Compositions and methods for enhanced protein production in Bacillus cells

Abstract

The present disclosure relates generally to compositions and methods for constructing and obtaining Bacillus sp. host cells (strains) with enhanced protein production phenotypes. Accordingly, certain embodiments of the present disclosure relate to genetically modified Bacillus sp. cells derived from a parent Bacillus sp. cell (e.g., a parent Bacillus cell that expresses / produces a protein of interest). Accordingly, certain embodiments are directed to engineered Bacillus sp. cells that express / produce an endogenous protein of interest or a heterologous protein of interest, wherein the engineered Bacillus sp. cells comprise a genetic modification of the yitMOP operon, a combined genetic modification of the yitMOP operon and the sdpABC operon, a genetic modification of the yitM gene, etc., and wherein the engineered Bacillus sp. cells produce increased amounts of a POI compared to the parental cells from which they are derived (i.e., when grown / cultured / fermented under identical conditions).

Description

[Technical field]

[0001] The present disclosure relates generally to the fields of bacteriology, microbiology, genetics, molecular biology, enzymology, industrial protein production, etc. More particularly, the present disclosure relates to compositions and methods for obtaining Bacillus sp. cells with increased protein production capabilities.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 933,536, filed Nov. 11, 2019, which is incorporated by reference in its entirety.

[0003] Sequence Listing Reference The contents of the electronic submission of the sequence listing text file entitled "NB41375-WO-PCT_SequenceListing.txt" was created on October 23, 2020, is 36KB in size, and is incorporated by reference in its entirety herein. [Background technology]

[0004] Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens are frequently used as microbial factories to produce industrially relevant proteins due to their excellent fermentation properties and high yields (e.g., up to 25 grams per liter of culture; Van Dijl and Hecker, 2013). For example, B. subtilis is well known for producing α-amylases (Jensen et al., 2000; Raul et al., 2014) and proteases (Brode et al., 1996) required for the food, textile, laundry, medical equipment cleaning, and pharmaceutical industries (Westers et al., 2004). These non-pathogenic Gram-positive bacteria produce proteins (e.g., lipopolysaccharides; LPS, also known as endotoxins) that are completely free of harmful by-products and have been awarded a "Qualified Presumption of Safety" (QPS) rating by the European Food Safety Authority, and many of their products have been awarded "Generally Recognized As Safe" (GRAS) status by the US Food and Drug Administration (Olempska-Beer et al., 2006; Earl et al., 2008; Caspers et al., 2010). Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, the production of proteins (e.g., enzymes, antibodies, receptors, etc.) in microbial host cells is of particular interest in the field of biotechnology. Similarly, the optimization of Bacillus sp. host cells to produce and secrete one or more proteins of interest is highly important, especially in the context of industrial biotechnology, where small improvements in protein yields are of crucial importance when the protein is to be industrially mass-produced. Thus, the ability to modify and engineer Bacillus sp. host cells for enhanced protein production is highly desirable in the art. As described below, the present disclosure addresses such a need in the art. More particularly, as presented, described and exemplified herein, certain embodiments of the present disclosure relate to methods and compositions for constructing Bacillus sp. host cells capable of producing increased amounts of a protein of interest. [Means for solving the problem]

[0006] The present disclosure relates generally to compositions and methods for constructing Bacillus sp. cells with enhanced protein production phenotypes. Certain embodiments of the present disclosure relate to genetically modified Bacillus sp. cells derived from a parent Bacillus sp. cell (e.g., a parent Bacillus cell that expresses / produces a protein of interest). For example, in certain embodiments, the modified Bacillus sp. cell of the present disclosure is derived from a parent Bacillus sp. cell that expresses / produces an endogenous protein of interest or a heterologous protein of interest, where the modified Bacillus sp. cell contains a genetic modification of the yitMOP operon and produces an increased amount of a POI compared to the parent cell from which it was derived (i.e., when grown / cultured / fermented under the same conditions). In certain other embodiments, the engineered Bacillus sp. cells of the present disclosure are derived from (obtained from) a parent Bacillus sp. cell that expresses / produces an endogenous protein of interest or a heterologous protein of interest, but where the engineered Bacillus sp. cell comprises a combined genetic modification of the yitMOP operon and the sdpABC operon, and where the engineered Bacillus sp. cell produces an increased amount of a POI as compared to the parent cell (i.e., when grown / cultured / fermented under the same conditions). In other embodiments, the engineered Bacillus sp. cells of the present disclosure are derived from a parent Bacillus sp. cell that expresses / produces an endogenous protein of interest or a heterologous protein of interest, but where the engineered Bacillus sp. cell comprises a genetic modification of the yitM gene, and where the engineered Bacillus sp. cell produces an increased amount of a POI compared to the parent cell (i.e., when grown / cultured / fermented under the same conditions).In yet other embodiments, the engineered Bacillus sp. cells of the present disclosure are derived from a parent Bacillus sp. cell that expresses / produces an endogenous protein of interest or a heterologous protein of interest, but where the engineered Bacillus sp. cell comprises a combined genetic modification of the yitM gene and the sdpABC operon, and where the engineered Bacillus sp. cell produces an increased amount of a POI compared to the parent cell (i.e., when grown / cultured / fermented under the same conditions). [Brief description of the drawings]

[0007] [Figure 1] 1 shows the nucleic acid sequence and encoded amino acid sequence of the native B. subtilis yitMOP operon. More specifically, FIG 1A presents the nucleic acid sequence of the yitM open reading frame (ORF; SEQ ID NO: 1) encoding the yitM polypeptide (SEQ ID NO: 2) and the yitO open reading frame (ORF; SEQ ID NO: 3) encoding the yitO polypeptide (SEQ ID NO: 4), and FIG 1B presents the nucleic acid sequence of the yitP open reading frame (ORF; SEQ ID NO: 5) encoding the yitP polypeptide (SEQ ID NO: 6).

[0008] [Diagram 2] This shows the beneficial effect of a modified B. subtilis strain (JL77) containing a combined deletion of the yitMOP and sdpABC cannibalism operons (ΔyitMOP+ΔsdpABC) on protein production during large-scale fermentation. More specifically, as presented in FIG. 2, cells of strains JL77 and CB24-12 were tested in fermenters under identical standard conditions, where the average increase in V42 protease production at the end of fermentation for strain JL77 was approximately a 10% increase compared to the amount of V42 protease produced by strain CB24-12 at the end of fermentation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] A brief description of biological sequences SEQ ID NO:1 is the nucleic acid sequence of the B. subtilis open reading frame (ORF) encoding the native YitM polypeptide of SEQ ID NO:2.

[0010] SEQ ID NO:2 is the amino acid sequence of the B. subtilis YitM polypeptide encoded by SEQ ID NO:1.

[0011] SEQ ID NO:3 is the ORF sequence of B. subtilis encoding the native YitO polypeptide of SEQ ID NO:4.

[0012] SEQ ID NO:4 is the amino acid sequence of the B. subtilis YitO polypeptide encoded by SEQ ID NO:3.

[0013] SEQ ID NO:5 is the ORF sequence of B. subtilis encoding the native YitP polypeptide of SEQ ID NO:6.

[0014] SEQ ID NO:6 is the amino acid sequence of the B. subtilis YitP polypeptide encoded by SEQ ID NO:5.

[0015] SEQ ID NO:7 is the nucleic acid sequence of primer oJKL228.

[0016] SEQ ID NO:8 is the nucleic acid sequence of primer oJKL229.

[0017] SEQ ID NO:9 is the nucleic acid sequence of primer oJKL232.

[0018] SEQ ID NO:10 is the nucleic acid sequence of primer oJKL223.

[0019] SEQ ID NO:11 is the nucleic acid sequence of primer oJKL230.

[0020] SEQ ID NO:12 is the nucleic acid sequence of primer oJKL231.

[0021] SEQ ID NO:13 is the nucleic acid sequence of primer oJKL226.

[0022] SEQ ID NO:14 is the nucleic acid sequence of primer oJKL227.

[0023] SEQ ID NO:15 is the nucleic acid sequence of primer M13F.

[0024] SEQ ID NO:16 is the nucleic acid sequence of primer M13R.

[0025] SEQ ID NO:17 is the nucleic acid sequence of primer oJKL246.

[0026] SEQ ID NO:18 is the nucleic acid sequence of primer 5'spec out.

[0027] SEQ ID NO:19 is the nucleic acid sequence of primer oJKL247.

[0028] SEQ ID NO:20 is the nucleic acid sequence of primer 3'spec out.

[0029] SEQ ID NO:21 is the nucleic acid sequence of primer oJKL220.

[0030] SEQ ID NO:22 is the nucleic acid sequence of primer oJKL221.

[0031] SEQ ID NO:23 is the nucleic acid sequence of primer oJKL224.

[0032] SEQ ID NO:24 is the nucleic acid sequence of primer oJKL225.

[0033] SEQ ID NO:25 is the nucleic acid sequence of primer oJKL222.

[0034] SEQ ID NO:26 is the nucleic acid sequence of primer oJKL223.

[0035] SEQ ID NO:27 is the nucleic acid sequence of primer oJKL218.

[0036] SEQ ID NO:28 is the nucleic acid sequence of primer oJKL2195.

[0037] SEQ ID NO:29 is the nucleic acid sequence of primer oJKL238.

[0038] SEQ ID NO:30 is the nucleic acid sequence of primer tet3'out.

[0039] SEQ ID NO:31 is the nucleic acid sequence of primer oJKL229.

[0040] SEQ ID NO:32 is the nucleic acid sequence of primer tet5'out.

[0041] SEQ ID NO:33 is the nucleic acid sequence of the Bacillus amyloliquefaciens open reading frame (ORF) encoding the native YitP polypeptide of SEQ ID NO:34.

[0042] SEQ ID NO:34 is the amino acid sequence of the B. amyloliquefaciens YitP polypeptide encoded by SEQ ID NO:33.

[0043] SEQ ID NO:35 is the ORF sequence of B. amyloliquefaciens encoding the native YitO polypeptide of SEQ ID NO:36.

[0044] SEQ ID NO:36 is the amino acid sequence of the B. amyloliquefaciens YitO polypeptide encoded by SEQ ID NO:35.

[0045] SEQ ID NO:37 is the ORF sequence of B. amyloliquefaciens encoding the native YitM polypeptide of SEQ ID NO:38.

[0046] SEQ ID NO:38 is the amino acid sequence of the B. amyloliquefaciens YitM polypeptide encoded by SEQ ID NO:37.

[0047] SEQ ID NO:39 is the DNA sequence of the promoter region of the B. amyloliquefaciens yitMOP operon.

[0048] SEQ ID NO:40 is the DNA sequence of the promoter region of the B. subtilis yitMOP operon.

[0049] SEQ ID NO:41 is the DNA sequence of the promoter region of the B. subtilis sdpABC operon.

[0050] SEQ ID NO:42 is the nucleic acid sequence of the B. subtilis open reading frame (ORF) encoding the native SdpA polypeptide of SEQ ID NO:43.

[0051] SEQ ID NO:43 is the amino acid sequence of the B. subtilis SdpA polypeptide encoded by SEQ ID NO:42.

[0052] SEQ ID NO:44 is the nucleic acid sequence of the B. subtilis open reading frame (ORF) encoding the native SdpB polypeptide of SEQ ID NO:45.

[0053] SEQ ID NO:45 is the amino acid sequence of the B. subtilis SdpB polypeptide encoded by SEQ ID NO:44.

[0054] SEQ ID NO:46 is the nucleic acid sequence of the B. subtilis open reading frame (ORF) encoding the native SdpC polypeptide of SEQ ID NO:47.

[0055] SEQ ID NO:47 is the amino acid sequence of the B. subtilis SdpC polypeptide encoded by SEQ ID NO:46.

[0056] The present disclosure relates generally to compositions and methods for constructing and obtaining Bacillus sp. cells with enhanced protein production phenotypes. Accordingly, certain embodiments of the present disclosure relate to genetically modified Bacillus sp. cells derived from a parent Bacillus sp. cell (e.g., a parent Bacillus cell that expresses / produces a protein of interest). In certain embodiments, the parent Bacillus sp. cell comprises an endogenous gene encoding a protein of interest (POI). In certain embodiments, the endogenous gene encodes a protease or amylase. In certain other embodiments, the parent Bacillus sp. cell comprises an expression cassette encoding a heterologous protein of interest (POI). For example, in certain embodiments, the engineered Bacillus sp. cells of the present disclosure are derived from a parent Bacillus sp. cell that expresses / produces an endogenous protein of interest or a heterologous protein of interest, but where the engineered Bacillus sp. cell contains a genetic modification of the yitMOP operon and produces an increased amount of a POI compared to the parent cell (i.e., when grown / cultured / fermented under the same conditions). In certain other embodiments, the engineered Bacillus sp. cells of the present disclosure are derived from a parent Bacillus sp. cell that expresses / produces an endogenous protein of interest or a heterologous protein of interest, where the engineered Bacillus sp. cell comprises a combined genetic modification of the yitMOP operon and the sdpABC operon, such that the engineered Bacillus sp. cell produces an increased amount of the POI as compared to the parent cell (i.e., when grown / cultured / fermented under the same conditions).In yet other embodiments, the modified Bacillus sp. cells of the present disclosure are derived from a parent Bacillus sp. cell that expresses / produces an endogenous protein of interest or a heterologous protein of interest, but where the modified Bacillus sp. cell comprises a genetic modification of the yitMOP gene and produces an increased amount of the POI compared to the parent cell (i.e., when grown / cultured / fermented under the same conditions). In other embodiments, the engineered Bacillus sp. cells of the present disclosure are derived from a parent Bacillus sp. cell that expresses / produces an endogenous protein of interest or a heterologous protein of interest, where the engineered Bacillus sp. cell comprises a combined genetic modification of the yitMOP operon (or yitM gene) and the sdpABC operon, such that the engineered Bacillus sp. cell produces an increased amount of the POI as compared to the parent cell (i.e., when grown / cultured / fermented under the same conditions).

[0057] definition In view of the disclosed modified Bacillus sp. cells and methods thereof described herein, the following terms and phrases are defined. Terms not defined herein should be given their commonly used meaning in the art.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the compositions and methods of the present invention belong. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the compositions and methods of the present invention, exemplary methods and materials are described below.

[0059] All publications and patents cited herein are hereby incorporated by reference in their entirety.

[0060] It is further noted that the claims may be drafted to exclude optional elements, and thus this statement is intended to serve as a prelude to the use of exclusive terminology such as "solely," "only," "excluding," "not including," or the use of any "negative" limitation or qualification in connection with the recitation of claim elements.

[0061] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the compositions and methods described herein. Any described method may be carried out in the order of events recited or in any other order which is logically possible.

[0062] As used herein, the phrase "host cell" refers to a cell that has the ability to act as a host or expression vehicle for a newly introduced DNA sequence. Thus, in certain embodiments of the present disclosure, the host cell is, for example, a Bacillus sp. cell or an E. coli cell.

[0063] As used herein, the terms "wild-type" and "native" are used interchangeably and refer to a gene, promoter, protein, protein mix, cell or strain found in nature.

[0064] As used herein, "Bacillus" or "Bacillus species" refers to "Bacillus sp." cells, as known to those of skill in the art, include all species within the genus "Bacillus," such as, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomic reorganization, and thus the genus is intended to include, but is not limited to, reclassified species, such as organisms such as "B. stearothermophilus," which is now designated "Geobacillus stearothermophilus."

[0065] As used herein, an "modified cell" refers to a recombinant (host) cell that contains at least one genetic modification that is not present in the "parent" host cell from which the modified cell is derived. For example, in certain embodiments, a "parent" cell is altered (e.g., by one or more genetic modifications introduced into the parent cell) to generate its "modified" (daughter) cell. In certain embodiments, a parent cell may be referred to as a "control cell," particularly when compared to or in relation to an "modified" Bacillus sp. (daughter) cell.

[0066] As used herein, when expression and / or production of a protein of interest (POI) in an "unmodified" (parent) cell is compared to expression and / or production of the same POI in an "modified" (daughter) cell, it will be understood that the "modified" and "unmodified" cells are grown / cultured / fermented under identical conditions (e.g., identical conditions of medium, temperature, pH, etc.).

[0067] As used herein, the wild-type Bacillus subtilis "yitMOP operon" encodes a "YitM polypeptide," a "YitO polypeptide," and a "YitP polypeptide."

[0068] As used herein, a wild-type B. subtilis "yitM" nucleic acid sequence encodes a wild-type B. subtilis "YitM" polypeptide comprising SEQ ID NO:2.

[0069] As used herein, a wild-type B. subtilis "yitO" nucleic acid sequence encodes a wild-type B. subtilis "YitO" polypeptide comprising SEQ ID NO:4.

[0070] As used herein, a wild-type B. subtilis "yitP" nucleic acid sequence encodes a wild-type B. subtilis "YitP" polypeptide comprising SEQ ID NO:6.

[0071] As used herein, the wild-type Bacillus amyloliquefaciens "yitMOP operon" encodes a "YitM polypeptide," a "YitO polypeptide," and a "YitP polypeptide."

[0072] As used herein, a wild-type B. amyloliquefaciens "yitM" nucleic acid sequence encodes a wild-type B. amyloliquefaciens "YitM" polypeptide comprising SEQ ID NO:38.

[0073] As used herein, a wild-type B. amyloliquefaciens "yitO" nucleic acid sequence encodes a wild-type B. amyloliquefaciens "YitO" polypeptide comprising SEQ ID NO:36.

[0074] As used herein, a wild-type B. amyloliquefaciens "yitP" nucleic acid sequence encodes a wild-type B. amyloliquefaciens "YitP" polypeptide comprising SEQ ID NO:34.

[0075] As used herein, the wild-type B. subtilis "sdpA" nucleic acid sequence (SEQ ID NO:42) encodes the wild-type B. subtilis "sdpA" polypeptide comprising SEQ ID NO:43.

[0076] As used herein, the wild-type B. subtilis "sdpB" nucleic acid sequence (SEQ ID NO:44) encodes the wild-type B. subtilis "SdpB" polypeptide comprising SEQ ID NO:45.

[0077] As used herein, the wild-type B. subtilis "sdpC" nucleic acid sequence (SEQ ID NO:46) encodes the wild-type B. subtilis "SdpC" polypeptide comprising SEQ ID NO:47.

[0078] As used herein, phrases such as "deficient in the production of a functional YitM protein," "deficient in the production of a functional YitO protein," "deficient in the production of a functional YitP protein," and the like, include modified (mutated) Bacillus sp. cells that do not produce detectable amounts of functional YitM protein, YitO protein, and / or YitP protein (i.e., compared to the parent cell when grown / cultured / fermented under the same conditions), or instead produce at least about 5% to about 99% less of one or more functional YitM, YitO, and / or YitP proteins (i.e., compared to the parent cell when cultured / fermented under the same conditions). For example, in certain embodiments, the modified Bacillus sp. cells of the present disclosure include a genetic modification that deletes, disrupts, inactivates, etc., the regulatory region of the yitMOP operon, thereby rendering the modified cell deficient in the production of a functional gene product (i.e., YitM, YitO, and / or YitP protein). Thus, in certain embodiments, the promoter region sequence of the B. amyloliquefaciens yitMOP operon comprises the nucleotide sequence set forth in SEQ ID NO:39, the promoter region sequence of the B. subtilis yitMOP operon comprises the nucleotide sequence set forth in SEQ ID NO:40, and the promoter region sequence of the B. subtilis sdpABC operon comprises the nucleotide sequence set forth in SEQ ID NO:41.

[0079] In certain other embodiments, the modified Bacillus sp. cell comprises a genetic modification that deletes, disrupts, inactivates, or interferes with the regulatory region of the yitM gene (e.g., promoter region sequence; SEQ ID NO:39, SEQ ID NO:40), thereby rendering the modified cell defective in producing a functional YitM protein.

[0080] As used herein, the plasmid referred to as "pUCyitMOP:spec" contains a spectinomycin resistance (specR) marker gene flanked by loxP sites. More specifically, the plasmid pUCyitMOP:spec was constructed to replace the entire yitMOP operon in B. subtilis with a spectinomycin resistance (specR) marker.

[0081] As used herein, a "Topo-specR vector" contains the spectinomycin adenyltransferase gene (specR) with its native promoter and terminator elements.

[0082] As used herein, the plasmid referred to as "pUCsdp::tet" contains upstream (5') and downstream (3') DNA sequence markers for replacing the entire sdpABC operon in B. subtilis with a tetracycline (tetR) marker flanked by loxP sites (herein a "loxP-tetR-loxP cassette"), where removal of the tetracycline marker results in a deletion of the sdpABC operon.

[0083] As used herein, a "loxP-tetR-loxP cassette" contains loxP recombination sequences that facilitate subsequent looping out of a tetracycline resistance cassette (Sauer, 1987; Sauer and Henderson 1988) and have been used to select for subsequent chromosomal integration.

[0084] As used herein, the plasmid designated "pUCLlacA-ganAB::tet" is used to delete the ganAB genes of B. subtilis and contains homology regions upstream (5') and downstream (3') of ganAB and a loxP-tetR-loxP cassette.

[0085] As used herein, "ME-3 protease" refers to a thermostable serine protease derived from Thermobifida cellulosilytica. For example, T. cellulosilytica ME-3 protease and variants thereof are described in WO 2018 / 118815, which is incorporated herein by reference in its entirety.

[0086] As used herein, the B. subtilis strain designated "ZM207" comprises a truncated alanine racemase gene (ΔalrA) and an introduced expression cassette encoding ME-3 protease. More specifically, the introduced expression cassette comprises a promoter sequence ("P") located upstream (5') and operably linked to an open reading frame (ORF) encoding ME-3 protease, which is operably linked to an ORF encoding alanine racemase (alrA), wherein the cassette is integrated within the B. subtilis aprE locus (aprE::P-ME-3-alrA).

[0087] As used herein, the genetically modified B. subtilis strain designated "ZM336" contains an introduced ME-3 protease expression cassette and a deleted yitMOP operon (ΔyitMOP), where the deleted yitMOP operon has been replaced with a spectinomycin resistance (specR) marker gene (i.e., ΔyitMOP). Thus, the ZM336 strain was constructed by introducing the deleted yitMOP operon (ΔyitMOP) into the parent ZM207 strain.

[0088] As used herein, the genetically modified B. subtilis strain designated "ZM268" contains a deletion of the yitMOP operon (ΔyitMOP) and the sdpABC operon (ΔsdpABC). After deletion of the yitMOP and sdpABC operons in an alanine racemase deletion strain (ΔalrA), an expression cassette encoding ME-3 protease (aprE::P-ME-3-alrA) was introduced to obtain strain ZM268.

[0089] As used herein, the genetically modified B. subtilis strain designated "ZM334" contains a deletion of the sdpABC operon (ΔsdpABC), where the deleted sdpABC operon has been replaced with a tetracycline resistance (tetR) marker gene (i.e., ΔsdpABC). Thus, the ZM334 strain was constructed by introducing the deleted sdpABC operon (ΔsdpABC) into the parent ZM207 strain.

[0090] As used herein, the genetically modified B. subtilis strain designated "ZM434" contains a deletion of the ΔyitM gene (ΔyitM), where the deleted yitM operon has been replaced with an erythromycin resistance (ermC) marker gene (i.e., ΔyitM). Thus, the ZM434 strain was constructed by introducing the deleted yitM (ΔyitM) into the parent ZM207 strain.

[0091] As used herein, "V42 protease" refers to a serine protease derived from Bacillus amyloliquefaciens subtilisin protease BPN'. For example, V42 protease and variants thereof are described in WO 2011 / 072099 (hereby incorporated by reference in its entirety).

[0092] As used herein, the B. subtilis strain designated "CB24-12" comprises an introduced expression cassette encoding a truncated alanine racemase gene (ΔalrA) and a V42 protease. More specifically, the introduced expression cassette comprises a promoter sequence ("P4") located upstream (5') and operably linked to an open reading frame (ORF) encoding alanine racemase (alrA), which is operably linked to an ORF encoding alanine racemase (alrA), wherein the cassette is integrated within the B. subtilis aprE locus (aprE::P4-V42-alrA).

[0093] As used herein, the genetically modified B. subtilis strain designated "JL77" (i.e., derived from the CB24-12 parent strain) contains identical expression cassettes encoding the V42 protease, a deleted yitMOP operon (ΔyitMOP), and a deleted sdpABC operon (ΔsdpABC). For example, the yitMOP operon and the sdpABC operon of the JL77 strain were deleted by homologous recombination with a cassette containing an antibiotic resistance marker. The yitMOP operon was replaced with a spectinomycin cassette flanked by loxP sequences. The spdABC operon was replaced with a tetracycline cassette flanked by loxP sequences. Upon transformation with a plasmid containing a cassette for expressing Cre recombinase, the antibiotic resistance marker is excised from the genome leaving one loxP sequence.

[0094] As used herein, the term "equivalent position" refers to an amino acid residue position after alignment with a particular polypeptide sequence, or a nucleotide position after alignment with a particular polynucleotide sequence.

[0095] The terms "modification" and "genetic modification" are used interchangeably and include: (a) the introduction, substitution, or removal of one or more nucleotides in a gene (or its ORF), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of a gene or its ORF; (b) gene disruption; (c) gene conversion; (d) gene deletion; (e) gene downregulation; (f) directed mutagenesis; and / or (g) random mutagenesis of any one or more genes disclosed herein.

[0096] As used herein, "gene disruption," "gene disruption," "gene inactivation," and "gene inactivation" are used interchangeably and refer broadly to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., YitM protein, YitO protein, YitP protein, etc.). Exemplary gene disruption methods include complete or partial loss of any portion of a gene, including a polypeptide coding sequence, promoter, enhancer, or other regulatory element, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and any combinations and variations thereof that disrupt / inactivate the target gene and substantially reduce or prevent production of a functional gene product (i.e., protein).

[0097] As used herein, the compound term "express / produce", e.g., in the phrase "the modified cell expresses / produces increased amounts of a protein of interest compared to the parent cell", is intended to include any process involved in the expression and production of a protein of interest in a host cell of the present disclosure.

[0098] As used herein, phrases such as "enhancing protein production" or "increasing protein production" refer to increased amounts of endogenous or heterologous proteins produced by the host cells of the present disclosure. In certain embodiments, the protein of interest is produced inside the host cell and secreted (or transported) into the culture medium. In certain embodiments, the protein of interest is produced (secreted) into the culture medium. Increased protein production can be detected, for example, as a higher maximum level of protein or enzyme activity (e.g., protease activity, amylase activity, cellulase activity, hemicellulase activity, etc.) or as total extracellular protein produced, as compared to the parent host cell.

[0099] As used herein, "nucleic acid" refers to DNA, cDNA and RNA of genomic or synthetic origin, whether representing a nucleotide or polynucleotide sequence, as well as fragments or portions thereof, and whether representing the sense or antisense strand, and whether double-stranded or single-stranded. It will be understood that, as a result of the degeneracy of the genetic code, many nucleotide sequences can code for a given protein.

[0100] The polynucleotides (or nucleic acid molecules) described herein are understood to include "genes," "vectors," and "plasmids."

[0101] Thus, the term "gene" refers to a polynucleotide that codes for a particular sequence of amino acids, including all or part of a coding sequence for a protein, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, that determine the conditions under which the gene is expressed. The transcribed region of a gene may include introns, untranslated regions (UTRs), including 5'-untranslated regions (UTRs) and 3'-UTRs, as well as the coding sequence.

[0102] As used herein, the term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of a coding sequence are generally determined by an open reading frame (hereinafter "ORF"), which usually begins with the ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0103] The term "promoter" as used herein refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' (downstream) of the promoter sequence. Promoters may be derived entirely from a native gene, or may be composed of different elements derived from different naturally occurring promoters, or may even comprise synthetic nucleic acid segments. Those skilled in the art understand that different promoters may direct the expression of genes in different cell types, or at different developmental stages, or in response to different environmental or physiological conditions. The promoters that most often cause the expression of genes in most cell types are generally referred to as "constitutive promoters". Furthermore, it is recognized that DNA fragments of different lengths may have the same promoter activity, since in most cases the exact boundaries of regulatory sequences are not completely clear.

[0104] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence (e.g., an ORF) if it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation.

[0105] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader (i.e., signal peptide) is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0106] As used herein, a "functional promoter sequence (or open reading frame thereof) that controls expression of a gene of interest linked to a gene of a protein coding sequence of interest" refers to a promoter sequence that controls transcription and translation of the coding sequence in Bacillus. For example, in certain embodiments, the present disclosure is directed to a polynucleotide that includes a 5' promoter (or a 5' promoter region or a tandem 5' promoter, etc.), where the promoter region is operably linked to a nucleic acid sequence encoding a protein of interest. Thus, in certain embodiments, the functional promoter sequence controls expression of a gene encoding a protein disclosed herein. In other embodiments, the functional promoter sequence controls expression of a heterologous gene (or endogenous gene) encoding a protein of interest in a Bacillus sp. cell.

[0107] As defined herein, a "suitable regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence that influences the transcription, RNA processing or stability or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0108] As defined herein, the term "introducing" as used in phrases such as "introducing into a bacterial cell" or "introducing at least one polynucleotide open reading frame (ORF), or gene thereof, or vector thereof, into a Bacillus sp. cell" includes methods known in the art for introducing polynucleotides into a cell, including, but not limited to, protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation, and the like (see, e.g., Ferrari et al., 1989).

[0109] As used herein, "transformed" or "transformation" refers to a cell being transformed by the use of recombinant DNA technology. Transformation typically occurs by inserting one or more nucleotide sequences (e.g., a polynucleotide, ORF, or gene) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence that does not naturally occur in the cell being transformed). For example, in certain embodiments of the present disclosure, a parent Bacillus sp. cell is modified (e.g., transformed) by introducing into the parent cell a polynucleotide construct comprising a promoter operably linked to a nucleic acid sequence encoding a protein of interest, thereby resulting in a modified Bacillus sp. (daughter) host cell derived from the parent cell.

[0110] As used herein, "transformation" refers to the introduction of foreign DNA into a host cell such that the DNA is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector. As used herein, "transforming DNA," "transforming sequence," and "DNA construct" refer to DNA used to introduce a sequence into a host cell or organism. The transforming DNA is the DNA used to introduce a sequence into a host cell or organism. This DNA can be generated in vitro by PCR or any other suitable technique. In some embodiments, the transforming DNA includes the incoming sequence, while in other embodiments, the transforming DNA further includes the incoming sequence flanked by homology boxes. In yet other embodiments, the transforming DNA includes other non-homologous sequences (i.e., stuffer sequences or flanking sequences) added to the ends. The ends can be closed such that the transforming DNA forms a closed circle, such as, for example, by insertion into a vector.

[0111] As used herein, the term "introduced" in the context of introducing a nucleic acid sequence into a cell refers to any method suitable for transferring a nucleic acid sequence into a cell. Such methods for introduction include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction (see, e.g., Ferrari et al., 1989).

[0112] As used herein, "incoming sequence" refers to a DNA sequence that is introduced into the Bacillus chromosome. In some embodiments, the incoming sequence is part of a DNA construct. In other embodiments, the incoming sequence encodes one or more proteins of interest. In some embodiments, the incoming sequence comprises a sequence that may or may not already be present in the genome of the cell to be transformed (i.e., it may be a homologous or non-homologous sequence). In some embodiments, the incoming sequence encodes one or more proteins of interest, genes and / or mutant or modified genes. In alternative embodiments, the incoming sequence encodes a functional wild-type gene or operon, a functional mutant gene or operon, or a non-functional gene or operon. In some embodiments, a non-functional sequence may be inserted into a gene to disrupt the function of the gene. In another embodiment, the incoming sequence comprises a selection marker. In yet another embodiment, the incoming sequence comprises two homology boxes.

[0113] As used herein, a "homology box" refers to a nucleic acid sequence that is homologous to a sequence in the Bacillus chromosome. More specifically, a homology box is an upstream or downstream region that has about 80-100% sequence identity, about 90-100% sequence identity, or about 95-100% sequence identity with the gene or some immediately adjacent coding region of the gene to be deleted, disrupted, inactivated, downregulated, etc., according to the present invention. These sequences direct where in the Bacillus chromosome the DNA construct is integrated and direct what part of the Bacillus chromosome is replaced by the incoming sequence. While not intending to limit the disclosure, a homology box can include from about 1 base pair (bp) to 200 kilobases (kb). Preferably, the homology box comprises about 1 bp to 10.0 kb; 1 bp to 5.0 kb; 1 bp to 2.5 kb; 1 bp to 1.0 kb, and 0.25 kb to 2.5 kb. The homology box may also comprise about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb, and 0.1 kb. In some embodiments, the 5' and 3' ends of the selectable marker are flanked by homology boxes, where the homology box comprises nucleic acid sequences that are immediately adjacent to the coding region of a gene.

[0114] As used herein, the phrase "selectable marker gene" or "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence that is expressible in a host cell and whose expression confers on the cell containing the expressed gene the ability to grow in the presence of a corresponding selection agent or in the absence of an essential nutrient. Examples of such selectable markers include, but are not limited to, antimicrobial agents. Thus, a selectable marker refers to a gene that provides an indication that a host cell has taken up an incoming DNA of interest or that some other reaction has occurred. Typically, a selectable marker is a gene that confers on a host cell an antimicrobial resistance or a metabolic advantage that allows cells containing foreign DNA to be distinguished from cells that have not received the exogenous sequence during transformation.

[0115] An "present selectable marker" is a marker that is located on the chromosome of the microorganism being transformed. The present selectable marker encodes a different gene than the selectable marker on the transforming DNA construct. Selectable markers are well known to those skilled in the art. As indicated above, markers include antimicrobial resistance markers (e.g., amplicons ... R , phleo R , spec R , kan R ,ery R , tet R , cmp R and neo R (See, e.g., Guerot-Fleury, 1995; Palmeros et al., 2000; and Trieu-Cuot et al., 1983). In some embodiments, the invention provides a chloramphenicol resistance gene (e.g., genes present on pC194, as well as resistance genes present in the genome of Bacillus sp.). This resistance gene is particularly useful in the present invention and in embodiments involving chromosomal amplification of chromosomally integrated cassettes and integrative plasmids (See, e.g., Albertini and Galizzi, 1985; Stahl and Ferrari, 1984). Other markers useful according to the invention include, but are not limited to, auxotrophic markers such as serine, lysine, tryptophan, and detection markers such as β-galactosidase or fluorescent proteins.

[0116] As defined herein, a host cell "genome," bacterial cell "genome," or Bacillus sp. "genome" includes chromosomal genes and extrachromosomal genes.

[0117] As used herein, the terms "plasmid," "vector," and "cassette" refer to extrachromosomal elements, usually in the form of circular double-stranded DNA molecules, that often carry genes that are not typically involved in the central metabolism of a cell. Such elements may be linear or circular, single-stranded or double-stranded, autonomously replicating sequences of DNA or RNA, genome-integrating sequences, phages, or nucleotide sequences from any source in which multiple nucleotide sequences have been joined or recombined into a unique configuration that allows the introduction of promoter fragments and DNA sequences for selected gene products, along with appropriate 3'-non-translated sequences, into a cell.

[0118] As used herein, a "transformation cassette" refers to a specific vector that contains a gene (or its ORF) and has elements in addition to a foreign gene that facilitate transformation of a particular host cell.

[0119] The term "vector" as used herein refers to any nucleic acid that can replicate (multiply) in a cell and can carry new genes or DNA segments into the cell. Thus, the term refers to a nucleic acid construct designed for transport between various host cells. Vectors include viruses, bacteriophages, proviruses, plasmids, phagemids, transposons, which are "episomes" (i.e., they can replicate autonomously or integrate into the chromosomes of the host organism), as well as artificial chromosomes, such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes).

[0120] "Expression vector" refers to a vector capable of incorporating and expressing heterologous DNA in a cell. Many prokaryotic and eukaryotic expression vectors are commercially available and known to those skilled in the art. The selection of an appropriate expression vector is within the knowledge of one of ordinary skill in the art.

[0121] The term "expression cassette" as used herein refers to a nucleic acid construct (i.e., a vector or vector element as described above) that is recombinantly or synthetically produced with a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, a chromosome, mitochondrial DNA, plastid DNA, a virus, or a nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, the DNA construct also includes a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. In certain embodiments, the DNA construct of the present disclosure includes a selectable marker and an inactivated chromosomal segment or gene segment or DNA segment as defined herein.

[0122] As used herein, a "targeting vector" is a vector that contains a polynucleotide sequence that is homologous to a region in a host cell chromosome into which the targeting vector is transformed and can drive homologous recombination at that region. For example, a targeting vector is used to introduce a mutation into a host cell chromosome by homologous recombination. In some embodiments, the targeting vector contains other non-homologous sequences (i.e., stuffer sequences or flanking sequences), for example, added to the ends. In some embodiments, the targeting vector contains elements to increase homologous recombination, including, but not limited to, RNA-guided endonucleases, DNA-guided endonucleases, and recombinases. The ends can be closed such that the targeting vector forms a closed circle, for example, by insertion into a vector.

[0123] As used herein, the term "plasmid" refers to a circular, double-stranded (ds) DNA construct that is used as a cloning vector and forms an extrachromosomal, self-replicating genetic element in many bacteria and some eukaryotes. In some embodiments, the plasmid is integrated into the genome of the host cell.

[0124] The term "protein of interest" (abbreviated "POI") as used herein refers to a protein of interest that is desired to be expressed in a Bacillus sp. host cell. Thus, a POI as used herein can be an enzyme, a substrate binding protein, a surfactant protein, a structural protein, a receptor protein, and the like. In certain embodiments, the engineered cells of the present disclosure produce an increased amount of a heterologous POI or an increased amount of an endogenous POI as compared to a parent cell. In certain embodiments, the increased amount of POI produced by the engineered cells of the present disclosure is at least a 0.5% increase, at least a 1.0% increase, at least a 5.0% increase, or greater than a 5.0% increase as compared to a parent cell.

[0125] Similarly, a "gene of interest" (abbreviated "GOI") as defined herein refers to a nucleic acid sequence (e.g., polynucleotide, gene or ORF) that encodes a POI. A "gene of interest" that encodes a "protein of interest" may be a naturally occurring gene, a mutated gene or a synthetic gene.

[0126] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of any length that contain amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein. Polypeptides may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term polypeptide also encompasses amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety. Also included within the scope of this definition are, for example, polypeptides that contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0127] In certain embodiments, the genes of the present disclosure are those encoding enzymes (e.g., acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lyase, lysase), endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, laccase, lipase, lyase, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methyl esterase, pectinolytic enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phytase, polygalacturonase, protease, peptidase, rhamno-galacturonase, ribonuclease, transferase, transport protein, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0128] As used herein, a "mutant" polypeptide refers to a polypeptide derived from a parent (or reference) polypeptide by one or more amino acid substitutions, additions, or deletions, typically by recombinant DNA techniques. A mutant polypeptide can differ from a parent polypeptide by a small number of amino acid residues and can be defined by the level of primary amino acid sequence homology / identity with the parent (reference) polypeptide.

[0129] Preferably, a variant polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% amino acid sequence identity with a parent (reference) polypeptide sequence. As used herein, a "variant" polynucleotide refers to a polynucleotide that encodes a variant polypeptide, which has a particular degree of sequence homology / identity with a parent polynucleotide or hybridizes to a parent polynucleotide (or its complement) under stringent hybridization conditions. Preferably, the variant nucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% nucleotide sequence identity with the parent (reference) polynucleotide sequence.

[0130] As used herein, "mutation" refers to any change or alteration in a nucleic acid sequence. There are several types of mutations, including point mutations, deletion mutations, silent mutations, frameshift mutations, splicing mutations, etc. Mutations can be made specifically (e.g., by site-directed mutagenesis) or randomly (e.g., by chemicals, repair minus passaging through bacterial strains).

[0131] As used herein, in reference to a polypeptide or sequence thereof, the term "substitution" refers to the replacement (ie, substitution) of one amino acid with another.

[0132] As defined herein, "endogenous gene" refers to a gene that is present in its natural location in the genome of an organism.

[0133] A "heterologous" gene, "non-endogenous" gene or "foreign" gene, as defined herein, refers to a gene (or ORF) that is not normally found in the host organism, but which has been introduced into the host organism by gene transfer. As used herein, the term "foreign" gene includes a native gene (or ORF) inserted into a non-native organism and / or a chimeric gene inserted into a native or non-native organism.

[0134] A "heterologous" nucleic acid construct or "heterologous" nucleic acid sequence, as defined herein, has a portion of sequence that is not native to the cell in which it is expressed.

[0135] As defined herein, a "heterologous control sequence" refers to a gene expression control sequence (e.g., a promoter or enhancer) that does not function in nature to regulate (control) the expression of a gene of interest. Generally, heterologous nucleic acid sequences are not endogenous (natural) to the cell or part of the genome in which they are present, but have been added to the cell by infection, transfection, transformation, microinjection, electroporation, etc. A "heterologous" nucleic acid construct may contain a control sequence / DNA coding (ORF) sequence combination that is the same or different from the control sequence / DNA coding sequence combination found in the native host cell.

[0136] The terms "signal sequence" and "signal peptide" as used herein refer to a sequence of amino acid residues that may be involved in the secretion or direct transport of a mature protein or a precursor form of a protein. A signal sequence is typically located at the N-terminus of a precursor or mature protein sequence. A signal sequence may be endogenous or foreign. A signal sequence is not usually present in a mature protein. A signal sequence is typically cleaved from a protein by a signal peptidase after the protein has been transported.

[0137] The term "derived from" includes the terms "originating from," "obtained from," "available from," and "made from," and generally indicates that one particular material or composition has characteristics that can be found its origin in another material or composition, or that can be described with reference to that other particular material or composition.

[0138] The term "homology" as used herein relates to homologous polynucleotides or homologous polypeptides. When two or more polynucleotides or two or more polypeptides are homologous, this means that the homologous polynucleotides or polypeptides have a "degree of identity" of at least 60%, more preferably at least 70%, even more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, and most preferably at least 98%. Whether two polynucleotide or polypeptide sequences have a sufficiently high degree of identity to be homologous as defined herein can be conveniently determined by aligning the two sequences using a computer program known in the art, for example, "GAP" provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman and Wunsch, (1970). GAP is used with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0139] As used herein, the term "percent identity" refers to the level of nucleic acid or amino acid sequence identity between nucleic acid sequences encoding a polypeptide or between the amino acid sequences of a polypeptide when aligned using a sequence alignment program.

[0140] As used herein, the term "specific productivity" refers to the total amount of protein produced per cell per unit time over a given period of time.

[0141] The terms "purified," "isolated," or "enriched," as defined herein, refer to a biomolecule (e.g., a polypeptide or polynucleotide) that has been altered from its native state by separation from some or all of the naturally occurring components with which it is naturally associated. Such isolation or purification can be accomplished by separation techniques known in the art, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation or other protein salting out, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove unwanted whole cells, cell debris, impurities, extraneous proteins, or enzymes in the final composition. Purified or isolated biomolecule compositions can then be supplemented with components that confer additional benefits, such as activators, anti-inhibitors, desirable ions, pH adjusting compounds, or other enzymes or chemicals.

[0142] As used herein, "homologous genes" refers to a pair of genes from different but usually related species that correspond to each other and are identical or highly similar to each other. The term encompasses genes that have been separated by speciation (i.e., the emergence of new species) (e.g., orthologous genes) and genes that have been separated by genetic duplication (e.g., paralogous genes).

[0143] As used herein, "ortholog" and "orthologous genes" refer to genes in different species that have arisen from a common ancestral gene (i.e., a homologous gene) by speciation. Typically, orthologs retain the same function during the course of evolution. Identification of orthologs is used for reliable prediction of gene function in newly sequenced genomes.

[0144] As used herein, "paralog" and "paralogous genes" refer to genes that are involved in duplication within a genome. Orthologs retain the same function throughout evolution, while paralogs give rise to new functions, although some functions are often related to the original. Examples of paralogous genes include, but are not limited to, genes encoding trypsin, chymotrypsin, elastase, and thrombin, all of which are serine proteinases and occur together in the same species.

[0145] As used herein, "homology" refers to sequence similarity or identity, with identity being preferred, which is determined using standard techniques known in the art (see, e.g., Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., 1984).

[0146] The term "hybridization" as used herein refers to the process by which a nucleic acid strand binds with its complementary strand by base pairing, as known in the art. A nucleic acid sequence is considered to be "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize to each other under medium to high stringency hybridization and wash conditions. Hybridization conditions are determined by the melting temperature (T m For example, "maximum stringency" is usually based on T m - 5°C (T m 5°C below T m and "intermediate stringency" is approximately 5-10°C below the T mand "low stringency" is T m This occurs when the temperature drops by about 20 to 25 degrees Celsius.

[0147] Functionally, maximum stringency conditions can be used to identify sequences with exact or near exact identity to the hybridization probe, while intermediate or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs. Medium to high stringency hybridization conditions are well known in the art. Examples of high stringency conditions include hybridization at about 42°C in 50% formamide, 5xSSC, 5xDenhardt's solution, 0.5% SDS and 100pg / mL denatured carrier DNA, followed by two washes at room temperature (RT) in 2xSSC and 0.5% SDS, and two additional washes at 42°C in 0.1xSSC and 0.5% SDS. An example of moderately stringent conditions includes overnight incubation at 37° C. in a solution containing 20% ​​formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg / mL denatured, fragmented salmon sperm DNA, followed by washing of the filter in 1×SSC at about 37-50° C. One of skill in the art will know how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

[0148] As used herein, "recombinant" includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid sequence, or that is derived from a cell that has been so modified. Thus, for example, a recombinant cell expresses a gene that is not found in the same form within the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise abnormally expressed, under-expressed, or not expressed at all as a result of deliberate human intervention. "Recombinant," "recombining," or producing a "recombinant" nucleic acid is generally the assembly of two or more nucleic acid fragments, which assembly gives rise to a chimeric gene.

[0149] As used herein, "flanking sequence" refers to any sequence that is upstream (5') or downstream (3') of the sequence under consideration (e.g., in gene ABC, gene B is flanked by gene sequences A and C). In certain embodiments, the incoming sequence is flanked on both sides by homology boxes. In other embodiments, the incoming sequence and the homology box comprise a unit that is flanked on both sides by stuffer sequences. In some embodiments, the flanking sequences are present on only one side (3' or 5'), but in preferred embodiments, they are present on both sides of the sequence that they are flanking. The sequence of each homology box is homologous to a sequence in the Bacillus chromosome. These sequences direct where in the Bacillus chromosome the new construct will be integrated and which part of the Bacillus chromosome will be replaced by the incoming sequence. In other embodiments, the 5' and 3' ends of the selectable marker are flanked by polynucleotide sequences that comprise a portion of an inactivated chromosomal segment. In some embodiments, flanking sequences are present on only one side (3' or 5'), while in other embodiments, they are present on both sides of the sequences they are flanking. In some embodiments, the homology boxes directly flank each other or lack intervening sequences (e.g., constructs D through F for genes DEF) such that when the constructs recombine in the genome, gene E will be removed from the genome.

[0150] II. Stress response of cannibalistic cells In their natural environment, microorganisms must constantly compete for nutrients. To defend their habitat against invading species, many bacteria produce and secrete antimicrobial peptides (AMPs) that interfere with the integrity and / or biosynthesis of their cell envelope (e.g., the action of AMPs often results in the arrest of cell growth, leading to cell lysis (Silver, 2003; Silver, 2006)). Thus, to defend against such AMP attacks, many bacteria induce complex cell envelope stress responses. For example, when faced with limiting carbon sources, B. subtilis initiates a survival strategy called sporulation, resulting in the formation of highly resistant endospores that allow B. subtilis to survive long periods of starvation. To avoid a deep commitment to this energy-demanding, irreversible process, B. subtilis uses another strategy, called "cannibalism," to delay sporulation for as long as possible (Gonzalez-Pastor et al., 2003). Cannibalism in B. subtilis generally involves the production and secretion of two known cannibalistic toxins (AMPs), sporulation delay protein (SDP) and sporulation killing factor (SKF), which can lyse susceptible siblings. Thus, it is believed that lysed sibling cells provide nutrients for subsequent cannibalism to be slowed down, or even prevent the sporulation they invade.

[0151] For example, as generally described by Gonzalez-Pastor et al. (2003), the B. subtilis skf operon contains eight genes (i.e., skfABCDEFGH), where the skfA gene encodes the AMP toxin SKF; and the B. subtilis sdp operon contains three genes (i.e., sdpABC), where the sdpC gene encodes the AMP toxin SPD. More recent work on the spdABC operon by Perez Morales et al. (2013) suggests that the encoded SdpA and SdpB proteins are essential for post-translational processing of the immature SpdC protein into the mature and active SDP toxin protein that is selected extracellularly. Wang et al. (2014) studied the effect of single and combined deletion of selected cell lysis genes (i.e., skfA, sdpC, xpf, or lytC) in B. subtilis, where the maximum number of intact cells was detected in cultures of the quadruple mutant (ΔlytCΔsdpCΔxpfΔskfA). As concluded by Wang et al. (2014), the production of intracellular and secreted recombinant proteins (nattokinase and β-galactosidase, respectively) was significantly elevated in cultures of the quadruple mutant LM2531 (ΔlytCΔsdpCΔxpfΔskfA) by deletion of the major PG hydrolase, the prophage-encoded xpf, and lytC, which encodes the cannibalism factors skfA and sdpC.

[0152] As described herein and presented in the Examples section below, Applicants have identified a novel cannibalism operon in B. subtilis, designated yitMOP, which encodes the YitM (SEQ ID NO:2), YitO (SEQ ID NO:4) and YitP (SEQ ID NO:6) proteins. More specifically, Applicants have identified the novel yitMOP operon as a highly expressed transcript in transcriptome analysis of B. subtilis strains expressing FNA protease.

[0153] Thus, as further described in Example 3 below, Applicants have constructed a parent B. subtilis cell (strain) designated ZM207 containing an introduced protease (ME-3) expression cassette and its modified B. subtilis cell (strains) designated ZM334, ZM336 and ZM434; where ZM334 cells contain the same protease (ME-3) expression cassette and a deleted sdpABC operon (ΔsdpABC), ZM336 cells contain the ME-3 protease expression cassette and a deleted yitMOP operon (ΔyitMOP), and ZM434 cells contain the ME-3 protease expression cassette and a deletion of the yitM gene (ΔyitM). ZM268 cells were constructed by introducing a protease (ME-3) expression cassette into a host containing a ME-3 protease expression cassette, a deleted yitMOP operon (ΔyitMOP), and a deleted sdpABC operon (ΔsdpABC), with a deletion of the yitMOP operon (ΔyitMOP), a deleted sdpABC operon (ΔsdpABC), and a deletion of the alanine racemase gene (ΔalrA).

[0154] As presented in Example 3, the parental (ZM207; control) and modified B. subtilis cells (ZM334, ZM336, ZM268 and ZM434) were fermented in shake flasks under identical conditions in Grant's II medium at 42° C. for 40 hours. Fermentation supernatants were then removed and ME-3 protease activity (Table 15) was measured. For example, as shown in Table 15, strains ZM334 (ΔsdpABC) and ZM336 (ΔyitMOP) produced increased amounts of ME-3 protease compared to strain ZM207 (control). Similarly, strain ZM268, which contains a combined deletion of the yitMOP and sdpABC operons (i.e., ΔyitMOP+ΔsdpABC) (Table 15), produces increased amounts of protease compared to the ZM207 parent strain, strain ZM334 (ΔsdpABC), and strain ZM336 (ΔyitMOP). Moreover, it was surprisingly observed that disruption or deletion of the yitM ORF alone was sufficient to elicit the beneficial phenotype of increased protein production, as strain ZM434 (ΔyitM) produced slightly more ME-3 protease than strain ZM336 (ΔyitMOP) (Table 15).

[0155] Similarly, as presented and described in Example 4 below, Applicant further constructed a parent B. subtilis strain designated CB24-12 containing an introduced protease (V42) expression cassette and a modified B. subtilis daughter cell protease expression cassette designated JL77 containing the same V42 protease expression cassette and a combined deletion of the yitMOP and sdpABC operons (ΔyitMOP+ΔsdpABC). As described in Example 4, B. subtilis strains CB24-12 and JL77 were evaluated for V42 protease production under large-scale fermentation conditions. For example, as presented in FIG. 2, strains CB24-12 and L77 were tested in fermenters under the same standard conditions, with each strain run twice, showing the average percent improvement in V42 protease production at the end of fermentation. Thus, as presented in FIG. 2, the average increase in V42 protease production at the end of fermentation for the JL77 strain was approximately a 10% increase compared to the amount of V42 protease produced by the CB24-12 strain at the end of fermentation.

[0156] These observations demonstrate that genetic modification of the yitMOP operon in Bacillus sp. cells results in an enhanced protein production phenotype compared to the parental Bacillus sp. cells (i.e., containing the native yitMOP operon). Furthermore, these observations also demonstrate that the combined genetic modification of the yitMOP operon and the sdpABC operon in Bacillus sp. cells further enhances the observed protein production phenotype compared to modified Bacillus sp. cells containing a genetic modification of the yitMOP operon alone (see, e.g., Example 3, Table 15 and Example 4, Figure 2).

[0157] Similarly, it was surprisingly observed herein that Bacillus sp. cells containing a genetic modification of the yitM gene alone demonstrated an enhanced protein production phenotype compared to Bacillus sp. cells containing a combined genetic modification of the yitMOP and sdpABC operons (ΔyitMOP+ΔsdpABC; Example 3, Table 15).

[0158] III.Molecular biology As explained above, certain embodiments of the present disclosure relate to modified parent Bacillus sp. cells derived from a parent Bacillus sp. cell that contains a native yitMOP operon. In particular embodiments, the modified Bacillus sp. cell contains a modified yitMOP operon. In certain other embodiments, the modified Bacillus sp. cell contains a disrupted or deleted yitM gene. In certain other embodiments, the modified Bacillus sp. cell of the present disclosure further contains a deleted spdABC operon. Certain embodiments relate to compositions and methods for genetically modifying a parent Bacillus sp. cell to generate modified B. licheniformis (daughter) cells therefrom.

[0159] Accordingly, certain embodiments of the present disclosure are directed to methods for genetically modifying a Bacillus sp. cell, the modification including, but not limited to, (a) introduction, substitution or removal of one or more nucleotides within a gene (or its ORF) or the introduction, substitution or removal of one or more nucleotides within a regulatory element required for transcription or translation of a gene or its ORF, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) site-directed mutagenesis, and / or (g) random mutagenesis. For example, genetic modification as used herein includes, but is not limited to, modification of one or more genes from a Bacillus sp. cell (e.g., one or more genes of the yitMOP operon and / or spdABC operon).

[0160] Thus, in certain embodiments, altered Bacillus cells of the present disclosure are constructed by reducing or eliminating expression of a gene using methods well known in the art, such as insertion, disruption, substitution, or deletion. The portion of the gene to be altered or inactivated can be, for example, a coding region, or a regulatory element required to express the coding region.

[0161] An example of such a regulatory or control sequence may be a promoter sequence or a functional portion thereof (i.e., a portion sufficient to affect the expression of a nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, and the like.

[0162] In certain other embodiments, modified Bacillus sp. cells are constructed by gene deletion to eliminate or reduce expression of at least one of the above genes of the present disclosure. Gene deletion techniques allow for partial or complete removal of genes, thereby eliminating their expression or expressing a non-functional (or reduced activity) protein product. In such methods, deletion of the gene can be accomplished by homologous recombination using a plasmid that has been constructed to contain adjacent 5' and 3' regions flanking the gene. The adjacent 5' and 3' regions can be introduced into the Bacillus cell on a temperature sensitive plasmid, such as pE194, associated with a second selectable marker at a permissive temperature that allows the plasmid to become established in the cell. The cells are then shifted to a non-permissive temperature to select for cells that have the plasmid integrated into the chromosome at one of the homologous flanking regions. Selection for integration of the plasmid is performed by selection for the second selectable marker. After integration, recombination events at the second homologous flanking region are stimulated by shifting the cells to a permissive temperature for several generations without selection. Cells are plated to obtain single colonies, which are tested for the loss of both selectable markers (see, e.g., Perego, 1993). Thus, one skilled in the art can easily identify nucleotide regions in the coding sequence of a gene and / or the non-coding sequence of a gene that are suitable for complete or partial deletion (e.g., by reference to the yitM, yitO, and yitP (nucleic acid) sequences and their encoded yitM, yitO, and yitP protein sequences).

[0163] In other embodiments, modified Bacillus cells of the present disclosure are constructed by introducing, substituting, or removing one or more nucleotides within genes or regulatory elements required for their transcription or translation. For example, nucleotides may be inserted or removed to introduce a stop codon, remove an initiation codon, or cause a frameshift in the open reading frame. Such modifications may be made by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art (see, for example, Botstein and Shortle, 1985; Lo et al., 1985; Higuchi et al., 1988; Shimada, 1996; Ho et al., 1989; Horton et al., 1989, and Sarkar and Sommer, 1990). Thus, in certain embodiments, genes of the present disclosure are inactivated by complete or partial deletion.

[0164] In another embodiment, modified Bacillus cells are constructed by the process of gene conversion (see, e.g., Iglesias and Trautner, 1983). For example, in gene conversion methods, a nucleic acid sequence corresponding to a gene is mutated in vitro to produce a defective nucleic acid sequence, which is then transformed into a parent Bacillus cell to produce the defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. It may be desirable for the defective gene or gene fragment to also encode a marker that can be used to select for transformants containing the defective gene. For example, the defective gene can be introduced into a non-replicating or temperature-sensitive plasmid in association with a selectable marker. Selection for integrating the plasmid is achieved by marker selection under conditions that do not allow the plasmid to replicate. Selection of a second recombination event resulting in gene replacement is achieved by examining colonies for loss of the selectable marker and acquisition of a mutated gene (Perego, 1993). Alternatively, the defective nucleic acid sequence may contain an insertion, substitution or deletion of one or more nucleotides of the gene, as described below.

[0165] In other embodiments, modified Bacillus cells are constructed by established antisense technology using a nucleotide sequence that is complementary to the nucleic acid sequence of a gene (Parish and Stoker, 1997). More specifically, expression of a gene by a Bacillus cell can be reduced (downregulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which can be transcribed in the cell and hybridize to the mRNA produced in the cell. Under conditions that allow the complementary antisense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is therefore reduced or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, etc., all of which are well known to those skilled in the art.

[0166] In other embodiments, modified Bacillus cells are created / constructed by CRISPR-Cas9 editing. For example, the gene encoding the yitM protein may be disrupted (or deleted or downregulated) by a guide RNA (e.g., Cas9) that recruits the endonuclease to a target sequence on the DNA and a nucleic acid-guided endonuclease that finds its target DNA by binding to either Cpfl or a guide DNA (e.g., NgAgo), where the endonuclease can generate a single-strand or double-strand break in the DNA. This targeted DNA break can become a substrate for DNA repair and recombine with the editing template provided to disrupt or delete the gene. For example, a gene encoding a nucleic acid-guided endonuclease (for this purpose, Cas9 from S. pyogenes) or a codon-optimized gene encoding a Cas9 nuclease is operably linked to a promoter active in a Bacillus cell and a terminator active in a Bacillus cell, thereby generating a Bacillus Cas9 expression cassette. Similarly, one or more target sites unique to a gene of interest can be readily identified by one of skill in the art. For example, to construct a DNA construct encoding a gRNA directed to a target site within a gene of interest using Streptococcus pyogenes Cas9, a variable targeting domain (VT) would contain the nucleotides of the target site that are 5' of the (PAM) protospacer adjacent motif (NGG), which nucleotides are fused to DNA encoding the Cas9 endonuclease recognition domain (CER) for S. pyogenes Cas9. Combination of the DNA encoding the VT domain with the DNA encoding the CER domain thereby generates DNA encoding the gRNA. Thus, a Bacillus expression cassette for a gRNA is made by operably linking the DNA encoding the gRNA to a promoter active in Bacillus cells and a terminator active in Bacillus cells.

[0167] In some embodiments, the DNA break induced by the endonuclease is repaired / replaced with the incoming sequence.For example, to precisely repair the DNA break generated by the above-mentioned Cas9 expression cassette and gRNA expression cassette, a nucleotide editing template is provided so that the DNA repair mechanism of the cell can utilize the editing template.For example, about 500 bp 5' of the targeting gene can be fused to about 500 bp 3' of the targeting gene to generate an editing template, which is used by the Bacillus host's mechanism to repair the DNA break generated by the RGEN.

[0168] The Cas9 expression cassette, the gRNA expression cassette and the editing template can be delivered simultaneously to cells using a number of different methods. Transformed cells are screened by PCR amplification of the target locus by amplifying the locus with forward and reverse primers. These primers can amplify the wild-type locus or the modified locus that has been edited by RGEN. These fragments are then sequenced using sequencing primers to identify edited colonies.

[0169] In yet other embodiments, the modified Bacillus cells are constructed by random or directed mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis (see, e.g., Hopwood, 1970) and translocation (see, e.g., Youngman et al., 1983). Genetic modification may be performed by subjecting a parent cell to mutagenesis and screening for mutant cells in which expression of the gene is reduced or eliminated. Mutagenesis, which may be directed or random, may be performed, for example, by the use of suitable physical or chemical mutagenizing agents, by the use of suitable oligonucleotides, or by subjecting the DNA sequence to PCR-generated mutagenesis. Moreover, this mutagenesis may be performed by using any combination of these mutagenesis methods.

[0170] Examples of physical or chemical mutagenic agents suitable for the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid and nucleotide analogs. When such agents are used, mutagenesis is typically carried out by incubating parent cells to be mutagenized under suitable conditions in the presence of the mutagenizing agent of choice, and selecting mutant cells that show reduced or no expression of the gene.

[0171] WO 2003 / 083125 discloses methods for modifying Bacillus cells, such as creating Bacillus deletion strains and DNA constructs using PCR fusion to bypass E. coli. WO 2002 / 14490 discloses methods for modifying Bacillus cells, including (1) construction and transformation of an integrative plasmid (pComK), (2) random mutagenesis of coding, signal and propeptide sequences, (3) homologous recombination, (4) increasing transformation efficiency by adding non-homologous flanks to the transforming DNA, (5) optimizing double crossover integration, (6) site-directed mutagenesis, and (7) markerless deletion.

[0172] Those skilled in the art are familiar with suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli and Bacillus species) (see, e.g., Ferrari et al., 1989; Saunders et al., 1984; Hoch et al., 1967; Mann et al., 1986; Holubova, 1985; Chang et al., 1979; Vorobjeva et al., 1980; Smith et al., 1986; Fisher et al., 1981 and McDonald, 1984). Indeed, methods such as transformation, including protoplast transformation and conjugation, transduction, and protoplast fusion, are known and suitable for use in the present disclosure. Transformation methods are particularly preferred for introducing the DNA constructs of the present disclosure into host cells.

[0173] In addition to commonly used methods, in some embodiments, the host cell is directly transformed (i.e., no intermediate cell is used to amplify or otherwise process the DNA construct prior to introduction into the host cell). Introduction of the DNA construct into the host cell includes those physical and chemical methods known in the art for introducing DNA into a host cell without insertion into a plasmid or vector. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, liposomes, and the like. In additional embodiments, the DNA construct is co-transformed with a plasmid without insertion into a plasmid. In further embodiments, the selection marker is deleted or substantially excised from the modified Bacillus strain by methods known in the art (e.g., Stahl et al., 1984; Palmeros et al., 2000). In some embodiments, degradation of the vector from the host chromosome leaves flanking regions in the chromosome while removing the unique chromosomal region.

[0174] Promoters and promoter sequences, their open reading frames (ORFs) and / or variants thereof for use in expressing genes in Bacillus cells are generally known to those of skill in the art. The promoter sequences of the present disclosure are generally selected such that they are functional in Bacillus cells. Certain exemplary Bacillus promoter sequences include, but are not limited to, the B. subtilis alkaline protease (aprE) promoter, the B. subtilis α-amylase promoter, the B. amyloliquefaciens α-amylase promoter, the neutral protease (nprE) promoter from B. subtilis, the mutant aprE promoter (e.g., WO 2001 / 51643) or any other promoter from B. licheniformis or other related Bacilli. Methods for screening and generating promoter libraries with a wide range of activity (promoter strength) in Bacillus cells are described in WO 2003 / 089604.

[0175] IV. Culturing the Modified Cells to Produce a Protein of Interest As generally described above, certain other embodiments relate to compositions and methods for constructing and obtaining Bacillus cells with an increased protein production phenotype. Accordingly, certain embodiments relate to methods for producing a protein of interest in a Bacillus cell by fermenting the cells in a suitable medium. Fermentation methods well known in the art can be applied to ferment the parent and modified (daughter) Bacillus cells of the present disclosure.

[0176] In some embodiments, the cells are cultured under batch or continuous fermentation conditions. Classical batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and does not change during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism. In this method, fermentation is allowed to occur without adding any components to the system. Typically, batch fermentation is considered to be "batch" with respect to the addition of the carbon source, and factors such as pH and oxygen concentration are often controlled. The metabolic products and biomass composition of a batch system are constantly changing until the point at which the fermentation is stopped. In a typical batch culture, cells may progress through a static lag phase to a high growth log phase and eventually to a stationary phase where the growth rate is reduced or stopped. If not treated, the cells in the stationary phase will eventually die. Generally, the cells in the log phase are responsible for the mass production of the product.

[0177] A suitable variation to the standard batch system is the "fed-batch fermentation" system. In this variation of the typical batch system, substrate is added gradually as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and when a limited amount of substrate in the medium is desired. In fed-batch systems, the actual substrate concentration is difficult to measure and is therefore estimated based on changes in measurable factors such as pH, dissolved oxygen, and partial pressure of waste gases such as CO2. Batch and fed-batch fermentation are common and known in the art.

[0178] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant high density where the cells are primarily in logarithmic growth phase. Continuous fermentation allows for the adjustment of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, the limiting nutrient, such as the carbon or nitrogen source, is maintained at a constant ratio while all other parameters can be adjusted. In other systems, many factors that affect growth can be continuously varied while the cell concentration, measured by the turbidity of the medium, is kept constant. Continuous systems attempt to maintain steady-state growth conditions. Thus, cell loss due to medium removal must be balanced against the cell growth rate during fermentation. Methods for adjusting nutrients and growth factors in continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[0179] In certain embodiments, the protein of interest expressed / produced by the Bacillus cells of the present disclosure can be recovered from the culture medium by separating the host cells from the medium by centrifugation or filtration, or, if necessary, by disrupting the cells and removing the supernatant from the cell fraction and cell debris. Typically, after clarification, the protein component of the supernatant or filtrate is precipitated by a salt, e.g., ammonium sulfate. The precipitated protein can then be solubilized and purified by various chromatographic methods, e.g., ion exchange chromatography, gel filtration.

[0180] In some embodiments, the cells are cultured under batch or continuous fermentation conditions. Classical batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and does not change during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism. In this method, fermentation is allowed to occur without adding any components to the system. Typically, batch fermentation is considered to be "batch" with respect to the addition of the carbon source, and factors such as pH and oxygen concentration are often controlled. The metabolic products and biomass composition of a batch system are constantly changing until the point at which the fermentation is stopped. In a typical batch culture, cells may progress through a static lag phase to a high growth log phase and eventually to a stationary phase where the growth rate is reduced or stopped. If not treated, the cells in the stationary phase will eventually die. Generally, the cells in the log phase are responsible for the mass production of the product.

[0181] A suitable variation to the standard batch system is the "fed-batch fermentation" system. In this variation of the typical batch system, substrate is added gradually as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and when a limited amount of substrate in the medium is desired. In fed-batch systems, the actual substrate concentration is difficult to measure and is therefore estimated based on changes in measurable factors such as pH, dissolved oxygen, and partial pressure of waste gases such as CO2. Batch and fed-batch fermentation are common and known in the art.

[0182] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant high density where the cells are primarily in logarithmic growth phase. Continuous fermentation allows for the adjustment of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, the limiting nutrient, such as the carbon or nitrogen source, is maintained at a constant ratio while all other parameters can be adjusted. In other systems, many factors that affect growth can be continuously varied while the cell concentration, measured by the turbidity of the medium, is kept constant. Continuous systems attempt to maintain steady-state growth conditions. Thus, cell loss due to medium removal must be balanced against the cell growth rate during fermentation. Methods for adjusting nutrients and growth factors in continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[0183] In certain embodiments, the protein of interest expressed / produced by the Bacillus cells of the present disclosure can be recovered from the culture medium by separating the host cells from the medium by centrifugation or filtration, or, if necessary, by disrupting the cells and removing the supernatant from the cell fraction and cell debris. Typically, after clarification, the protein component of the supernatant or filtrate is precipitated by a salt, e.g., ammonium sulfate. The precipitated protein can then be solubilized and purified by various chromatographic methods, e.g., ion exchange chromatography, gel filtration.

[0184] V. Protein of Interest The protein of interest (POI) of the present disclosure may be any endogenous or heterologous protein, or a mutant of such a POI. The protein may contain one or more disulfide bridges or may be a protein whose functional form is monomeric or multimeric, i.e., the protein has a quaternary structure and is composed of multiple identical (homologous) or non-identical (heterologous) subunits, where the POI or mutant POI is preferably a protein with a property of interest.

[0185] For example, in certain embodiments, the modified Bacillus cells of the present disclosure produce at least about 0.5% more, at least about 1% more, at least about 5% more, at least about 6% more, at least about 7% more, at least about 8% more, at least about 9% more, or at least about 10% or more of the POI compared to the unmodified (parent) cell.

[0186] In certain embodiments, the modified Bacillus cells of the present disclosure exhibit increased specific productivity (Qp) of the POI compared to the (unmodified) parent Bacillus cell. For example, detection of specific productivity (Qp) is a suitable method for assessing protein production. Specific productivity (Qp) is calculated according to the following equation: "Qp = gP / gDCW·hr" (where "gP" is grams of protein produced in the tank, "gDCW" is grams of dry cell weight (DCW) in the tank, and "hr" is the fermentation time (hours) from the time of inoculation, which includes the production time and the growth time.) can be determined using

[0187] Thus, in certain other embodiments, the modified Bacillus cells of the present disclosure comprise an increase in specific productivity (Qp) of at least about 0.1%, at least about 1%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% or more compared to the unmodified (parent) cell.

[0188] In certain embodiments, the POI or a variant thereof is an acetyl esterase, an aminopeptidase, an amylase, an arabinase, an arabinofuranosidase, a carbonic anhydrase, a carboxypeptidase, a catalase, a cellulase, a chitinase, a chymosin, a cutinase, a deoxyribonuclease, an epimerase, an esterase, an α-galactosidase, a β-galactosidase, an α-glucanase, a glucan lyase, a glycan ... lysases), endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methyl esterase, pectin degrading enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phytase, polygalacturonase, protease, peptidase, rhamno-galacturonase, ribonuclease, transferase, transport protein, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0189] Thus, in certain embodiments, the POI or variant POI thereof is an enzyme selected from Enzyme Code (EC) EC1, EC2, EC3, EC4, EC5 or EC6.

[0190] In certain other embodiments, the modified Bacillus cells of the present disclosure comprise an expression construct encoding an amylase. A wide variety of amylase enzymes and variants thereof are known to those of skill in the art. For example, WO 2006 / 037484 and WO 2006 / 037483 describe mutant α-amylases with improved solvent stability, WO 1994 / 18314 discloses oxidatively stable α-amylase variants, WO 1999 / 19467, WO 2000 / 29560 and WO 2000 / 60059 disclose Termamyl-like α-amylase variants, and WO 2008 / 112459 discloses mutant α-amylases having improved solvent stability, WO 2006 / 037483 ... sp. 707, WO 1999 / 43794 discloses maltogenic α-amylase variants, WO 1990 / 11352 discloses hyperthermostable α-amylase variants, and WO 2006 / 089107 discloses α-amylase variants with granular starch hydrolyzing activity.

[0191] There are a variety of assays known to those of skill in the art to detect and measure the activity of intracellularly and extracellularly expressed proteins.

[0192] WO 2014 / 164777 discloses a Ceralpha α-amylase activity assay useful for detecting the amylase activity described herein.

[0193] V. Exemplary Embodiments Non-limiting embodiments of the present disclosure include, but are not limited to, the following.

[0194] 1. A method for producing increased amounts of an endogenous protein of interest (POI) in an engineered Bacillus sp. cell, comprising the steps of: (a) obtaining a parent Bacillus sp. cell that produces the endogenous POI; (b) genetically modifying the parent Bacillus sp. cell by deleting or disrupting the yitMOP operon; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under the same conditions.

[0195] 2. A method for producing increased amounts of an endogenous protein of interest (POI) in an engineered Bacillus sp. cell, comprising the steps of: (a) obtaining a parent Bacillus sp. cell that produces the endogenous POI; (b) genetically modifying the parent Bacillus sp. cell by deleting or disrupting the yitMOP and sdpABC operons; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under the same conditions.

[0196] 3. A method for producing increased amounts of an endogenous protein of interest (POI) in an engineered Bacillus sp. cell, comprising the steps of: (a) obtaining a parent Bacillus sp. cell that produces the endogenous POI; (b) genetically modifying the parent Bacillus sp. cell by deleting or disrupting the regulatory region of the yitMOP operon, thereby rendering the engineered cell deficient in producing functional YitM, YitO and / or YitP proteins; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under the same conditions.

[0197] 4. A method for producing increased amounts of an endogenous protein of interest (POI) in an engineered Bacillus sp. cell, comprising the steps of: (a) obtaining a parent Bacillus sp. cell that produces the endogenous POI; (b) genetically modifying the parent cell by disrupting or deleting the yitM gene; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under the same conditions.

[0198] 5. A method for producing increased amounts of an endogenous protein of interest (POI) in an engineered Bacillus sp. cell, comprising the steps of: (a) obtaining a parent Bacillus sp. cell that produces the endogenous POI; (b) genetically modifying the parent Bacillus sp. cell by deleting or disrupting the regulatory region of the yitM gene, thereby rendering the engineered cell deficient in producing a functional YitM protein; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under the same conditions.

[0199] 6. The method of any one of embodiments 1-5, wherein the Bacillus sp. cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.

[0200] 7. The method of embodiment 6, wherein the Bacillus sp. cell is B. subtilis.

[0201] 8. The method of embodiment 6, wherein the Bacillus sp. cell is B. amyloliquefaciens.

[0202] 9. The method of any one of embodiments 1-5, wherein the POI is an enzyme.

[0203] 10. The method of embodiment 7, wherein the enzyme is a protease or an amylase.

[0204] 11. The method of any one of embodiments 1 to 3, wherein the yitMOP operon encodes a YitM polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO:2 or SEQ ID NO:38, a YitO polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO:4 or SEQ ID NO:36, and a YitP polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO:6 or SEQ ID NO:34.

[0205] 12. The method of embodiment 4 or embodiment 5, wherein the yitM gene comprises at least 60% to about 99% sequence identity with the yitM gene of SEQ ID NO: 1 or SEQ ID NO: 37.

[0206] 13. The method of embodiment 4 or embodiment 5, wherein the yitM gene encodes a yitM polypeptide comprising at least 60% to about 99% sequence identity with the yitM polypeptide of SEQ ID NO: 2 or SEQ ID NO: 38.

[0207] 14. The method of any one of embodiments 3-5, wherein the modified cell further comprises a deletion or disruption of the sdpABC operon.

[0208] 15. A method for producing increased amounts of a heterologous protein of interest (POI) in an engineered Bacillus sp. cell, comprising: (a) constructing a parent Bacillus sp. cell containing an introduced expression cassette encoding the heterologous POI; (b) genetically modifying the parent cell by deleting or disrupting the yitMOP operon; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the heterologous POI compared to the parent cell when fermented under the same conditions.

[0209] 16. A method for producing increased amounts of a heterologous protein of interest (POI) in an engineered Bacillus sp. cell, comprising: (a) constructing a parent Bacillus sp. cell containing an introduced expression cassette encoding the heterologous POI; (b) genetically modifying the parent Bacillus sp. cell by deleting or disrupting the yitMOP and sdpABC operons; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the heterologous POI compared to the parent cell when fermented under the same conditions.

[0210] 17. A method for producing increased amounts of a heterologous protein of interest (POI) in an engineered Bacillus sp. cell, comprising: (a) constructing a parent Bacillus sp. cell containing an introduced expression cassette encoding the heterologous POI; (b) genetically modifying the parent Bacillus sp. cell by deleting or disrupting the regulatory region of the yitMOP operon, thereby rendering the engineered cell deficient in producing functional YitM, YitO and / or YitP proteins; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the heterologous POI compared to the parent cell when fermented under the same conditions.

[0211] 18. A method for producing increased amounts of a heterologous protein of interest (POI) in an engineered Bacillus sp. cell, comprising: (a) constructing a parent Bacillus sp. cell containing an introduced expression cassette encoding the heterologous POI; (b) genetically modifying the parent cell by disrupting or deleting the yitM gene; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the heterologous POI compared to the parent cell when fermented under the same conditions.

[0212] 19. A method for producing increased amounts of a heterologous protein of interest (POI) in an engineered Bacillus sp. cell, comprising: (a) constructing a parent Bacillus sp. cell containing an introduced expression cassette encoding the heterologous POI; (b) genetically modifying the parent Bacillus sp. cell by deleting or disrupting the regulatory region of the yitM gene, thereby rendering the engineered cell deficient in producing a functional YitM protein; and (c) fermenting the engineered cell under conditions suitable for producing the POI, wherein the engineered cell produces increased amounts of the heterologous POI compared to the parent cell when fermented under the same conditions.

[0213] 20. The method of any one of embodiments 15-19, wherein steps (a) and (b) are carried out simultaneously.

[0214] 21. Bacillus species 20. The method of any one of embodiments 15-19, wherein the B. sp. cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.

[0215] 22. The method of embodiment 21, wherein the Bacillus sp. cell is B. subtilis.

[0216] 23. The method of embodiment 21, wherein the Bacillus sp. cell is B. amyloliquefaciens.

[0217] 24. The method of any one of embodiments 15-19, wherein the heterologous POI is an enzyme.

[0218] 25. Enzymes include acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lyase 25. The method of embodiment 24, wherein the enzyme is selected from the group consisting of: endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methyl esterase, pectin degrading enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phytase, polygalacturonase, protease, peptidase, rhamno-galacturonase, ribonuclease, transferase, transport protein, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0219] 26. The method of any one of embodiments 15 to 17, wherein the yitMOP operon encodes a YitM polypeptide comprising at least 80% sequence identity to SEQ ID NO:2 or SEQ ID NO:38, a YitO polypeptide comprising at least about 80% sequence identity to SEQ ID NO:4 or SEQ ID NO:36, and a YitP polypeptide comprising at least about 80% sequence identity to SEQ ID NO:6 or SEQ ID NO:34.

[0220] 27. The method of embodiment 18 or embodiment 19, wherein the yitM gene comprises at least 60% to about 99% sequence identity with the yitM gene of SEQ ID NO: 1 or SEQ ID NO: 37.

[0221] 28. The method of embodiment 18 or embodiment 19, wherein the yitM gene encodes a YitM polypeptide comprising at least 60% to about 99% sequence identity with the YitM polypeptide of SEQ ID NO: 2 or SEQ ID NO: 38.

[0222] 29. The method of any one of embodiments 17-19, wherein the modified cell further comprises a deletion or disruption of the sdpABC operon.

[0223] 30. A genetically modified Bacillus sp. cell produced by the method of any one of embodiments 1-5 or a genetically modified Bacillus sp. cell produced by the method of any one of embodiments 15-19.

[0224] 31. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell that produces an endogenous protein of interest (POI), wherein the modified cell contains a deleted or disrupted yitMOP operon, and wherein the modified cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under identical conditions to produce the POI.

[0225] 32. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell that produces an endogenous protein of interest (POI), wherein the modified cell comprises a deleted or disrupted yitMOP operon and a deleted or disrupted sdpABC operon, wherein the modified cell produces an increased amount of the endogenous POI compared to the parent cell when fermented under identical conditions to produce the POI.

[0226] 33. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell that produces an endogenous protein of interest (POI), wherein the modified cell contains a deleted or disrupted regulatory region of the yitMOP operon that renders the modified cell deficient in producing functional YitM, YitO and / or YitP proteins, and wherein the modified cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under identical conditions to produce the POI.

[0227] 34. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell that produces an endogenous protein of interest (POI), wherein the modified cell contains a deleted or disrupted yitM gene, and wherein the modified cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under identical conditions.

[0228] 35. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell that produces an endogenous protein of interest (POI), wherein the modified cell contains a deleted or disrupted regulatory region of the yitM gene that renders the modified cell defective in producing a functional YitM protein, and wherein the modified cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under identical conditions.

[0229] 36. Bacillus species The modified cell of any one of embodiments 31 to 35, wherein the sp. cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.

[0230] 37. The modified cell of embodiment 36, wherein the Bacillus sp. cell is B. subtilis.

[0231] 38. The modified cell of embodiment 36, wherein the Bacillus sp. cell is B. amyloliquefaciens.

[0232] 39. The modified cell of any one of embodiments 29-33, wherein the POI is an enzyme.

[0233] 40. The modified cell of embodiment 39, wherein the enzyme is a protease or an amylase.

[0234] 41. The modified cell of any one of embodiments 31 to 33, wherein the yitMOP operon encodes a YitM polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 38, a YitO polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 36, and a YitP polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO: 6 or SEQ ID NO: 34.

[0235] 42. The modified cell of embodiment 34 or embodiment 35, wherein the yitM gene comprises at least 60% to about 99% sequence identity with the yitM gene of SEQ ID NO: 1 or SEQ ID NO: 37.

[0236] 43. The modified cell of embodiment 34 or embodiment 35, wherein the yitM gene encodes a YitM polypeptide comprising at least 60% to about 99% sequence identity with the YitM polypeptide of SEQ ID NO: 2 or SEQ ID NO: 38.

[0237] 44. The modified cell of any one of embodiments 33-35, wherein the modified cell further comprises a deletion or disruption of the sdpABC operon.

[0238] 45. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell containing an introduced expression cassette encoding a heterologous protein of interest (POI), wherein the modified cell contains a deleted or disrupted yitMOP operon, and wherein the modified cell produces increased amounts of the heterologous POI compared to the parent cell when fermented under identical conditions for producing the POI.

[0239] 46. ​​A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell containing an introduced expression cassette encoding a heterologous protein of interest (POI), wherein the modified cell contains a deleted or disrupted yitMOP operon and a deleted or disrupted sdpABC operon, wherein the modified cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under identical conditions to produce the POI.

[0240] 47. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell containing an introduced expression cassette encoding a heterologous protein of interest (POI), wherein the modified cell contains a deleted or disrupted regulatory region of the yitMOP operon that renders the modified cell defective in producing functional YitM, YitO and / or YitP proteins, and wherein the modified cell produces increased amounts of endogenous POI compared to the parent cell when fermented under identical conditions to produce the POI.

[0241] 48. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell containing an introduced expression cassette encoding a heterologous protein of interest (POI), wherein the modified cell contains a deleted or disrupted yitM gene, and wherein the modified cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under identical conditions.

[0242] 49. A genetically modified Bacillus sp. cell derived from a parent Bacillus sp. cell containing an introduced expression cassette encoding a heterologous protein of interest (POI), wherein the modified cell contains a deleted or disrupted regulatory region of the yitM gene that renders the modified cell defective in producing a functional YitM protein, and wherein the modified cell produces increased amounts of the endogenous POI compared to the parent cell when fermented under identical conditions.

[0243] 50. Bacillus species The modified cell of any one of embodiments 45 to 49, wherein the sp. cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.

[0244] 51. The modified cell of embodiment 50, wherein the Bacillus sp. cell is B. subtilis.

[0245] 52. The modified cell of embodiment 50, wherein the Bacillus sp. cell is B. amyloliquefaciens.

[0246] 53. The modified cell of any one of embodiments 45-49, wherein the POI is an enzyme.

[0247] 54. Enzymes include acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lyase 54. The method of embodiment 53, wherein the enzyme is selected from the group consisting of: endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methyl esterase, pectin degrading enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phytase, polygalacturonase, protease, peptidase, rhamno-galacturonase, ribonuclease, transferase, transport protein, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0248] 55. The modified cell of any one of embodiments 45 to 47, wherein the yitMOP operon encodes a YitM polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 38, a YitO polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 36, and a YitP polypeptide having at least 60% to about 99% sequence identity with SEQ ID NO: 6 or SEQ ID NO: 34.

[0249] 56. The modified cell of embodiment 48 or embodiment 49, wherein the yitM gene comprises at least 80% sequence identity with the yitM gene of SEQ ID NO: 1 or SEQ ID NO: 37.

[0250] 57. The modified cell of embodiment 48 or embodiment 49, wherein the yitM gene encodes a YitM polypeptide comprising at least 80% sequence identity to the YitM polypeptide of SEQ ID NO: 2 or SEQ ID NO: 38.

[0251] 58. The modified cell of any one of embodiments 47-49, wherein the modified cell further comprises a deletion or disruption of the sdpABC operon.

[0252] 59. The method of any one of embodiments 3, 5, 17 or 19, wherein the deleted or disrupted regulatory region is a promoter sequence.

[0253] 60. The method of embodiment 59, wherein the promoter sequence comprises at least 60% to about 99% sequence identity with a nucleotide sequence selected from SEQ ID NO: 39, SEQ ID NO: 39 and SEQ ID NO: 40.

[0254] 61. The modified cell of any one of embodiments 33, 35, 47 or 49, wherein the deleted or disrupted regulatory region is a promoter sequence.

[0255] 62. The modified cell of embodiment 61, wherein the promoter sequence comprises at least 60% to about 99% sequence identity with a nucleotide sequence selected from SEQ ID NO: 39, SEQ ID NO: 39 and SEQ ID NO: 40. EXAMPLES

[0256] Certain aspects of the present disclosure can be further understood in light of the following examples, which should not be construed as limiting. Modifications of materials and methods will be apparent to those skilled in the art.

[0257] Example 1 Deletion of the yitMOP operon in Bacillus cells As generally defined and described above, a novel B. subtilis cannibalism operon, designated yitMOP, was identified as a highly expressed transcript by transcriptome analysis of B. subtilis strains expressing FNA protease. More specifically, as presented in FIG. 1A and FIG. 1B, the B. subtilis yitMOP operon encodes the YitM protein (SEQ ID NO:2), the YitO protein (SEQ ID NO:4) and the YitP protein (SEQ ID NO:6). This example describes the deletion of the yitMOP operon in parent B. subtilis cells. For example, a plasmid designated "pUCyitMOP:spec" was constructed to delete the yitMOP operon in B. subtilis to generate modified B. subtilis (daughter) cells. For example, the pUCyitMOP:spec plasmid was constructed to replace the entire yitMOP operon in B. subtilis with a spectinomycin resistance (specR) marker gene flanked by loxP sites (herein a "loxP-specR-loxP cassette"). Thus, the upstream (5') sequence was amplified from B. subtilis (strain 168) genomic DNA (gDNA) using the oJKL228 (SEQ ID NO:7) and oJKL229 (SEQ ID NO:8) primers shown in Table 1 below. [Table 1]

[0258] The downstream (3') homology sequence was amplified from B. subtilis (strain 168) gDNA using the oJKL232 (SEQ ID NO:9) and oJKL233 (SEQ ID NO:10) primers shown in Table 2 below. [Table 2]

[0259] The loxP-specR-loxP cassette was amplified from the Topo-specR vector using oJKL230 (SEQ ID NO:11) and oJKL231 (SEQ ID NO:12) shown in Table 3 below. The Topo-specR vector contains the spectinomycin adenyltransferase gene (specR) with its native promoter and terminator elements, which was cloned into the Invitrogen Topo vector blunt and used to transform E. coli TOP10 cells. [Table 3]

[0260] The pUC vector backbone was amplified from the pUCL fragment obtained from the GeneArt Seamless Cloning Kit using the oJKL226 (SEQ ID NO: 13) and oJKL227 (SEQ ID NO: 14) primers shown in Table 4 below, according to the manufacturer's instructions. [Table 4]

[0261] The vector fragment was assembled using the Gibson cloning kit and used to transform E. coli. The upstream (5') and downstream (3') homology sections of the vector were sequence verified using the forward (M13F; SEQ ID NO: 15) and reverse (M13R; SEQ ID NO: 16) primers shown in Table 5 below. [Table 5]

[0262] The sequence confirmed vector was isolated and linearized with AatII, where the linearized vector was then used to transform B. subtilis cells. The deletion was then verified using PCR, where the upstream (5') integration junction was confirmed using the oJKL246 (SEQ ID NO:17) and 5'spec out (SEQ ID NO:18) primers shown in Table 6 below. [Table 6]

[0263] The downstream integration junction was confirmed using the oJKL247 (SEQ ID NO: 19) and 3'spec out (SEQ ID NO: 20) primers shown in Table 7 below. [Table 7]

[0264] Example 2 Deletion of the sdpABC operon This example describes the construction of a plasmid designated "pUCsdp::tet" and its introduction into B. subtilis cells. More specifically, the pUCsdp::tet plasmid, containing upstream (5') and downstream (3') DNA sequences, was constructed to replace the entire sdpABC operon in B. subtilis with a tetracycline (tetR) marker flanked by loxP sites (herein a "loxP-tetR-loxP cassette"), where removal of the tetracycline marker results in a deletion of the sdpABC operon.

[0265] Therefore, the upstream (5') sdpABC sequence was amplified from B. subtilis gDNA using the oJKL220 (SEQ ID NO:21) and oJKL221 (SEQ ID NO:22) primers shown in Table 8 below. [Table 8]

[0266] The downstream (3') sequence was amplified from B. subtilis gDNA using the oJKL224 (SEQ ID NO:23) and oJKL225 (SEQ ID NO:24) primers shown in Table 9 below. [Table 9]

[0267] The loxP-tetR-loxP cassette was amplified from plasmid pUCLlacA-ganAB::tet using the oJKL222 (SEQ ID NO:25) and oJKL223 (SEQ ID NO:26) primers shown in Table 10 below. [Table 10]

[0268] The pUC19 vector backbone was amplified from the GeneArt Linear pUC19L vector from Life Technologies using the oJKL218 (SEQ ID NO:27) and oJKL219 (SEQ ID NO:28) primers shown in Table 11 below, according to the manufacturer's instructions. [Table 11]

[0269] The upstream, downstream, loxP-tetR-loxP and pUC19 amplicons were fused together using the NEB Gibson kit according to the manufacturer's instructions. The sequence verified plasmid was then linearized with AatII and used to transform B. subtilis cells. Deletion of the genomic sdp operon (Δsdp:loxP-tetR-loxP) was confirmed using cPCR, where the upstream integration site was confirmed using the oJKL238 (SEQ ID NO:29) and tet3'out (SEQ ID NO:30) primers shown in Table 12 below. [Table 12]

[0270] The downstream site was confirmed using the oJKL239 (SEQ ID NO: 31) and tet5'out (SEQ ID NO: 32) primers shown in Table 13 below. [Table 13]

[0271] Shown below in Table 14, and further described in Example 3 below, are the strain names and short descriptions of the B. subtilis cells (strains) constructed herein. [Table 14]

[0272] Example 3 Production of heterologous proteases in engineered B. subtilis cells This example describes the production of a heterologous protein of interest (POI) in B. subtilis cells. More specifically, the parent (ZM207) and modified B. subtilis strains were constructed as described in Examples 1 and 2 above (see, e.g., Table 14) and fermented in shake flasks under identical conditions in Grant's II medium at 42° C. for 40 hours. A daughter B. subtilis strain designated ZM434(ΔyitM) was constructed by transforming genomic DNA of strain BKE11040 (trpC2 yitM::erm) obtained from the Bacillus Genetics Stock Center into the parent strain (ZM207).

[0273] Fermentation supernatants were subsequently removed and ME-3 protease activity was determined using an AAPF-assay (see, e.g., WO 2018 / 118815, incorporated herein by reference in its entirety) and monitored by the change in absorbance at 405 nm. The slope of the change in absorbance (mOD / min) reflects ME-3 protease activity and is reported herein for comparison. More specifically, Table 15 below presents the average ME-3 titer and standard error of multiple (n≧3) biological replicates. [Table 15]

[0274] For example, both the ZM334 (ΔsdpABC) and ZM336 (ΔyitMOP) daughter strains produced increased amounts of protease compared to the ZM207 parent strain, as shown in Table 15. Similarly, the modified ZM268 strain (Table 15) containing a combined deletion of the yitMOP and sdpABC operons (i.e., ΔyitMOP+ΔsdpABC) produces increased amounts of ME-3 protease compared to the ZM207 (control), ZM334 (ΔsdpABC) and ZM336 (ΔyitMOP) strains.

[0275] Moreover, it was surprisingly observed that disruption or deletion of the yitM ORF alone was sufficient to elicit the beneficial phenotype of increased protein production, as the ZM434(ΔyitM) daughter strain produced slightly more ME-3 protease than the ZM336(ΔyitMOP) daughter strain (Table 15).

[0276] Example 4 Production of heterologous proteases in engineered B. subtilis cells This example describes the production of a heterologous protease (V42) in parent and engineered B. subtilis cells under large-scale fermentation conditions. More specifically, as described herein, the B. subtilis strain designated CB24-12 contains an introduced expression cassette comprising a promoter sequence ("P4") located upstream (5') and operably linked to an open reading frame (ORF) encoding the V42 protease, which is operably linked upstream (5') to an ORF encoding an alanine racemase (alrA), where the cassette is integrated within the B. subtilis aprE locus (aprE::P4-V42-alrA). Similarly, a B. subtilis strain designated "JL77" contains introduced expression cassettes encoding the V42 protease (aprE::P4-V42-alrA) and a combined deletion of the yitMOP and sdpABC operons (ΔyitMOP+ΔsdpABC).

[0277] Thus, cells from strains JL77 and CB24-12 were tested in large-scale fermentors under standard conditions, with each strain run in duplicate, to show the average percent improvement in V42 protease production at the end of fermentation (EOF), as shown in Figure 2. For example, as presented in Figure 2, the average percent increase in V42 protease production at the end of fermentation for strain JL77 was approximately 10% compared to the amount of V42 protease produced by strain CB24-12 at the end of fermentation. 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Claims

1. 1. A method for producing increased amounts of a protein of interest (POI) in an engineered Bacillus sp. cell, comprising: (a) obtaining a parent Bacillus sp. cell that produces a POI; (b) genetically modifying the parent Bacillus sp. cell by disrupting or deleting the yitMOP operon; and (c) fermenting the modified cells under suitable conditions to produce the POI. Including, The modified cells produce an increased amount of the POI as compared to the parent cells when cultured under the same conditions.

2. 1. A method for producing increased amounts of a protein of interest (POI) in an engineered Bacillus sp. cell, comprising: (a) obtaining a parent Bacillus sp. cell that produces a POI; (b) genetically modifying the parent Bacillus sp. cell by disrupting or deleting a regulatory region of the yitMOP operon, thereby rendering the modified cell defective in the production of functional YitM, YitO and / or YitP proteins; and (c) fermenting the modified cells under suitable conditions to produce the POI; The modified cell produces an increased amount of the POI as compared to the parent cell when fermented under the same conditions.

3. 1. A method for producing increased amounts of a protein of interest (POI) in an engineered Bacillus sp. cell, comprising: (a) obtaining a parent Bacillus sp. cell that produces a POI; (b) genetically modifying the parent cell by disrupting or deleting the yitM gene; and (c) fermenting the modified cells under suitable conditions to produce the POI; The modified cell produces an increased amount of the POI as compared to the parent cell when fermented under the same conditions.

4. The method of any one of claims 1 to 3, wherein the Bacillus sp. cell further comprises a disruption or deletion of the sdpABC operon.

5. The Bacillus sp. cells include B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. erythrocytes ...

5. The method according to any one of claims 1 to 4, wherein the B. circulans, B. lautus and B. thuringiensis are selected from the group consisting of B. circulans, B. lautus and B. thuringiensis.

6. The method according to any one of claims 1 to 3, wherein the POI is an enzyme.

7. The enzymes include acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucanlysase, endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, 7. The method of claim 6, wherein the enzyme is selected from the group consisting of enzymes, isomerases, laccases, ligases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenol oxidases, phytases, polygalacturonases, proteases, peptidases, rhamno-galacturonases, ribonucleases, transferases, transport proteins, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

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