherichia coli (APEC), Salmonella Lubbock, Cryptococcus pyogenes, Shiga toxin-producing Escherichia coli, enterotoxigenic Escherichia coli, Campylobacter coli and Lawsonia intracellularis.

[0186] In some aspects, the composition is used to treat at least one of inflammatory bowel disease, obesity, liver abscess, rumen acidosis, leaky gut syndrome, piglet diarrhea, necrotic enteritis, coccidiosis, salmon ricketsialsepticemia, and foodborne diseases.

[0187] The composition may further include one or more components or additives. The one or more components or additives may be components or additives that facilitate administration, such as by stabilizers or carriers, or by additives that enable administration to animals, such as by any suitable mode of administration, including aerosol or spray form, in water, feed, or injection. Administration to animals may be by any known or standard technique. These measures include oral administration, gastric intubation, or bronchial nasal spray. The compositions disclosed herein may be administered by immersion, intranasal administration, intramammary administration, topical administration, mucosal administration, or inhalation administration. When the animal is a bird, it may be administered in ovo or by spray inhalation for treatment.

[0188] The composition may include a carrier in which the bacteria or any other components of this type are suspended or dissolved. This carrier may be any solvent or solid, or may be encapsulated in a material that is nontoxic to the animal being inoculated and compatible with the organism. Suitable pharmaceutical carriers include liquid carriers, such as physiological saline and other nontoxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose, which may also be incorporated into the feed of farm animals. When used for administration via the bronchus, the composition is preferably present in the form of an aerosol. Dyes may be added to the composition herein, including promoting chewing or confirming whether the animal has taken in or inhaled the composition.

[0189] When administered to animals (including farm animals), oral or injection administration may be included. Oral administration may include pills, tablets or pastes, or as a powder or solution in feed or drinking water. The method of administration generally depends on the species being fed or administered, the number of animals being fed or administered, and other factors such as available handling facilities and the risk of stress on the animals.

[0190] The required dosage will vary and will be sufficient to induce an immune response or produce the desired or desired biological or phenotypic change or response. Routine experimentation will determine the required amount. Increasing amounts or multiple doses may be implemented and used as needed.

[0191] In one embodiment of the present invention, the bacterial strain is administered in a dose expressed as CFU / g or colony forming units / gram. In one embodiment, the dose is between 1x10 3 Up to 1x10 9 In one embodiment, the dosage is in the range of 1x10 4 to 1x10 6 In one embodiment, the dose is 5x10 4 to 1x10 6 In one embodiment, the dose is 5x10 4 to 6x10 5 In one embodiment, the dose is in the range of 7x10 4to 3x10 5 In one embodiment, the dosage is about 50K, 75K, 100K, 125K, 150K, 200K, 300K, 400K, 500K, 600K CFU / g.

[0192] Administration of the compositions disclosed herein may include co-administration with a vaccine or therapeutic compound. Administration of the vaccine or therapeutic compound may include administration before, simultaneously with, or after administration of the compositions disclosed herein.

[0193] Suitable vaccines according to this embodiment include vaccines that help prevent coccidiosis.

[0194] In some embodiments, the above methods are administered to animals in the absence of antibiotics.

[0195] Antigen is a kind of material that can induce immune response, especially the production of antibody, such as protein or peptide.In immunology, antigen is a kind of molecule or molecular structure that can be present in the outside of pathogen, by antigen-specific antibody or B cell antigen receptor combination, for example.The existence of antigen in vivo can trigger immune response usually.In many systems, defined can trigger immune response, particularly antigen or peptide or peptide sequence of protective or neutralizing immune response.The basis of vaccine is that one or more antigens are presented to animal or host from pathogenic factor, so that animal or host has immune response and produces antibody for pathogenic factor.This immune response and these recovered antibodies are used to protect host or animal from further infection of pathogenic factor, disease or ailment that pathogenic factor causes.In embodiments of the present invention, the vaccine that this paper provides and envisions can and is used to produce mucosa, system and cellular immunity for one or more pathogenic factors.

[0196] Antigens can include all or part of a protein. In particular, an antigen can be an antigenic portion or fragment of a full-length protein. An antigen can be a non-natural fragment of a protein. The delivery platform can be used to express one or more antigens of a specific pathogen. Multiple antigens can be expressed, for example, from a single self-amplifying RNA. Multiple antigens of a pathogenic agent or pathogen can be expressed by a single modified Bacillus subtilis 105 strain.

[0197] Coccidiosis is a common poultry disease caused by Eimeria spp. Coccidiosis is controlled by adding coccidiostats to feed or administering vaccines containing low doses of virulent or attenuated Eimeria oocysts. However, drug resistance and uneven dosing of these Eimeria species, resulting in variable immunity, have prompted efforts to develop improved and recombinant Eimeria vaccines and other approaches to stimulate immunity and address coccidiosis.

[0198] Eimeria is a genus of parasites that includes various species that can cause coccidiosis in cattle, poultry, dogs (especially puppies), cats (especially kittens), and smaller ruminants (including sheep and goats). Species of this genus infect a variety of hosts. The most common Eimeria species that cause coccidiosis in cattle are E. bovis, E. zuernii, and E. auburnensis.

[0199] This paper has demonstrated that antigens capable of generating an immune response can be delivered via a live cell delivery platform and using the SAM vector of the present invention. Coccidia vaccine (poultry): Salmonella typhimurium is modified to deliver cross-protective antigens that cover Eimeria tenella, Eimeria maxima, and Eimeria acervulina as part of the SAM payload. Eimeria tenella elongation factor-1α, EtAMAl, EtAMA2, Eimeria tenella 5401, Eimeria acervulina lactate dehydrogenase antigen gene, Eimeria maxima surface antigen gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Eimeria common antigen 14-3-3 antigen are delivered and expressed in an applicable system.

[0200] There are various peptides or proteins that are independently used as therapeutic biomolecules. Among them are anti-infective or antimicrobial peptides, which can be used to block or treat infection by pathogenic factors or bacteria.

[0201] Plants and animals secrete a variety of antimicrobial peptides to combat foreign viral, bacterial, or fungal attacks (Boman, HG (2003) J. Intern. Med. 254 (3): 197-215). These form part of the innate immune response to infection, which is short-lived and rapid compared to humoral immunity. These peptides are heterogeneous in length, sequence, and structure, but most are small, cationic, and amphipathic (Zasloff, M. (2002) Nature 415 (6870): 389-395). Exemplary known antimicrobial peptides of this type are listed in the Antimicrobial Database (aps.unmc.edu / AP / main.php; Wang Z and Wang G (2004) NAR 32: D590-D592), and the contents and disclosures of this site are incorporated herein by reference in their entirety. Although the cell wall may be the primary target, several lines of evidence suggest that antimicrobial peptides act by lysing bacterial membranes. Cells become permeable after exposure to the peptides, and their membrane potential decreases accordingly. Protamine or polycationic amino acid peptides containing combinations of one or more repeating units of cationic amino acids such as arginine (R), tryptophan (W), lysine (K), or even synthetic polyarginine, polytryptophan, and polylysine have been shown to kill microbial cells.

[0202] A cell wall degrading enzyme is an enzyme that degrades cell wall components, including peptidoglycans, such as murein and pseudomurein, chitin, and teichoic acid. Cell wall degrading enzymes may include, but are not limited to, amidases, muramamidases, endopeptidases, and glucosaminidase. Phage lytic enzymes are cell wall degrading antimicrobial enzymes encoded by bacteriophages in bacteria. Lytic enzymes are peptidoglycan hydrolases that break bonds in the bacterial wall, rapidly hydrolyzing covalent bonds necessary for the integrity of the peptidoglycan, leading to bacterial lysis and the concomitant release of progeny phage. Bacterial phage lytic enzymes have been shown to be useful for the assessment and specific treatment of various types of infections in subjects via various routes of administration. Phage-associated lytic enzymes have been identified and cloned from various bacterial phages, each of which has been shown to be effective in killing specific bacterial strains.

[0203] The present invention is widely applicable to the development of effective immunostimulatory compositions, immunoboosting compositions and vaccines for bacterial, fungal, parasitic or viral disease preparations, wherein local immunity is important and may be the first line of defense. Such vaccines may be applicable to hatchery or field vaccine programs, particularly on farms and in raised animals. Viral vaccines for DNA or RNA viruses can be produced. The present invention also contemplates vaccines for preventing pathogenic fungi, protozoa and parasitic infections. The present invention provides therapeutic vaccines and prophylactic vaccines, for example, wherein antibodies or portions thereof are administered and expressed (for example, for animals suffering from disease or infection) by a delivery platform, and wherein proteins or antigens are administered and expressed by a delivery platform, and for stimulating the immune prophylactic vaccine of an animal.

[0204] Any examples or figures given herein shall not in any way be considered as limiting, restricting or explicitly defining any term (singular) or term (plural) used therein. Instead, these examples or figures will be considered as being described with respect to a specific embodiment and are illustrative only. One of ordinary skill in the art will understand that any term or term used in these examples or figures will encompass other embodiments that may or may not be given herein or elsewhere in the specification, and all such embodiments are intended to be included within the scope of the term or term. Language designating such non-limiting examples and figures includes, but is not limited to: "for example," "for instance," "eg," and "in one embodiment." In this specification, various parameter groups comprising multiple members are described. Within a set of parameters, each member can be combined with any one or more of the other members to form additional subgroups. For example, if the members of a group are a, b, c, d, and e, then the additional subgroups specifically contemplated include any one, two, three, or four of the members, such as a and c; a, d, and e; b, c, d, and e; etc.

[0205] In this specification, quantities are defined by ranges and their lower and upper limits. Each lower limit can be combined with each upper limit to define a range. A lower limit and an upper limit should each be treated as a separate element. Two lower limits or two upper limits can be combined to define a range.

[0206] Preservation Information

[0207] Bacillus subtilis strain "ELA191105" was deposited on June 19, 2020, with the American Type Culture Collection (ATCC), ATCC Patent Collection, 10801 University Boulevard, Manassas, Va., 20110, USA, under the Budapest Treaty. The patent deposit number for this deposit is PTA-126786.

[0208] During the pendency of this application, the deposit will be available to such persons as the Patent and Trademark Office determines to be entitled to receive it under 37 C.F.R. § 1.14 and 35 U.S.C. § 122. Upon the admission of any embodiment of this application, all restrictions on public access to the variety will be irrevocably lifted.

[0209] The deposit will be maintained in the ATCC depository, a public depository, for a period of 30 years, or 5 years after the last request, or the life of the patent, whichever is longer, and will be replaced if the deposit becomes invalid during this period.

[0210] The present disclosure may be better understood with reference to the following embodiments. The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, and / or methods claimed herein, and are intended to be illustrative only and not to limit the present disclosure. It should be understood that other embodiments and uses will be apparent to those skilled in the art, and the present disclosure is not limited to these specific illustrative examples or preferred embodiments.

[0211] Example 1

[0212] Isolation and Characterization of Bacillus subtilis Strains

[0213] The sample was isolated from a chicken cecal sample. Spores were isolated by heating the sample to 90°C for 10 minutes or treating it with ethanol to a final concentration of 50% for 1 hour. The treated sample was plated on LB medium, and the resulting colonies were purified by three consecutive transfers onto LB agar plates. The identity of the isolate was confirmed by amplifying the 16S rRNA gene and then performing DNA Sanger sequencing of the PCR amplicon.

[0214] The inhibitory effect of ELA191105 on bacterial strains was tested. Table 1 summarizes the inhibition results of Bacillus subtilis strain 105 on other isolated bacterial strains.

[0215] Table 1

[0216]

[0217] Enterobacteriaceae, BRRS

[0218] Note: *Clostridium perfringens strain 15

[0219] Antibiotic susceptibility: The antibiotic susceptibility of strain ELA191105 was tested. ELA191105 was sensitive to chloramphenicol, gentamicin, tetracycline, erythromycin, clindamycin, streptomycin, kanamycin, and vancomycin.

[0220] Growth medium: Growth experiments were conducted using xylan and banana starch as the sole growth medium. ELA191105 was able to grow on these substrates as the sole growth medium.

[0221] Sporulation: ELA191105 was tested for sporulation. ELA191105 was sporulated in a tested sporulation medium (Difco sporulation medium, DSM) and the culture was grown at 37°C for 72 hours.

[0222] Digestive Enzyme Assays: ELA191105 was tested for amylase and protease activity according to the protocol described by Latorre, JD, 2016. Briefly, overnight cultures of the Bacillus isolate were spotted on agar plates containing soluble starch and skim milk, respectively, for amylase and protease assays. The plates were incubated at 37°C for 48 hours. Clearance zones caused by protease activity were visualized directly, while clearing zones caused by amylase activity were visualized by flooding the plate surface with 5 mL of Gram's iodine solution. Protease activity of ELA191105 was determined using the protease assay, and amylase and protease activities were observed. β-mannanase activity of ELA191105 was also tested, demonstrating that the strain is capable of digesting galactomannan.

[0223] Cytotoxicity Assay: ELA191105 was tested for cytotoxicity against African green monkey kidney cells (Vero cells). Cytotoxicity was determined using the LDH cytotoxicity assay. Positive control: Bacillus cereus DSM 31 (ATCC 14579) (cytotoxicity 78.6%); negative control: Bacillus licheniformis ATCC 14580 (cytotoxicity -0.1%); experimental control: Bacillus subtilis 747 (Correlink™ strain) (8.7% cytotoxicity; non-toxic). The ELA191105 strain showed no cytotoxicity against Vero cells. The percent cytotoxicity was less than 10.

[0224] Genome analysis: The genome of ELA191105 strain was sequenced, and some genome characteristics are as follows: contigs: 3; coverage: 117 times; %GC: 43%; length (Mbp): 4.089.

[0225] ELA191105 has genes that are not present in other Bacillus strains for genome comparison. Some unique genes include metabolic enzymes (phosphosulfolactic acid synthase, ethanolamine / propylene glycol utilization, malate / lactate dehydrogenase); antioxidant (prokaryotic glutathione synthetase); transporter (organic anion transporter polypeptide (OATP) family); and digestive enzyme (α-amylase). Details on the unique genes and metabolic analysis of ELA191105 and exemplary antimicrobial peptides, secondary metabolite genes, including comparisons with other Bacillus strains, are provided in USSN 63 / 083697, filed September 25, 2020, and USSN 63 / 241369, filed September 7, 2021, each of which is incorporated herein by reference.

[0226] The genomic nucleic acid sequence of strain 105 (ELA191105) is provided as SEQ ID NO: 1 as the complete genome sequence and as SEQ ID NOs: 2-6.

[0227] Table 2 summarizes some of the digestive enzymes identified in the genome analysis of Bacillus subtilis strain 105.

[0228] Table 2

[0229]

[0230] Strain ELA191105 contains genes encoding bacteriocins, specifically bacitracin A, lipastatin, surfacin, bacillobactin, and bacilysin. Furthermore, strain ELA191105 contains two clusters of terpene-derived metabolites and one cluster of polyketide-derived metabolites.

[0231] Example 2

[0232] Global metabolomics analysis

[0233] A global metabolomic analysis of Bacillus subtilis strain (ELA191105) was performed. Strains were grown individually, and the resulting cell pellets and supernatants were analyzed to identify metabolites. Strains were grown in minimal or complete medium at 37°C for 24 hours. Fresh medium (without cells) was used as a control sample. Metabolites in the supernatant represent molecules secreted by the cells. Minimal medium: M9 salts containing 0.5 g casamino acids / L and 1% glucose. M9 salts contain 6.78 g / L disodium phosphate (anhydrous), 3 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, and 1 g / L ammonium chloride. Complete medium: Bacillus broth (per liter): 30 g peptone, 30 g sucrose, 8 g yeast extract, 4 g KH2PO4; 1.0 g MgSO4; and 25 mg MnSO4.

[0234] Using Hamilton's automated MicroLab The samples were prepared systematically. For QC purposes, several recovery standards were added before the first step of the extraction process. The samples were extracted with methanol under vigorous shaking for 2 minutes (Glen Mills GenoGrinder 2000) to precipitate proteins and dissociate small molecules bound to proteins or trapped in the precipitated protein matrix, and then centrifuged to recover chemically distinct metabolites. The resulting extracts were divided into five fractions: two for analysis by two independent reversed-phase (RP) / UPLC-MS / MS methods using positive-mode electrospray ionization (ESI), one for analysis by RP / UPLC-MS / MS using negative-mode ESI, another for analysis by HILIC / UPLC-MS using negative-mode ESI, and one for future use. The samples were briefly placed in The organic solvent was removed by centrifugation (Zymark). The sample extracts were stored under nitrogen overnight before analysis.

[0235] Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS): All methods used a Waters ACQUITY ultra-high performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high-resolution / accurate mass spectrometer connected to a heated electrospray ionization (HESI-II) source and an Orbitrap mass analyzer operating at 35,000 mass resolution. The sample extracts were dried and then reconstituted in a solvent compatible with each of the four methods. Each reconstitution solvent contained a series of fixed concentrations of standards to ensure consistency in injection and chromatography. An aliquot was analyzed using acidic positive ion conditions to optimize chromatography for more hydrophilic compounds. In this method, the extracts were gradient eluted from a C18 column (Waters UPLC BEHC18-2.1x100mm, 1.7μm) using water and methanol containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). The second aliquot was also analyzed using acidic cationic conditions, but the chromatography was optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the above C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA, and operated at an overall higher organic content. The third aliquot was analyzed using a separate dedicated C18 column using basic negative ion optimized conditions. The basic extract was gradient eluted from the column using methanol and water, followed by elution with 6.5mM ammonium bicarbonate at pH 8. The fourth aliquot was analyzed by negative ionization after elution from a HILIC column (Waters UPLC BEH Amide 2.1x150mm, 1.7μm) using a gradient consisting of water and acetonitrile and 10mM ammonium formate (pH 10.8). MS analysis was performed using dynamic exclusion, alternating between MS and data-dependent MSn scans. The scan range was approximately 70-1000 m / z.

[0236] The data were analyzed using global non-targeted metabolic profiling. The data were analyzed using Welch's t-test and principal component analysis (PCA). PCA is a mathematical procedure that reduces the dimensionality of the data while retaining most of the variation within the dataset. This approach allows for a visual assessment of similarities and differences between samples (growth conditions, including media type and strains present). Dissimilar groups should be grouped separately and vice versa.

[0237] Metabolite Quantification and Block Correction: Peak values ​​were quantified as area under the curve (AUC) detector ion counts. For studies spanning multiple days, a data adjustment step was performed to correct for block variations caused by instrument tuning differences between days while preserving intra-day variance. Essentially, each compound was corrected for the day block of the balanced run by registering the daily median equal to one (1.00) and adjusting each data point proportionally (termed "block correction"). For studies that did not require analysis beyond a single day, no raw data adjustment was required, other than scaling for data visualization.

[0238] Metabolites were identified as strain-specific if their secreted metabolite values ​​were at least 1.5-fold greater than those of other strains or individual isolates of the control strain. Metabolites unique to a strain community were determined using a cutoff value of >1.5-fold compared to the values ​​of the respective metabolites secreted by individual isolates of the community. In complete medium, 231 metabolites were identified for strain ELA 191105, while 111 metabolites were identified in minimal medium, for a total of 272 metabolites. Overall, strain ELA 191105 had 77 unique metabolites compared to the other Bacillus strains used in the analysis, 45 of which had values ​​above the 2-fold threshold.

[0239] Strain ELA191105 was cultured alone in minimal or complete medium, and the supernatants were analyzed for secreted metabolites. Table 3 provides an exemplary list of metabolites secreted by the strains. Unless otherwise stated, metabolites were at least 1.5-fold more abundant than the medium control.

[0240] Table 3

[0241]

[0242]

[0243]

[0244] A- The secretion amount of metabolite is at least 2 times greater than that of the culture medium control; B- The secretion amount of metabolite is at least 3 times greater than that of the culture medium control; C- The secretion amount of metabolite is at least 5 times greater than that of the culture medium control.

[0245] Strain ELA191105 was cultured alone in complete medium, and the supernatant was analyzed for secreted metabolites. Table 4 provides an exemplary list of metabolites secreted by the strains. Unless otherwise stated, metabolites were at least 1.5-fold more abundant than the medium control.

[0246] Table 4

[0247]

[0248]

[0249] A - metabolite secreted at least 2-fold more than the medium control; B - metabolite secreted at least 3-fold more than the medium control; C - metabolite secreted at least 5-fold more than the medium control.

[0250] Strain ELA191105 was cultured in minimal and complete medium, and the supernatants were analyzed for secreted metabolites. An exemplary list of metabolites uniquely secreted by strain 105 is as follows: betaine A, carboxyethyl GABA A, 3-methylhistidine A, saccharoine, pipecolic acid, N,N-dimethyl-5-aminovalerate AB, N-butyryl-phenylalanine A, tryptophan A, N-butyryl-leucine, 2-hydroxy-4-(methylthio)butyric acid A, S-methylcysteine ​​A, ornithine, N-methylproline A, N,N,N-trimethyl-alanine proline betaine (TMAP) A, N-monomethylarginine A, guanidine acetic acid, putrescine, cysteinylglycine A，B，C , cyclo(glycine-phenylalanine), tryptophanylglycine, pyruvate A、B , mannose, N-acetylmuramate A , eicosene amide (20:1) A，B，C , deoxycamitine A , 2S, 3R dihydroxybutyrate, chiro-inositol A，B , choline, glycerylphosphorylcholine (GPC) A , 1-palmitoyl-GPE (16:0) A , 1-linoleylglycerol (18:2), 3-hydroxy-3-methylglutaric acid, 3-ureidopropionic acid, (3′-5′)-uridine uridine, nicotinamide riboside, trigonelline (N′-nicotinate methyl ester), oxalate (acetate disodium) A , pyridoxine (vitamin B6), maltol, histidine betaine (hercynine), 2,6-dihydroxybenzoic acid, pentose acid, N-acetylserine R，A , N-acetylthreonine R , N-acetylglutamine R，A , 1-methylhistidine R，A，B , N-acetylhistidine R，A , trans-urocanic acid R，A , N6 acetyl lysine R , N-(5-aminopentyl)acetamide R，A，B , N-acylphenylalanine R，A , phenyllactic acid (PLA) R，A , 3-(4-hydroxyphenyl)lactic acid (HPLA) R，A，B 、Isovaleric acid (C5) R，A，B , N-acetyl isoleucineR，A，B，C , N-acetyl valine R，A , N-acetylmethionine R , S-adenosylmethionine (SAM) R , 2-hydroxy-4-(methylthio)butyric acid R , S-methylcysteine R，A , N-acetylarginine R , acetylagmatine R，A , glutathione, oxidized (GSSG) R，A , 2-hydroxybutyric acid / 2-hydroxyisobutyric acid R，A , γ-glutamylhistidine R，A , glucuronate R，A，B , aconitic acid [cis or trans] R , 2-methyl citrate R , 2R, 3R-dihydroxybutyrate R，A，B , 5-aminoimidazole-4-carboxamide R，A，B，C , N-carbamate R，A , dihydroorotic acid R , orotidine R，A，B，C , thymine R，A，B , (3′-5t) adenosylguanosine R，A，B , Nicotinamide riboside R 、NAD+ R，A , pyridoxamine, pyridoxamine phosphate A and high citrate.

[0251] R - metabolite secreted when grown in complete medium; A - metabolite at least 2-fold more than the other two strains; B - metabolite at least 3-fold more than the other two strains; C - metabolite at least 5-fold more than the other two strains.

[0252] The 16S rRNA sequence of strain ELA191105 is shown below:

[0253] Full-length 16S-rRNA sequence

[0254] >Bacillus subtilis ELA191105 (BSUB_00009) (SEQ ID NO: 34)

[0255]

[0256]

[0257] Example 3

[0258] Bacillus metabolite and genome analysis

[0259] Metabolite analysis was performed on strain ELA1901105 (also referred to as strain 105). Table 5 provides an analysis of the presence or absence of certain natural antibiotics / antimicrobials or bacteriocins in strain 105 (ELA1901105).

[0260] Table 5

[0261]

[0262] Small peptides have powerful biological activities, ranging from antibiotics to immunosuppression. Some of these peptides are synthesized by nonribosomal peptide synthetases (NRPSs) (Challis GL and Naismith JH (2004) Cur Opin Struct Biol 14(6):748-756). Although the vast majority of peptide bond formation is catalyzed by the ribosome, NRPSs are important for the catalysis of peptide bond formation. Some of the best-known examples of molecules made by NRPSs illustrate the importance of NRPS systems. The antibiotic vancomycin and its analogs have very complex structures formed by NRPSs and associated enzymes. In fact, almost all peptide-based antibiotics are produced by NRPSs. Bacterial chelation of iron is essential for their survival and is often a determinant of virulence in pathogens. NRPSs synthesize macrocyclic compounds with extremely high iron affinity, such as enterobactins. The immunosuppressant cyclosporine and the potent antitumor compound bleomycin are both made by NRPSs. Molecules made by NRPSs are generally cyclic, have a high density of non-protein amino acids, and often contain amino acids connected by bonds other than peptide or disulfide bonds. NRPSs are now recognized as very large proteins that, despite the apparent complexity of their products, consist of a series of repeated enzymes fused together.

[0263] Nonribosomal peptide synthetases are modular enzymes that catalyze the synthesis of important peptide products from a variety of standard and nonproteinogenic amino acid substrates. Within a single module are multiple catalytic domains responsible for introducing individual residues. After the amino acid is activated and covalently linked to an integrated carrier protein domain, substrates and intermediates are delivered to adjacent catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain, which catalyzes the release of the peptide product. (Miller BR and Gulick AM (2016) Methods Mol Biol 1401:3-29).

[0264] The Bacillus strain 105 used in the present invention includes many NRPSs and predicted proteins expected to be synthesized by NRPSs. Some of these proteins are as follows: NRPS, NRPS, NRP, beta lactone, CDPS, head-to-tail, sec-peptide, transAT-PKS, PKS-like, T3PKS, transAT-PKS-like, NRPS, terpene, terpene, T3PKS.

[0265] The presence of certain predicted proteins and secondary metabolites is indicated by the predicted numbers of such proteins provided in parentheses below Table 6.

[0266] Table 6

[0267]

[0268]

[0269] By analysis of the whole genome sequence, no plasmid was identified in strain ELA1 901105 (also referred to as strain 105).

[0270] The predicted antioxidant proteins were further analyzed based on sequence analysis of the Bacillus strains. Some of the results are shown in Table 7 below.

[0271] Table 7

[0272] Antioxidant prediction. Putative antioxidant genes in the genomes of 105 Bacillus subtilis strains.

[0273]

[0274]

[0275] Toxin or antitoxin prediction analysis showed that strain ELA191105 (strain 105) includes the antitoxin EndoAI corresponding to Uniprot ID DP96621 and the endonuclease EndoA corresponding to Uniprot ID P96622.

[0276] Digestive enzymes include enzymes that cleave cell wall or cell membrane components, particularly bacterial enzymes. For example, lysine is a cell wall hydrolase commonly found on and encoded by bacteriophages. Lysine activity can be divided into two categories based on specificity for bonds within the peptidoglycan: glycosidases that hydrolyze bonds within the amino sugar moiety and amidases that hydrolyze amide bonds that cross-link the backbone peptide. (Fischetti VA et al. (2006) Nat Biotechnol 24(12):1508-11). The digestive enzymes predicted in Bacillus strain 105 based on sequence analysis are provided in Table 8 below.

[0277] Various other components of strain 105, particularly antimicrobial resistance genes, were evaluated as shown in Table 9 below.

[0278] Table 8 Predicted digestive enzymes identified in the genome of Bacillus subtilis PTA-86 (percent identity > 50, percent alignment length > 90)

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285] Table 9 - Putative antibiotic resistance genes identified in Bacillus subtilis PTA-86 (strain ELA191105) by genomic analysis (percent identity >80, percent coverage >95)

[0286]

[0287]

[0288] Example 4

[0289] Safety and multiomic characterization of host-derived Bacillus strains as potential probiotic, production, and in vivo delivery strains

[0290] Isolate and screen host-derived Bacillus strains to obtain desired probiotic properties, as well as safety and stability as production or in vivo delivery strains. Phenotypic, genomic, and metabolomic analyses of Bacillus subtilis (Bs ATCC PTA126786 (ELA191105, strain 105)) have demonstrated promising probiotic properties, safety, and stability.

[0291] Microbial feed ingredients, also known as direct-fed microorganisms (DFMs) or probiotics, have attracted considerable interest as an alternative to AGPs to support improved production efficiency. Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (5). Probiotics are thought to exert their benefits through the following mechanisms: aiding nutrition and digestion, competitive exclusion of pathogens, modulation of the immune system and digestive tract microbiota, improvement of epithelial integrity, and / or production of small molecule metabolites that are beneficial to the host (6, 7). In addition to the above-mentioned probiotic effects, microorganisms used as probiotics or ingested by animals must survive environmental and processing challenges before reaching their target sites in the animal body. These include low acidity in the upper gastrointestinal tract (GIT), bile acid toxicity, and heat exposure during the production of bacteria-containing feeds and feed pelleting applications.

[0292] Endospore-forming Bacillus species offer advantages over traditional probiotic strains because Bacillus spores are able to withstand harsh environments such as high temperature, desiccation, and acidic pH, resulting in improved viability during production and feed pelleting, improved stability within the animal’s GIT, and extended product shelf life. Bacillus strains have been widely used to support improved production parameters (8-11). Once inside the GIT, spores germinate into metabolically active vegetative cells (12-15). Within the genus Bacillus, commonly used species include Bacillus subtilis, Bacillus coagulans, Bacillus clausii, Bacillus amyloliquefaciens, and Bacillus licheniformis (16). Bacillus strains have also been utilized and are known to produce commercial enzymes, antimicrobial peptides, and small metabolites that may provide health benefits to the host by supporting improved feed digestion, suppressing undesirable organisms, and maintaining a healthy gut microbiome and immune system (reviewed in (17)).

[0293] To fill the knowledge gap regarding the genomic and phenotypic characterization of Bacillus DFMs, we utilized DNA sequencing and omics technologies to comprehensively identify, screen, and characterize Bacillus strains to assess their safety and efficacy as probiotic candidates. Detailed strain characterization using a multi-omics approach can reveal correlations between strain properties and the effects of administration on the host, support the possible mechanisms of action of probiotic strains, identify biomolecules (i.e., peptides, enzymes, metabolites) that can be used in place of live bacteria, and facilitate the rational design of strains to maximize their positive effects on the host and / or deliver biomolecules to the host.

[0294] Materials and methods

[0295] Microbial Strains and Growth Conditions—Bacillus strains were routinely grown in lysing broth (LB) and incubated overnight at 37°C with shaking at 200 rpm. Avian pathogenic Escherichia coli (APEC) serotypes O2, O18, and O78 and Clostridium perfringens NAH 1314-JP1011 were obtained from the Elanco pathogen library. Salmonella enterica serovar Typhimurium ATCC 14028 was purchased from the American Type Culture Collection (ATCC, Manassas, VA). E. coli strains and S. typhimurium were routinely grown in LB, while Clostridium perfringens was grown in anaerobic brain heart infusion (BHI) broth supplemented with yeast extract (5.0 g / L) and L-cysteine ​​(0.5 g / L). For growth in liquid culture, colonies from the corresponding agar plates were inoculated into 10 mL test tubes containing liquid culture medium, and the test tubes were cultured in a shaking incubator in an anaerobic chamber (Sheldon Manufacturing Co., Cornellius, OR) at 37° C. and 200 rpm for E. coli and S. typhimurium, and at 39° C. for static culture of C. perfringens. The anaerobic chamber contained a mixture of N2:CO2:H2 (87.5:10:2.5, v / v / v).

[0296] Vero cell growth conditions— Vero cells were obtained from Elanco cell culture and maintained in a 5% FBS-containing medium (Cytiva, Marlborough, MA) and gentamicin (Opti-5-Gent) (Life Technologies, Carlsbad, CA). Serum-free cell culture medium was prepared similarly using basal medium containing Earle's balanced salt solution (MEM / EBSS), 10% fetal bovine serum (FBS), 1% nonessential amino acids, and 1% L-glutamine instead of FBS. To generate wells containing 100% confluent cells for cytotoxicity assays, Vero cells grown for two to three days were plated into 96-well flat-bottom tissue culture plates (Fisher Scientific, Waltham, MA), with each well containing 1×10 4 The cells were then cultured on plates in a CO2 incubator (37°C; % CO2 maintained at 5±1%) for 48-72 hours.

[0297] Isolation and identification of Bacillus

[0298] Bacillus Isolation - Bacillus species were isolated from the cecal contents of healthy chickens aged 30-42 days raised on poultry research farms in Arkansas, Georgia, and Indiana, USA, using a high-throughput separation platform and the classical isolation method described previously. (GALT Corporation, San Carlos, CA) protocols. For both methods, Bacillus spores were selected from the starting cecal contents by heating at 95°C for 5 minutes or treating with ethanol before the isolation protocol. For the latter, frozen cecal samples of the Elanco library stored in BHI containing 20% ​​glycerol were thawed and an equal amount of tryptone soy broth (TSB) medium was added and mixed. An equal amount of absolute ethanol was added to the sample to a final concentration of 50%, and the mixture was incubated at 30°C for one hour. The ethanol-treated samples were then used for isolation. For Bacillus spp., isolation was performed using conventional methods, and 10-fold serial dilutions of the treated cecal samples were performed to ensure recovery of isolated colonies on agar plates. Each colony was purified by three serial passages on agar plates.

[0299] Strain Identification - For initial strain identification, Bacillus cell lysates were sent to the TACGen genome sequencing facility (Richmond, CA) for strain identification. Strain identity was confirmed by Sanger sequencing of a partial length amplified region of the 16S ribosomal RNA (rRNA) gene using primers 27F (5'AGA GTT TGA TCMTGG CTC AG 3') and 1492R (5'CGG TTA CCT TGT TAC GAC TT3'). The resulting 16S rRNA sequences were then searched against the NCBI 16S rRNA database using BLAST with an e-value cutoff of <10-20 and a sequence identity percentage value of >95%. Strain identification of selected isolates was further confirmed by orthogonal analysis as described in the following sections: Genome-Based Strain Identification and Comparative Genomic Analysis.

[0300] In vitro microbial inhibition test - Bacillus strains were screened based on their antimicrobial activity against five microorganisms, namely APEC serotypes O2, O18, O78, Salmonella typhimurium ATCC 14028, and Clostridium perfringens NAH 1314-JP1011. The assay was modified according to the protocol described in (23) and performed in duplicate.

[0301] The assay was modified according to the protocol described in (113) and repeated. Briefly, 10 μl of cold stock of Bacillus was inoculated into 2 mL of 0.5x LB in a 15 mL round-bottom shaker tube. The culture was incubated at 37°C for 48 h while shaking at 200 rpm. For APEC strains and S. typhimurium, 50 μl of frozen stock was inoculated into 5 mL of LB in a 15 mL round-bottom shaker tube. The culture was incubated overnight at 37°C while shaking at 200 rpm. Once the pathogens were grown overnight in liquid culture, 1.0 x 10 5 cfu / ml overnight culture was inoculated into freshly prepared LB soft agar (0.8% w / v) and cooled in a water bath set at 45°C after autoclaving. 5 mL of molten agar was aliquoted into each well of a 6-well cell culture plate (2 wells for each Bacillus strain plus a negative control). The soft agar was solidified and air-dried for 3-4 hours. On this agar, 5 μl of a 48-hour Bacillus culture was applied to the center of each well. The plate was inverted and incubated at 37°C for 24 hours overnight, and the inhibition zone was observed and recorded.

[0302] For the C. perfringens screening, 5 mL of molten LB agar (1.5%, w / v) was aliquoted into each well of a 6-well cell culture plate and allowed to solidify overnight. 5 μl of a 48-hour Bacillus culture was then spotted in the center of each well. The plates were inverted and incubated aerobically at 37°C overnight. A C. perfringens NAH 1314-JP1011 colony was inoculated into liquid BYC broth and incubated in an anaerobic chamber at 39°C overnight. Freshly prepared BYC soft agar (0.8%, w / v) was autoclaved and cooled in a water bath set at 45°C. After cooling, 1.0 x 10 5 An overnight Clostridium perfringens culture was inoculated into molten soft agar at 100 cfu / ml and mixed on a stir plate. A 5 mL aliquot of molten agar was placed on top of each well of a 6-well cell culture plate containing a Bacillus spore. As a negative control, Clostridium perfringens cultured on molten agar was poured onto LB agar without Bacillus spores. After solidification, the plate was inverted and incubated anaerobically at 39°C for 24 hours. The zone of inhibition was then observed and recorded.

[0303] Enzyme activity - The β-mannanase assay was adapted from the protocol described by Cleary, B. et al. (24). Amylase and protease activity were determined according to the protocol described in (23). The β-mannanase assay was performed according to the protocol described by Cleary, B. et al. (114). Amylase and protease assays followed the protocol described in (113). To test for β-mannanase activity, Bacillus strains were cultured in 5 ml of LB medium in a 15 ml culture tube at 37°C overnight while shaking at 200 rpm. Then, 5 μl of the 24 h Bacillus culture was spotted in duplicate on the center of an LB agar plate containing 100 mM CaCl2. The agar plates were incubated at 37°C overnight. Fresh soft agar containing azo-carob galactomannan (0.5%, w / v) and agar (0.7%, w / v) dissolved in 50 mM Tris-HCl pH 7.0 buffer was autoclaved and cooled in a water bath set at 45°C. After cooling, the soft agar matrix was overlaid on agar plates containing Bacillus colonies until each colony was surrounded by the matrix. The plates were incubated at 37°C overnight and allowed to incubate for 48 hours. The clearance zone caused by β-mannanase activity was directly observed and recorded.

[0304] For the amylase assay, agar plates containing the following ingredients (units, g / L) were used: trypsin, 10, soluble starch, 3, KH2PO4, 5, yeast extract, 10, and Noble agar, 15. An overnight culture of a Bacillus isolate in 0.5xLB was used as an inoculum. The Bacillus culture was spotted on the above-mentioned plates containing soluble starch, and the inoculated plates were incubated at 37°C for 48 hours. The clearing zone due to amylase activity was observed by flooding the surface of the plate with 5 mL of Gram's iodine solution.

[0305] To test protease activity, use agar plates containing the following ingredients (units, g / L): skim milk, 25, noble agar, 25. Use an overnight culture of a Bacillus isolate in 0.5xLB as an inoculum. Spot the Bacillus culture onto the above plates containing soluble starch, and incubate the inoculated plates at 37°C for 24 hours. Clearance zones caused by protease activity can be directly visualized.

[0306] Cytotoxicity assay - Cytotoxicity assays were performed on Bacillus culture supernatants according to the protocol described in the EFSA guidance (25). Culture supernatants of Bacillus cereus ATCC 14579 and Bacillus licheniformis ATCC 14580 were used as positive and negative controls, respectively. Bacillus strains were cultured overnight in 5 mL of brain heart infusion (BHI) liquid medium at 30°C. This overnight culture was used as an inoculum for 5 mL of fresh LB, and the inoculated medium was then incubated at 30°C for 6 hours without shaking. The expected cell density was at least 108 CFU / mL. The culture was then centrifuged at 1700 x g for 1 hour to produce a cell-free culture supernatant.

[0307] 200 μL serum-free medium was added to 100% confluent Vero cells grown in 96-well plates generated according to the protocol described in Materials and Methods. The cells were then exposed to 100 μL of cell-free culture supernatant of Bacillus. The mixture was incubated for 3 hours in a 37°C CO2 incubator (5% v / v CO2 headspace, Thermo Scientific, Waltham, Massachusetts). The corresponding cell-free culture supernatant was used in the control wells. Bacillus cereus and Bacillus licheniformis were used as positive and negative controls, respectively, and 0.1% Triton-X, 100 μL was used as a positive cytotoxicity control. This assay was performed with three technical replicates and three biological replicates.

[0308] At the end of the incubation period, the culture supernatant was collected by centrifugation at 300 x g for 5 minutes. The culture supernatants from the wells of technical replicates were combined. Four microliters of culture supernatant were used for the lactate dehydrogenase assay (Sigma-Aldrich, St. Louis, MO) according to the protocol described in (115) in a total volume of 100 μL. The reaction was monitored at 37°C for 10 minutes at an absorbance of 450 nm to measure the production of NADH from NAD+ as a product of the lactate dehydrogenase reaction. The percentage of cytotoxicity was calculated by the following formula.

[0309]

[0310] The A450nm value is the average of three biological replicates. Cytotoxicity percentage values ​​above 20 are considered cytotoxic. If the cytotoxicity percentage of the positive control Bacillus cereus is less than 40 or the bacteriotoxicity percentage of the negative control Bacillus licheniformis is higher than 20, the assay is repeated.

[0311] Antimicrobial Susceptibility Assessment - Antimicrobial susceptibility analysis of Bacillus spp. was performed for tetracycline, chloramphenicol, streptomycin, kanamycin, erythromycin, vancomycin, gentamicin, ampicillin, and clindamycin according to the U.S. Food and Drug Administration (FDA) guidance on resistance in Bacillus spp. as direct-fed microorganisms. Bacillus spp. strains were sent to Microbial Research, Inc. (Fort Collins, CO) for analysis according to the protocol of the Clinical Laboratory Standards Institute (CLSI) document VET01 (26). Briefly, MIC plates were prepared using cation-adjusted MH broth (MHB) and the antimicrobials were serially diluted 2-fold to obtain final concentrations ranging from 0.06 to 32 μg / mL. Growth of Bacillus spp. was monitored in the presence of nine different dilutions of the antimicrobials. Susceptibility was interpreted as the absence of growth in the presence of the antimicrobials at concentrations below the cutoff values ​​for the respective antimicrobials as described in the EFSA guidance ( Figure 2 A) For quality control, the following organisms were used as controls: Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213.

[0312] Whole genome sequencing, assembly, and annotation

[0313] Genomic DNA Isolation - High molecular weight genomic DNA of Bacillus was extracted using the phenol:chloroform:isoamyl alcohol (PCI) method as previously described (27). Bacterial cells were harvested by centrifugation at 7000 x g for 10 minutes from an overnight culture of Bacillus grown in 25 mL LB supplemented with 0.005% Tween 80 in a 50 mL sterile Falcon tube (Fisher Scientific, Waltham, MA). The resulting cell pellet was resuspended in 0.75 mL of 1X Tris-EDTA (TE) buffer (Life Technologies, Carlsbad, CA), pH 8, containing Tris-HCl and EDTA at final concentrations of 10 and 1 mM, respectively, in a 2 mL Eppendorf tube (Fisher Scientific, Waltham, MA). To lyse the cells, lysozyme (Sigma-Aldrich, St. Louis, MO) was added at a final concentration of 7 mg / mL, and the mixture was incubated at 37°C for 1 hour. Then, SDS and proteinase K (Sigma-Aldrich, St. Louis, MO) were added to the mixture at a final concentration of 2% and 400 μg / mL, respectively, and the lysate was incubated at 60°C for 1 hour. To remove RNA from the cell lysate, 10 μL of RNase (ThermoFisher Scientific, Waltham, MA) was added, and the mixture was incubated at 37°C for 30 minutes. An equal volume of PCI (25:24:1, v / v / v) mixture was added to the supernatant and mixed by carefully inverting the tube 5-10 times. The aqueous phase containing the DNA was separated from the organic phase by centrifugation at 12,000×g for 15 minutes, and the top aqueous layer was collected in a fresh 2 mL Eppendorf tube. An equal volume of chloroform:isoamyl alcohol (24:1, v / v) was added to the aqueous phase containing the DNA and mixed by carefully inverting the tube. The mixture was centrifuged at 12,000×g for 10 minutes. DNA was pelleted from the aqueous layer by adding one-tenth volume of sodium acetate (3 M, pH 5.2) followed by centrifugation at 16,000 x g for 20 minutes. The DNA pellet was washed three times with ice-cold 70% ethanol, air-dried, and resuspended in 0.5 mL of 1X TE buffer.

[0314] PacBio long-read genome sequencing - Bacterial genomic DNA samples were shipped on dry ice to DNA Link, Inc. (San Diego, CA) for whole-genome sequencing using the PacBio RSII platform. Briefly, 20 kb DNA fragments were generated by shearing genomic DNA using covaris G tubes according to the manufacturer's recommended protocol (Covaris, Woburn, MA). Smaller fragments were purified using the AMpureXP bead purification system (Beckman Coulter, Berry, CA). For library preparation, 5 μg of genomic DNA was used. SMRTbell TM Template Prep Kit1.0( SMRTbell libraries were constructed using a BluePippin size selection system (Sage Science, Beverly, MA). Small fragments were removed using the BluePippin size selection system (Sage Science, Beverly, MA). The remaining DNA samples were used for large insert library preparation. Sequencing primers were annealed to the SMRTbell template and the DNA / polymerase binding kit P6 ( Menlo Park, CA) to bind DNA polymerase to the complex. After polymerase binding reaction, the DNA polymerase was isolated using MagBeads kit ( Menlo Park, CA) binds the MagBead to the library complex. This polymerase-SMRTbell-adaptor complex is loaded into a zero-mode waveguide. The SMRTbell library consists of two cell( Menlo Park, CA) using DNA Sequencing Kit 4.0 with C4 chemistry ( Sequencing was performed using The sequencing platform captured 1 × 240-minute video for each SMRT cell.

[0315] Genome assembly, annotation, and feature prediction—The genome was assembled by DNA link, Inc. using HGAP.3. Genome annotation was performed using a custom annotation pipeline by combining multiple prediction tools. Coding sequences, transfer RNAs, and transmembrane RNAs were predicted and annotated using Prokka (28–30). Ribosome binding site (RBS) prediction was performed using RBSFinder (31). TranstermHP was used to predict R-factor-independent terminators (TTS) (32). Ribosomal RNAs and other functional RNAs, such as riboswitches and noncoding RNAs, were annotated using Infernal (33). Operons were predicted based on the raw genome sequence information using Rockhopper version 2.0.3 using default parameters (34). Insertion sequence prediction was performed using ISEscan version 1.7.2.1 (40). Prediction was performed using PhiSpy version 4.2.6, which combines similarity-based and composition-based strategies (41).

[0316] Genome-Based Strain Identification and Comparative Genomic Analysis—Assembled microbial genomes were taxonomically labeled using CAMITAX (35). CAMITAX is a scalable workflow that combines taxonomic assignment based on genomic distance, 16S ribosomal RNA gene, and gene homology with phylogenetic positioning. OrthoFinder version 2.3.1 (36) was used to determine orthologous relationships (37).

[0317] Phylogenetic Analysis—The phylogenetic relationships of the genomes were explored using UBCG version 3.0 with default settings ( 38 ). This software tool used a set of 92 single-copy core genes that are commonly found in all bacterial genomes. These genes were then aligned and linked using UBCG with default parameters. Node robustness was estimated using the gene support index (GSI), which is defined as the number of individual gene trees that presented the same node among the total genes used. Maximum likelihood phylogenetic trees were inferred using FastTree version 2.1.10 in GTR+CAT mode ( 39 ).

[0318] Patent Deposits of Bacillus amyloliquefaciens ATCC PTA-126784 and PTA-126785, and Bacillus subtilis ATCC PTA-126786 - The Bacillus amyloliquefaciens ATCC PTA-126784 and PTA-126785 and Bacillus subtilis ATCC PTA-126786 strains are deposited with the ATCC Culture Collection (Manassas, VA). For simplicity, the Bacillus amyloliquefaciens ATCC PTA-126784 and PTA-126785, and Bacillus subtilis ATCC PTA-126786 strains are referred to as Ba PTA84 and Ba PTA75, and Bs PTA86, respectively.

[0319] Global Untargeted Metabolomics Analysis - Bacillus strains Bs-PTA86, Ba-PTA84, and Ba-PTA85 were grown as three single strain cultures and then analyzed as consortia of two strains (Ba-PTA84 and PTA85) or three strains (Bs-PTA86, Ba-PTA84, and Ba-PTA85) in 5 mL of incomplete or complete liquid medium. For growth in minimal medium, medium containing 1X M9 salts and a final concentration of 0.5% (w / v) glucose was used. Complete medium contained the following (in g / L): peptone 30, sucrose 30, yeast extract 8, KH2PO4 4, MgSO4 1, and MnSO4 0.025. Cultures were grown overnight at 37°C. Bacillus cells were pelleted by centrifugation at 10,000 x g for 10 minutes, and the cell pellets were washed three times with ice-cold PBS. The resulting cell pellets and cell-free supernatants were stored at −80°C and sent to Metabolon Inc. (Durham, NC) for global untargeted metabolomics analysis. A detailed description of the metabolomics analysis is provided in the Supplementary Methods.

[0320] In vivo evaluation of Bacillus DFM in improving growth performance in broiler chickens

[0321] Spore formation—Bacillus spores were generated using a modified protocol described in (42). Bacillus was grown in liquid Difco sporulation medium containing 8.0 g / L nutrient broth (BD Difco, Franklin, NJ, USA), 1 g / L KCl, and 0.12 g / L MgSO4·7H2O. The pH of the mixture was adjusted to 7.6 by adding NaOH. After pH adjustment and sterilization of the medium at 121°C using an autoclave, 1 mL of the following sterile stock solutions of minerals were added to the broth medium: 1.0 M CaCl2, 0.01 M MnSO4, and 1.0 mM FeSO4. Sterile glucose solution was also added to the medium mixture to a final concentration of 5.0 g / L. A single colony was taken from the agar plate and inoculated into 100 mL of sporulation medium. The culture was incubated overnight at 37°C while shaking at 200 rpm. This culture was used as a seed culture for 1 L of liquid medium. All growth was performed in ventilated baffled flasks. The cultures were incubated at 37°C while shaking at 200 rpm for at least 72 hours. The presence of spores was monitored using a brightfield microscope. Spores were harvested at 17,000 rpm and washed three times with pre-cooled sterile distilled water. The spores were then resuspended in 30 mL of pre-cooled sterile distilled water, and the spore suspension was mixed with irradiated ground rice hulls (Rice Hull Specialty Products, Stuttgart, Arkansas) and dried at 60°C for 3-4 hours to eliminate vegetative cells. To determine spore inclusion complexes in rice hulls, 0.25 g of spore-containing material was heat-treated at 90°C for 5 minutes. 1 mL of water was added to the material and soaked for 15-30 minutes. The suspension was vortexed for 30 seconds and serially diluted 10-fold on agar plates for colony counts.

[0322] Study Design

[0323] On study day 0 (SD), 2500 one-day-old male broiler chickens (Cobb 500) were randomly assigned to two treatment groups. The control group received the basal diet alone, while the treatment groups received the basal diet plus 1.5 x 10 5 CFU of BaPTA84. The control group consisted of 30 pens with 50 birds each, and the BaPTA84 group consisted of 20 pens with 50 birds each.

[0324] Chickens were housed in pens in an environmentally controlled room with ad libitum access to the treated diet and water. The basal diet was formulated to be isonutritional and meet or exceed the recommended nutrient requirements for broiler chickens. Feeding was conducted over four study periods: Starter Phase I (SD 0-12); Grower Phase II (SD 12-26); Finisher Phase III (SD 26-35); and Exit Phase IV (SD 35-42). The diets did not contain antibiotics, anticoccidial agents, or growth promoters and were fed to the birds in the form of a mash at all stages.

[0325] Bird weights (pen weights) were measured and recorded at SD 0, 12, 26, 35, and 42 hours. Feed distribution and weights were recorded during each feeding period. Overall health status, mortality, and ambient temperature of the birds were recorded daily.

[0326] Statistical analysis

[0327] The experimental unit is the column. All statistical analyses used SAS was performed using the SAS system, version 9.4 (SAS Institute, Cary, NC). All tests were performed using one-sided tests with a significance level of P < 0.05 to compare the control group with the treatment group.

[0328] The target performance variables for each feeding period and overall included live final body weight (LFBW), average daily gain (ADG), average daily intake (ADFI), feed to gain ratio (GF), feed efficiency (FCR), mortality, and European Broiler Index (EBI). These variables were calculated and evaluated for each study phase (starting phase, growing phase, finishing phase, exit phase, and overall phase (SD0-42)) with or without adjustment for mortality.

[0329] Microbiome analysis of cecal contents in chickens treated with Ba PTA84

[0330] DNA Extraction, Library Preparation, and Sequencing - Total DNA was extracted from cecal content samples using a lysis and purification kit (Shoreline Biome, Farmington, CT) according to the manufacturer's protocol. The resulting DNA was used as a template for library preparation using Shoreline Biome's V4 16S DNA Purification and Library Preparation Kit (Shoreline Biome, Farmington, CT). Briefly, the V4 region of the 16S rRNA gene was PCR amplified using the extracted DNA and primers 515F (5'GTGGCCAGCMGCCGCGGTAA (SEQ ID NO: 35)) and 806R (5'GGACTACHVHHHTWTCTAAT (SEQ ID NO: 36)). The resulting amplicons were then sequenced on the Illumina iSeq platform using a 2x150 bp paired-end kit. To increase diversity, PhiX 50 pM was added to the amplicon library at a final concentration of 5%.

[0331] Bioinformatics Analysis - Forward and reverse reads were processed with cutadapt (version 2.5) (43) to remove primer sequences. Read pairs without primer sequences or with primer mismatches exceeding 15% were discarded. The DADA2 pineline (version 1.12.1) (44) was used to generate a count matrix of amplicon sequence variants (ASVs) between samples. Due to the short iSeq read length, forward and reverse reads were trimmed to a length of 110 bp and merged with the justConcatenate option of DADA2. The DADA2 parameters maxN = 0, truncQ = 2, rm.phix = TRUE, and maxEE = 2 were used. Taxonomic labels were assigned to each ASV using the DADA2 assignment taxonomy method and the Silva version 138 database (45). The diversity and richness of each sample were quantified from the ASV matrix using the Simpson, Shannon, and Chao indices (46-48), and the different treatments were compared using the Mann-Whitney U test. The microbiome structures of the different treatments were compared using PERMANOVA and ANOSIM analyses based on the Bray-Curtis dissimilarity between samples. PERMANOVA and ANOSIM were performed using code from the scikit-bio python package (49). Principal component analysis of the Bray-Curtis dissimilarity matrix was used to analyze sample clustering according to treatment group.

[0332] Global Untargeted Metabolomics Analysis - Metabolomics studies were performed using an untargeted UPLC-MS / MS approach at Metabolon Inc. using a Waters ACQUITY ultra-high performance liquid chromatography (Waters, Milford, MA) coupled to a QE high-resolution / accurate mass spectrometer (Themo Scientific, Waltham, MA) with a heated electrospray ionization (HESI-II) source and an orbitrap mass analyzer, operated at a mass resolution of 35,000. The sample was dried, reconstituted, and aliquoted into four aliquots for the following analyses: a) Analysis of hydrophilic compounds using a C18 column (Waters UPLC BEH C18-2.1 x 100 mm, 1.7 μm) in water and methanol containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA) under acidic positive ion conditions. b) Using a similar system as described above, the mobile phase used was methanol, acetonitrile, water, 0.05% PFPA, and 0.01% FA, performed in a total organic environment to analyze more hydrophobic compounds. c) Basic negative ions were analyzed using a C18 column with a mobile phase of methanol and water containing 6.5 mM ammonium bicarbonate at pH 8. d) Negative ionization was performed after elution from a HILIC column (Waters UPLC BEH Amide 2.1 x 150 mm, 1.7 μm) using a gradient consisting of water containing 10 mM ammonium formate at pH 10.8 and acetonitrile. MS analysis covered approximately m / z 70-1000.

[0333] Metabolite compounds were identified by comparison with purified standards and a metabolite library of recurrent unknown metabolites. Identification was based on retention indices within a narrow RI window for the proposed identification, accurate mass matches to the library of + / - 10 ppm, and MS / MS forward and reverse scores.

[0334] Data from cell pellets and culture supernatants were analyzed separately. The raw intensity values ​​of each identified metabolite were rescaled by dividing them by the median intensity of the sample. Missing values ​​for a given metabolite and sample were imputed by assigning the minimum value of the metabolite in the sample. The scaled and imputed data were Log10 transformed for subsequent analysis. Principal component analysis (PCA) was used to analyze the similarity of metabolic profiles between samples. For supernatant samples, secreted metabolites were identified by comparing the scaled and estimated intensities with the corresponding metabolites in the culture medium control. A 1.5-fold increase in scaled intensity on the culture medium was used to define secreted metabolites. A similar 1.5-fold increase was used to define uniquely secreted metabolites between a single strain and the remaining two strains, or between a strain consortium and the corresponding single strain.

[0335] result

[0336] Isolation and identification of Bacillus spp. from healthy animals

[0337] Bacillus strains were isolated from the cecal contents and feces of healthy chickens. The taxonomic identity of the isolates was confirmed by 16S rRNA amplicon sequencing. These isolates belonged to 30 different Bacillus species, the most popular of which were B. velezensis, B. amyloliquefaciens, B. haynesii, B. pumilus, B. subtilis, and B. licheniformis.

[0338] For safety reasons, the Bacillus isolates selected for further screening included only those species that are listed as DFMs in the official publications of the Association of American Feed Control Officials, Inc. (AAFCO) because they have been reviewed by the U.S. Food and Drug Administration’s Center for Veterinary Medicine and found to present no safety concerns when used in direct-fed microbial products (50) and have been assigned Qualified Permitted Safety (QPS) status according to the European Food Safety Authority’s (EFSA) Panel of Experts on Biological Hazards (3). These are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, and Bacillus licheniformis.

[0339] In vitro screening of probiotic properties of Bacillus strains

[0340] Bacillus strains were tested to determine their effects on selected microorganisms and their ability to secrete selected enzymes (23). For the former, Gram-negative and Gram-positive microorganisms (Escherichia coli O2, O18, and O78, and Clostridium perfringens NAH 1314-JP1011) and Salmonella enterica serovar Typhimurium ATCC 14028 were used. For the latter, plate-based assays were performed to measure the secretion of amylase, protease, and β-mannanase.

[0341] A total of 266 Bacillus strains were first screened against E. coli O2, and 71% of the strains showing positive E. coli O2 inhibition were selected for a second round of assays against E. coli O18, followed by E. coli O78, Salmonella typhimurium, and finally Clostridium perfringens JP1 011. The top eight candidate Bacillus strains were selected based on their cumulative inhibition scores, and the selection data for the included Bacillus subtilis (Bs) isolate, Bs PTA86 (ELA191105, also referred to as strain 105), are provided in Table 10.

[0342] Table 10 In vitro pathogen inhibition and digestive enzyme activity of Bacillus subtilis PTA86

[0343]

[0344] aPathogen inhibition is scored based on the size of the clearance zone as follows: 0, no inhibition; 1, 2, 3, and 4, clearance zone values ​​of 0-0.9, 1.0-1.9, 2.0-2.9, and 3.0-4.0 mm, respectively. The clearance zone value is defined as the distance from the outside of the Bacillus colony to the end of the pathogen growth inhibition zone.

[0345] b Relative digestive enzyme activity was measured as relative enzyme activity (REA), which was calculated as the ratio between the diameter of the enzyme-active clearance zone and the diameter of the Bacillus colony.

[0346] The cumulative inhibition score was calculated as the sum of the inhibition score values ​​of the Bacillus strain against the five microorganisms tested. The average cumulative inhibition score of BsPTA86 was 5.5.

[0347] The candidate Bacillus strains were evaluated for their ability to secrete enzymes. Bacillus strains are known to produce a variety of enzymes (51, 52). In vitro plate-based assays for protease, amylase, and β-mannanase activities showed that Bs PTA86 exhibited amylase, protease, and β-mannanase activities.

[0348] Safety assessment of Bacillus strains

[0349] To assess the safety of Bacillus species as microbial feed ingredients, the antimicrobial susceptibility of candidate Bacillus species to medically relevant antimicrobial drugs was tested. Microbial feed ingredients should not carry or be able to transfer antimicrobial resistance genes to other digestive tract microorganisms. Given the increase in multidrug-resistant bacteria, this is particularly important in the context of human use of medically relevant antimicrobial drugs. Antimicrobial susceptibility testing of Bacillus strain BS PTA86 showed that it was sensitive to all antibiotics tested, particularly clindamycin, chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, vancomycin, and ampicillin (data not shown).

[0350] To determine the potential toxicity of Bacillus strains to host cells, culture supernatants of Bacillus species were tested for cytotoxicity against Vero cells according to (25). Cytotoxicity assays were performed by monitoring lactate dehydrogenase (LDH) from damaged Vero cells as described (53). The results showed that the tested Bacillus strains were non-cytotoxic, with toxicity levels well below 20%, which is considered cytotoxic according to the European Food Safety Authority guidelines (data not shown). Bs PTA86 had the lowest cytotoxicity level of 5% among the strains evaluated.

[0351] Selection of Bacillus species as candidate microorganisms for direct feeding

[0352] Based on its performance in microbial inhibition, enzyme activity, antimicrobial susceptibility, and low toxicity to Vero cells, strain Bs PTA86 was selected for more detailed characterization using genomic and metabolomic approaches described in the following sections.

[0353] Untargeted global metabolomic analysis of cell pellets and culture supernatants of Bs PTA86

[0354] Non-targeted metabolomics analysis was performed on cell pellets and culture supernatants of Bs PTA86 to assess differences in metabolite profiles. Cells were cultured as a single strain in complete and minimal media. Named metabolites were identified in the supernatant and pellet samples, respectively. Thus, strain Bs PTA86 (ELA191105) secretes metabolites and includes unique intracellular metabolites compared to other Bacillus strains. Detailed information and details (including table listings) about the unique metabolites of ELA191105, including comparisons with other Bacillus strains, are provided in USSN 63 / 083697, filed September 25, 2020, and USSN 63 / 241369, filed September 7, 2021, each of which is incorporated herein by reference.

[0355] Genomic characteristics of Bs PTA86

[0356] The genome of Bs PTA86 was sequenced by PacBio sequencing. The genomic properties and annotations for different features are summarized in Table 11. The whole genome sequence was deposited in DDBJ / ENA / GenBank under BioProject Nos. PRJNA701126 and PRJNA701127. The genome sequence of strain Bs PTA 86 is included and provided in USSN 63 / 083697, filed on September 25, 2020, and USSN 63 / 241369, filed on September 7, 2021, each of which is incorporated herein by reference. The genomic nucleic acid sequence of strain Bs PTA86 (ELA191105) is also provided in SEQ ID NO: 1 and SEQ ID NOs: 2-6.

[0357] Table 11 Summary of genome assembly and annotation of Bacillus

[0358]

[0359] Phylogenetic analysis of Bs PTA86 - Phylogenetic relationships of the genome were explored using UBCG version 3.0, which uses a set of 92 single-copy core genes commonly found in all bacterial genomes. The Bs PTA86 genome was compared with the genomes of strains of Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus subtilis, as well as Lactobacillus reuteri as an outgroup (accession numbers: AL009126, CP000560, CP002627, CP002634, CP002927, HE617159, HG5144499, JMEF01000001, CP005997, CP009748, CP009749, CP011115, LHCC01000001, CP014471, and QVMX01000001). Bs PTA86 is most closely related to Bacillus subspecies subtilis 168 (ATCC 23857, DSM 23788).

[0360] Genome Analysis Bs PTA86 - The assembled genome sequence of Bacillus strain 105 was annotated for potential probiotic properties, such as enzymes, antioxidants, bacteriocins, and secondary metabolites, as well as the presence of genes of potential safety concern, such as genes encoding toxins, virulence factors, and antimicrobial resistance genes. Detailed descriptions of each of these characteristics are described below.

[0361]

[00145] Selected Enzyme Analysis - Table 12 illustrates the presence of genes encoding selected digestive enzymes identified in the Bacillus sp. PTA86 genomes. All three Bacillus sp. genomes encode lipase, β-phytase, α-amylase, endo-1,4-β xylanase A, β-glucanase, β-glucanase, β-mannanase, pectate lyase, and α-galactosidase. Bs PTA86 carries two copies of the β-mannanase gene. β-mannanase catalyzes the hydrolysis of β-1,4-linkages in glucomannan, releasing manno-oligosaccharides (24, 54). This enzyme, along with phytase, xylanase, and amylase, is added as a feed ingredient to improve feed digestibility (55-57). Bs-PTA86 has pullulanase, oligo-1,6-glucosidase, and glycogen degrading enzymes such as 1,4-α-glucan branching enzyme.

[0362] Table 12

[0363]

[0364] Secondary metabolites - The secondary metabolite cluster accounts for 12% of the Bacillus PTA86 genome. Table 13 shows the corresponding clusters of the Bacillus Bs PTA86 genome encoding 10 clusters. More than half of the clusters are contributed by antimicrobial peptide (AMP) biosynthesis genes (Table 14). The Bs PTA86 genome has subtilisin A, a cyclic antimicrobial peptide effective against some Gram-positive and Gram-negative bacteria, such as Listeria, Enterococcus faecalis, Porphyromonas gingivalis, Klebsiella rhizophila, Streptococcus pyogenes and Shigella sonnei, Pseudomonas aeruginosa and Staphylococcus aureus (58-60). For non-ribosomally synthesized AMPs, Bs PTA86 carries phosphatidylcholine, surfactin, bacillibactin and bacilysin. Table 14 provides a list and comparison of some antimicrobial peptides, and Table 15 provides the digestive enzymes provided by strain Bs PTA86.

[0365] Table 13 Secondary metabolite gene cluster of Bs PTA86

[0366]

[0367] *Abbreviations: RiPP, ribosomally synthesized and post-translationally modified peptide; NRPS, non-ribosomal peptide synthetase; PKS, polyketide synthase; T3PKS, type III polyketide synthase; trans-Type 3-PKS; AT-PKS, trans-acyltransferase polyketide synthase. Acyltransferase PKS.

[0368] Table 14 antimicrobial peptides

[0369]

[0370]

[0371] Table 15 Putative digestive enzymes in the genome of Bacillus subtilis PTA-85

[0372]

[0373]

[0374]

[0375]

[0376] Safety-related genes

[0377] To search for genes encoding known virulence factors, toxins, and antimicrobial resistance (AMR), we applied a screening approach using cutoff values ​​of sequence identity and coverage above 80% and 70%, respectively, according to EFSA guidelines (61). Based on the analysis, no genes encoding known virulence factors or toxins were identified in Bacillus strain Bs PTA86.

[0378] Table 16 shows the genes encoding putative genes for antimicrobial resistance (AMR). The Bs PTA86 genome carries putative genes encoding macrolide 2′ phosphotransferase (mphK), ABC-F type ribosomal protection protein (vmlR), streptothricin-N-acetyltransferase (satA), tetracycline efflux protein (tet(L)), aminoglycoside 6-adenylyltransferase (aadK) (29), and rifamycin inactivating phosphotransferase (rphC). The aadK gene of B. subtilis was originally discovered in a susceptible derivative of the Marburg 168 strain. Heterologous expression of this gene in an E. coli plasmid resulted in a resistance phenotype to rifamycins, indicating that high gene copy numbers are required to confer resistance (30).

[0379] Table 16 Putative antimicrobial resistance genes in Bacillus subtilis PTA-86 (strain ELA191105)

[0380]

[0381]

[0382] Antioxidants, Adhesion, and Folate Biosynthesis

[0383] Genes encoding primary oxidoreductases such as superoxide dismutase and catalase that scavenge reactive oxygen species were found in the genomes of the three Bacillus species listed in Table 17. Genes for the thioredoxin system and bacillithiolate biosynthesis were also identified. The genome of Bs PTA86 encodes thioredoxin reductase and Trx of Bs PTA66. The thioredoxin system maintains cellular redox homeostasis (62). Interestingly, despite the lack of a glutathione-glutaredoxin system, several glutathione transport genes were found, suggesting that redox proteins (possibly bacillithiolates) may be transported to the extracellular environment, thereby maintaining the redox potential of the surrounding environment. Two bacillithiolate biosynthesis genes (63), bshA and B, were identified in the genome of Bs PTA86, Table 17.

[0384] Table 17 Putative genes encoding antioxidants in the genomes of 105 Bacillus strains

[0385]

[0386]

[0387] One of the key desirable characteristics of candidate probiotics is the ability to adhere to epithelial cells. Two genes identified in all three strains putatively encode proteins involved in mucus, epithelial cell adhesion, and are known to be involved in host immune regulation and unwanted microbial aggregation, providing the strain with stability and the ability to compete with other unwanted resident digestive tract bacteria, thereby enabling efficient colonization in the intestine and exclusion of pathogens (64, 65). Two genes were identified in all three genomes, each encoding the elongation factor Tu and a 60 kDa chaperone protein, which are involved in the adhesion of Bacillus species to the intestinal epithelium.

[0388] Probiotics have multiple health benefits for the host, including the production of vitamins. We used the Enzyme Commission (EC) numbers associated with the folate biosynthetic pathway to search for key components of the folate production pathway in Bacillus strains. Analysis of the genome sequences of the Bacillus strains identified genes involved in para-aminobenzoic acid (PABA) synthesis in all three strains (Table 18). However, strain Ba PTA84 had a frameshift mutation in the pabB gene. The enzymes necessary to convert chorismate to PABA were present in all three Bacillus probiotic strains. The Bacillus probiotic strains also contained genes for the de novo DHPPP biosynthetic pathway. Previous studies have shown that the Bacillus subtilis genome contains all pathway components and has been engineered for folate production (66-68).

[0389] Table 18 Genes involved in the folate biosynthesis pathway in the probiotic Bacillus sp.

[0390]

[0391]

[0392] Screening for Prophages, Insertions, and Transposases - Strain BsPTA-86 was scanned for the presence of mobile genetic elements such as prophages, insertion sequences (IS), and transposases. BsPTA86 has four transposases and two copies of the IS21 insertion sequence.

[0393] discuss

[0394] A clear understanding of the physiology and safety of probiotics or live delivery strains, as well as their interactions with the target host and the host digestive tract microbiota, is crucial for the rational development of next-generation probiotics or live delivery strains with improved safety, efficacy, and reproducibility. Here, we employed an integrated multi-omics, biochemical, and microbiological approach to select and characterize Bacillus strains for improving the growth performance of poultry.

[0395] Bacillus isolates were screened in vitro for their activity against certain pathogens and their ability to secrete digestive enzymes. The top candidates were further selected based on their safety profile (i.e., antimicrobial resistance and cytotoxicity levels). Selected isolates underwent genomic and metabolomic analysis to further investigate potential host benefit properties and possible health / safety issues. This bottom-up approach ensured that the best candidates were selected at each screening step. Strains that did not meet safety criteria were not selected. Only the best candidates that met the phenotypic selection criteria were advanced to the next step of screening. Genomic analysis of the top Bacillus strains helped establish a link between phenotypic observations and genomic features.

[0396] Host-adapted Bacillus strains. We expected that host-adapted Bacillus strains would function better as probiotics in the host environment than strains isolated from other sources, and therefore, we targeted our isolations to those from animal GIT contents or fecal samples of healthy animals (8). As previously reported (8, 22), a higher diversity of isolates was obtained from ethanol-treated samples compared to heat-treated samples. Although Bacillus spores have general characteristics of heat resistance, the composition of the spore core, cortex, shell, and membrane determines the degree of heat resistance of the spores (10, 69, 70), resulting in different responses of spores to heat stress.

[0397] Desirable probiotic properties. As the use of antibiotics in poultry farms continues to decrease, driven by regulations and some consumer preferences, the development of microbial feed additives will help maintain poultry health in the face of undesirable microorganisms. Our screening results showed that Bacillus controlled the growth of undesirable Escherichia coli O2, O18, and O78, Clostridium perfringens, and Salmonella typhimurium. APEC strains cause colibacillosis, which is a major problem in commercial production (74, 75). Colibacillosis occurs when APEC derived from fecal matter are transferred to the lung epithelium during fecal aerosolization. Therefore, reducing the APEC load in feces (potential impact of Bacillus in feed) can help reduce the incidence of bacteriovorus disease (76, 77). Clostridium perfringens is a pathogen that causes necrotic enteritis in poultry (78) by producing α-toxin and NetB (79, 80). Necrotic enteritis is a multifactorial disease that costs poultry farmers $6 billion annually (81). Salmonella typhimurium is a commensal bacterium in the poultry gut and a major cause of salmonellosis in humans. Consumption of poultry products containing Salmonella can promote this infection (82, 83). The ability of Bacillus to inhibit the growth of these undesirable organisms may be due to the production of AMPs (bacteriocins). Genomic analysis of BsPTA86 revealed that the genome encodes different AMPs (Table 14).

[0398] Bacillus spp. are known to secrete enzymes that benefit the host, such as cellulases, xylanases, amylases, proteases, β-mannanases, and phytases (23, 51, 84). These enzymes, when fed to animals, improve digestion of low-calorie diets or reduce intestinal inflammation by breaking down non-starch polysaccharides (NSP). Some NSPs are anti-nutritional factors that increase digestive tract viscosity and slow feed residence time in the digestive tract, thereby reducing nutrient absorption (85). The accumulation of undigested NSPs can lead to the growth of pathogens, causing subclinical infectious challenges (86, 87). In response to NSPs, the production of proinflammatory cytokines requires a large amount of energy that would otherwise be conserved for growth, thereby reducing feed efficiency and growth performance (reviewed in (88)). Bs PTA-86 showed considerable protease, amylase, and β-mannanase activities. Our genomic analysis supported these activities, showing that Bs has genes encoding amylase, protease, β-mannanase, and phytase.

[0399] Notably, genomic analyses have revealed other potential benefits for animals from candidate Bacillus strains. Genes encoding a variety of antioxidant proteins have been identified, including superoxide dismutase, catalase, thioredoxin, methionine sulfoxide, and bacilliothiol. When expressed and secreted in the GIT, these proteins can provide protection against oxidative stress (89-91). Oxidative stress occurs in the GIT when the levels of free radicals generated by reactive oxygen / nitrogen species (RO / NS) are much higher than the levels of antioxidant proteins available to neutralize these toxic compounds (57). This event is triggered by a variety of factors, including nutritional or environmental heat stress, or pathological factors that ultimately reduce the growth performance and quality of meat and eggs (57).

[0400] The putative functions of probiotics include reducing potential pathogens, immunomodulation, removing harmful metabolites in the digestive tract and / or providing bioactive or other regulatory metabolites. Probiotics that produce folic acid are able to better digest nutrients and recycle energy. Probiotic strains that produce folic acid may have a potential protective effect against cancer, inflammation, stress and digestive system disorders (66,92-95). Several studies exploring the commercial use of probiotic strains for folic acid production have been reported (92,96,97). Genes encoding essential enzymes in the folic acid biosynthetic pathway have also been found in the genomes of three Bacillus strains. The products of these pathways provide important cofactors that, once secreted, are absorbed by the host, thereby improving health (92,96,97).

[0401] Safety profile. In addition, candidate Bacillus DFMs must have an acceptable safety profile as expected by regulatory agencies. Some Bacillus species are known to produce AMPs and enterotoxins that may have deleterious effects on host cells (25). Cytotoxicity evaluation of Bacillus strains showed that Bacillus did not cause cytotoxicity in Vero cells. In addition, genomic analysis of Bs PTA 86 showed that enterotoxins and other known virulence factors were absent in the subject Bacillus. Another important safety criterion is that the Bacillus genome must be free of transferable antimicrobial resistance genotypes (100). The data showed that the tested Bacillus isolates were susceptible to the tested antimicrobial drugs with apparent MIC values ​​below the recommended cutoff values. Genomic analysis of three Bacillus species. Putative genes conferring resistance to tetracycline, lincosamides, and strepthrothricin were identified. Putative genes conferring resistance to rifampicin and macrolides were found in the genome of Bs PTA86. However, these genes have been reported to be present in Ba and Bs isolates from the environment (101, 102), suggesting that these genes may be intrinsic to Ba and Bs strains. Furthermore, the absence of transferable mobile genetic elements such as transposons and insertion sequences near these genes suggests that the risk of horizontal transfer of these genes to other digestive tract microorganisms is very low and poses little risk to public health safety.

[0402] Metabolomic analysis. It is well known that probiotic strains secrete beneficial metabolites as microbial fermentation byproducts, such as short-chain fatty acids (SCFAs), which contribute to mucus secretion, mucosal epithelial integrity, immune cell regulation, and serve as an energy source for colonocytes (103, 104). To investigate the potential host-beneficial metabolite secretion, we performed a global untargeted metabolomic analysis of BsPTA86. A specific target metabolite was 1-kestose, which was identified in the culture supernatant of the strain. 1-kestose is the smallest fructooligosaccharide (FOS) and is a trisaccharide molecule composed of one glucose and two fructose residues linked by glycosidic bonds. Kestose is a prebiotic that, upon consumption, can enrich the growth of intestinal commensal bacteria, such as Bifidobacterium, Lactobacillus, and Faecalibacterium prausnitzii, promoting digestive tract health (105). An antioxidant molecule, thioproline, was identified in the culture supernatant of BsPTA86. Thioproline has been reported to inhibit human carcinogenesis and has shown promise as a nitrite scavenger (106). Pyridoxine (vitamin B6) has been found in the culture supernatant of Bs PTA86. Bs PTA86 may secrete betaine and choline. These molecules are methyl donors required for acetylcholine and phosphatidylcholine biosynthesis, neurotransmission, and cell membrane integrity, respectively (107). When betaine was added to the feed, the growth performance of chickens during heat stress was improved (108,109). The addition of choline has been associated with a reduction in FCR in broiler chickens (110).

[0403] References

[0404] 1.FAO-UN(2018)The future of food and agriculture.Alternative pathways to 2050.in Supplementar Material(Food and Agriculture Organization of the United Nations).

[0405] 2.US-FDA(2013)New animal drugs and new animal drug combinationproducts administered in or on medicated feed or drinking water of food-producing animals:recommendations for drug sponsors for voluntarily aligningproduct use conditions with GFI#209.(Department of Health and HumanServices).

[0406] 3.EFSA Panel on Biological Hazards(BIOHAZ)et al.(2020)Update of thelist of QPS-recommended biological agents intentionally added to food or feedas notified to EFSA 13:suitabilityof taxonomic units notified to EFSA untilSeptember 2020.

[0407] 4.P.R.Moore,Evenson,A.,Luckey,T.D.,McCoy.E.,Elvehjem,C.A.Hart,E.B..Use of sulfasuxidine,streptothricin,and streptomycin in nutritionalstudies with the chick.Journal of Biological Chemistry 2,437-441(1946).

[0408] 5.C.Hill et al.,The International Scientific Association forProbiotics and Prebiotics consensus statement on the scope and appropriateuse of the term probiotic.Nature Reviews Gastroenterology&Hepatology 11,506-514(2014).

[0409] 6.R.Jha,R.Das,S.Oak,P.Mishra,Probiotics(Direct-Fed Microbials)inPoultry Nutrition and Their Effects on Nutrient Utilization,Growth and LayingPerformance,and Gut Health:A Systematic Review.Animals(Basel)10(2020).

[0410] 7.A.Grant,C.G.Gay,H.S.Lillehoj,Bacillus spp.as direct-fed microbialantibiotic alternatives to enhance growth,immunity,and gut health inpoultry.Avian Pathol 47,339-351(2018).

[0411] 8.T.M.Barbosa,C.R.Serra,R.M.La Ragione,M.J.Woodward,A.O.Henriques,Screening forBacillus isolates in the broiler gastrointestinal tract.ApplEnviron Microbiol 71,968-978(2005).

[0412] 9.X.Guo,D.Li,W.Lu,X.Piao,X.Chen,Screening of Bacillus strains aspotential probiotics and subsequent confirmation of the in vivo effectivenessof Bacillus subtilis MA139 in pigs.Antonie Van Leeuwenhoek 90,139-146(2006).

[0413] 10.P.Setlow,Spores of Bacillus subtilis:their resistance to andkilling by radiation,heat and chemicals.Journal of Applied Microbiology 101,514-525(2006).

[0414] 11.S.Shivaramaiah et al.,Evaluation of Bacillus species as potentialcandidates for direct-fed microbials in commercial poultry.Poult Sci 90,1574-1580(2011).

[0415] 12.M.Bernardeau,M.J.Lehtinen,S.D.Forssten,P.Nurminen,Importance ofthe gastrointestinal life cycle of Bacillus for probiotic functionality.JFood Sci Technol 54,2570-2584(2017).

[0416] 13.J.D.Latorre et al.,Evaluation of germination,distribution,andpersistence of Bacillus subtilis spores through the gastrointestinal tract ofchickens.Poult Sci 93,1793-1800(2014).

[0417] 14.T.T.Hoa et al..Fate and dissemination of Bacillus subtilis sporesin a murine model.Applied and environmental microbiology 67.3819-3823(2001).

[0418] 15.G.Casula.S.M.Cutting,Bacillus probiotics:spore germination in thegastrointestinal tract.Applied and environmental microbiology 68,2344-2352(2002).

[0419] 16.S.Mingmongkolchai,W.Panbangred,Bacillus probiotics:an alternativeto antibiotics for livestock production.J Appl Microbiol 124,1334-1346(2018).17.A.Q.Grant,C.G.Gay,H.S.Lillehoj,Bacillus spp.as direct-fed microbialantibiotic alternatives to enhance growth,immunity,and gut health inpoultry.Avian Pathology 47,339-351(2018).

[0420] 18.Y.Ma et al.,Supplemental Bacillus subtilis DSM 32315 manipulatesintestinal structure and microbial composition in broiler chickens.Sci Rep 8,15358(2018).

[0421] 19.J.S.Weese,H.Martin,Assessment of commercial probiotic bacterialcontents and label accuracy.Can Vet J 52,43-46(2011).

[0422] 20.J.S.Weese,Microbiologic evaluation of commercial probiotics.J AmVet Med Assoc 220,794-797(2002).

[0423] 21.D.H.Green et al.,Characterization of two Bacillus probiotics.ApplEnviron Microbio165,4288-4291(1999).

[0424] 22.J.R.Koransky,S.D.Allen,V.R.Dowell,Use of ethanol for selectiveisolation of sporeforming microorganisms.Applied and EnvironmentalMicrobiology 35,762-765(1978).

[0425] 23.J.D.Latorre et al.,Evaluation and Selection of Bacillus SpeciesBased on Enzyme Production,Antimicrobial Activity,and Biofilm Synthesis asDirect-Fed Microbial Candidates for Poultry.Front Vet Sci 3,95(2016).

[0426] 24.B.V.McCleary,A simple assay procedure for β-d-mannanase.Carbohydrate Research 67,213-221(1978).

[0427] 25. GREFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Gabriele Aquilina, Giovanna Azimonti, Vasileios Bampidis, Maria de Lourdes Bastos, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Gerhard Flachowsky, Jürgen Gropp, Boris Kolar, Maryline Kouba, Marta López-Alonso, Secundino López Puente, Alberto Mantovani, Baltasar Mayo, Femando Ramos, Maria Saarela, Roberto Edoardo Villa, Robert John Wallace, Pieter Wester, Boet Glandorf, Lieve Herman, Sirpa K`irenlampi,Jaime Aguilera,MontserratAnguita,Rosella Brozzi,Jaume Galobart(2018)Guidance on the characterizationof microorganisms used as feed additives or as production organisms.(EFSA(European Food Safety Authority)).

[0428] 26.GLSfaHWorld(2021)Performance Standards for AntimicrobialSusceptibility Testing.(CLSI),p 352.

[0429] 27.J.Rao et al.,Tn2008-driven carbapenem resistance in Acinetobacterbaumannii isolates from a period of increased incidence of infections in aSouthwest Virginia hospital(USA).J Glob Antimicrob Resist 12.79-87(2018).

[0430] 28.D.Hyatt et al.,Prodigal:prokaryotic gene recognition andtranslation initiation site identification.BMC Bioinformatics 11,119(2010).

[0431] 29.D.Laslett,B.Canback,ARAGORN,a program to detect tRNA genes andtmRNA genes in nucleotide sequences.Nucleic Acids Res 32,11-16(2004).

[0432] 30.T.Seemann,Prokka:rapid prokaryotic genomeannotation.Bioinformatics 30,2068-2069(2014).

[0433] 31.B.E.Suzek,M.D.Ermolaeva,M.Schreiber,S.L.Salzberg,A probabilisticmethod for identifying start codons in bacterial genomes.Bioinformatics 17,1123-1130(2001).

[0434] 32.C.L.Kingsford.K.Ayanbule.S.L.Salzberg,Rapid,accurate,computationaldiscovery of Rho-independent transcription terminators illuminates theirrelationship to DNA uptake.Genome Biol 8,R22(2007).

[0435] 33.E.P.Nawrocki,D.L.Kolbe,S.R.Eddy,Infernal 1.0:inference of RNAalignments.Bioinformatics 25,1335-1337(2009).

[0436] 34.B.Tjaden,A computational system for identifying operons based onRNA-seq data.Methods.176,62-70(2020).

[0437] 35.A.Bremges,A.Fritz,A.C.McHardy,CAMITAX:Taxon labels for microbialgenomes.GigaScience 9(2020).

[0438] 36.D.M.Emms,S.Kelly,OrthoFinder:phylogenetic orthology inference forcomparative genomics.Genome Biol 20,238(2019).

[0439] 37.E.W.Sayers et al.,Database resources of the National Center forBiotechnology Information.Nucleic Acids Res 49,Dl0-dl7(2021).

[0440] 38.S.I.Na et al.,UBCG:Up-to-date bacterial core gene set and pipelinefor phylogenomic tree reconstruction.J Microbiol 56,280-285(2018).

[0441] 39.M.N.Price,P.S.Dehal,A.P.Arkin,FastTree 2--approximately maximum-likelihood trees for large alignments.PLoS One 5,e9490(2010).

[0442] 40.Z.Xie,H.Tang.ISEScan:automated identification of insertionsequence elements in prokaryotic genomes.Bioinformatics 33,3340-3347(2017).

[0443] 41.S.Akhter,R.K.Aziz,R.A.Edwards,PhiSpy:a novel algorithm for findingprophages in bacterial genomes that combines similarity-and composition-basedstrategies.Nucleic Acids Res 40,e126(2012).

[0444] 42.W.N.P.Setlow,Molecular Biological Methods forBacillus.C.H.S.Cutting.Ed.(John Wiley,New York.1990).

[0445] 43.M.Martin,Cutadapt removes adapter sequences from high-throughputsequencing reads.2011 17,3(2011).

[0446] 44.B.J.Callahan et al.,DADA2:High-resolution sample inference fromIllumina amplicon data.Nat Methods 13,581-583(2016).

[0447] 45.P.Yilmaz et al.,The SILVA and″All-species Living Tree Project(LTP)”taxonomic frameworks.Nucleic acids research 42,D643-D648(2014).

[0448] 46.E.H.Simpson,Measurement of Diversity.Nature 163,688-688(1949).

[0449] 47.C.E.Shannon, The mathematical theory of communication.1963.MDComput 14,306-317(1997).

[0450] 48.A.Chao,Nonparametric Estimation of the Number of Classes in aPopulation.Scandinavian Journal of Statistics 11,265-270(1984).

[0451] 49.Anonymous(http: / / scikit-bio.org / .

[0452] 50.The Association of American Feed Control Officials,Inc.,2021Official Publication(FASS Inc.,Champaign,IL).

[0453] 51.I.Danilova,M.Sharipova,The Practical Potential of Bacilli andTheir Enzymes for Industrial Production.Frontiers in Microbiology 11(2020).

[0454] 52.F.J.Contesini,R.R.d.Melo,H.H.Sato,An overview of Bacillusproteases:from production to application.Critical Reviews in Biotechnology38,321-334(2018).

[0455] 53.A.Fagerlund,T. A.K.Storset,P.E.Granum,S.P.Hardy,Bacilluscereus Nhe is a pore-forming toxin with structural and functional propertiessimilar to the ClyA(HlyE,SheA)family of haemolysins,able to induce osmoticlysis in epithelia.Microbiology(Reading)154,693-704(2008).

[0456] 54.M.Saeed et al.,The Role of fβ-Mannanase(Hemicell)in ImprovingPoultry Productivity,Health and Environment.Brazilian Journal of PoultryScience 21(2019).

[0457] 55.H.Fayek.M.Abou El-Ella,M.Attia,Y.Mady(1995)Evaluation of usingsome feed additives in layer diets,in Sci.Conf.Anim.Nutr,pp 313-321.

[0458] 56.Y.Zhou,Z.Jiang,D.Lv.T.Wang,Improved energy-utilizing efficiency byenzyme preparation supplement in broiler diets with different metabolizableenergy levels.Poult Sci 88,316-322(2009).

[0459] 57.K.Sherif,Performance of broiler chicksfed plant protein dietssupplemented with commercial enzymes.Journal of Animal and Poultry Production34,2819-2834(2009).

[0460] 58.K.Babasaki,T.Takao,Y.Shimonishi,K.Kurahashi,Subtilosin A,a newantibiotic peptide produced by Bacillus subtilis 168:isolation,structuralanalysis,and biogenesis.J Biochem 98,585-603(1985).

[0461] 59.C.E.Shelburne et al.,The spectrum of antimicrobial activity of thebacteriocin subtilosin A.Journal of Antimicrobial Chemotherapy 59,297-300(2007).

[0462] 60.T.Stein,S.Düsterhus,A.Stroh,K.D.Entian,Subtilosin production bytwo Bacillus subtilis subspecies and variance of the sbo-alb cluster.ApplEnviron Microbiol 70,2349-2353(2004).

[0463] 61.欧洲食品安全局(EFSA),EFSA关于食物链中有意使用的微生物全基因组序列分析要求的声明。《EFSA杂志》,第1 - 15页(2021年)。

[0464] 62.Y. 迈耶、B.B. 布坎南、F. 维尼奥尔斯、J.-P. 雷希尔德,硫氧还蛋白和谷氧还蛋白:氧化还原生物学中的统一元素。《遗传学年度评论》43卷,第335 - 367页(2009年)。

[0465] 63.A. 加巴拉等人,芽孢硫醇的生物合成与功能,芽孢杆菌中一种主要的低分子量硫醇。《美国国家科学院院刊》107卷,第6482 - 6486页(2010年)。

[0466] 64.H. 安特尔曼、C. 沙夫、M. 赫克,枯草芽孢杆菌的磷酸盐饥饿诱导蛋白:蛋白质组学和转录分析。《细菌学杂志》182卷,第4478 - 4490页(2000年)。

[0467] 65.B. 桑切斯、P. 布雷索利耶、M.C. 乌尔达西,益生菌中的分泌蛋白:对肠道表面的粘附、宿主免疫调节以及与宿主的分子相互作用。《FEMS免疫学与医学微生物学》54卷,第1 - 17页(2008年)。

[0468] 66.T.Zhu et al.,Engineering of Bacillus subtilis for enhanced totalsynthesis of folic acid.Appl Environ Microbiol 71,7122-7129(2005).

[0469] 67.V.de Crecy-Lagard,Variations in metabolic pathways createchallenges for automated metabolic reconstructions:Examples from thetetrahydrofolate synthesis pathway.Comput Struct Biotechnol J 10,41-50(2014).

[0470] 68.A.R.Salem,J.R.Pattison.M.A.Foster,Folic acid and the methylationofhomocysteine by Bacillus subtilis.Biochem J 126,993-1004(1972).

[0471] 69.S.Brul et al.,Challenges and advances in systems biology analysisof Bacillus spore physiology;molecular differences between an extreme heatresistant spore forming Bacillus subtilis food isolate and a laboratorystrain.Food Microbiology 28,221-227(2011).

[0472] 70.E.M.Berendsen,M.H.Zwietering,O.P.Kuipers,M.H.J.Wells-Bennik,Twodistinct groups within the Bacillus subtilis group display significantlydifferent spore heat resistance properties.Food Microbiology 45,18-25(2015).

[0473] 71.F.Van Immerseel,J.I.Rood,R.J.Moore,R.W.Titball,Rethinking ourunderstanding of the pathogenesis of necrotic enteritis in chickens.TrendsMicrobiol 17,32-36(2009).

[0474] 72.K.Grave,M.C.Kaldhusdal,H.Kruse,L.M.Hart,K.Flatlandsmo,What hashappened in norway after the ban ofavoparcin?Consumption of antimicrobials bypoultry.Prev Vet Med 62,59-72(2004).

[0475] 73.L.Hughes,P.Hermans,K.Morgan,Risk factors for the use ofprescription antibiotics on UK broiler farms.J Antimicrob Chemother 61,947-952(2008).

[0476] 74.F.Dziva,M.P.Stevens,Colibacillosis in poultry:unravelling themolecular basis of virulence of avian pathogenic Escherichia coli in theirnatural hosts.Avian Pathol 37,355-366(2008).

[0477] 75.M.Mellata,Human and avian extraintestinal pathogenic Escherichiacoli:infections,zoonotic risks,and antibiotic resistance trends.FoodbornePathog Dis 10,916-932(2013).

[0478] 76.H.Duan et al.,Transmission identification of Escherichia coliaerosol in chicken houses to their environments using ERIC-PCR.Sci China CLife Sci 51,164-173(2008).

[0479] 77.Y.C.Chien,C.J.Chen,T.H.Lin,S.H.Chen,Y.C.Chien.Characteristics ofmicrobial aerosols released from chicken and swine feces.J Air Waste ManagAssoc 61,882-889(2011).

[0480] 78.K.K.Cooper,J.G.Songer,Necrotic enteritis in chickens:A paradigm ofenteric infection by Clostridium perfringens type A.Anaerobe 15,55-60(2009).

[0481] 79.A.L.Keyburn et al.,NetB,a new toxin that is associated with aviannecrotic enteritis caused by Clostridium perfringens.PLoS Pathog 4,e26(2008).

[0482] 80.A.L.Keyburn et al.,Alpha-toxin ofClostridium perfringens is not anessential virulence factor in necrotic enteritis in chickens.Infect Immun 74,6496-6500(2006).

[0483] 81.B.Wade,A.Keyburn,The true cost of necrotic enteritis.World Poult31,16-17(2015).

[0484] 82.M.B.Batz,S.Hoffmann,J.G.Morris,Jr.,Ranking the disease burden of14 pathogens in food sources in the United States using attribution data fromoutbreak investigations and expert elicitation.J Food Prot 75,1278-1291(2012).

[0485] 83.S.L.Foley,A.M.Lynne,Food animal-associated Salmonella challenges:pathogenicity and antimicrobial resistance.J Anim Sci 86,El 73-187(2008).84.S.Singh,B.K.Bajaj,Bioprocess optimization for production ofthermoalkali-stable protease from Bacillus subtilis K-1 under solid-statefermentation.Preparative Biochemistry&Biotechnology 46,717-724(2016).

[0486] 85.A.Raza,S.Bashir,R.Tabassum,An update on carbohydrases:growthperformance and intestinal health of poultry.Heliyon 5,e01437-e01437(2019).

[0487] 86.M.Kaldhusdal,Necrotic enteritis as affected by dietaryingredients.World Poultry 16,42-43(2000).

[0488] 87.F.Van Immerseel et al.,Clostridium perfringens in poultry:anemerging threat for animal and public health.Avian Pathol 33,537-549(2004).

[0489] 88.G.Cardoso Dal Pont,M.Farnell,Y.Farnell,M.H.Kogut,Dietary Factorsas Triggers of Low-Grade Chronic Intestinal Inflammation inPoultry.Microorganisms 8,139(2020).

[0490] 89.Y.Wang et al.,Bacillus amyloliquefaciens SC06 alleviates theoxidative stress of IPEC-1 via modulating Nrf2 / Keapl signaling pathway anddecreasing ROS production.Appl Microbiol Biotechnol 101,3015-3026(2017).

[0491] 90.A.Abudabos,A.Alyemni,H.Zakaria,Effect of Two Strains of Probioticson the Antioxidant Capacity,Oxidative Stress,and Immune Responses ofSalmonella-Challenged Broilers.Brazilian Journal of Poultry Science 18,175-180(2016).

[0492] 91.T.Inatomi,K.Otomaru,Effect of dietary probiotics on the sementraits and antioxidative activity of male broiler breeders.Scientific Reports8,5874(2018).

[0493] 92.M.Rossi,A.Amaretti,S.Raimondi,Folate production by probioticbacteria.Nutrients 3,118-134(2011).

[0494] 93.J.Zhang et al.,Screening of folate-producing lactic acid bacteriaand modulatory eftects of folate-biofortified yogurt on gut dysbacteriosis offolate-deficient rats.Food Funct 11,6308-6318(2020).

[0495] 94.C.S.Fuchs et al.,The Influence of Folate and Multivitamin Use onthe Familial Risk of Colon Cancer in Women.Cancer Epidemiology Biomarkers&;Prevention 11,227-234(2002).

[0496] 95.E.White,J.S.Shannon,R.E.Patterson,Relationship between vitamin andcalcium supplement use and colon cancer.Cancer Epidemiol Biomarkers Prey 6,769-774(1997).

[0497] 96.M.Schallmey,A.Singh,O.P.Ward,Developments in the use of Bacillusspecies for industrial production.Can J Microbiol 50,1-17(2004).

[0498] 97.U.Sauer,D.C.Cameron,J.E.Bailey,Metabolic capacity of Bacillussubtilis for the production of purine nucleosides,riboflavin,and folicacid.Biotechnology and Bioengineering 59,227-238(1998).

[0499] 98.F.M.Asrar,D.L.O′Connor,Bacterially synthesized folate andsupplemental folic acid are absorbed across the large intestine of piglets.JNutr Biochem 16,587-593(2005).

[0500] 99.L.H.Duc,H.A.Hong,T.M.Barbosa,A.O.Henriques,S.M.Cutting,Characterization of Bacillus probiotics available for human use.Applied andenvironmental microbiology 70,2161-2171(2004).

[0501] 100.V.Ciorba,A.Odone,L.Veronesi,C.Pasquarella,C.Signorelli,Antibioticresistance as a major public health concern:epidemiology and economicimpact.Ann Ig 27,562-579(2015).

[0502] 101.D.B.Adimpong et al.,Antimicrobial susceptibility of Bacillusstrains isolated from primary starters for African traditional breadproduction and characterization of the bacitracin operon and bacitracinbiosynthesis.Appl Environ Microbiol 78,7903-7914(2012).

[0503] 102.R.W.Phelan et al.,Tetracycline resistance-encoding plasmid fromBacillus sp.strain#24,isolated from the marine sponge Haliclona simulans.ApplEnviron Microbiol 77,327-329(2011).

[0504] 103.L.R.Lopetuso,F.Scaldaferri,V.Petito,A.Gasbarrini,CommensalClostridia:leading players in the maintenance of gut homeostasis.Gutpathogens 5,1-8(2013).

[0505] 104.H.M.Hamer et al.,Review article:the role of butyrate on colonicfunction.Aliment Pharmacol Ther 27,104-119(2008).

[0506] 105.J.A.Patterson,J.I.Orban,A.L.Sutton,G.N.Richards,Selectiveenrichment of Bifidobacteria in the intestinal tract of broilers by thermallyproduced kestoses and effect on broiler performance.Poult Sci 76,497-500(1997).

[0507] 106.J.A.Patterson et al.,Thioproline formation as a driver offormaldehyde toxicity in Escherichia coli.Biochem J 477,1745-1757(2020).

[0508] 107.R.R.Zempleni J,McCormick DB,Suttie JW.Handbook of vitamins.(CRCPress,Boca Raton(FL),ed.4th.2007).

[0509] 108.W.Liu et al.,Effects of Dietarv Betaine on Growth Performance,Digestive Function,Carcass Traits,and Meat Quality in Indigenous Yellow-Feathered Broilers under Long-Term Heat Stress.Animals(Basel)9(2019).

[0510] 109.S.He,S.Zhao,S.Dai,D.Liu,S.G.Bokhark Effects of dietary betaine ongrowth performance,fat deposition and serum lipids in broilers subjected tochronic heat stress.Anim Sci J 86,897-903(2015).

[0511] 110.G.S.Santiago et al.,Dietary choline affects field performance andbroiler leg deviations.Livestock Science 240,104127(2020).

[0512] 111.Y.Shang,S.Kumar,B.Oakley,W.K.Kim,Chicken Gut Microbiota:Importance and Detection Technology.Front Vet Sci 5,254(2018).

[0513] 112.A.-R.Al-Fatafiah,A.Abdelqader,Effects of dietary Bacillussubtilis on heat-stressed broilers performance,intestinal morphology andmicrofiora composition.Animal Feed Science and Technology 198,279-285(2014).

[0514] 113.J.D.Latorre et al.,Evaluation and Selection of Bacillus SpeciesBased on Enzyme Production,Antimicrobial Activity,and Biofilm Synthesis asDirect-Fed Microbial Candidates for Poultry.Front Vet Sci 3,95(2016).

[0515] 114.B.V.McCleary,A simple assay procedure forβ-d-mannanase.Carbohydrate Research 67,213-221(1978).

[0516] 115. GREFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Gabriele Aquilina, Giovanna Azimonti, Vasileios Bampidis, Maria de Lourdes Bastos, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Gerhard Flachowsky, Jürgen Gropp, Boris Kolar, Maryline Kouba, Marta López-Alonso, Secundino López Puente, Alberto Mantovani, Baltasar Mayo, Fernando Ramos, Maria Saarela, Roberto Edoardo Villa, Robert John Wallace, Pieter Wester, Boet Glandorf, Lieve Herman, Sirpa Jaime Aguilera,Montserrat Anguita,Rosella Brozzi,Jaume Galobart(2018)Guidance on the characterization ofmicroorganisms used as feed additives or as production organisms.(EFSA(European Food Safety Authority))。

[0517] Example 5

[0518] Bacillus strain 105 (BSUB105; PTA-126786 or PTA-86) was analyzed and certain classes of genes or secondary metabolic pathways unique to this strain were identified. Some of the results, such as bacteriocin predictions, secondary metabolites, and carbohydrate metabolism enzymes, were provided in earlier examples and tables. Unique proteins (predicted proteins for which no equivalent or homologous protein-encoding genes were identified in other compared Bacillus strains through consensus searches) were predicted based on strain sequence comparisons and evaluation of the protein sequences of genes from Bacillus subtilis 105 (BSUB105; PTA-126786 or PTA-86). Strain 105 includes four subtilisin genes, pullulanase (which helps break down branched carbohydrates into simple carbohydrates), a cyclodextrin-binding protein, nine genes involved in sporulation, the β-galactosidase YesZ and GanA genes, and the oxidoreductase YimC. Unique genes encoded based on the genomic sequence of strain Bs-PTA 86 are included and provided in USSN 63 / 083697, filed September 25, 2020, and USSN 63 / 241369, filed September 7, 2021, each of which is incorporated herein by reference.

[0519] Example 6

[0520] Application of Bacillus subtilis 105 strain as a live delivery platform and production system

[0521] Bacillus subtilis strain 105, also known as ELA191105 (PTA-126786), has been selected as a useful and suitable strain for the development and use of food-grade and pharmaceutical protein production.

[0522] A comparison was conducted between Bacillus subtilis strain 105 (also known as ELA191105 (PTA-126786)) and Bacillus subtilis species 168. Genomic analysis and comparison showed that the Bacillus subtilis 168 genome includes 1,109 genes involved in 1,681 reactions, 1,376 metabolites, 243 crossovers, and two compartments. The Bacillus subtilis strain 105 (ELA191105) genome includes 1,077 genes involved in 1,462 reactions, 1,253 metabolites, 153 crossovers, and two compartments.

[0523] The metabolic genes unique to strain 105 relative to strain 168, particularly the proteins encoded therein, are shown in Table 19 below.

[0524] Table 19 - Metabolic genes unique to Bacillus subtilis 105 strain

[0525]

[0526]

[0527] Bacillus subtilis strain 105, also known as ELA191105 (PTA-126786), provides a useful and applicable strain for the development and use of food-grade and pharmaceutical protein production, and as an in vivo delivery platform for the delivery and production of useful biomolecules and proteins, including homologous and heterologous proteins, in animal hosts.

[0528] Table 20. Development and application of Bacillus subtilis #105 as a production and in vivo delivery strain

[0529]

[0530]

[0531]

[0532]

[0533] Example 7

[0534] Strain 105 was modified to improve competence

[0535] Competence is a physiological state that enables cells, including bacterial cells, to take up and internalize extracellular DNA. In practice, only a small subset of bacterial cells, such as Bacillus subtilis cells, become competent upon entering the stationary growth phase. Specifically, Bacillus subtilis becomes competent when the competence transcription factor ComK reaches a certain threshold level (Maamar and Dubnau, 2005; Smits et al., 2005). ComK is a master regulator of competence that activates approximately 100 genes involved in DNA recombination, repair, binding, and uptake (Berka et al., 2002; Hamoen et al., 2002), cell division (Hamoen, 2011), and its own promoter in a positive feedback loop (van Sinderen and Venema, 1994). When Bacillus subtilis cells enter the stationary growth phase due to nutrient deprivation and high cell density, they begin to differentiate into distinct subpopulations. Some of them become motile (Nishihara and Freese, 1975), while others form biofilms (Vramakis et al., 2008), secrete degradative enzymes and antibiotics (González-Pastor et al., 2003), or eventually sporulate (Rudner and Losick, 2001; Piggot and Hilberr, 2004). Another small subpopulation differentiates into competent cells capable of taking up extracellular DNA (Dubnau, 1991a; Dubnau and Provedi, 2000).

[0536] Improving the transformation efficiency of Bacillus subtilis is beneficial, including in complete medium. It has been reported that a supercompetent Bacillus strain was constructed using a genetically modified comKS encoding gene cassette, specifically under the control of the mannitol-inducible PmtlA promoter (Rahmer R et al. (2015) Front Microbiol 6:1431; doi:10.3389 / fmicb.2015.01431). This cassette resulted in overexpression of comK and comS and increased the transformation efficiency of Bacillus subtilis with plasmid DNA by more than 6-fold compared to wild-type Bacillus subtilis without the cassette.

[0537] Bacillus subtilis strain 105 was genetically modified to increase competence by generating a modified strain 105 that overexpresses comK and comS. In one method, an expression cassette containing the PxylA promoter from Bacillus subtilis strain 105 was ligated to the comK coding sequence, followed by the in-frame conS coding sequence. ComK and comS were produced under the control of the PxylA promoter. The PxylA promoter is a xylose-inducible promoter.

[0538] An exemplary expression cassette comprising a promoter (PxlA promoter from Bacillus subtilis strain 105), a ComK coding sequence, and a ComS coding sequence (SEQ ID NO: 37) is provided below.

[0539]

[0540] Bold: PxylA promoter from Bacillus subtilis #105

[0541] ComK coding sequence

[0542] Bold Underline : ComS coding sequence

[0543] The ComK coding sequence of strain 105 is provided below (SEQ ID NO: 38):

[0544]

[0545]

[0546] The ComS coding sequence of strain 105 is provided below (SEQ ID NO: 39):

[0547]

[0548] The sequence of the xylose-inducible promoter PxylA_Bs105 from strain 105 is as follows (SEQ ID NO: 40):

[0549]

[0550] To improve the transformation efficiency of Bacillus subtilis #105, the native ComK gene CDS was deleted and replaced with ComKS under a xylose-inducible promoter (xylA). Figure 4 A schematic diagram of the integration and replacement strategy is provided. PCR and sequencing confirmed that the ComKS expression cassette was correctly integrated into the genome. Flanking PCR first confirmed the correct integration of the ComKS expression cassette, and then colony PCR showed the complete ComKS insert in the genome (data not shown).

[0551] Evaluation of the ComKS system showed that it improves competence. Engineering of this inducible ComKS system increased transformation efficiency by approximately 100-fold (from 2-3 colonies to 200-400 colonies per 500 ng of DNA).

[0552] Other natural or non-natural promoters can be used for the comKcomS inducible expression cassette. In some embodiments, the natural or non-natural promoter is inducible and allows for controlled and timed competence, including under specific growth conditions, with the addition of specific culture media, or at different or specific bacterial cell growth stages (e.g., growth phase versus stationary growth phase, etc.). Other exemplary inducible promoters include the strain 105 mannitol-inducible promoter. The sequence of the mannose-inducible promoter from Bacillus subtilis strain 105 is as follows (SEQ ID NO: 41):

[0553]

[0554]

[0555] Example 8

[0556] Signal peptide for secretion of heterologous proteins in Bacillus subtilis

[0557] In cases where one or more target biomolecules or proteins of interest are secreted proteins or must be secreted by the bacterial delivery cell to be active or to reach a relevant location in the host system or organ or tissue, a secretion signal or signal sequence can be incorporated to promote secretion of the molecule or protein. In Bacillus subtilis, protein export is typically accomplished through the Sec-type secretory pathway, which controls over 90% of the secreted proteins of Bacillus subtilis.

[0558] The N-terminal sequence of a secreted protein carries a specific secretion signal called a signal peptide. After synthesis, the nascent peptide with the signal peptide is recognized by components of the Sec-type secretory pathway and transported across the membrane into the extracellular medium. The signal peptide can be a key factor in determining the optimal pathway for a target protein and how it is secreted across the membrane.

[0559] By analyzing global proteomics data, exemplary and suitable secretion signal peptides for strain 105 were identified. Several secretion signals are provided below. These can be fused to existing or encoded protein sequences, including in conjunction with high-expression or inducible promoters. These signal sequences can be used in expression cassettes and / or integrated with coding nucleic acid sequences to provide for effective and efficient secretion of biomolecules or heterologous proteins. The signal sequence is fused in frame to the coding sequence.

[0560] The Bacillus subtilis strain 105 β-mannanase secretion signal is as follows (SEQ ID NO: 42):

[0561]

[0562] This sequence encodes a secretory signal peptide with the following amino acid sequence:

[0563]

[0564] The Bacillus subtilis strain 105 pel secretion signal is as follows (SEQ ID NO: 45):

[0565]

[0566] This sequence encodes a secretory signal peptide with the following amino acid sequence:

[0567]

[0568] The Bacillus subtilis strain 105 dacC secretion signal is as follows (SEQ ID NO: 48):

[0569]

[0570]

[0571] The sequence encodes a secretory signal peptide of the following amino acid sequence (SEQ ID NO: 49): MKKSIKLYVAVLLLFVVASVPYMHQAALA

[0572] Alternative secretion signal sequences are known and available in the art. For example, Fu et al. evaluated the extracellular production of α-amylase (AmyS) from Bacillus stearothermophilus by generating and screening a high-capacity signal peptide library in Bacillus subtilis (Fu, G et al. (2018) J Agric Food Chem 66: 13141-13151). A total of 173 Sec-type signal peptides from Bacillus subtilis were fused. The target protein was linked to the target protein by a sequence-independent, polymerase chain reaction-based cloning method without the use of restriction endonucleases or ligases (You, C.; Zhang, XZ; Zhang, YH (2012) Appl Environ Microbiol 78 (5): 1593-5). The resulting multimeric plasmid library DNA containing different signal peptides was transformed into Bacillus subtilis, and a high-throughput method based on starch-iodine was used to screen constructs with high extracellular α-amylase activity. Signal peptides optimized for secretory expression of AmyS were identified and validated through high-density fermentation. A number of signal peptides were identified as candidates for improving secretory expression of the α-amylase AmyS in Bacillus subtilis. These signal peptides are listed in Table 21 below and can be used in strain 105. In fact, the amino acid sequence of the pel secretion signal of strain 105 corresponds in sequence to the pel signal sequence in the table below.

[0573] Table 21 - Signal peptides for protein secretion based on amylase secretion assessment

[0574]

[0575]

[0576] Those skilled in the art will recognize that, due to the redundancy of the genetic code, multiple nucleic acid sequences may encode the above-mentioned peptides.

[0577] Example 9

[0578] Strain 105 was modified to produce a non-spore-forming strain

[0579] Bacillus subtilis is a Gram-positive, endospore-forming microorganism that holds Qualified Prescribed Safety (QPS) status from the European Food Safety Authority (EFSA) based on its apathogenicity and lack of exotoxin and endotoxin production (Hohmann HP et al. (2016) Industrial Biotechnology: Microorganisms pp. 221-297). However, B. subtilis strains often produce spores in response to physical and chemical stimuli, thereby ceasing growth and resulting in nutrient waste and reduced yield. Spores occur naturally in B. subtilis cultures, helping the bacterium resist physical and chemical stimuli and supporting its terrestrial life. Spores are a dormant state during which enzyme or chemical product synthesis and secretion cease. Spores are more resistant to environmental extremes than vegetative cells and can germinate, resuming vegetative growth under appropriate nutrient conditions. Several reports have described various methods for preventing B. subtilis sporulation during fermentation of various target products. For example, deletion of the initial regulatory sporulation gene spo0A results in enhanced maintenance metabolism (Tannler S et al. (2008) Microb Cell Factories 7:9-19), increased rates of glucose consumption and acetate formation (Fischer and Sauer, 2005 Nat. Genet. 37 636-640), and abolishes polymyxin production in Bacillus subtilis BSK4-0A (Park et al. (2012) Appl. Environ. Microbiol. 78, 4194-4199).

[0580] Wang et al. engineered several non-spore-forming Bacillus subtilis strains by knocking out individual spore-forming genes (spo0A, spoIIIE, and spoIVB) involved in various stages of sporulation (Wang et al. (2020) Metabolic Engineering 62:235-248). The spo0A-null non-spore-forming mutants were particularly efficient in producing secondary metabolites such as surfactin.

[0581] Bacillus subtilis strain 105 (ELA191105) was modified and engineered to delete or otherwise inactivate the Spo0A and / or SpoIVB coding sequences. Figure 1 The Spo0A and SpoIVB loci are shown, with SpoIVb and SpoA encoded by tandemly positioned sequences. Both genes can be deleted by single deletions of the coding regions using overlapping sequences at the ends or outside of the SpoIVb and Spo0A coding sequences. The Spo0A and SpoIVB sequences used for deletion to generate the non-sporulating and modified Bacillus subtilis strain 105 are as follows:

[0582] >Spo0A and SpoIVB sequences used for deletion to generate non-spore-forming Bacillus subtilis #105 (SEQ ID NO: 65)

[0583]

[0584]

[0585]

[0586] Using the ComKS-inducible strain described in Example 7 as a basis (this genetically modified strain has enhanced competence), a non-spore-forming version of Bacillus subtilis #105 was generated.

[0587] A non-spore-forming version of B. subtilis #105 was generated by deleting the Spo0A and SpoIVB coding sequences and confirmed by PCR and sequencing. Junction PCR confirmed the correct deletion of the sporulation genes in B. subtilis strain 105 (data not shown).

[0588] Example 10

[0589] Strain 105 promoter for expression of heterologous proteins or sequences

[0590] By analyzing genomic and global proteomic data, a suitable promoter for Bacillus subtilis strain 105 was identified. The promoter was engineered upstream of a nucleic acid encoding one or more biomolecules or heterologous proteins. The promoter is used in an expression cassette suitable for generating genetically modified bacteria capable of producing biomolecules or heterologous proteins and / or expressing the desired biomolecules or heterologous proteins for delivery to a host animal in need. The expression cassette will contain a suitable promoter, a heterologous coding sequence encoding the desired biomolecule or heterologous protein, and a transcription terminator. The biomolecule or heterologous coding sequence may also contain a secretion signal sequence, a cell wall anchor sequence, and / or a detectable peptide tag. It is noteworthy that multiple promoters in series are utilized and suitable for some constructs. Several copies of these promoters can be used in series to further increase expression. Suitable promoter sequences selected from strain 105 include:

[0591] Bacillus subtilis #105 tuf promoter (SEQ ID NO: 66)

[0592]

[0593] Bacillus subtilis #105 SigX promoter (SEQ ID NO: 67)

[0594]

[0595] Bacillus subtilis #105 groS promoter (SEQ ID NO: 68)

[0596]

[0597]

[0598] Bacillus subtilis #105 ftsH promoter (SEQ ID NO: 69)

[0599]

[0600] Example 11

[0601] Strain 105 was modified to inactivate or delete the protease

[0602] In cases where bacterial host cells must secrete one or more target biomolecules or proteins of interest, inherent environmental proteases, including native bacterial proteases produced by the delivery bacteria, can reduce the amount and extent of available active and full-length biomolecules or proteins. Expression of recombinant secretory proteins in Bacillus subtilis is inefficient, with yields lower than expected or even unsuccessful due to degradation of secretory proteins by extracellular proteases (Westers L, Westers H, Quax WJ (2004) Biochim Biophys Acta 1694: 299-310; https: / / doi.org / 10.1016 / j.bbamcr.2004.02.011). Bacillus subtilis has eight natural extracellular proteases, designated NprE, AprE, Epr, Bpr, Mpr, NprB, Vpr, and WprA (Jeong H et al. (2018) Microbiol Resour Announc 7:e01380-18; doi.org / 10.1128 / MRA.01380-18). To improve the stability and / or systemic activity of secreted proteins, extracellular protease-deficient mutants were constructed.

[0603] Although one factor limiting the use of Bacillus subtilis as an expression host is its production of at least eight extracellular proteases, researchers have also reported that some proteases sometimes favor secretion of foreign proteins. Therefore, to maximize foreign protein production, proteases can be selectively inactivated. Consequently, optimal protease-deficient hosts are constructed by inactivating the least favorable proteases.

[0604] Zhao and colleagues investigated the inactivation of various and combined proteases in Bacillus subtilis and evaluated the production of non-native proteins by protease mutants, specifically α-amylase (AmyM) (Corallociccus sp.), methylparathion hydrolase (MPH) (Plesiomonas sp.), and chlorothalonil hydrodehalogenase (Chd) (Pseudomonas sp.) (Zhao L. et al. (2019) Biotechnology and Engineering 116:2052-2060). The study showed that the subtilis proteases AprE and NprE contributed the majority of the extracellular protease activity. The remaining significant protease activities were fulfilled by Epr, NprB, Bpr, Vpr, WprA, and Mpr, which is consistent with previous reports (Ferrari, Jarnagin, and Schmidt (1993) Bacillus subtilis and other Gram-positive bacteria 263:917-937). For the production of secreted AmyM and Chd proteins, NprE, AprE, and Epr protease-deficient mutants and NprE, AprE, NprB, Vpr, and WprA-deficient mutants, respectively, were shown to provide optimal production. In summary, it is clear that the secretion level of target proteins can be improved by inactivating extracellular proteases.

[0605] The sequences of eight natural extracellular proteases NprE, AprE, Epr (Epr1 and Epr2), Bpr, Mpr, NprB, Vpr and WprA from Bacillus subtilis strain 105 are described below. These sequences are targeted by inactivation or deletion using recombinant techniques and genetic manipulation of the 105 genomes. For example, deletion can be achieved by inserting a flanking nucleic acid sequence that replaces selected and targeted protease sequences using the N-terminal and C-terminal genomic sequences provided below and / or using the N-terminal or C-terminal sequences that are connected to a heterologous or selective sequence. Recombination and gene replacement can be selected and / or detected using means and methods known and generally acknowledged to those skilled in the art.

[0606] Gene encoding Bacillus #105 protease

[0607]

[0608]

[0609] The encoded nprE protease is (SEQ ID NO: 71):

[0610]

[0611]

[0612]

[0613]

[0614]

[0615] The encoded vpr protease is: (SEQ ID NO: 74)

[0616]

[0617]

[0618]

[0619]

[0620]

[0621] The encoded nprB protease is (SEQ ID NO: 79):

[0622]

[0623]

[0624] The encoded aprE protease is: (SEQ ID NO: 81)

[0625]

[0626] Using the ComKS inducible strain described in Example 7 as a basis (this genetically modified strain has enhanced competence), protease encoding genes such as nprE and vpr were deleted from Bacillus subtilis #105. Deletion of the wprA, nprB and aprE genes of Bacillus subtilis #105 was also performed.

[0627] The protease encoding genes nprE and vpr were deleted from B. subtilis #105 and confirmed by PCR and sequencing (data not shown).

[0628] Example 12

[0629] Integration of nucleic acids or expression cassettes encoding biomolecules or heterologous proteins into the genome of Bacillus subtilis strain 105

[0630] The sequence or coding region encoding the desired biomolecule or heterologous protein can be integrated into the chromosome of the genetically modified microorganism of Bacillus subtilis strain 105. This is an alternative to expressing one or more biomolecules or heterologous proteins on a plasmid, which may result in stability issues and copy number problems that may limit the suitability for biomolecule or foreign protein delivery.

[0631] One of the strategies for introducing new genes into bacterial hosts is homologous recombination between double-stranded DNAs of identical sequences. The frequency of recombination can depend on the length of homology and host factors. Antibiotic resistance genes are used to construct integration vectors and integration steps to promote and select recombination events and integration. If a plasmid (integration vector) containing two DNA fragments (fragment A and fragment B) homologous to certain parts of the chromosome (A and B sequences on the chromosome) and a target gene (gene X) is placed between these two fragments, a double crossover event will result in gene X being integrated into the chromosome between fragments A and B. The original DNA sequence of the chromosome between A and B will be replaced by gene X. The integration site is determined by the sequences of A and B. The accuracy of recombination is achieved by pairing the complementary DNA chains from the plasmid and the chromosome.

[0632] In the case of a single crossover event, the entire plasmid will be integrated into either A or B. Then, in the case of a second crossover, the plasmid sequence will be eliminated from the chromosome, and the X gene will be integrated between sequences A and B. Homologous recombination is used in step (1) to integrate the entire plasmid (integrating vector) into the chromosome (single crossover), and then in step (2) further recombination events remove all foreign DNA from the chromosome, including the plasmid replication origin and antibiotic resistance genes (double crossover). The initial integration step (1) can be monitored by the acquisition of antibiotic resistance, and the second step of removal of plasmid / vector sequences can be monitored by the loss of antibiotic resistance. Complete and proper integration is confirmed by PCR of the target integration site region of the strain chromosome and sequencing across this region.

[0633] Chromosomal integration can be achieved using suicide vectors.Suicide vectors contain an origin of replication for replication in E. coli, a drug resistance marker for selection, and an expression cassette flanked by nucleic acids homologous to specific regions of the chromosome.

[0634] For chromosomal integration, one or more Bacillus subtilis genes can be interrupted by inserting an expression cassette (notably, this can also be used to inactivate a Bacillus subtilis gene and replace it with a gene or nucleic acid encoding a target biomolecule or heterologous protein). The heterologous sequence can be integrated into the genome of strain 105. For example, the location of non-essential genes can be screened or selected for integration. Integration can be achieved by replacing a non-essential gene with another target sequence, such as a sequence encoding a biotherapeutic molecule, polypeptide, antigen, therapeutic molecule, immunomodulatory molecule, antibody, or fragment thereof (including VHH antibodies or Nanobodies, etc.).

[0635] Suitable and applicable integration sites include genes for α-amylase (amyE), nprE, apr, and wprA. The nprE, apr, and wprA genes encode proteases, and integration at these gene sites replaces the respective genes for integration of the target heterologous sequence and also inactivates the proteases. The gene maps of amyE, nprE, apr, and wprA on the genome of Bacillus subtilis strain 105 are shown in FIG. Figure 2 The native strain 105 sequences of nprE, apr, and wprA, respectively, on the Bacillus subtilis strain 105 genome are provided in Example 11 above.

[0636] α-Amylase is an enzyme that hydrolyzes the α-bonds of large α-linked polysaccharides. Deletion or inactivation of the amyE gene encoding α-amylase in Bacillus subtilis is well tolerated and does not adversely affect bacterial growth. Gene integration at the amyE locus is utilized and is further described and provided in the Examples. The amyE gene sequence from Bacillus subtilis strain 105 is provided below:

[0637]

[0638]

[0639] One or more biomolecules or heterologous proteins can be integrated into the Bacillus subtilis 105 strain genome for production or delivery via the modified Bacillus subtilis 105 strain. Integration can occur at one or more sites within the Bacillus subtilis 105 strain genome. For example, a first construct providing one or more of the first group of one or more biomolecules or heterologous proteins can be integrated at a site selected from amyE, nprE, apr, and wprA, and a second construct providing one or more of the second group of one or more biomolecules or heterologous proteins can be integrated at a site selected from amyE, nprE, apr, or wprA. A first construct providing one or more of the first group of one or more biomolecules or heterologous proteins can be integrated at the amyE site, and a second construct providing one or more of the second group of one or more biomolecules or heterologous proteins can be integrated at a site selected from nprE, apr, and wprA. Other suitable genes and sites for integration are also contemplated and can be selected from one or more naturally occurring lytic enzymes and / or antimicrobial peptides, as provided in Example 15.

[0640] Example 13

[0641] Loss of native lytic enzymes and / or antimicrobial peptides

[0642] Bacillus subtilis strain 105 is modified to lack one or more natural lytic enzymes and / or antimicrobial peptides. These one or more deletions are used to reduce the genome size of the Bacillus subtilis strain. It can also remove the potential antimicrobial activity of the strain, which may be detrimental to their growth or colonization. The reduced genome is used to enable the insertion of larger coding cassettes or heterologous sequences that are used to encode or produce biomolecules or homologous or heterologous sequences. In addition, the reduced genome size can promote improved and / or faster or more efficient growth of the bacteria. This further helps to improve the expression and production of biomolecules or heterologous proteins by the modified bacteria.

[0643] For example, native Bacillus subtilis strain 105 lytic enzymes for deletion include one or more of the following:

[0644]

[0645]

[0646] Example 14

[0647] Production of γ-polyglutamic acid

[0648] γ-Polyglutamic acid (poly-γ-glutamic acid; (γ-PGA)) is a naturally occurring biopolymer made from repeating units of L-glutamic acid, D-glutamic acid, or both. Because some bacteria can biosynthesize γ-PGA in large quantities from renewable biomass, γ-PGA is considered a promising biobased chemical and has been widely used in the food, medical, and wastewater industries due to its biodegradability, nontoxicity, and nonimmunogenicity. As a biodegradable, water-soluble, edible, and nontoxic biopolymer, γ-PGA and its derivatives can be safely used in a wide range of applications, including thickeners, humectants, bitterness relief agents, cryoprotectants, sustained-release materials, drug carriers, heavy metal absorbents, and animal feed additives. Peptidoglycan-bound γ-PGA can protect bacterial cells from bacteriophage infection and block the entry of antibodies into bacteria. Furthermore, γ-PGA can be used as an oral therapeutic for diabetes in dogs and cats. Dietary γ-PGA has been shown to have a blood sugar-lowering effect.

[0649] Figure 3A The pathway for poly-gamma-glutamic acid biosynthesis is shown. The natural locus for PGA production in Bacillus subtilis strain 105 is shown in FIG. Figure 3B The native Bacillus subtilis locus includes capC, capB, and capA encoded by a single promoter. The sequence of the CapABC locus is as follows:

[0650] CapABC locus sequence (SEQ ID NO: 87)

[0651]

[0652]

[0653]

[0654] Bacillus subtilis strain 105 is modified to produce increased amounts of poly-gamma-glutamic acid. Bacillus subtilis strain 105 is modified to produce inducible poly-gamma-glutamic acid. The strain 105 capABC locus is modified to add an inducible promoter in place of the native promoter. The strain 105 capABC locus is modified to replace the native promoter with one or more promoters, including tandem promoters. Exemplary promoters are provided in Example 10. In an alternative approach, additional capABC loci are integrated into the genomic sequence of strain 105, the loci including alternative, inducible, or tandem promoters, to provide for enhanced or increased or inducible production of proteins encoding the capABC loci.

[0655] Genes suitable for integration sites include amyE, nprE, apr, and wprA. The nprE, apr, and wprA genes encode proteases, and integration at these gene sites replaces the respective genes for integration of the target heterologous sequence and also inactivates the proteases. The gene maps of amyE, nprE, apr, and wprA on the genome of Bacillus subtilis strain 105 are shown in FIG. Figure 2 The native strain 105 sequences of nprE, apr, and wprA, respectively, on the B. subtilis strain 105 genome are provided above in Example 11. The amyE sequence is provided in Example 12 as SEQ ID NO: 111.

[0656] Example 15

[0657] Production of other biomolecules and / or heterologous proteins

[0658] The native Bacillus subtilis strain 105 was genetically modified to express a variety of biomolecules and heterologous proteins. Several classes of biomolecules and heterologous proteins are provided and described below.

[0659] I. Lysine

[0660] The desired biomolecule can be a biomolecule with anti-infective activity. The anti-infective activity can be the lysis of pathogenic bacteria by a lytic enzyme (e.g., a lytic enzyme from a bacteriophage) that is specific for a particular genus of pathogenic bacteria. Suitable exemplary lytic enzymes are known and available to those skilled in the art. Phage-associated lytic enzymes have been identified and cloned from various bacteriophages, each of which has been shown to effectively kill specific bacterial strains. U.S. Patents 7,402,309, 7,638,600, and published PCT application WO 2008 / 018854 provide different phage-associated lytic enzymes that can be used as antibacterial agents for treating or reducing anthrax infection. U.S. Patent 7,569,223 describes a lytic enzyme for Streptococcus pneumoniae. Lysine useful for enterococci (E. faecalis and E. faecium, including vancomycin-resistant strains) is described in U.S. Patent 7,582,291. Lysines are unique in that they are generally bacterial species specific and do not affect or kill normal intestinal bacterial flora, so the normal flora is likely to remain largely intact (MJ Loessner et al. (1995) Mol Microbiol 16:1231-41). Targeting bacterial pathogens that colonize the gastrointestinal tract using Bacillus strain 105 modified to produce one or more lysines specific to these digestive or intestinal pathogens is an application of this system and method.

[0661] Lytic enzymes for expression by the genetically modified Bacillus subtilis strain 105 may include PlyCM, a lytic enzyme targeted to Clostridium perfringens, encoded by the following sequence: (SEQ ID NO: 88)

[0662]

[0663] CP025C, a lytic enzyme targeting Clostridium perfringens, is encoded by the following sequence: (SEQ ID NO: 89)

[0664]

[0665]

[0666] Lysostaphin is an antimicrobial lytic peptide originally isolated from Staphylococcus aureus. It acts as a bacteriocin (bactericidal) against various bacteria, particularly Staphylococci (Kumar, JK (2008) Appl. Microbiol. Biotechnol. 80:555-561; do Carmo de Freire Bastos, M et al (2010) Pharmaceuticals 3:39-1161; doi:10.3390 / ph3041139). The cell wall-degrading activity of lysostaphin is primarily due to glycylglycine endopeptidase activity, which is capable of lysing many Staphylococcal strains. Like many lysine and antimicrobial lytic peptides, the lysostaphin molecule consists of two distinct domains: (i) an N-terminal peptidase domain, responsible for the protein's catalytic activity, and (ii) a C-terminal targeting domain (CWT), which is associated with peptidoglycan substrate binding. The C-terminal 92 amino acid residues of lysostaphin are dispensable for enzymatic activity but are necessary and sufficient to direct lysostaphin to the cell wall envelope of S. aureus. The amino acid sequence of mature lysostaphin is as follows:

[0667]

[0668] II antimicrobial peptides

[0669] Many peptides with intrinsic antimicrobial activity have been described. Antimicrobial peptides (AMPs) are alternatives to traditional antibiotics. Bacteriocins are a group of antimicrobial peptides produced by bacteria that are able to control clinically relevant susceptible and resistant bacteria. Bacteriocins are protein or peptide toxins produced by bacteria to inhibit the growth of similar or closely related strains. They are diverse in structure, function and ecology. A wide range of antimicrobial peptides are secreted in plants and animals to combat foreign viral, bacterial or fungal attacks (Boman, HG (2003) J. Intern. Med. 254 (3): 197-215). These form part of the innate immune response to infection, which is short-term and fast-acting compared to humoral immunity.

[0670] AMPs are found in all domains of life, not just bacteria, and are part of innate immunity, serving as the first line of defense against infection (Zasloff M. (2002) Nature 415:389-95). Despite their diverse origins and sequences, many AMPs, particularly cationic antimicrobial peptides, typically have a significant proportion of hydrophobic amino acids (=>30%), an overall positive charge (+2 to +11), and are relatively short, consisting of 10-50 amino acids (Hancock REW, Sahl HG (2006) Nat Biotech 24:1551-157). Proteins or polycationic amino acid peptides containing combinations of one or more repeating units of cationic amino acids, such as arginine (R), tryptophan (W), lysine (K), and even synthetic polyarginine, polytryptophan, and polylysine, have been shown to kill microbial cells. These peptides cross the plasma membrane to facilitate the uptake of various biopolymers or small molecules (Mitchell DJ et al. (2002) J Peptide Res 56 (5): 318-325). Based on these properties, AMPs are able to fold into amphipathic three-dimensional structures and are generally classified based on their secondary structure as α-helical, β-sheet, or peptides with extended / random helical structures. To date, most characterized AMPs belong to the α-helical or β-sheet peptide families (Takahashi D, Shukla SK, Prakash O, Zhang G. (2010) Biochimie pp. 1236±1241; Nguyen LT, Haney EF, Vogel HJ. (2011) Trends in Biotechnology pp. 464-472).

[0671] A.Methicillin

[0672] Mesacillin is a peptide with antimicrobial activity, a bacteriocin. Several mesacillin peptides have been identified and characterized from Lactobacillus species, particularly Lactobacillus reuteri. The probiotic and direct-feed microorganisms Lactobacillus reuteri strains 3630 and 3632, and the mesacillin peptides produced therefrom, are described and detailed in WO 2020 / 163398, published on August 13, 2020; US 2022 / 0088094, published on March 24, 2022; and US 2022 / 0125860, published on April 28, 2022.

[0673] Bacillus subtilis strain 105 was modified to produce the antimicrobial peptide mericidin, which was partially identified from Bacillus reuteri strain 3632. The nucleic acid encoding mericidin (mericidin-E1) is: (SEQ ID NO: 91)

[0674]

[0675] The nucleic acid encodes a polypeptide: (SEQ ID NO: 92)

[0676]

[0677] The nucleic acid encoding another mericidin (mericidin-E2) is: (SEQ ID NO: 93)

[0678]

[0679] The nucleic acid encodes a polypeptide: (SEQ ID NO: 94)

[0680]

[0681] B. Cathepsins and CAP18

[0682] Cathelicidins represent a new family of gene-encoded antimicrobial peptides in vertebrates that play a key role in the host's immune response to microbial infection (Reddv, K et al. (2004) Int J Antimicrob Agents 24: 536-547, doi: 10.1016 / J.jantimicag.2004.09.05). Due to their potent antimicrobial activity and bacterial resistance, cathelicidin-derived peptides are considered potential alternatives to traditional antibiotics (Hancock RE and Sahl HG (2006) Nat Biotechnol 24: 1551-1557, doi: 10.1038 / nbtl267). They generally have broad-spectrum antibacterial activity against bacteria, including clinically isolated drug-resistant strains, enveloped viruses, fungi, and even parasites (Giacometti, A et al. (2003) J Antimicrob Chemother 51:843-847, doi:10.1093 / jac / dkg149; Rapala-Kozik M et al. (2015) Infect Immun 83:2518-2530, doi:10.1128 / IAI.00023-15; Tripathi S et al. (2014) J Leukoc Biol 96:931-938, doi:10.1189 / jlb.4A1113-604RR).

[0683] Cathestatins are generally characterized by an N-terminal signal peptide, a highly conserved cathelin domain, followed by a C-terminal mature peptide with significant structural variation (Zanetti, M et al. (2000) Adv Exp Med Biol 479:203-218, doi: 10.1007 / b112037). Most cathepsin inhibitors are hydrophobic and cationic, which endows these small peptides with a unique antibacterial mechanism that is different from traditional antibiotics. That is, cathepsin inhibitors easily adhere to negatively charged bacterial membranes and form lipophilic anchors within minutes, inducing membrane destruction and cell death, limiting the chance of drug resistance through bacterial gene mutation (Reddy, K et al. (2004) Int J Antimicrob Agents 24: 53-547, doi: 10.1016 / J.ijantimicag.2004.09.005; Ling et al. (2014) PloS ONE 9, e93216, doi: 10.1371 / joumal.pone.0093216)

[0684] Recent evidence suggests that in addition to their antimicrobial effects, cathepsin inhibitors also have anti-inflammatory activity during pathogen infection (Bowdish, DM et al. (2005) J Leukocyte Biol 77:451-459, doi:10.1189 / jlb.0704380; Finlay BB and Hancock RE (2004) Nat Rev Microbiol 2:497-504, doi:10.1038 / nrmicro908). Cathepsin-derived peptides have great potential as medical coating materials and antimicrobial agents for controlling various infections (Ong ZY et al. (2013) Adv Funct Mater 23:3682-3692, doi:10.1002 / marc.201300538; Shukla A et al. (2010) Biomaterials 31:2348-2357, doi:10.1016 / j.biomaterials.2009.11.082).

[0685] A cathepsin inhibitor (Hc-CATH) with potent bactericidal activity has been described from the sea snake (Hymenoptera cyanocephalans). It consists of 30 residues and primarily adopts an α-helical conformation (Wei L et al. (2015) J Biol Chem 290:16633-16652, doi:10.1074 / jbc.M115.624645). Peptide variants and hybrid peptides of Hc-CATH have also been described (Yu H et al. (2017) Nature Scientific Reports 7:2600; DOI:10.1038 / s41598-017-0250-2).

[0686] CAP18 was originally isolated from rabbit neutrophils and has antibacterial activity against a variety of pathogens, is highly thermostable, and has no hemolytic activity in vitro (Ebbensgaard A, Mordhorst H, Overgaard MT, Nielsen CG, Aarestrup FM, Hansen EB. (2015) PLoS One 10: e0144611). In addition, a recent study evaluated the potential therapeutic effect of CAP18 on redmouth disease caused by Yersinia ruckeri in juvenile rainbow trout by oral or intraperitoneal injection, and compared with untreated fish, injection of CAP18 into juvenile rainbow trout before exposure to Yersinia ruckeri was associated with lower mortality (Chettri JK, Mehrdana F, Hansen EB, Ebbensgaard A, Overgaard MT, Lauritsen AH et al. (2017) J Fish Dis. 40: 97 ± 104). CAP18 has the potential to serve as a lead peptide for further development and optimization.

[0687] Cathepsin inhibitor is an 18kDa cationic antimicrobial protein (CAP18) originally isolated from rabbit granulocytes. The C-terminal 37 amino acids of rabbit CAP18 constitute the lipopolysaccharide binding domain. Synthetic CAP18 106-142 It has been shown to have broad antimicrobial activity against both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium (Larrick JW et al. (1993) Antimicrobial Agents Chemotherapy 37(12): 2534-2539).

[0688] Rabbit CAP1837 amino acid peptide has the sequence: (SEQ ID NO: 95)

[0689]

[0690] The antibacterial activity of human CAP18 peptide has been evaluated (Larrick JW et al. (1995) Immunotechnology 1: 65-72). Human CAP 18, or cathepsin inhibitor peptide, also known as LL37, has been shown to regulate immunity by recruiting neutrophils, monocytes, and T cells during bacterial infection (Ciornei CD et al. (2005) Agents Chemother 49: 2845-2850, doi: 10.1128 / AAC.49.7.2845-2850.2005; De Y et al. (2000) J Exp Med 192: 1069-1074, doi: 10.1084 / jem.192.7.1069). Human CAP18 peptide LL37 has the following sequence:

[0691]

[0692] Other antimicrobial cathepsin inhibitors from different species include BMAP28 (CATHL5; bovine), Bac7 (CATHL3; bovine rumen), k9Cath (canine), and PMAP36 (porcine).

[0693] III antibodies—including single-chain or single-domain antibodies

[0694] Nanobodies (also known as VHHs) are small, low molecular weight, single domain, heavy chain-only antibodies found in camels. Due to their small size, the genes for these proteins are easily cloned in plasmids. Therefore, by using molecular cloning techniques, nanobodies against various antigens can be presented in the systemic circulation. Bacillus subtilis strain 105 is modified to include a heterologous coding region encoding a desired biomolecule, which can be a nanobody, or can encode one or more nanobodies. The desired biomolecule can be a biomolecule with anti-infective activity. The anti-infective activity can be a toxin that inhibits or neutralizes the production of pathogens. The inhibition or neutralization can be accomplished with single-chain antibodies.

[0695] For example, lactobacillus has been described as an expression system for single-chain antibodies against host adhesion factors (WO 2012 / 019054). Lactobacillus reuteri strains 3630 and 3632 are described and detailed as probiotic strains in WO 2020 / 163398 published on August 13, 2020, and corresponding US 2022 / 0088094 published on March 24, 2022, and US 2022 / 0125860 published on April 28, 2022. Live delivery systems based on Lactobacillus reuteri strains 3630 or 3632 are described and detailed in PCT / US2020 / 016522 filed on April 2, 2020, and disclosed as WO 2020 / 163284 on August 13, 2020. This application describes native bacterial promoters, signal sequences and vectors suitable for expression, and bacterial genomic sites / genes for integration to generate stable modified strains. Recombinant lactobacilli (Lactobacillus reuteri strain 3630 and Lactobacillus reuteri strain 3632) delivering nanobodies against NetB and alpha toxin of Clostridium perfringens have been described and shown to provide protection against necrotic enteritis in poultry (Gangaiah D et al. Microbiology Open 2022; 11: e1270, doi.org / 10.1002 / mb03.1270).

[0696] A. Clostridium perfringens toxin antibodies

[0697] The toxins targeted by the single-chain antibodies include Clostridium perfringens alpha toxin and NetB. Camelid heavy chain-only (VHH) antibodies were generated against Clostridium perfringens alpha toxin and NetB. Briefly, two llama calves were immunized with recombinant alpha toxin or NetB variant W262A, respectively. Both immunogens were non-hemolytic. The immunized camels were boosted twice with toxin peptides. On days 44 and 72 after the initial immunization, blood samples were collected and RNA was isolated for phage library construction. The phage library was screened for binding activity to each of the two toxins. The candidate antibodies were sequenced and further screened in bioassays.

[0698] alpha toxin antibodies

[0699] Alpha toxin causes membrane damage to a variety of erythrocytes and cultured cells. It is preferentially active against phosphatidylcholine (PC or phosphatidylcholine) and sphingomyelin (SM), two major components of the outer leaflet of eukaryotic cell membranes. The N-terminal domain is fully active against phosphatidylcholine but lacks sphingomyelinase activity and is not hemolytic or cytotoxic. The C-terminal domain has no enzymatic activity, but the interaction between the N-terminal and C-terminal domains is essential for conferring sphingomyelinase, hemolytic, and cytotoxic activity to the toxin. Although alpha toxin is a potent hemolysin, lysis of erythrocytes has been seen only in experimental animals following intravenous injection of the toxin or in the setting of Clostridium difficile sepsis.

[0700] The ability of VHH antibodies against α-toxin to inhibit the phosphatase activity of α-toxin was determined by measuring their effect on egg yolk lipoproteins. Fresh egg yolks were centrifuged (10,000 x g, 20 minutes at 4°C) and diluted 1:10 in PBS. Before adding 10% egg yolk emulsion, a two-fold dilution series of VHHs (two wells per dilution, 5 μM starting concentration) was added to a constant amount of α-toxin (5 μg / ml recombinant α-toxin or 3.33 x 10 -4 The ability of VHHs to neutralize alpha-toxin activity was assessed by preincubation with 100 U / μl alpha-toxin (Sigma, P7633) at 37°C for 30 minutes. Serum from calves immunized with recombinant alpha-toxin was used as a positive control, starting at a 1:4 dilution. After incubation at 37°C for 1 hour, absorbance at 650 nm (A650) was measured. Alpha-toxin activity is indicated by an increase in turbidity, which results in an increase in absorbance.

[0701] The control serum was able to neutralize the phosphatidylcholine phosphatase activity of both commercially available and recombinant α-toxins. An eight-fold dilution of the antiserum (equivalent to 3.12% serum) was able to completely neutralize the α-toxin selective endoenzyme activity of the recombinant α-toxin, while only the highest dilution of the antiserum (equivalent to 25% serum) was able to completely neutralize the phosphatidylcholine phosphatase activity of the commercially available α-toxin. Differences in inhibitory ability were observed between the five candidate VHH antibodies. VHH EAT-1F3 had no effect on the phosphatidylcholine phosphatase activity of any α-toxin. The neutralizing abilities of EAT-1A2 and EAT-1C8 were very similar and were the same for both recombinant and commercially available α-toxins. The maximum inhibitory ability was maintained up to a 32-fold dilution of the VHH (0.16 μM VHH). However, neither EAT-1A2 nor EAT-1C8 was able to completely neutralize the phosphatidylcholine phosphatase activity, resulting in 40% to 50% residual phosphatidylcholine phosphatase activity. Two other VHHs, EAT-1F2 and EAT-1G4, showed differences in their neutralization potency against recombinant and commercial α-toxin. EAT-1F2 had high neutralization potency against recombinant α-toxin but was unable to completely neutralize commercial α-toxin, resulting in approximately 25% residual phosphatase activity. Compared to EAT-1F2, EAT-1G4 was able to 100% neutralize the phosphatase activity of commercial α-toxin but had a weaker ability to neutralize recombinant α-toxin.

[0702] Neutralization of the hemolytic activity of α-toxin by VHH antibodies against α-toxin was determined by measuring their effect on sheep erythrocytes. Similar to the inhibition of α-toxin phosphatase activity, the ability to neutralize hemolytic activity was determined by incubating a two-fold dilution series of VHH antibodies (two wells per dilution, starting at 5 μM) with a constant amount of α-toxin (6.25 x 10 -5 U / μl α-toxin (Sigma, P7633) was pre-incubated for 30 minutes to assess the effect. As a positive control, serum from calves immunized with recombinant α-toxin was used, starting at a 1:4 dilution. After incubation at 37°C for 1 hour, the plates were centrifuged to pellet intact erythrocytes. The supernatant was transferred to a new 96-well plate and the A 550 Alpha-toxin activity is indicated by an increase in absorbance caused by the release of hemoglobin from erythrocytes.

[0703] The ability of VHH antibodies to inhibit the hemolytic activity of α-toxin was determined using only commercially available α-toxin, as recombinant α-toxin does not show hemolytic activity. Control serum diluted up to 16 times (equivalent to 1.56% serum) completely inhibited α-toxin hemolysis. In contrast, none of the candidate VHHs had an effect on the hemolytic activity of α-toxin. Because the control serum contained polyclonal antibodies and the VHHs were monoclonal, the combined effect of all five VHHs on α-toxin was determined (1 μM of each VHH at the highest dilution, equivalent to a total of 5 μM of VHHs). The combined VHHs had no effect on α-toxin hemolysis.

[0704] Based on the above results, VHH antibodies EAT-1F2 and EAT-1G4 were selected for further characterization and expression. The peptide sequence of EAT-1F2 is: (SEQ ID NO: 97)

[0705]

[0706] The peptide sequence of EAT-1G4 is: (SEQ ID NO: 98)

[0707]

[0708] Those skilled in the art will recognize that, due to the redundancy of the genetic code, multiple nucleic acid sequences may encode the above peptides. Even so, an exemplary sequence encoding EAT-1F2 is: (SEQ ID NO: 99)

[0709]

[0710] An exemplary sequence encoding EAT-1G4 is: (SEQ ID NO: 100)

[0711]

[0712] NetB antibody

[0713] NetB is a heptameric β-pore-forming toxin that forms a single channel in planar phospholipid bilayers. NetB activity is influenced by membrane fluidity and cholesterol, which enhances NetB oligomerization and plays a key role in pore formation. NetB has high hemolytic activity against avian erythrocytes.

[0714] The neutralization of NetB hemolytic activity by Camelidae VHH antibodies against NetB was determined by measuring NetB-mediated lysis of chicken erythrocytes. The ability to neutralize NetB hemolytic activity was assessed by preincubating a two-fold dilution series of VHH antibodies (two wells per dilution, starting at a concentration of 5 μM) with a constant amount of NetB toxin (20 μg recombinant NetB) for 30 minutes at 37°C before adding 1% chicken erythrocytes. The avirulent NetB variant W262A was included as a negative control because this variant did not show hemolytic activity. A positive control serum from a rabbit immunized with recombinant NetB (wild-type NetB) was used, starting at a 1:4 dilution. After incubation for 1 hour at 37°C, the plates were centrifuged to pellet intact erythrocytes. The supernatant was transferred to a new 96-well plate and the A 550 NetB activity is indicated by the increase in absorbance caused by the release of hemoglobin from erythrocytes.

[0715] Control serum neutralized the hemolytic activity of NetB. VHH antibodies ENB-1F4 and ENB-1F10 had no effect on NetB hemolysis. ENB-1B9 had moderate inhibitory potency, while ENB-1D11 and ENB-1A4 neutralized NetB hemolysis at 4- to 8-fold dilutions (1.25 μM to 0.625 μM VHH).

[0716] Based on the above results, VHH antibodies ENB-1A4 and ENB-1D11 were selected for further characterization and bacterial expression. The peptide sequence of ENB-1A4 is: (SEQ ID NO: 101)

[0717]

[0718] The peptide sequence of ENB-1D11 is: (SEQ ID NO: 102)

[0719]

[0720] Those skilled in the art will recognize that due to the redundancy of the genetic code, multiple nucleic acid sequences can encode the above peptides. Even so, an exemplary sequence encoding ENB-1A4 is: (SEQ ID NO: 103)

[0721]

[0722] An exemplary sequence encoding ENB-1G4 is: (SEQ ID NO: 104)

[0723]

[0724]

[0725] IV delivery of antigens as therapeutic vaccines

[0726] The delivery of antigens in immunomodulatory, immunostimulatory or vaccine strategies is an important and feasible application of the platform of the present invention. Bacillus subtilis strain 105 is modified and used to produce antigens that can be used as immunogenic polypeptides to stimulate an immune response and promote immunity, such as immunity against infection by a pathogenic agent or pathogen in an animal. In some embodiments, Bacillus subtilis strain 105 is modified and used to produce one or more or a combination of related antigens that are used alone or collectively as immunogenic polypeptides to stimulate an immune response and promote immunity, particularly enhanced immunity or a broader, more effective immune response against a pathogenic agent or pathogen, including in applications as an immunogen, immunostimulant or vaccine.

[0727] Avian coccidiosis is a common poultry disease caused by Eimeria. Eimeria is a genus of parasites that includes various species that can cause coccidiosis in cattle, poultry, dogs (especially puppies), cats (especially kittens), and smaller ruminants such as sheep and goats. Eimeria infects a wide range of hosts. The most common Eimeria species that cause coccidiosis in cattle are E. bovis, E. zurei, and E. obos. It is estimated that as few as 50,000 infective oocysts in a young, susceptible calf can cause severe disease. Eimeria infection is particularly devastating to the poultry industry, resulting in losses exceeding $1.5 billion annually in the United States. The economically most important species in poultry are E. tenella, E. acervulina, and E. maxima.

[0728] To produce and provide immunogenic compositions or coccidial vaccines for poultry, Bacillus subtilis strain 105 was modified to deliver cross-protective antigens covering Eimeria parasites, including Eimeria tenella, Eimeria maxima, and Eimeria acervulina. Eimeria antigens, including Eimeria tenella elongation factor-1α, EtAMA1, EtAMA2, Eimeria tenella 5401, Eimeria acervulina lactate dehydrogenase antigen genes, Eimeria maxima surface antigen genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Eimeria common antigen 14-3-3, were cloned into plasmids or integrated into the genome of strain 105 as target applicable genes. Expression of the Eimeria antigens delivered by Bacillus subtilis 105 in poultry provides vectored delivery of immunogens to stimulate an immune response and provide protection or immunity against Eimeria in animals.

[0729] Coccidia antigen sequence:

[0730]

[0731]

[0732]

[0733] Em surface antigen (SEQ ID NO: 108)

[0734]

[0735] EtAMA1 (extracellular domain) (SEQ ID NO: 109)

[0736]

[0737]

[0738] Example 16

[0739] Engineering bacteria to include biosynthetic gene clusters (BCGs)

[0740] A biosynthetic gene cluster (BGC) is a group of genes in bacteria that work together to produce or generate one or more molecules or proteins, or in some instances, a protein complex, that provides one or more activities or related activities and / or provides related or ultimate functions. The clustering of a group of genes can allow or enable the timed and coordinated synthesis of, for example, proteins involved in a pathway. The proteins can be under the control of multiple promoters, or transcribed by a single promoter or promoter group.

[0741] A. Engineering of a Polyketide Synthase (PKS) Biosynthetic Cluster from Lactobacillus reuteri to Bacillus subtilis

[0742] Polyketide synthases (PKSs) are secondary metabolites produced by biosynthetic gene clusters (BGCs) that assemble simple molecules into complex metabolites with potential therapeutic value. The gut microbiome encodes several BGCs that produce secondary metabolites that directly interact with the host immune system. Of particular importance are BGCs that encode AhR-activating metabolites. AhR is a ligand-activated transcription factor that recognizes environmental pollutants, dietary compounds (i.e., glucosinolates and flavonoids), and microbially derived secondary metabolites (i.e., indole-3-carbinol). Upon ligand binding, AhR translocates to the nucleus to induce target gene expression. The role of AhR in the metabolism of environmental toxins has been extensively studied, but recent focus has shifted to its role in regulating the adaptive and innate immune systems. AhR is a ligand-activated transcription factor that plays a key role in a variety of diseases, including ameliorating intestinal inflammation. Ozcam et al. have shown that some strains of Lactobacillus reuteri can activate the aryl hydrogen receptor (AhR) and that this activation is associated with the presence of a PKS gene cluster and its metabolites (Ozcam M et al. (2019) Appl Environ Microbiology 85(10):e01661-18). Strains with the PKS biosynthesis gene cluster activate AhR and produce a bright orange pigment. Deletion of the PKS gene cluster results in a loss of the ability to activate the AhR receptor. AhR activation by Lactobacillus reuteri has been shown to alleviate Escherichia coli-induced mastitis in mice (Zhao (2021) PLOS Pathogens 17(17):e1009774) and may be an effective approach to combat mastitis in other animals, particularly lactating animals such as cattle. AhR activity and AhR-expressing microbiota communications are influenced by multiple factors, including regulation of immune tolerance and response, intestinal homeostasis, carcinogenesis, and intestinal barrier integrity (Dong F and Perdew GH (2020) Gut Microbes doi.org / 10.1080 / 19490976.202.1859812). AhR has been implicated in various inflammatory and immune-mediated diseases, such as atopic dermatitis.

[0743] Bacillus subtilis strain 105 was modified to introduce a biosynthetic gene cluster from Lactobacillus reuteri that encodes a polyketide synthase that provides and acts as an AhR activating metabolite. In particular, the Lactobacillus reuteri strain is 3632 (ATCC PTA-126788). The metabolites of Lactobacillus reuteri appear to give the strain an orange pigmentation, primarily associated with the cell envelope. Lactobacillus reuteri strain 3632, including its full genome nucleotide sequence, is described in detail in WO 2020 / 163398A1 by Kumar et al., published on August 13, 2020, and the corresponding U.S. publications are US 2022 / 0088094, published on March 24, 2022, and US 2022 / 0125860, published on April 28, 2022, the entire contents of which are incorporated herein by reference. In one particular aspect, Bacillus subtilis strain 105 is modified to introduce a PKS cluster from Lactobacillus reuteri to efficiently produce candidate AhR activating metabolites. In particular, Lactobacillus reuteri strain 3632 (ATCC PTA-126788) contains BGCs encoding a full set of proteins required for the synthesis and production of AhR activating metabolites. Lactobacillus reuteri strain 3632, including its full genome nucleic acid sequence, is described in detail in WO 2020 / 163398A1 by Kumar et al., published on August 13, 2020, and the corresponding U.S. publications are US 2022 / 0088094, published on March 24, 2022, and US2022 / 0125860, published on April 28, 2022, the entire contents of which are incorporated herein by reference.

[0744] The PKS gene cluster of Lactobacillus reuteri strain 3632 is encoded on a 165 kb conjugative plasmid. The biosynthetic gene cluster (BGC) contains 15 genes that encode the complete set of proteins required for the synthesis of AhR-activating metabolites (Table 22). This gene cluster was introduced into Bacillus subtilis strain 105 to enable the modified Bacillus subtilis to synthesize and produce active and potent AhR-activating metabolites.

[0745] Table 22 Genes involved in the synthesis and secretion of guanycin

[0746]

[0747]

[0748] Please note that LREU3632_02265 shown in Table 22 may not be required.

[0749] The PKS gene cluster from Lactobacillus reuteri 3632 was engineered into Bacillus subtilis #105 to efficiently produce (and secrete) AhR-activating metabolites. Chromosomal integration of the BGC cluster was confirmed by PCR and sequencing. The final strain was free of any antibiotic markers.

[0750] In the first initial step (i), the transcriptional regulator pksI gene was deleted from the PKS gene cluster, as it may not be essential. Then (2) the remaining gene cluster and pathway genes were cloned as controls. Using the cloned wild-type AhRPKS BGC, three promoters were inserted—two PxylA promoters and one Physpank promoter—to control gene expression, such as Figure 5 As shown. Physpank and pxl promoters were introduced as promoters for the emrY and fabF2 genes (these genes are encoded in opposite directions on the gene cluster, and the promoters are located on both sides and promote gene expression in opposite directions). A third promoter, pxlA, was introduced in front of and upstream of the fabZ3 gene. The cloned gene cassette construct was sequenced in its entirety to confirm all components and promoters. The cassette was then inserted into a suitable vector, designated in this case as the Bacillus BCG expression vector ( Figure 6 The cassette is flanked by left and right amylase gene amyE arms for homologous recombination and integration at the amylase gene amy E in Bacillus subtilis strains. Bacillus subtilis strain 105 is transformed with the vector under conditions that promote homologous recombination and integration, and PCR screening is then used to screen for the full-length insert to verify the left, right, and middle portions of the Ahr PKS BGC.

[0751] The PKS encoding BGC was successfully engineered into Bacillus subtilis #105 and confirmed by PCR and sequencing (data not shown). Flanking PCR confirmed the correct and complete integration of the PKS gene cluster in the Bacillus subtilis 105 genome (data not shown).

[0752] Bacillus subtilis 105 was genetically modified to integrate a PKS gene cassette into its bacterial genome. Extracts and supernatants from the modified strain were evaluated. These were evaluated in synergy with native Lactobacillus reuteri strain 3632, which expresses an AhR activation product derived from its native PKS gene cassette. The AhR activation product was evaluated in in vitro efficacy assessments against several AhR-responsive cell lines, including HepG2-Lucia (human HepG2 liver carcinoma; Invivogen) and HT-29-Lucia (human HT29 colon carcinoma; Invivoen).

[0753] The AhR activity of the engineered strains was evaluated as follows: The strains were cultured overnight in LB medium. TMAhR cells (Invivogen, hpgl-ahr) were used to assess AhR activity in cell pellets and filter-sterilized culture supernatants. TM AhR cells were grown in MEM (Thermo Fisher, 616965-026) and 10% iFCS at 37°C and 5% CO2. For selection purposes, the medium was supplemented with 100 μg / ml bleomycin (Invivogen, ant-zn-5). FICZ (6-formylindole [3, 2-b] carbazole), an AhR agonist, and L-kynurenine were used as positive controls. The assay also included a medium control and a Bacillus subtilis 105 (ELA191105) parent strain control. The test material was diluted in 40 ml of complete growth medium at a concentration twice the expected final concentration and 40 μl of 2,2 x 10 5 Cells / ml. Culture supernatants were harvested at different time points (over 48 hours) and subjected to luciferase assays. To measure luciferase activity in 384 wells, 10 μl of culture supernatant / well sample was added to 15 μl / well of Quanti-Luc / 50 ml ddH2O (not 25 ml) in coelenterazine-utilizing luciferase detection medium (Invivogen rep-qlc). The reaction was monitored at room temperature using a BioTek microplate reader.

[0754] The in vivo efficacy of AhR metabolites produced by the Bsub-integrated PKS cassette was evaluated using a mouse atopic dermatitis (AD) animal model (Martel BC et al. (2017) Yale J Biol and Med 90:389-402). The AhR activator tapinaraf is in clinical development for the treatment of human psoriasis and atopic dermatitis (AD) (Bissonnette R et al. (2021) J Am Acad Dermatol 84:1059-1067; Lebwohl M et al. (2020) Skin J Cutane Med 4(6):s75). Tapinarof is a secondary metabolite of Photorhabdus luminescens.

[0755] The complete sequence of the PKS cluster and the 5 kb flanking regions on both sides of the BGC, corresponding to a total of 12969 bp, is shown below (SEQ ID NO: 110):

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765] The amino acid sequences of the PKS gene cluster proteins indicated in Table 22 and encoded by the gene cluster are shown below. Also indicated are annotations providing their positions in the above 12969 bp cluster sequence and corresponding genomic map positions.

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776] B. Engineering of the merantidin biosynthetic cluster from Lactobacillus reuteri to Bacillus subtilis

[0777] The lantibiotic peptide merantidin is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by Lactobacillus reuteri and Bacillus amyloliquefaciens. It exhibits antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus, and has potential therapeutic relevance. Merantidin's structure and bioactivity derive from a unique combination of lanthionine ring structures, making it interesting from the perspective of lantibiotic engineering. Lantibiotics are a class of polycyclic peptide antibiotics containing the characteristic thioether amino lanthionine or methyl lanthionine, as well as the unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid. They are ribosomally synthesized and post-translationally modified peptides. These peptides primarily act by disrupting the membrane integrity of the target organism. The production of active lantibiotics in bacteria is typically mediated by gene clusters. Bacterial production of lantibiotics requires a series of steps, including the formation of prolantibiotics, dehydration and cross-linking reactions, leader cleavage, and secretion, and the proteins / enzymes / transporters involved in these necessary steps are encoded and / or regulated by gene clusters.

[0778] Bacillus subtilis strain 105 is modified to introduce the merantisin cluster from Lactobacillus reuteri, thereby efficiently producing merantisin. In one specific aspect, Bacillus subtilis strain 105 is modified to introduce the merantisin cluster from Lactobacillus reuteri, thereby efficiently secreting merantisin. In particular, Lactobacillus reuteri strain 3632 (ATCC PTA-126788) contains BGCs that encode a full set of proteins required for merantisin production. Lactobacillus reuteri strain 3632, including its full genome nucleic acid sequence, is described in detail in WO 2020 / 163398A1 by Kumar et al., published on August 13, 2020, the entire contents of which are incorporated herein by reference.

[0779] Lactobacillus reuteri strain 3632 encodes / produces two mericidins, designated mericidin-1 and mericidin-2, having the amino acid sequences described below:

[0780] Methicillin 1:

[0781]

[0782] Meisacidin 2:

[0783]

[0784] The mericidin cluster from Lactobacillus reuteri strain 3632 is encoded on a 165 kb conjugative plasmid. The BGC contains eight genes that encode the full set of proteins required for the synthesis of mericidin (Table 23). This gene cluster was introduced into Bacillus subtilis strain 105 to enable the modified Bacillus subtilis strain to synthesize and produce activated and potent mericidin.

[0785] Table 23 Genes involved in the synthesis and secretion of mesaccharin

[0786]

[0787] Genes from the merapidine biosynthetic gene cluster (BGC) from lactobacilli, specifically from Lactobacillus reuteri strain 3632, were engineered into Bacillus subtilis #105 to efficiently produce (secrete) merapidine. Chromosomal integration of the BGC cluster was confirmed by PCR and sequencing. The resulting strain was free of any antibiotic markers.

[0788] Three constructs were generated for integration into the Bacillus subtilis 105 genome. In the first initial step and construct, (i) the merantigen pathway gene cluster and the wild-type merantigen pathway were cloned without a promoter as a control sequence. The sequenced fragments were inserted into the Bacillus BGC genomic integration vector ( Figure 6 ), including amyE sequences in the left and right amyE arms for homologous recombination. The cassette is flanked by the left and right amylase gene amyE arms for homologous recombination and integration at the amylase gene amy E in the Bacillus subtilis strain. The construct was then transformed into Bacillus subtilis #105 and PCR was used to screen for full-length insertion to verify the left, right and middle parts of the merantigen BGC. Similarly, using the cloned wild-type merantigen BGC, two promoters were inserted. Construct (2) was generated, comprising: the Physpank promoter in front of (upstream of) LagD and the Pxl promoter in front of the lanthione synthase C-like sequence. The remaining genes in the cassette were retained in tandem ( Figure 7 A). The entire cloning pathway has been sequence confirmed. The fragment was then inserted into the Bacillus BGC genomic integration vector ( Figure 6 ) were homologous recombination was performed in the third step and the third construct (3), the lagD sequence was deleted and a single Pxl promoter was connected in front of the lanthione synthase C-like sequence. The remaining genes in the cassette were retained in tandem ( Figure 7 B). The cloned gene cassette construct is sequenced in its entirety to confirm all components and promoters. The cassette is then inserted into a suitable vector, designated in this case as a Bacillus BCG expression vector ( Figure 6The cassette is flanked by left and right amylase gene amyE arms for homologous recombination and integration at the amylase gene amy E in Bacillus subtilis strains. Bacillus subtilis strain 105 is transformed with the vector under conditions that promote homologous recombination and integration, and PCR screening is then used to screen for the full-length insert to verify the left, right, and middle portions of the Ahr PKSBGC.

[0789] The BGC encoding mericidin was successfully engineered into Bacillus subtilis #105 and confirmed by PCR and sequencing (data not shown). Junctional PCR confirmed that the mericidin BGC was correctly integrated into the Bacillus subtilis #105 genome (data not shown).

[0790] The engineered strains were evaluated for merantidote activity as follows. The strains were cultured overnight in tryptic soy broth. Filter-sterilized culture supernatants were evaluated for merantidote activity by measuring bacterial growth inhibition and / or bactericidal activity using Staphylococcus aureus as an indicator organism. The culture supernatant was serially diluted two-fold in 50 μl of tryptic soy broth and 50 μl of a 5% broth solution containing approximately 1 × 10 5 Staphylococcus aureus (S. aureus) at a concentration of 10 cells / ml was cultured aerobically at 37°C for 24-48 hours. Following incubation, the minimum inhibitory concentration (MIC) of the test material was recorded. Culture supernatant from the parent strain and antibiotics (e.g., oxacillin, vancomycin, linezolid, tetracycline) served as negative and positive controls, respectively.

[0791] The full-length sequence of the mesacidine cluster and the 5 kb flanking regions on both sides of the BGC, corresponding to a total of 8742 bp, is shown below (SEQ ID NO: 24).

[0792]

[0793]

[0794]

[0795]

[0796]

[0797]

[0798] The amino acid sequences of the cluster proteins encoded by the gene clusters indicated in Table 23 are shown below. Annotations providing their positions within the above 8742 bp cluster sequence and corresponding to map positions are also indicated.

[0799]

[0800]

[0801]

[0802]

[0803]

[0804]

[0805] Without departing from its spirit or essential characteristics, the present invention may be embodied in other forms or implemented in other ways. Therefore, the present disclosure is considered in all respects to be illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes within the meaning and range of equivalents are intended to be embraced therein.

[0806] Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

Claims

1. A modified Bacillus for producing or delivering one or more biomolecules or heterologous proteins in vivo, wherein the bacterium comprises a Bacillus subtilis strain 105 (ELA191105) genetically modified in one or more aspects selected from the following: (a) genetically modified to enhance competence; (b) genetically modified to reduce or block sporulation; (c) genetically modified to delete or inactivate one or more native proteases; and (d) genetically modified to include a nucleic acid encoding one or more biomolecules or heterologous proteins.

2. The modified Bacillus according to claim 1, wherein the Bacillus subtilis strain 105 comprises the nucleic acid sequence of SEQ ID NO: 1, or its genomic sequence comprises at least 95%, 97%, 98%, or 99% identity to SEQ ID NO:

1.

3. The modified Bacillus according to claim 1, wherein the Bacillus subtilis strain corresponds to ATCC deposit PTA-126786 strain, or has at least 95%, 97%, 98%, or 99% identity in genome sequence to the sequence of ELA191105 corresponding to ATCC deposit PTA-26786.

4. The modified Bacillus according to claim 1, wherein the Bacillus subtilis strain 105 comprises the nucleic acid sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6, or comprises a nucleic acid having at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 1, 2, 3, 4, 5 or 6 in the genomic sequence.

5. The modified Bacillus according to any one of claims 1 to 4, wherein in (a), the bacterium is modified to overexpress comK, comS, or comK and comS to increase competence. The modified Bacillus according to claim 5 , wherein the gene cassette encoding comK and comS is integrated into the Bacillus subtilis genome.

7. The modified Bacillus according to claim 5 or 6, wherein the competence is increased and the transformation efficiency of the strain is improved by at least at least 20 times; 50 times; 50 times or more; 60 times; 80 times; 80 times or more; 90 times; 100 times; or 100 times or more.

8. The modified Bacillus according to any one of claims 1 to 4, wherein in (b), the bacterium is modified to delete or inactivate one or more native genes encoding Spo0A, SpoIVB, or SpoA and SpoIVB.

9. The modified Bacillus according to any one of claims 1 to 4, wherein in (c), the bacterium is modified to delete or inactivate one or more native proteases or genes encoding one or more native proteases selected from NprE, AprE, Epr1, Epr2, Bpr, Mpr, NprB, Vpr and WprA.

10. The modified Bacillus according to claim 9, wherein the bacterium is modified to delete or inactivate genes encoding native proteases NprE and Vpr.

11. The modified Bacillus according to claim 9, wherein the bacterium is modified to delete or inactivate genes encoding native proteases AprE, NprB and WprA.

12. The modified Bacillus according to any one of claims 1 to 11, which is further genetically modified to delete or inactivate one or more native lytic enzymes or antimicrobial peptides. 13 . The modified Bacillus according to claim 12 , wherein one or more natural lytic enzymes or antimicrobial peptides selected from the group consisting of xpf, lytC1, lytC2 and sdpC are deleted or inactivated.

14. The modified Bacillus according to any one of claims 1 to 13, which is further genetically modified to delete or inactivate one or more native genes encoding virulence factors, toxins or antimicrobial resistance (AMR).

15. The modified Bacillus according to claim 14, wherein the one or more virulence factors, toxins or antimicrobial resistance (AMR) are selected from macrolide 2′ phosphotransferase (mphK), ABC-F type ribosomal protection protein (vmlR), streptothricin-N-acetyltransferase (satA), tetracycline efflux protein (tet(L)), aminoglycoside 6-adenylyltransferase (aadK) (29) and rifamycin inactivating phosphotransferase.

16. The modified Bacillus for producing or delivering one or more biomolecules or heterologous proteins in vivo according to any one of claims 1 to 15, wherein the modified Bacillus comprises a Bacillus subtilis isolate having at least one gene knockout selected from the group consisting of spo0A, spoIIIE, spoIVB, NprE, AprE, NprB, Vpr, and WprA; and one or more heterologous genes encoding one or more biomolecules or heterologous proteins operably linked to one or more promoters selected from the group consisting of a tuf promoter, a sigx promoter, a gros promoter, a fish promoter, a PxylA promoter, a mannose-inducible promoter, and a Physpank promoter.

17. A modified Bacillus for use in the production or in vivo delivery of one or more biomolecules or heterologous proteins according to any one of claims 1 to 15, wherein the modified Bacillus comprises: a Bacillus subtilis strain 105 isolate modified to overexpress comK, comS, or comK and comS to increase competence; a Bacillus subtilis strain 105 isolate having a knockout of at least one gene selected from the group consisting of spo0A, spoIIIE, spoIVB, NprE, AprE, NprB, Vpr, and WprA; and a Bacillus subtilis strain 105 isolate modified to contain one or more heterologous genes encoding one or more biomolecules or heterologous proteins operably linked to one or more promoters selected from the group consisting of tuf promoter, sigx promoter, gros promoter, ftsh promoter, PxylA promoter, a mannose-inducible promoter, and Physpank promoter.

18. The modified Bacillus according to claim 16 or 17, wherein the one or more promoters are selected from SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 40 and SEQ ID NO:

41.

19. The modified Bacillus according to any one of claims 1 to 18, wherein one or more heterologous genes encoding one or more biomolecules or heterologous proteins are integrated into the genome of the host Bacillus subtilis strain 105.

20. The modified Bacillus according to claim 19, wherein the one or more heterologous genes encoding one or more biomolecules or heterologous proteins are integrated at one or more gene positions in the genome of the host Bacillus subtilis strain 105, wherein the gene positions are selected from amyE, NprE, AprE, Epr1, Epr2, Bpr, Mpr, NprB, Vpr and WprA.

21. The modified Bacillus according to any one of claims 1 to 20, wherein the one or more biomolecules or heterologous proteins are selected from the group consisting of anti-infective agents, antibacterial agents, antipathogenic agents, immunomodulatory factors or immunomodulators, antigens, antibodies, growth-promoting biomolecules, probiotics, and bio-based chemicals.

22. The modified Bacillus of claim 21, wherein the one or more biomolecules or heterologous proteins are antibacterial agents.

23. The modified Bacillus of claim 22, wherein the one or more antimicrobial agents are one or more lysins or lytic peptides.

24. The modified Bacillus according to claim 23, wherein the one or more lysins or lytic peptides are PlyCM, CP025C, lysostaphin, or native Bacillus subtilis 105 lytic enzyme.

25. The modified Bacillus of claim 22, wherein the one or more antimicrobial agents are one or more antimicrobial peptides (AMPs).

26. The modified Bacillus according to claim 25, wherein the one or more antimicrobial peptides (AMPs) are mesaccharin or cathelicidin peptides.

27. The modified Bacillus according to claim 26, wherein the one or more antimicrobial peptides (AMPs) are CAP18 peptides.

28. The modified Bacillus of claim 21, wherein the one or more biomolecules or heterologous proteins are one or more antibodies or fragments thereof.

29. The modified Bacillus according to claim 28, wherein the one or more antibodies or fragments thereof are one or more single chain antibodies, domain antibodies, VHH antibodies or nanobodies.

30. The modified Bacillus according to claim 29, wherein the one or more single-chain antibodies, domain antibodies, VHH antibodies or nanobodies are one or more single-chain antibodies, domain antibodies, VHF antibodies or nanobodies against pathogenic bacteria.

31. The modified Bacillus according to claim 30, wherein the one or more antibodies are one or more VHH antibodies or Nanobodies against Clostridium perfringens.

32. The modified Bacillus according to claim 31, wherein the one or more antibodies are one or more VHH antibodies or Nanobodies against Clostridium perfringens alpha toxin and NetB.

33. The modified Bacillus of claim 32, wherein the one or more VHH antibodies are selected from the group consisting of SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 101, and SEQ ID NO:

102.

34. The modified Bacillus of claim 21, wherein the one or more biomolecules or heterologous proteins are one or more antigens, and wherein the antigens are capable of stimulating an immune response against a parasite, a bacterium, or a virus.

35. The modified Bacillus of claim 34, wherein the one or more biomolecules or heterologous proteins are one or more antigens capable of stimulating an immune response against the Eimeria parasite.

36. The modified Bacillus according to claim 35, wherein the one or more antigens are selected from Eimeria tenella elongation factor-1α, EtAMA1, EtAMA2, Eimeria tenella 5401, Eimeria acervulina lactate dehydrogenase antigen gene, Eimeria maxima surface antigen gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Eimeria common antigen 14-3-3.

37. The modified Bacillus of claim 36, wherein the one or more antigens are Eimeria antigens encoded by one or more of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO:

109.

38. The modified Bacillus according to any one of claims 1-20, wherein one or more heterologous genes encoding one or more biomolecules or heterologous proteins are provided on a biosynthetic gene cluster (BGC), and wherein the BGC or a portion thereof is integrated into the host Bacillus subtilis strain 105 genome.

39. The modified Bacillus according to claim 38, wherein the biosynthetic gene cluster (BGC) is PKSBGC or mesaccharin BGC.

40. The modified Bacillus according to claim 39, wherein the PKS-BGC is capable of producing a metabolite that activates AhR.

41. The modified Bacillus according to claim 39, wherein the mericidin BGC is capable of producing one or more mericidin polypeptides SEQ ID NO: 22 or SEQ ID NO: 23 that are capable of inhibiting or killing one or more bacteria or viruses.

42. The modified Bacillus of claim 39, wherein the PKS BGC comprises the nucleic acid set forth in SEQ ID NO: 110 or comprises a nucleic acid encoding one or more polypeptides selected from SEQ ID NOs: 7-21.

43. The modified Bacillus according to claim 39, wherein the masacidin BGC comprises the nucleic acid set forth in SEQ ID NO: 24 or comprises a nucleic acid encoding one or more polypeptides selected from SEQ ID NOs: 25-32.

44. The modified Bacillus of claim 21, wherein the one or more biomolecules or heterologous proteins are bio-based chemicals.

45. The modified Bacillus according to claim 44, wherein the bio-based chemical is gamma-polyglutamic acid (γ-PGA) 46. The modified Bacillus of claim 45, wherein the γ-PGA is encoded by a CapABC locus and the Bacillus subtilis strain 105 is modified to produce increased amounts of γ-PGA by integrating at least one additional copy of the CapABC locus into the Bacillus subtilis strain 105 genome.

47. The modified Bacillus of claim 46, wherein at least one additional copy of the CapABC locus is integrated in the Bacillus subtilis strain 105 genome at one or more loci selected from the group consisting of amyE, nprE, apr, and wprA.

48. The modified Bacillus according to any one of claims 1 to 47, wherein the one or more heterologous genes encoding one or more biomolecules or heterologous proteins comprise a native Bacillus subtilis 105 strain signal sequence or other bacterial strain signal sequence, and the signal sequence is used to secrete the one or more biomolecules or heterologous proteins from the modified bacterium.

49. The modified Bacillus according to claim 48, wherein the signal sequence for secretion of the native Bacillus subtilis 105 strain or other strains is selected from SEQ ID NO: 43, SEQ ID NO-44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49 and SEQ ID NO: 50-64.

50. A live delivery platform comprising a genetically modified Bacillus for producing one or more biomolecules or heterologous proteins in an animal, wherein the modified Bacillus comprises a genetically modified Bacillus subtilis strain 105 (ELA191105), the ELA191105 being modified to contain a nucleic acid encoding one or more biomolecules or heterologous proteins, the one or more biomolecules or heterologous proteins being produced and delivered when the modified Bacillus is administered to the animal.

51. The living body delivery platform according to claim 50, wherein the bacteria comprises Bacillus subtilis strain 105 (ELA191105) genetically modified in one or more aspects selected from the following: (a) genetically modified to enhance competence; (b) genetically modified to reduce or block sporulation; (c) genetically modified to delete or inactivate one or more native proteases; and (d) genetically modified to include a nucleic acid encoding one or more biomolecules or heterologous proteins.

52. The living delivery platform according to claim 50 or 51, wherein the Bacillus subtilis strain 105 comprises the nucleic acid sequence of SEQ ID NO: 1, or comprises at least 95%, 97%, 98%, or 99% identity to SEQ ID NO: 1 in the genomic sequence.

53. The living delivery platform of claim 50 or 51, wherein the Bacillus subtilis strain corresponds to ATCC deposit PTA-126786 strain, or has at least 95%, 97%, 98%, or 99% identity in genome sequence to the sequence of ELA191105 corresponding to ATCC deposit PTA-26786.

54. The living delivery platform of any one of claims 50-53, wherein the Bacillus subtilis is genetically modified to contain a nucleic acid encoding one or more biomolecules or heterologous proteins and comprises an expression cassette; The expression cassette comprises one or more of the following: The promoter of transcriptional expression, a nucleic acid sequence encoding a signal sequence for secretion, at least one heterologous coding region encoding a desired biomolecule or heterologous protein, and Terminators for the termination of translation and transcription.

55. The living body delivery platform according to claim 54, wherein the promoter for transcriptional expression is one or more promoters selected from tuf promoter, sigx promoter, gros promoter, ftsh promoter, PxylA promoter, mannose-inducible promoter and Physpank promoter.

56. The living body delivery platform of claim 55, wherein the one or more promoters are selected from SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 40, and SEQ ID NO:

41.

57. The living body delivery platform of claim 54, wherein the nucleic acid sequence encoding the signal sequence for secretion encodes at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 44 amino acids, at least 50 amino acids, at least 55 amino acids, at least 60 amino acids, or at least 65 amino acids.

58. The living body delivery platform according to claim 54, wherein the nucleic acid sequence encoding the signal sequence for secretion encodes the signal sequence for secretion in a natural Bacillus subtilis 105 strain or other strains, and the signal sequence comprises a sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49 and SEQ ID NOs: 50-64.

59. The living delivery platform of any one of claims 50-58, wherein the expression cassette or at least one heterologous coding region encoding a desired biomolecule or heterologous protein is integrated into the host Bacillus subtilis strain 105 genome.

60. The living delivery platform according to claim 59, wherein the expression cassette or the at least one heterologous coding region encoding a desired biomolecule or heterologous protein is integrated into the genome of the host Bacillus subtilis strain 105 at one or more gene positions selected from the group consisting of amyE, NprE, AprE, Epr1, Epr2, Bpr, Mpr, NprB, Vpr and WprA.

61. The living body delivery platform according to any one of claims 50 to 59, wherein the desired biomolecule or heterologous protein is selected from anti-infective agents, antibacterial agents, antipathogenic agents, immunomodulatory factors or immunomodulators, antigens, antibodies, growth-promoting biomolecules, probiotics, and bio-based chemicals.

62. A method of reducing colonization of an animal by pathogenic bacteria, parasites or viruses, the method comprising treating the animal with the modified Bacillus of any one of claims 1-49 or with the live delivery platform of any one of claims 50-60.

63. The method of claim 61, wherein the animal is a bird, a human, or a non-human mammal.

64. The method of claim 61, wherein the pathogenic bacteria is selected from the group consisting of Salmonella, Clostridium, Campylobacter, Staphylococcus, Streptococcus, and Escherichia coli.

65. The method of claim 61, wherein the pathogenic parasite is Eimeria.

66. The method of any one of claims 61-64, wherein the modified Bacillus or the living delivery platform is administered orally, parenterally, nasally, or transmucosally.

67. The method of any one of claims 61-64, wherein the animal is a bird, and wherein the treatment is administered in ovo.

68. Use of the modified Bacillus of any one of claims 1-49 or the living delivery platform of any one of claims 50-60 in therapy.

69. Use of the modified Bacillus sp. of any one of claims 1-49 or the living delivery platform of any one of claims 50-60 in reducing colonization of animals by pathogenic bacteria, parasites or viruses.

70. Use of the modified Bacillus according to any one of claims 1 to 49 or the living delivery platform according to any one of claims 50 to 60 in the preparation of a medicament for reducing the colonization of an animal by pathogenic bacteria, parasites or viruses.

Images