Compositions and methods for enhanced protein production in Bacillus licheniformis

Abstract

The present disclosure generally relates to compositions and methods for constructing and / or obtaining B. licheniformis cells (e.g., protein-producing hosts) that comprise enhanced protein production capabilities. Accordingly, certain embodiments relate to genetically modified B. licheniformis strains derived from a parent Bacillus licheniformis strain that produce increased amounts of one or more proteins of interest.

Description

[Technical Field]

[0001] The present disclosure relates generally to the fields of bacteriology, microbiology, genetics, molecular biology, enzymology, industrial protein production, etc. Accordingly, certain embodiments of the present disclosure relate to compositions and methods for constructing Bacillus licheniformis cells / strains with enhanced protein production phenotypes.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 961,234, filed January 15, 2020, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing Reference The contents of the electronic submission of the text file of the Sequence Listing entitled "NB41684-WO-PCT_SequenceListing.txt", which is incorporated herein by reference in its entirety, was created on January 7, 2021, and is 425KB in size. [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-amylase (Jensen et al., 2000; Raul et al., 2014) and protease (Brode et al., 1996), which are 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 earned the European Food Safety Authority's "Qualified Presumption of Safety" (QPS) rating, and many of their products have earned the US Food and Drug Administration's "Generally Recognized As Safe" (GRAS) rating (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 host cells for the production and secretion of one or more proteins of interest is highly important, especially in the context of industrial biotechnology, where even small improvements in protein yield can be crucial when proteins are produced industrially on a large scale. More particularly, B. licheniformis is a host cell for Bacillus species of high industrial importance; therefore, the ability to modify and engineer B. licheniformis host cells for enhanced / increased protein expression / production is highly desirable for constructing new and improved B. licheniformis production strains. Accordingly, the present disclosure relates to a highly desirable and unmet need for obtaining and constructing B. licheniformis cells (e.g., protein-producing host cells) with increased protein production capabilities. [Means for solving the problem]

[0006] The present disclosure generally relates to compositions and methods for obtaining B. licheniformis cells (e.g., protein-producing hosts) that contain enhanced protein production capabilities. Accordingly, certain embodiments of the present disclosure relate to methods for constructing such modified B. licheniformis cells / strains that produce increased amounts of one or more proteins of interest.

[0007] Accordingly, certain embodiments of the present disclosure are directed to a method for producing increased amounts of an endogenous protein of interest (POI) in an engineered Bacillus licheniformis cell, comprising: (a) obtaining a parent B. licheniformis cell that expresses the POI and modifying the parent cell by introducing therein a polynucleotide comprising a native prsA promoter operably linked to a native prsA open reading frame (ORF); and (b) fermenting the engineered cell of step (a) under conditions suitable for production of the POI, wherein the engineered cell produces increased amounts of the POI compared to the parent cell when fermented under the same conditions. In certain embodiments of the method, the introduced polynucleotide of step (a) comprises a native prsA promoter comprising at least 95% sequence identity to SEQ ID NO:100. In other embodiments of the method, the introduced polynucleotide of step (a) comprises a native prsA ORF comprising at least 90% sequence identity to SEQ ID NO:101. In other embodiments, the introduced polynucleotide encodes a native prsA protein that comprises about 90% sequence identity to SEQ ID NO: 155. In certain preferred embodiments, the parent cell comprises an endogenous (wild-type) prsA gene encoding a native prsA protein, but the introduced polynucleotide thereby encodes a second copy of the prsA protein that comprises about 90% sequence identity to SEQ ID NO: 155. In other embodiments, the introduced polynucleotide of step (a) is integrated into the genome of the modified B. licheniformis cell. In yet other embodiments of this method, the protein of interest (POI) is a protease or amylase. In other embodiments, the modified cell comprises a deleted or disrupted dltA gene that comprises at least 90% sequence identity to SEQ ID NO: 122. In other embodiments, the modified cell comprises a deleted or disrupted rghR2 gene that comprises at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.In other embodiments, the engineered cell comprises a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122 and a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.

[0008] In certain other embodiments, the disclosure relates to a method for producing increased amounts of a heterologous protein of interest (POI) in an engineered Bacillus licheniformis cell, comprising: (a) introducing into a parent B. licheniformis cell a polynucleotide comprising (i) an expression cassette encoding the POI and (ii) a native prsA promoter operably linked to a native prsA open reading frame (ORF); and (b) fermenting the engineered cell of step (a) under conditions suitable for production of the POI, wherein the engineered cell produces increased amounts of the POI compared to the parent cell when fermented under the same conditions. In certain embodiments of the method, the introduced polynucleotide of step (a)(ii) comprises a native prsA promoter comprising at least 95% sequence identity to SEQ ID NO: 100. In certain other embodiments, the introduced polynucleotide of step (a)(ii) comprises a native prsA ORF comprising at least 90% sequence identity to SEQ ID NO: 101. In yet other embodiments of the method, the endogenous prsA gene encodes a native prsA protein that comprises about 90% sequence identity to SEQ ID NO: 155. In certain other embodiments, the introduced polynucleotide of step (a)(ii) is integrated into the genome of the modified B. licheniformis cell. In certain preferred embodiments, the parent cell comprises an endogenous (wild-type) prsA gene encoding a native prsA protein, wherein the introduced polynucleotide of step (a)(ii) thereby encodes a second copy of the prsA protein that comprises about 90% sequence identity to SEQ ID NO: 155. In particular embodiments, the protein of interest (POI) is a protease or amylase. In other embodiments, the modified cell comprises a deleted or disrupted dltA gene that comprises at least 90% sequence identity to SEQ ID NO: 122. In other embodiments, the modified cell comprises a deleted or disrupted rghR2 gene that comprises at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.In certain preferred embodiments, the modified cell comprises a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO:122 and a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO:121 or SEQ ID NO:158.

[0009] Other embodiments of the present disclosure are directed to modified B. licheniformis cells / strains derived from a parent Bacillus licheniformis cell / strain containing an endogenous prsA gene encoding a native prsA protein. Accordingly, in certain embodiments, the modified B. licheniformis cells of the present disclosure contain an introduced polynucleotide comprising a native prsA promoter operably linked to a native prsA open reading frame (ORF). In certain embodiments, the introduced polynucleotide comprises a native prsA promoter comprising at least 95% sequence identity to SEQ ID NO: 100. In other embodiments, the introduced polynucleotide comprises a native prsA ORF comprising at least 90% sequence identity to SEQ ID NO: 101. In yet other embodiments, the introduced polynucleotide encodes a native prsA protein comprising about 90% sequence identity to SEQ ID NO: 155. In certain other embodiments, an introduced polynucleotide encoding a native prsA protein is integrated into the genome of the modified B. licheniformis cell. In another embodiment, the modified cell comprises a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122. In another embodiment, the modified cell comprises a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158. In a preferred embodiment, the modified cell comprises a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122 and a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158. In certain other embodiments, the modified cell comprises an introduced expression construct encoding a heterologous protein of interest (POI). In other embodiments, the heterologous POI is a protease or amylase. Accordingly, certain embodiments of the present disclosure are directed to obtaining, isolating, purifying, etc., a protein of interest produced by the modified B. licheniformis cells of the present disclosure.

[0010] Accordingly, certain other embodiments of the present disclosure are directed to modified B. licheniformis cells that produce increased amounts of a protein of interest (POI) relative to the parent Bacillus licheniformis cells from which they are derived. Accordingly, in certain embodiments, the disclosure relates to an engineered B. licheniformis cell that produces an increased amount of a protein of interest (POI) relative to a parent Bacillus licheniformis cell, wherein the engineered cell is derived from a parent B. licheniformis cell that expresses the POI, the engineered cell comprises an introduced polynucleotide comprising a native prsA promoter operably linked to a native prsA open reading frame (ORF), and comprises a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158, and the engineered cell produces an increased amount of the POI relative to the parent strain when fermented under identical conditions. In another embodiment, the engineered Bacillus licheniformis cell comprises a deleted or disrupted dltA gene that comprises at least 90% sequence identity to SEQ ID NO: 122. In yet another embodiment, the native prsA promoter comprises at least 95% sequence identity to SEQ ID NO: 100. In certain other embodiments, the native prsA ORF comprises at least 90% sequence identity to SEQ ID NO: 101. In other embodiments, the native prsA protein comprises about 90% sequence identity to SEQ ID NO: 155. In particular embodiments, the protein of interest (POI) is a protease or amylase. Accordingly, certain other embodiments of the present disclosure are directed to obtaining, isolating, purifying, etc., a protein of interest produced by an engineered B. licheniformis cell.

[0011] In another embodiment, the disclosure relates to an engineered B. licheniformis cell that produces an increased amount of a protein of interest (POI) relative to a parent B. licheniformis cell, wherein the engineered cell is derived from a parent B. licheniformis cell that expresses the POI, the engineered cell comprises an introduced polynucleotide comprising a native prsA promoter operably linked to a native prsA open reading frame (ORF), and a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122, and the engineered cell produces an increased amount of the POI relative to the parent strain when fermented under the same conditions. In another embodiment, the engineered cell further comprises a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158. In yet another embodiment, the native prsA promoter comprises at least 95% sequence identity to SEQ ID NO: 100. In certain other embodiments, the native prsA ORF comprises at least 90% sequence identity to SEQ ID NO: 101. In other embodiments, the native prsA protein comprises about 90% sequence identity to SEQ ID NO: 155. In particular embodiments, the protein of interest (POI) is a protease or amylase. Accordingly, certain other embodiments of the present disclosure are directed to obtaining, isolating, purifying, etc., a protein of interest produced by an engineered B. licheniformis cell.

[0012] A brief description of biological sequences SEQ ID NO: 1 is the amino acid sequence encoding the native S. pyogenes Cas9 protein.

[0013] SEQ ID NO:2 is a nucleic acid sequence encoding the Cas9 protein of SEQ ID NO:1, which nucleic acid sequence has been codon-optimized for expression in a Bacillus sp. cell.

[0014] SEQ ID NO: 3 is the amino acid sequence of a synthetic N-terminal nuclear localization signal (NLS).

[0015] SEQ ID NO: 4 is the amino acid sequence of a synthetic C-terminal nuclear localization signal (NLS).

[0016] SEQ ID NO: 5 is the amino acid sequence of a synthetic deca-histidine tag.

[0017] SEQ ID NO: 6 is the aprE promoter sequence of B. subtilis.

[0018] SEQ ID NO: 7 is the nucleic acid sequence of the synthetic terminator.

[0019] SEQ ID NO: 8 is the nucleic acid sequence of the forward primer.

[0020] SEQ ID NO: 9 is the nucleic acid sequence of the reverse primer.

[0021] SEQ ID NO: 10 is the nucleic acid sequence of the synthetic pKB320 backbone.

[0022] SEQ ID NO: 11 is the nucleic acid sequence of the synthetic pKB320.

[0023] SEQ ID NO: 12 is the nucleic acid sequence of the primer.

[0024] SEQ ID NO: 13 is the nucleic acid sequence of the primer.

[0025] SEQ ID NO: 14 is the nucleic acid sequence of the primer.

[0026] SEQ ID NO: 15 is the nucleic acid sequence of the primer.

[0027] SEQ ID NO: 16 is the nucleic acid sequence of the primer.

[0028] SEQ ID NO: 17 is the nucleic acid sequence of the primer.

[0029] SEQ ID NO: 18 is the nucleic acid sequence of the primer.

[0030] SEQ ID NO: 19 is the nucleic acid sequence of the primer.

[0031] SEQ ID NO: 20 is the nucleic acid sequence of the primer.

[0032] SEQ ID NO: 21 is the nucleic acid sequence of the primer.

[0033] SEQ ID NO: 22 is the nucleic acid sequence of the primer.

[0034] SEQ ID NO: 23 is the nucleic acid sequence of the primer.

[0035] SEQ ID NO: 24 is the nucleic acid sequence of the primer.

[0036] SEQ ID NO:25 is the nucleic acid sequence of the synthetic pRF694.

[0037] SEQ ID NO:26 is the nucleic acid sequence of synthetic pRF801.

[0038] SEQ ID NO:27 is the nucleic acid sequence of the synthetic pRF806.

[0039] SEQ ID NO: 28 is the nucleic acid sequence of B. licheniformis target site 1 (TS1).

[0040] SEQ ID NO: 29 is the nucleic acid sequence of B. licheniformis target site 2 (TS2).

[0041] SEQ ID NO: 30 is the open reading frame (ORF) sequence of serA1 of B. licheniformis.

[0042] SEQ ID NO: 31 is the PAM sequence of target site 1 containing the nucleotides "AGG."

[0043] SEQ ID NO: 32 is the nucleic acid sequence encoding variable targeting (VT) site 1.

[0044] SEQ ID NO: 33 is a synthetic nucleic acid sequence encoding the CER domain.

[0045] SEQ ID NO: 34 is a synthetic guide RNA (gRNA) sequence targeting site 1.

[0046] SEQ ID NO: 35 is the nucleic acid sequence of the synthetic spac promoter.

[0047] SEQ ID NO: 36 is the nucleic acid sequence of the synthetic t0 terminator.

[0048] SEQ ID NO: 37 is the nucleic acid sequence of serA1 homology arm 1 of B. licheniformis.

[0049] SEQ ID NO: 38 is the sequence of the synthetic serA1 homology arm 1 forward primer.

[0050] SEQ ID NO: 39 is the sequence of the reverse primer of synthetic serA1 homology arm 1.

[0051] SEQ ID NO: 40 is the nucleic acid sequence of serA1 homology arm 2 of B. licheniformis.

[0052] SEQ ID NO: 41 is the sequence of the synthetic serA1 homology arm 2 forward primer.

[0053] SEQ ID NO: 42 is the sequence of the synthetic serA1 homology arm 2 forward primer.

[0054] SEQ ID NO: 43 is an expression cassette encoding the target site 1 (TS1) gRNA.

[0055] SEQ ID NO: 44 is a synthetic serA1 deletion edit template.

[0056] SEQ ID NO: 45 is the open reading frame (ORF) sequence of rghR1 of B. licheniformis.

[0057] SEQ ID NO: 46 is the PAM sequence of target site 2, which contains the nucleotides "CGG."

[0058] SEQ ID NO: 47 is a synthetic guide RNA (gRNA) sequence targeting site 2.

[0059] SEQ ID NO: 48 is the nucleic acid sequence of homology arm 1 of rghR1 of B. licheniformis.

[0060] SEQ ID NO: 49 is the sequence of the synthetic rghR1 homology arm 1 forward primer.

[0061] SEQ ID NO: 50 is the sequence of the reverse primer of synthetic rghR1 homology arm 1.

[0062] SEQ ID NO: 51 is the nucleic acid sequence of homology arm 2 of rghR1 of B. licheniformis.

[0063] SEQ ID NO: 52 is the sequence of the synthetic rghR1 homology arm 2 forward primer.

[0064] SEQ ID NO: 53 is the sequence of the reverse primer of synthetic rghR1 homology arm 2.

[0065] SEQ ID NO: 54 is an expression cassette encoding the target site 2 (TS2) gRNA.

[0066] SEQ ID NO: 55 is a synthetic rghR1 deletion edit template.

[0067] SEQ ID NO: 56 is the amino acid sequence encoding the Cas9(Y155H) mutant protein.

[0068] SEQ ID NO: 57 is the forward primer sequence of synthetic Y155H.

[0069] SEQ ID NO: 58 is the reverse primer sequence of synthetic Y155H.

[0070] SEQ ID NO:59 is the nucleic acid sequence of the synthetic pRF827.

[0071] SEQ ID NO: 60 is an expression cassette encoding the mutant Cas9(Y155H) protein of SEQ ID NO: 56.

[0072] SEQ ID NO: 61 is the nucleic acid sequence of the synthetic pRF856.

[0073] SEQ ID NO: 62 is the nucleic acid sequence of the synthetic pRF862.

[0074] SEQ ID NO: 63 is the sequence of a fragment of synthetic Y155H.

[0075] SEQ ID NO: 64 is the forward primer sequence of the synthetic Y155H fragment.

[0076] SEQ ID NO: 65 is the reverse primer sequence for the synthetic Y155H fragment.

[0077] SEQ ID NO: 66 is the sequence of a fragment of synthetic pRF694.

[0078] SEQ ID NO: 67 is the forward primer sequence for a fragment of synthetic pRF694.

[0079] SEQ ID NO: 68 is the reverse primer sequence for a fragment of synthetic pRF694.

[0080] SEQ ID NO: 69 is the nucleic acid sequence of the synthetic pRF869.

[0081] SEQ ID NO: 70 is the open reading frame (ORF) sequence of rghR2 of B. licheniformis.

[0082] SEQ ID NO: 71 is the synthetic rghR2 stop It is a fragment of.

[0083] SEQ ID NO: 72 is the synthetic rghR2 stop It is an editorial template.

[0084] SEQ ID NO: 73 is an expression cassette encoding the rghR2 gRNA.

[0085] SEQ ID NO: 74 is the forward primer for the synthetic fragment.

[0086] SEQ ID NO: 75 is the reverse primer for the synthetic fragment.

[0087] SEQ ID NO: 76 is the forward primer for the backbone of synthetic pRF862.

[0088] SEQ ID NO: 77 is the reverse primer for the backbone of synthetic pRF862.

[0089] SEQ ID NO: 78 is the nucleic acid sequence of the synthetic pRF879.

[0090] SEQ ID NO: 79 is the nucleic acid sequence of the target site and PAM of pRF879 of B. licheniformis.

[0091] SEQ ID NO: 80 is the sequence of the synthetic pRF879 editing template.

[0092] SEQ ID NO:81 is the nucleic acid sequence of the synthetic pRF946.

[0093] SEQ ID NO: 82 is the nucleic acid sequence of the target site and PAM of pR946 of B. licheniformis.

[0094] SEQ ID NO: 83 is the sequence of the synthetic pR946 editing template.

[0095] SEQ ID NO: 84 is the nucleic acid sequence of synthetic pZM221.

[0096] SEQ ID NO: 85 is the nucleic acid sequence of the target site and PAM of pZM221.

[0097] SEQ ID NO: 86 is the sequence of the synthetic pZM221 editing template.

[0098] SEQ ID NO: 87 is the open reading frame (ORF) sequence of LysA from B. licheniformis.

[0099] SEQ ID NO: 88 is the nucleic acid sequence of the synthetic pBl.comK.

[0100] SEQ ID NO: 89 is the nucleic acid sequence of a synthetic spectinomycin marker.

[0101] SEQ ID NO: 90 is the nucleic acid sequence of xylR of B. subtilis.

[0102] SEQ ID NO: 91 is the nucleic acid sequence of xylAp of B. subtilis.

[0103] SEQ ID NO: 92 is the nucleic acid sequence of synthetic comK.

[0104] SEQ ID NO: 93 is the nucleic acid sequence of synthetic cat_prsA.

[0105] SEQ ID NO: 94 is the nucleic acid sequence upstream of cat of B. licheniformis.

[0106] SEQ ID NO: 95 is the nucleic acid sequence of the cat promoter of B. licheniformis.

[0107] SEQ ID NO: 96 is the nucleic acid sequence of catH of B. licheniformis.

[0108] SEQ ID NO: 97 is the nucleic acid sequence of a synthetic double terminator.

[0109] SEQ ID NO: 98 is the nucleic acid sequence of the catH terminator of B. licheniformis.

[0110] SEQ ID NO: 99 is the nucleic acid sequence of the spoVG terminator of B. subtilis.

[0111] SEQ ID NO: 100 is the nucleic acid sequence of the prsA promoter of B. licheniformis.

[0112] SEQ ID NO: 101 is the open reading frame (ORF) sequence of prsA of B. licheniformis.

[0113] SEQ ID NO: 102 is the nucleic acid sequence of the amyL terminator of B. licheniformis.

[0114] SEQ ID NO: 103 is the nucleic acid sequence downstream of cat of B. licheniformis.

[0115] SEQ ID NO: 104 is the nucleic acid sequence of the compound forward primer.

[0116] SEQ ID NO: 105 is the nucleic acid sequence of the synthetic reverse primer.

[0117] SEQ ID NO: 106 is the nucleic acid sequence of the verification of synthetic prsA (second copy).

[0118] SEQ ID NO: 107 is the synthetic primer sequence.

[0119] SEQ ID NO: 108 is the synthetic primer sequence.

[0120] SEQ ID NO: 109 is the synthetic primer sequence.

[0121] SEQ ID NO: 110 is the nucleic acid sequence encoding the B. licheniformis deleted catHP and catH.

[0122] SEQ ID NO: 111 is a synthetic prsA (second copy) expression cassette in the cat catH deletion.

[0123] SEQ ID NO: 112 is the synthetic catH (second copy) deletion verification PCR product.

[0124] SEQ ID NO: 113 is the synthetic forward primer sequence.

[0125] SEQ ID NO: 114 is the synthetic reverse primer sequence.

[0126] SEQ ID NO: 115 is the validation PCR product of synthetic dltA-2.

[0127] SEQ ID NO: 116 is the synthetic dltA-2 parental validation PCR product.

[0128] SEQ ID NO: 117 is the synthetic forward primer sequence.

[0129] SEQ ID NO: 118 is the synthetic reverse primer sequence.

[0130] SEQ ID NO: 119 is the synthetic rghR2 deletion verification PCR product.

[0131] SEQ ID NO: 120 is the deletion verification PCR product of the B. licheniformis parental rghR2.

[0132] SEQ ID NO: 121 is the parental rghR2 locus of B. licheniformis.

[0133] SEQ ID NO: 122 is the parental dltA locus of B. licheniformis.

[0134] SEQ ID NO: 123 is the parental cat locus of B. licheniformis.

[0135] SEQ ID NO: 124 is a synthetic cat 2xprsA locus.

[0136] SEQ ID NO: 125 is the synthetic dltA-2 locus.

[0137] SEQ ID NO: 126 is the amino acid sequence of the amylase 1 protein of B. licheniformis.

[0138] SEQ ID NO:127 is the synthetic serA1-amylase1 cassette.

[0139] SEQ ID NO: 128 is the synthetic p3 promoter sequence.

[0140] SEQ ID NO: 129 is the synthetic modified aprE 5'-UTR sequence.

[0141] SEQ ID NO: 130 is the nucleic acid sequence of B. licheniformis encoding the amyL signal sequence.

[0142] SEQ ID NO:131 is the nucleic acid sequence of B. licheniformis encoding the amylase 1 protein of SEQ ID NO:126.

[0143] SEQ ID NO: 132 is the synthetic lysA amylase 1 cassette.

[0144] SEQ ID NO: 133 is the nucleic acid sequence of the synthetic lysA parent locus.

[0145] SEQ ID NO: 134 is the nucleic acid sequence of B. licheniformis encoding lysA.

[0146] SEQ ID NO: 135 is the synthetic p2 promoter sequence.

[0147] SEQ ID NO: 136 is the amino acid sequence of the amylase 2 protein.

[0148] SEQ ID NO:137 is a synthetic serA1-amylase2 cassette.

[0149] SEQ ID NO: 138 is the rmI promoter sequence of B. subtilis.

[0150] SEQ ID NO: 139 is the aprE 5'-UTR sequence of B. subtilis.

[0151] SEQ ID NO:140 is a synthetic nucleic acid sequence encoding the amylase 2 protein of SEQ ID NO:136.

[0152] SEQ ID NO: 141 is a synthetic amyL or lysA amylase 2 cassette.

[0153] SEQ ID NO: 142 is the parent locus of the synthetic amyL.

[0154] SEQ ID NO: 143 is the amino acid sequence of the amylase 3 protein.

[0155] SEQ ID NO:144 is the synthetic serA1 amylase 3 cassette.

[0156] SEQ ID NO:145 is a synthetic nucleic acid sequence encoding the amylase 3 protein of SEQ ID NO:143.

[0157] SEQ ID NO: 146 is a synthetic LysA amylase 3 cassette.

[0158] SEQ ID NO: 147 is the amino acid sequence of the amylase 4 protein.

[0159] SEQ ID NO:148 is the synthetic serA1 amylase 4 cassette.

[0160] SEQ ID NO:149 is a synthetic nucleic acid sequence encoding the Amylase 4 protein of SEQ ID NO:147.

[0161] SEQ ID NO: 150 is the synthetic lysA amylase 4 cassette.

[0162] SEQ ID NO: 151 is the amino acid sequence of the amylase 5 protein.

[0163] SEQ ID NO: 152 is the synthetic serA1 amylase 5 cassette.

[0164] SEQ ID NO:153 is a synthetic nucleic acid sequence encoding the Amylase 5 protein of SEQ ID NO:151.

[0165] SEQ ID NO: 154 is the synthetic lysA amylase 5 cassette.

[0166] SEQ ID NO: 155 is the amino acid sequence of the native B. licheniformis prsA protein.

[0167] SEQ ID NO: 156 is the amino acid sequence of the native B. licheniformis RghR2 protein.

[0168] SEQ ID NO: 157 is the amino acid sequence of the mutant B. licheniformis RghR2 protein.

[0169] SEQ ID NO:158 is the nucleic acid sequence of a mutant rghR2 gene of B. licheniformis that encodes the mutant RghR2 protein of SEQ ID NO:157. DETAILED DESCRIPTION OF THE INVENTION

[0170] The present disclosure generally relates to compositions and methods for obtaining B. licheniformis cells (e.g., protein-producing hosts) that comprise enhanced protein production capabilities. Certain embodiments of the present disclosure relate to genetically modified B. licheniformis cells / strains derived from a parent Bacillus licheniformis cell / strain. Accordingly, certain other embodiments of the present disclosure are directed to methods for constructing such modified B. licheniformis cells / strains that produce increased amounts of one or more proteins of interest.

[0171] For example, certain embodiments of the present disclosure are directed to methods for producing increased amounts of a protein of interest (POI) in an engineered Bacillus licheniformis cell, comprising: (a) modifying a parent B. licheniformis cell to express the POI by introducing therein a polynucleotide comprising a native prsA promoter operably linked to a native prsA open reading frame (ORF); and (b) fermenting the engineered cell under conditions suitable for production of the POI, wherein the engineered cell produces increased amounts of the POI compared to the parent cell when fermented under the same conditions. In certain embodiments, the engineered cell further comprises a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122 and / or a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121. In certain embodiments, the protein of interest (POI) is an enzyme. In certain embodiments, the enzyme is a protease or an amylase.

[0172] Other embodiments of the present disclosure are directed to modified B. licheniformis cells / strains derived from a parent Bacillus licheniformis cell / strain that contains an endogenous prsA gene encoding a native prsA protein. Thus, in certain embodiments, the modified B. licheniformis cells of the present disclosure contain an introduced polynucleotide comprising a native prsA promoter operably linked to a native prsA open reading frame (ORF). In certain embodiments, the introduced polynucleotide encodes a native prsA protein that contains about 90% sequence identity to SEQ ID NO: 155. In other embodiments, the modified cells contain a deleted or disrupted dltA gene that contains at least 90% sequence identity to SEQ ID NO: 122 and / or a deleted or disrupted rghR2 gene that contains at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.

[0173] Accordingly, certain embodiments of the present disclosure are directed to obtaining, isolating, purifying, etc., a protein of interest produced by the modified B. licheniformis cells of the present disclosure.

[0174] I. Definition In view of the disclosed modified B. licheniformis 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.

[0175] 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. All publications and patents cited herein are incorporated herein by reference in their entirety.

[0176] It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a prelude to the use of exclusive terminology such as "solely," "only," "excluding," or "not including" in connection with the recitation of claim elements, or the use of a "negative" limitation or qualification thereof.

[0177] It will be apparent to those skilled in the art upon reading this disclosure that 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 recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0178] As used herein, the term "Bacillus" includes all species within the genus "Bacillus" known to those skilled in the art, such as B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, and B. alkalophilus. Examples of Bacillus species include, but are not limited to, 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. Thus, the genus is intended to include organisms such as reclassified species, for example, but not limited to, "B. stearothermophilus," which is now referred to as "Geobacillus stearothermophilus."

[0179] As used herein, a "parent cell" refers to an "unmodified cell" (eg, an unmodified B. licheniformis parent cell).

[0180] As used herein, "modified cell" or "daughter cell" may be used interchangeably and refer to a recombinant B. licheniformis cell that contains at least one genetic modification that is not present in the "parent cell" from which the modified cell is derived.

[0181] In certain embodiments, "unmodified" B. licheniformis (parent) cells may be referred to as "control cells," particularly when compared to or in relation to "modified" B. licheniformis (daughter) cells.

[0182] As used herein, when the expression and / or production of a protein of interest (POI) in an "unmodified" (parent) cell is compared to the 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.).

[0183] As used herein, "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 a Bacillus sp. or E. coli cell.

[0184] As used herein, a "native B. licheniformis prsA promoter" of the present disclosure comprises about 95% sequence identity to SEQ ID NO: 100. In certain embodiments, a native B. licheniformis prsA promoter comprises about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 100.

[0185] As used herein, a "native B. licheniformis prsA open reading frame (ORF)" comprises about 90% or greater sequence identity to SEQ ID NO: 101. In certain embodiments, a native B. licheniformis prsA ORF comprises about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 101.

[0186] The prsA gene of Bacillus subtilis has been described by Kontinen and Sarvas (1993) and in WO 1994 / 019471, which suggest that the prsA gene is involved in protein secretion (i.e., encodes a component of the cellular secretory machinery), where the prsA gene product is a membrane-associated lipoprotein.

[0187] As used herein, a "native B. licheniformis prsA protein" comprises about 90% or greater sequence identity to SEQ ID NO: 155 and comprises peptidyl-propyl cis-trans isomerase activity (EC 5.2.1.8). In certain embodiments, a native B. licheniformis prsA protein comprises about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 155.

[0188] As used herein, "a parent B. licheniformis cell comprises an endogenous (wild-type) prsA gene encoding a native prsA protein," and thus, when a polynucleotide encoding a prsA protein comprising about 90% sequence identity to SEQ ID NO: 155 is introduced into an engineered B. licheniformis cell of the present disclosure, the introduced polynucleotide can be referred to herein as a second prsA copy. For example, an engineered B. licheniformis cell of the present disclosure comprising an introduced polynucleotide encoding a prsA protein comprising about 90% sequence identity to SEQ ID NO: 155 can be referred to herein as a two-copy prsA (engineered) B. licheniformis cell, comprising a first endogenous (wild-type) prsA gene encoding a native prsA protein and a second introduced polynucleotide encoding the prsA protein.

[0189] In B. subtilis, the dlt operon contains five ORFs (dltA, dltB, dltC, dltD, and dltE) that encode proteins designated DltA, DltB, DltC, DltD, and DltE, respectively (May et al., 2005). For example, as described by May et al. (2005), the DltA protein is a D-alanyl carrier protein ligase involved in the incorporation of D-alanine:D-Ala into lipoteichoic acid in the cell wall.

[0190] As used herein, a "dltA gene" comprises about 90% sequence identity to SEQ ID NO: 122. In certain embodiments, the dltA gene comprises about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 155.

[0191] The rghR gene of B. subtilis encodes a transcriptional regulatory protein designated RghR, which has been described in the art as a repressor of rapG, rapH (Hayashi et al., 2006), and rapD (Ogura and Fujita, 2007). In contrast, as recently described in WO 2018 / 156705, B. licheniformis encodes two homologs of the RghR transcriptional regulatory protein designated RghR1 and RghR2. As described below, certain embodiments of the present disclosure relate to B. licheniformis cells that contain an altered (e.g., deleted or disrupted) rghr2 gene.

[0192] As used herein, a "B. licheniformis rghR2 gene" suitable for the genetic modifications described herein refers to a wild-type B. licheniformis (B. licheniformis) gene encoding a native RhgR2 protein that has about 90% sequence identity to SEQ ID NO: 156 (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 156). The mutant RhgR2 protein may be a mutant B. licheniformis rghR2 gene (SEQ ID NO: 121) encoding a mutant B. licheniformis rghR2 protein having about 90% sequence identity to SEQ ID NO: 157 (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 157). For example, as shown in SEQ ID NO: 157, the mutant RhgR2 protein contains a hexa-amino acid repeat of "Ala-Ala-Ala-Ile-Ser-Arg" at amino acid residues 36-41 of SEQ ID NO: 157, but the hexa-amino acid repeat is not present in the native RghR2 protein (i.e., amino acid residues 1-134 of SEQ ID NO: 156).

[0193] Thus, in certain other embodiments, the rghR2 gene, or its open reading frame, comprises about 90% sequence identity with a native rghR2 gene (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 121); or about 90% sequence identity with a mutant rghR2 gene (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 158).

[0194] As used herein, the parent B. licheniformis strain designated "BF140" or "BF140(ΔserA_ΔlysA)" contains a deletion of the serA gene (ΔserA) and a deletion of the lysA gene (ΔlysA).

[0195] As used herein, the modified B. licheniformis strain designated "BF561" or "BF561 (second copy prsA)" was derived from the parent strain BF140, but now contains an introduced second copy of the wild-type B. licheniformis prsA gene that encodes the native prsA protein.

[0196] As used herein, "BF598" or "BF598(ΔdltA_2 nd The modified B. licheniformis strain, designated "BF598 (copy prsA)," was derived from strain BF561, but now modified BF598 further contains a deletion of the dltA gene of B. licheniformis.

[0197] As used herein, "BF602" or "BF602(ΔrghR2_2 nd The modified B. licheniformis strain, designated "BF602 (copy prsA)," was derived from strain BF561, but now contains a deletion of the rghR2 gene of B. licheniformis.

[0198] As used herein, "BF613" or "BF613(ΔrghR2_ΔdltA_2 nd The modified B. licheniformis strain, designated BF598 (ΔdltA_2 nd copy prsA) strain, but now modified BF613 further contains a deletion of the rghR2 gene of B. licheniformis.

[0199] As used herein, "Amylase 1" is the naturally occurring B. licheniformis α-amylase commonly referred to in the art as AmyL, and comprises the amino acid sequence of SEQ ID NO:126.

[0200] As used herein, "amylase 2" is a variant Bacillus sp. α-amylase comprising SEQ ID NO: 136 generally as described in WO 2018 / 184004, which is incorporated herein by reference in its entirety.

[0201] As used herein, "amylase 3" is a mutant Cytophaga sp. α-amylase comprising SEQ ID NO: 143, generally as described in WO 2014 / 164777; WO 2012 / 164800, and WO 2014 / 164834, each of which is incorporated herein by reference in its entirety.

[0202] As used herein, "amylase 4" is a mutant Cytophaga sp. α-amylase comprising SEQ ID NO: 147 generally as described in WO 2014 / 164777; WO 2012 / 164800 and WO 2014 / 164834 (each of which is incorporated herein by reference in its entirety).

[0203] As used herein, "Amylase 5" is generally a variant Bacillus sp. 707 alkaline α-amylase comprising SEQ ID NO: 151, as described in WO 2008 / 153805 and U.S. Patent Application Publication No. 2014 / 0057324, each of which is incorporated herein by reference in its entirety.

[0204] As used herein, the mutant Cas9 protein herein referred to as "Cas9 Y155H" is described in WO 2019 / 118463, which is incorporated by reference in its entirety.

[0205] As used herein, the terms "modification" and "genetic modification" are used interchangeably and include the following: (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 / control 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.

[0206] As used herein, "increased amount," e.g., when used in phrases such as "the modified host cell expresses / produces increased amounts of one or more proteins of interest compared to the (unmodified) parent host cell," specifically refers to the "increased amount" of any protein of interest (POI) expressed / produced in the modified host cell, but always with reference to the (unmodified) parent B. licheniformis cell expressing / producing the same POI, where the modified and unmodified cells are grown / cultured / fermented under the same conditions (e.g., the same conditions of medium, temperature, pH, etc.). For example, the increased amount of POI may be an endogenous Bacillus sp. POI or a heterologous POI expressed in the modified Bacillus sp. cell of the present disclosure.

[0207] As used herein, "increasing" protein production or "increased" protein production refers to an increased amount of a protein (e.g., a protein of interest) produced. The protein may be produced inside the host cell or secreted (or exported) 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 compared to the parent host cell.

[0208] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid molecule of the present disclosure. Expression can also refer to the translation of mRNA into a polypeptide. Thus, the term "expression" includes any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0209] As used herein, "nucleic acid" refers to a nucleotide or polynucleotide sequence, whether representing the sense or antisense strand, which may be double-stranded or single-stranded, and fragments or portions thereof, and to DNA, cDNA, and RNA of genomic or synthetic origin. It will be understood that, as a result of the degeneracy of the genetic code, more than one nucleotide sequence can encode a given protein.

[0210] The polynucleotides (or nucleic acid molecules) described herein are understood to include "genes," "vectors," and "plasmids."

[0211] Thus, the term "gene" refers to a polynucleotide that encodes a specific sequence of amino acids, including all or part of a protein's coding sequence, 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, and the coding sequence.

[0212] As used herein, the term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of its encoded protein product. The boundaries of the 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.

[0213] As used herein, the term "promoter" 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 from different naturally occurring promoters, or may even include synthetic nucleic acid segments. Those skilled in the art will appreciate that different promoters can direct the expression of a gene in different cell types, at different developmental stages, or in response to different environmental or physiological conditions. Promoters that most frequently cause gene expression in most cell types are generally referred to as "constitutive promoters." Furthermore, because the exact boundaries of regulatory sequences are not completely defined in most cases, it is recognized that DNA fragments of different lengths can have the same promoter activity.

[0214] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so 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., when the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either a sense or antisense orientation.

[0215] 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.

[0216] As used herein, a "functional promoter sequence (or open reading frame thereof) controlling expression of a gene of interest linked to a gene coding sequence for a protein of interest" refers to a promoter sequence that controls the transcription and translation of the coding sequence in Bacillus. For example, in certain embodiments, the present disclosure is directed to a polynucleotide comprising a 5' promoter (or 5' promoter region or tandem 5' promoters, etc.), where the promoter region is operably linked to a nucleic acid sequence (e.g., ORF) encoding a protein.

[0217] As used herein, "suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0218] As used herein, the term "introducing" as used in phrases such as "introducing into bacterial cells" or "introducing at least one polynucleotide open reading frame (ORF), or gene thereof, or vector thereof, into B. licheniformis cells" includes methods known in the art for introducing polynucleotides into cells, 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).

[0219] As used herein, "transformed" or "transformation" refers to a cell that has been transformed through the use of recombinant DNA technology. Transformation typically occurs by inserting one or more nucleotide sequences (e.g., polynucleotides, ORFs, or genes) into a cell. The inserted nucleotide sequences may be heterologous nucleotide sequences (i.e., sequences that do not naturally occur in the cell being transformed). Thus, "transformation" generally refers to the introduction of exogenous DNA into a host cell such that the DNA is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector.

[0220] As used herein, "transforming DNA," "transforming sequence," and "DNA construct" refer to DNA used to introduce a sequence into a host cell or organism. Transforming DNA is 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.

[0221] As used herein, "gene disruption" or "gene disruption" are used interchangeably and broadly refer to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., a protein). Thus, as used herein, gene disruption includes, but is not limited to, frameshift mutations, premature stop codons (i.e., so that a functional protein is not produced), substitutions that eliminate or reduce the activity of internal deletions of proteins (so that a functional protein is not produced), insertions that disrupt coding sequences, mutations that remove the operable link between the native promoter and open reading frame required for transcription, etc.

[0222] As used herein, "incoming sequence" refers to a DNA sequence that is introduced into the chromosome of a Bacillus sp. 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 heterologous 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 can be inserted into a gene to disrupt the function of the gene. In another embodiment, the incoming sequence comprises a selectable marker. In yet another embodiment, the incoming sequence comprises two homology boxes.

[0223] As used herein, a "homology box" refers to a nucleic acid sequence that is homologous to a sequence within a Bacillus chromosome. More specifically, a homology box is an upstream or downstream region that shares about 80-100% sequence identity, about 90-100% sequence identity, or about 95-100% sequence identity with the immediately adjacent coding region of a gene or portion of a gene that has been deleted, disrupted, inactivated, downregulated, etc., according to the present invention. These sequences direct where a DNA construct will integrate within the Bacillus chromosome and direct which portion of the Bacillus chromosome the incoming sequence will replace. While not intended to limit the present disclosure, a homology box can include a length of 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, wherein the homology box comprises nucleic acid sequences that are immediately adjacent to the coding region of a gene.

[0224] As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence that is expressible in a host cell and in which expression of the selectable marker confers on cells containing the expressed gene the ability to grow in the presence of a corresponding selection agent or in the absence of an essential nutrient.

[0225] As used herein, the terms "selectable marker" and "selection marker" refer to a nucleic acid (e.g., a gene) that can be expressed in a host cell, allowing for easy selection of those hosts that contain the vector. Examples of such selectable markers include, but are not limited to, antimicrobial agents. Thus, the term "selectable marker" refers to a gene that provides an indication that a host cell has taken up incoming DNA of interest or that some other reaction has occurred. Typically, a selectable marker is a gene that confers antimicrobial resistance or a metabolic advantage to the host cell, allowing cells containing foreign DNA to be distinguished from cells that have not received the exogenous sequence during transformation.

[0226] A "present selectable marker" is a marker 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), and the like. 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 present invention provides a chloramphenicol resistance gene (e.g., genes present on pC194 and resistance genes present within the genome of Bacillus licheniformis). 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 present invention include, but are not limited to, auxotrophic markers such as serine, lysine, tryptophan, and the like, and detectable markers such as β-galactosidase.

[0227] As defined herein, the "genome" of a host cell, the "genome" of a bacterial (host) cell, or the "genome" of a Bacillus sp. (host) cell includes chromosomal genes and extrachromosomal genes.

[0228] As used herein, the terms "plasmid," "vector," and "cassette" refer to extrachromosomal elements that frequently carry genes that are typically not part of the cell's central metabolism and are usually in the form of circular double-stranded DNA molecules. Such elements may be linear or circular, single- or double-stranded, DNA or RNA autonomously replicating sequences, genome-integrating sequences, phage, or nucleotide sequences from any source in which multiple nucleotide sequences have been joined or recombined into a unique structure that allows the introduction of promoter fragments and DNA sequences for selected gene products, along with appropriate 3' untranslated sequences, into cells.

[0229] 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 becomes integrated into the genome of the host cell; in some embodiments, the plasmid is present in the parent cell and lost in the daughter cells.

[0230] As used herein, "transformation cassette" refers to a specific vector containing a gene (or its ORF) and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.

[0231] As used herein, the term "vector" refers to any nucleic acid that can replicate (multiply) within a cell and 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 "episomal" (i.e., capable of autonomous replication or integration into the chromosomes of the host organism), viruses, bacteriophages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes, such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes).

[0232] An "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.

[0233] As used herein, the terms "expression cassette" and "expression vector" refer to nucleic acid constructs (i.e., they are vectors or vector elements as described above) that are recombinantly or synthetically produced with a set of specific nucleic acid elements that allow for transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or 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 for 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.

[0234] As used herein, a "targeting vector" is a vector that contains a polynucleotide sequence homologous to a region in a host cell chromosome into which the targeting vector is transformed and is capable of driving homologous recombination at that region. For example, targeting vectors are used to introduce mutations into a host cell chromosome by homologous recombination. In some embodiments, the targeting vector contains other non-homologous sequences (i.e., stuffer or flanking sequences), e.g., added to the ends. The ends can be closed, e.g., by insertion into a vector, such that the targeting vector forms a closed circle. For example, in certain embodiments, a parent B. licheniformis (host) cell is modified (e.g., transformed) by introducing one or more "targeting vectors" therein.

[0235] As used herein, the term "protein of interest" or "POI" refers to a polypeptide of interest that is desirably expressed in the modified B. licheniformis (daughter) host cells, where the POI is expressed at an increased level (i.e., compared to the "unmodified" (parent) cell). Thus, as used herein, a POI can be an enzyme, substrate-binding protein, surfactant protein, structural protein, receptor protein, etc. In certain embodiments, the modified cells of the present disclosure produce an increased amount of a heterologous protein of interest or an endogenous protein of interest compared to the parent cell. In certain embodiments, the increased amount of a protein of interest produced by the modified 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 more than a 5.0% increase compared to the parent cell.

[0236] Similarly, "gene of interest" or "GOI" as defined herein refers to a nucleic acid sequence (e.g., polynucleotide, gene, or ORF) that encodes the POI. A "gene of interest" that encodes a "protein of interest" may be a naturally occurring gene, a mutated gene, or a synthetic gene.

[0237] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of any length comprising amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein. Polypeptides can be linear or branched, contain modified amino acids, or 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 component. Also included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0238] 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, endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, and the like. The present invention relates to a method for producing a protein encoding a commercially relevant protein of industrial interest such as an enzyme, a soluble fiber ...

[0239] As used herein, a "variant" polypeptide refers to a polypeptide derived from a parent (or reference) polypeptide by one or more amino acid substitutions, additions, or deletions, generally by recombinant DNA techniques. A variant polypeptide may differ from the parent polypeptide by a small number of amino acid residues and may be defined by the level of primary amino acid sequence homology / identity with the parent (reference) polypeptide.

[0240] 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 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 "variant polynucleotide" 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 at least 99% nucleotide sequence identity with the parent (reference) polynucleotide sequence.

[0241] 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 chemical agents, repair minus passaging through bacterial strains).

[0242] As used herein, the term "substitution" in reference to a polypeptide or sequence thereof means the replacement (ie, substitution) of one amino acid with another.

[0243] As defined herein, "endogenous gene" refers to a gene that is present in its natural location in the genome of an organism.

[0244] As defined herein, a "heterologous" gene, "non-endogenous" gene, or "foreign" gene refers to a gene (or ORF) that is not normally found in the host organism, but that 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.

[0245] As defined herein, a "heterologous regulatory sequence" refers to a gene expression control sequence (e.g., a promoter or enhancer) that does not naturally function 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 can contain regulatory sequence / DNA coding (ORF) sequence combinations that are the same as or different from regulatory sequence / DNA coding sequence combinations found in the native host cell.

[0246] As used herein, the terms "signal sequence" and "signal peptide" 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 generally located at the N-terminus of the precursor or mature protein sequence. A signal sequence may be endogenous or exogenous. A signal sequence is usually not present in the mature protein. A signal sequence is typically cleaved from a protein by a signal peptidase after the protein has been exported.

[0247] The term "derived from" includes the terms "originating from," "obtained from," "obtainable from," and "made from," and generally indicates that one particular material or composition finds its origin in another material or composition, or has characteristics that can be described with reference to that other particular material or composition.

[0248] The term "homology" as used herein refers 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 considered homologous as defined herein can be conveniently determined by aligning the two sequences using a computer program known in the art, such as "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)). For DNA sequence comparison, GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0249] As used herein, the term "percent identity" refers to the level of nucleic acid or amino acid sequence identity between nucleic acid sequences encoding polypeptides or between the amino acid sequences of polypeptides when aligned using a sequence alignment program.

[0250] As used herein, "specific productivity" refers to the total amount of protein produced per cell per unit time over a given period of time.

[0251] As defined herein, the terms "purified," "isolated," or "enriched" 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.

[0252] As used herein, the term "ComK polypeptide" refers to the product of the comK gene; a transcription factor that acts as the final autoregulatory control switch before competence development; and is involved in activating the expression of later competence genes involved in DNA binding and uptake and recombination (Liu and Zuber, 1998; Hamoen et al., 1998). An exemplary ComK nucleic acid is shown in SEQ ID NO: 92.

[0253] 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 a cell derived from a cell 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, where the assembly gives rise to a chimeric gene.

[0254] As used herein, "flanking sequence" refers to any sequence upstream or downstream 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 homology box comprise a unit flanked on both sides by stuffer sequences. In some embodiments, flanking sequences are present on only one side (3' or 5'), but in preferred embodiments, they are present on both sides of the flanking sequence. The sequence of each homology box is homologous to a sequence within the Bacillus chromosome. These sequences direct where in the Bacillus chromosome the novel construct will integrate and which portion 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 comprising a portion of an inactivating chromosomal segment. In some embodiments, a flanking sequence is present on only one side (3' or 5'), while in other embodiments, it is present on both sides of the sequence it is flanking.

[0255] II. Engineered Bacillus Licheniformis Cells Containing an Enhanced Protein Production Phenotype Generally, as described in the Examples section below, Applicants constructed and introduced a series of host modifications into a parent B. licheniformis strain. More specifically, as shown in the Examples below (see, e.g., Table 18), the parent B. licheniformis strain used in this Example contained a deletion of the serA1 gene (SEQ ID NO:30) and the lysA gene (SEQ ID NO:87) and was designated BF140(ΔserA_ΔlysA). Applicants subsequently introduced (1) a second copy of the wild-type B. licheniformis prsA gene encoding the native prsA protein (designated BF561; second copy prsA), (2) a deletion of the B. licheniformis dltA gene (designated BF598; ΔdltA_2 nd copy prsA), (3) a deletion of the rghR2 gene in B. licheniformis (ghR2_2, designated BF602) nd copy prsA) and (4) a combined deletion of the rghR2 and dltA genes of B. licheniformis (designated BF613; ΔrghR2 ΔdltA 2 nd The desired genetic modifications, including a copy of prsA, were introduced into the parent B. licheniformis strain (BF140).

[0256] After construction of the above engineered strains, a series of α-amylase expression cassettes were introduced into the engineered B. licheniformis strains (BF561, BF598, BF602, and BF613) and the parent B. licheniformis strain (BF140). More specifically, as presented in Example 4 below, two copies of five different α-amylase expression cassettes (i.e., "Amylase 1," "Amylase 2," "Amylase 3," "Amylase 4," and "Amylase 5") were introduced into the B. licheniformis strains.

[0257] As further described in Example 5 below, parental (BF140) and modified (BF561, BF598, BF602, and BF613) B. licheniformis strains containing two copies of the expression cassettes for amylases 1-5 were assayed for amylase production (see, e.g., Table 19). For example, all five amylases tested from a diverse group of α-amylases were expressed in a modified background of native BF613 containing a deleted dltA-2 (ΔdltA-2) allele (SEQ ID NO: 125), a deleted rghR2 (ΔrghR2) allele (SEQ ID NO: 80), and a deleted rghR2 (ΔrghR2_ΔdltA_2) allele (SEQ ID NO: 80) compared to the unmodified parent host BF140. nd The figures show an improvement in the production of α-amylase in a modified BF602 background (ΔrghR2_2) containing a deleted rghR2 (ΔrghR2) allele (SEQ ID NO: 80) and a second copy of the native prsA gene controlled by the native prsA promoter (SEQ ID NO: 124). nd The improvement in α-amylase production in the BF613 engineered host (ΔrghR2_2) is nearly as good as the productivity improvement seen in the BF613 engineered host. This observation suggests that for some amylases, the productivity improvement is due to the combination of these two alleles (i.e., ΔrghR2_2 nd This suggests that only the presence of the ΔdltA-2 allele is required for this improvement.

[0258] III.Molecular biology As generally described above, certain embodiments of the present disclosure relate to engineered B. licheniformis (daughter) cells derived from parent Bacillus licheniformis cells. More particularly, certain embodiments of the present disclosure relate to engineered Bacillus (daughter) cells and methods for generating and constructing such engineered Bacillus (host) cells (e.g., protein-producing host cells, cell factories) with increased protein production capacity, increased secondary metabolite production capacity, etc.

[0259] In certain embodiments, the modified B. licheniformis cells of the present disclosure comprise an introduced second copy of a gene or ORF encoding a native prsA protein. In other embodiments, the modified B. licheniformis cells of the present disclosure comprise a deleted dltA gene. In certain other embodiments, the modified B. licheniformis cells of the present disclosure comprise an introduced second copy of a gene or ORF encoding a native prsA protein and a deleted dltA gene. In other embodiments, the modified B. licheniformis cells of the present disclosure comprise a deleted rghR2 gene. In certain other embodiments, the modified B. licheniformis cells of the present disclosure comprise an introduced second copy of a gene or ORF encoding a native prsA protein and a deleted rghR2 gene. In other embodiments, the modified B. licheniformis cells of the disclosure comprise a deleted dltA gene and a deleted rghR2 gene. In certain other embodiments, the modified B. licheniformis cells of the disclosure comprise an introduced second copy of a gene or ORF encoding a native prsA protein, a deleted dltA gene, and a deleted rghR2 gene.

[0260] Accordingly, certain embodiments of the present disclosure provide compositions and methods for genetically modifying (altering) a parent Bacillus cell of the present disclosure to generate an engineered Bacillus cell that produces increased amounts of an endogenous and / or heterologous protein of interest compared to the engineered Bacillus cell, and more particularly, compared to the (unmodified) parent B. licheniformis cell.

[0261] Accordingly, certain embodiments of the present disclosure are directed to methods of genetically modifying Bacillus cells, wherein the modification comprises (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 the 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.

[0262] In certain embodiments, the altered Bacillus cells of the present disclosure are constructed by reducing or eliminating expression of the above-defined genes 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, the coding region or a regulatory element required for expression of the coding region.

[0263] 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, a leader sequence, a propeptide sequence, a signal sequence, a transcription terminator, a transcription activator, and the like.

[0264] In certain other embodiments, modified Bacillus cells are constructed by gene deletion to eliminate or reduce expression of at least one of the above-described genes of this disclosure. Gene deletion techniques allow for the partial or complete removal of genes, thereby eliminating their expression or expressing a non-functional (or reduced activity) protein product. In such methods, gene deletion can be accomplished by homologous recombination using a plasmid constructed to contain adjacent 5' and 3' regions flanking the gene. The flanking 5' and 3' regions can be introduced into Bacillus cells 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 cells. The cells are then shifted to a non-permissive temperature to select for cells with the plasmid integrated into their chromosomes 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 readily 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.

[0265] 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 can be inserted or removed to introduce a stop codon, remove a start codon, or cause a frameshift in the open reading frame. Such modifications can be made by site-directed mutagenesis or PCR-generated mutagenesis, according to methods known in the art (see, e.g., 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.

[0266] In another embodiment, modified Bacillus cells are constructed by the process of gene transformation (see, e.g., Iglesias and Trautner, 1983). For example, in gene transformation, a nucleic acid sequence corresponding to a gene is mutated in vitro to generate a defective nucleic acid sequence, which is then transformed into a parent Bacillus cell to generate the defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. It may also be desirable for the defective gene or gene fragment to 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 for a second recombination event resulting in gene replacement is achieved by examining colonies for loss of the selectable marker and acquisition of the 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.

[0267] In other embodiments, modified Bacillus cells are constructed using established antisense technology, using a nucleotide sequence complementary to the nucleic acid sequence of a gene (Parish and Stoker, 1997). More specifically, gene expression by Bacillus cells 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 in which the complementary antisense nucleotide sequence can hybridize to the mRNA, the amount of translated protein 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, and the like, all of which are well known to those skilled in the art.

[0268] In other embodiments, modified Bacillus cells are produced / constructed by CRISPR-Cas9 editing. For example, genes encoding proteins of interest can be edited or destroyed (or deleted or downregulated) by a nucleic acid-guided endonuclease that finds its target DNA by binding to a guide RNA (e.g., Cas9) and Cpf1 or a guide DNA (e.g., NgAgo), which recruits the endonuclease to a target sequence on the DNA, where the endonuclease can generate single- or double-strand breaks in the DNA. This targeted DNA break can become a substrate for DNA repair and recombine with the provided editing template 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 Bacillus cells and a terminator active in Bacillus cells, thereby generating a Bacillus Cas9 expression cassette. Similarly, one or more target sites unique to a gene of interest are 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, the variable targeting domain (VT) would include the nucleotides of the target site 5' to a (PAM) protospacer adjacent motif (TGG), the nucleotides of which are fused to DNA encoding the Cas9 endonuclease recognition domain (CER) for S. pyogenes Cas9. The DNA encoding the gRNA is generated by combining the DNA encoding the VT domain with the DNA encoding the CER domain. Thus, a Bacillus expression cassette for the gRNA is prepared by operably linking the DNA encoding the gRNA to a promoter active in Bacillus cells and a terminator active in Bacillus cells.

[0269] In certain embodiments, the DNA break induced by the endonuclease is repaired / replaced using 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 use the editing template.For example, about 500 bp of the 5' side of the targeting gene can be fused to about 500 bp of the 3' side of the targeting gene to generate an editing template, and this template is used by the Bacillus host's mechanism to repair the DNA break generated by RGEN.

[0270] The Cas9 expression cassette, gRNA expression cassette, and editing template can be co-delivered into filamentous fungal cells using a number of different methods (e.g., protoplast fusion, electroporation, natural competence, or induced competence). Transformed cells are screened by PCR amplification of the target locus using forward and reverse primers. These primers can amplify the wild-type locus or the modified locus edited by RGEN. These fragments are then sequenced using sequencing primers to identify edited colonies.

[0271] 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 can be performed by subjecting parent cells to mutagenesis and screening for mutant cells in which expression of the gene is reduced or eliminated. Mutagenesis, which can be directed or random, can be performed, for example, by using suitable physical or chemical mutagenizing agents, by using suitable oligonucleotides, or by subjecting the DNA sequence to PCR-generated mutagenesis. Furthermore, the mutagenesis can be performed using any combination of these mutagenesis methods.

[0272] Examples of physical or chemical mutagenizing 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 exhibit reduced or no expression of the gene.

[0273] In certain other embodiments, the modified Bacillus cell comprises a deletion of an endogenous gene. In other embodiments, the modified Bacillus cell comprises a disruption of an endogenous gene. In certain embodiments, the polynucleotide disruption cassette of the present disclosure comprises a marker gene.

[0274] In other embodiments, the altered Bacillus cell comprises a down-regulated endogenous gene. For example, in certain embodiments, down-regulating one or more of the genes defined above comprises deleting or disrupting regulatory elements upstream or downstream of the gene.

[0275] WO 2003 / 083125 discloses methods for modifying Bacillus cells, such as the creation of Bacillus deletion strains and DNA constructs using PCR fusion to bypass E. coli.

[0276] WO 2002 / 14490 discloses methods for modifying Bacillus cells, including (1) construction and transformation of an integrating 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.

[0277] Those skilled in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli and Bacillus spp.) (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 protoplast transformation and conjugation, transduction, and transformation, including 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.

[0278] In addition to commonly used methods, in some embodiments, host cells are directly transformed (i.e., no intermediate cells are used to amplify or otherwise process the DNA construct before 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 being inserted into the plasmid. In further embodiments, the selectable marker is deleted or substantially excised from the modified Bacillus strain by methods known in the art (e.g., Stahl et al., 1984 and Palmeros et al., 2000). In some embodiments, degradation of the vector from the host chromosome leaves flanking regions within the chromosome while removing the native chromosomal region.

[0279] 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 skilled in the art. The promoter sequences of the present disclosure are generally selected so that they are functional in Bacillus cells (e.g., B. licheniformis cells, B. subtilis cells, etc.). Certain exemplary Bacillus promoter sequences are shown in Table 6. Similarly, promoters useful for driving gene expression in Bacillus cells include, but are not limited to, the alkaline protease (aprE) promoter of B. subtilis (Stahl et al., 1984), the α-amylase promoter of B. subtilis (Yang et al., 1983), the α-amylase promoter of B. amyloliquefaciens (Tarkinen et al., 1983), the neutral protease (nprE) promoter from B. subtilis (Yang et al., 1984), a mutant aprE promoter (WO 2001 / 51643), or any other promoter from B. licheniformis or other related Bacillus species. In certain other embodiments, the promoter is a ribosomal protein promoter or a ribosomal RNA promoter (e.g., the rrnI promoter) as disclosed in U.S. Patent Application Publication No. 2014 / 0329309. Methods for screening and generating promoter libraries with a wide range of activity (promoter strength) in Bacillus cells are described in WO 2003 / 089604.

[0280] IV. Cultivating Bacillus Cells to Produce a Protein of Interest In other embodiments, the present disclosure provides methods for increasing protein productivity of modified bacterial cells compared to (i.e., relative to) unmodified (parent) cells. In certain embodiments, the present disclosure is directed to methods for producing a protein of interest (POI) comprising fermenting / culturing modified bacterial cells, wherein the modified cells secrete the POI into the culture medium. Fermentation methods well known in the art can be applied to ferment modified and unmodified Bacillus cells of the present disclosure.

[0281] In some embodiments, 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 remains unchanged during the fermentation. At the start of the fermentation, the medium is inoculated with the desired organism. In this manner, fermentation is allowed without adding any components to the system. Typically, batch fermentation is considered "batch" with respect to the addition of a carbon source, and control of factors such as pH and oxygen concentration is frequently performed. The metabolite and biomass composition of a batch system changes constantly until the fermentation is stopped. In a typical batch culture, cells progress through a static lag phase to a high-growth logarithmic phase and may eventually progress to a stationary phase where growth rate decreases or stops. If not treated, cells in the stationary phase eventually die. Generally, cells in the logarithmic phase are responsible for the majority of product production.

[0282] A suitable variation on 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 cellular metabolism and when a limited amount of substrate is desired in the medium. 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 the partial pressure of waste gases such as CO2. Batch and fed-batch fermentation are common and known in the art.

[0283] 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, a limiting nutrient, such as the carbon or nitrogen source, is maintained at a fixed ratio, while all other parameters are adjustable. In other systems, multiple factors affecting growth can be continuously varied while the cell concentration, as measured by medium turbidity, remains constant. Continuous systems attempt to maintain steady-state growth conditions. Therefore, 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 product formation rates, are well known in the art of industrial microbiology.

[0284] Thus, in certain embodiments, the POI produced by the transformed (modified) host cells can be recovered from the culture medium by conventional procedures, for example, by separating the host cells from the medium by centrifugation or filtration, or, if necessary, by disrupting the cells and removing the supernatant from cell debris and cellular debris. Typically, after clarification, the protein component of the supernatant or filtrate is precipitated with a salt, for example, ammonium sulfate. The precipitated protein may then be solubilized and purified by various chromatographic methods, for example, ion exchange chromatography, gel filtration, etc.

[0285] V. Proteins of Interest Produced by Engineered (Host) Cells The protein of interest (POI) of the present disclosure may be any endogenous or heterologous protein, or may be a variant 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, wherein the POI or variant thereof is preferably a protein with a property of interest.

[0286] For example, as described in the Examples below, the engineered Bacillus cells of the present disclosure produce increased amounts of endogenous and / or heterologous proteins of interest. Thus, in certain embodiments, the engineered cells of the present disclosure express an endogenous POI, a heterologous POI, or a combination of one or more such POIs. For example, in certain embodiments, the engineered Bacillus (daughter) cells of the present disclosure produce increased amounts of an endogenous POI compared to the parent Bacillus cell. In other embodiments, the engineered Bacillus (daughter) cells of the present disclosure produce increased amounts of a heterologous POI compared to the parent Bacillus cell.

[0287] Thus, in certain embodiments, the altered Bacillus (daughter) cells of the present disclosure produce an increased amount of POI compared to the parental Bacillus (control) cells, where the increase in POI is at least about 0.01%, at least about 0.10%, at least about 0.50%, at least about 1.0%, at least about 2.0%, at least about 3.0%, at least about 4.0%, at least about 5.0%, or more than 5.0%. In certain embodiments, the increase in POI is determined by testing for enzyme activity and / or by testing / quantifying its specific productivity (Qp). Similarly, one of skill in the art can utilize other routine methods and techniques known in the art to detect, test, measure, etc., the expression or production of one or more proteins of interest.

[0288] 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, detecting specific productivity (Qp) is a suitable method for assessing protein production. Specific productivity (Qp) can be calculated using the following equation: "Qp = gP / gDCW·hr" where "gP" is the grams of protein produced in the tank, "gDCW" is the 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 growth time.

[0289] 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.

[0290] In certain embodiments, the POI or variant thereof is an acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lysate, or a combination thereof. 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 acetylesterase, pectin depolymerase, pectin methylesterase, pectinolytic enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phytase, polygalacturonase, protease, peptidase, rhamnogalacturonase, ribonuclease, transferase, transport protein, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0291] 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.

[0292] For example, in certain embodiments, the POI may be selected from the group consisting of EC 1.10.3.2 (e.g., laccase), EC 1.10.3.3 (e.g., L-ascorbic acid oxidase), EC 1.1.1.1 (e.g., alcohol dehydrogenase), EC 1.11.1.10 (e.g., chloride peroxidase), EC 1.11.1.17 (e.g., peroxidase), EC 1.1.1.27 (e.g., L-lactate dehydrogenase), EC 1.1.1.47 (e.g., glucose 1-dehydrogenase), EC 1.1.3.X (e.g., glucose oxidase), EC 1.1.3.10 (e.g., pyranose oxidase), EC 1.13.11.X (e.g., dioxygenase), EC 1.13.11.12 (e.g., linoleic acid 13S-lipoxygenase), EC 1.1.3.13 (e.g., alcohol oxidase), EC 1.14. and EC 1.1.99.18 (e.g., cellobiose dehydrogenase), EC 1.1.99.29 (e.g., pyranose dehydrogenase), EC 1.2.1.X (e.g., fatty acid reductase), EC 1.2.1.10 (e.g., acetaldehyde dehydrogenase), EC 1.5.3.X (e.g., fructosylamine reductase), EC 1.8.1.X (e.g., disulfide reductase), and EC 1.8.3.2 (e.g., thiol oxidase).

[0293] In certain embodiments, the POI is selected from the group consisting of EC 2.3.2.13 (e.g., transglutaminase), EC 2.4.1.X (e.g., hexosyltransferase), EC 2.4.1.40 (e.g., alternasucrase), EC 2.4.1.18 (e.g., 1,4 α-glucan branching enzyme), EC 2.4.1.19 (e.g., cyclomaltodextrin glucanotransferase), EC 2.4.1.2 (e.g., dextrin dextranase), EC 2.4.1.20 (e.g., cellobiose phosphorylase), EC 2.4.1.25 (e.g., 4-α-glucanotransferase), EC 2.4.1.333 (e.g., 1,2-β-oligoglucan phospho ...40 (e.g., alternasucrase), EC 2 transferase enzymes, including, but not limited to, EC2 (transferase) enzymes selected from EC 2.4.1.4 (e.g., amylosucrase), EC 2.4.1.5 (e.g., dextransucrase), EC 2.4.1.69 (e.g., galactoside 2-α-L-fucosyltransferase), EC 2.4.1.9 (e.g., inulosucrase), EC 2.7.1.17 (e.g., xylulokinase), EC 2.7.7.89 (formerly EC 3.1.4.15, e.g., [glutamine synthetase]-adenylyl-L-tyrosine phosphorylase), EC 2.7.9.4 (e.g., α-glucan kinase), and EC 2.7.9.5 (e.g., phosphoglucan kinase).

[0294] In other embodiments, the POI is selected from the group consisting of EC 3.1.XX (e.g., esterases), EC 3.1.1.1 (e.g., pectinases), EC 3.1.1.14 (e.g., chlorophyllases), EC 3.1.1.20 (e.g., tannases), EC 3.1.1.23 (e.g., glycerol-ester acyl hydrolases), EC 3.1.1.26 (e.g., galactolipases), EC 3.1.1.32 (e.g., phospholipase A1), EC 3.1.1.4 (e.g., phospholipase A2), EC 3.1.1.6 (e.g., acetylesterases), EC 3.1.1.72 (e.g., acetylesterases), EC 3.1.1.82 (e.g., acetylesterases), EC 3.1.1.92 (e.g., acetylesterases), EC 3.1.1.102 (e.g., acetylesterases), EC 3.1.1.112 (e.g., acetylesterases), EC 3.1.1.132 (e.g., phospholipase A1), EC 3.1.1.14 (e.g., phospholipase A2), EC 3.1.1.152 (e.g., acetylesterases), EC 3.1.1.162 (e.g., acetylesterases), EC 3.1.1.172 (e.g., acetylesterases), EC 3.1.1.182 (e.g., acetylesterases), EC 3.1.1.192 (e.g., acetylesterases), xylan esterase), EC 3.1.1.73 (e.g., feruloyl esterase), EC 3.1.1.74 (e.g., cutinase), EC 3.1.1.86 (e.g., rhamnogalacturonan acetyl esterase), EC 3.1.1.87 (e.g., fumosin B1 esterase), EC 3.1.26.5 (e.g., ribonuclease P), EC 3.1.3.X (e.g., phosphohydrolase), EC 3.1.30.1 (e.g., Aspergillus nuclease S1), EC 3.1.30.2 (e.g., Serratia marcescens nuclease), marcescens nuclease), EC 3.1.3.1 (e.g., alkaline phosphatase), EC 3.1.3.2 (e.g., acid phosphatase), EC 3.1.3.8 (e.g., 3-phytase), EC 3.1.4.1 (e.g., phosphodiesterase I), EC 3.1.4.11 (e.g., phosphoinositide phospholipase C), EC 3.1.4.3 (e.g., phospholipase C), EC 3.1.4.4 (e.g., phospholipase D), EC 3.1.6.1 (e.g., arylsulfatase), E C3.1.8.2 (e.g., diisopropyl-fluorophosphatase), EC3.2.1.10 (e.g., oligo-1,6-glucosidase), EC3.2.1.101 (e.g., mannan endo-1,6-α-mannosidase), EC3.2.1.11 (e.g., α-1,6-glucan-6-glucanohydrolase), EC3.2.1.131 (e.g., xylan α-1,2-glucuronosidase), EC3.2.1.132 (e.g., chitosan N-acetylglucosaminohydrolase), EC3.2.1.139 (e.g., α-glucuronidase), EC 3.2.1.14 (e.g., chitinase), EC 3.2.1.151 (e.g., xyloglucan-specific endo-β-1,4-glucanase), EC 3.2.1.155 (e.g., xyloglucan-specific exo-β-1,4-glucanase), EC 3.2.1.164 (e.g., galactan endo-1,6-β-galactosidase), EC 3.2.1.17 (e.g., lysozyme), EC 3.2.1.171 (e.g., rhamnogalacturonan hydrolase), EC 3.2.1.174 (e.g., Rhamnogalacturonan rhamnohydrolase), EC 3.2.1.2 (e.g., β-amylase), EC 3.2.1.20 (e.g., α-glucosidase), EC 3.2.1.22 (e.g., α-galactosidase), EC 3.2.1.25 (e.g., β-mannosidase), EC 3.2.1.26 (e.g., β-fructofuranosidase), EC 3.2.1.37 (e.g., xylan 1,4-β-xylosidase), EC 3.2.1.39 (e.g., glucan endo-1,3-β-D-glucosidase), EC 3.2.1.40 (e.g., α- L-rhamnosidase), EC 3.2.1.51 (e.g., α-L-fucosidase), EC 3.2.1.52 (e.g., β-N-acetylhexosaminidase), EC 3.2.1.55 (e.g., α-N-arabinofuranosidase), EC 3.2.1.58 (e.g., glucan 1,3-β-glucosidase), EC 3.2.1.59 (e.g., glucan endo-1,3-α-glucosidase), EC 3.2.1.67 (e.g., galacturan 1,4-α-gacturonidase), EC 3.2.1.68 (e.g., isoamylase), EC 3.2.1 .7 (e.g., 1-β-D-fructan fructanohydrolase), EC 3.2.1.74 (e.g., glucan 1,4-β-glucosidase), EC 3.2.1.75 (e.g., glucan endo-1,6-β-glucosidase), EC 3.2.1.77 (e.g., mannan 1,2-(1,3)-α-mannosidase), EC 3.2.1.80 (e.g., fructan β-fructosidase), EC 3.2.1.82 (e.g., exo-poly-α-galacturonidase), EC 3.2.1.83 (e.g., κ-carrageenase), EC 3.2.1.89 (e.g., arabinogalactan endo-1,4-β-galactosidase), EC 3.2.1.91 (e.g., cellulose 1,4-β-cellobiosidase), EC 3.2.1.96 (e.g., mannosyl-glycoprotein endo-β-N-acetylglucosaminidase), EC 3.2.1.99 (e.g., arabinan endo-1,5-α-L-arabinanase), EC 3.4.XX (e.g., peptidase), EC 3.4.11.X (e.g., aminopeptidase), EC 3.4.11.1 (e.g., leucyl aminopeptidase), EC 3.4.1 1.18 (e.g., methionyl aminopeptidase), EC 3.4.13.9 (e.g., Xaa-Pro dipeptidase), EC 3.4.14.5 (e.g., dipeptidyl-peptidase IV), EC 3.4.16.X (e.g., serine-type carboxypeptidase), EC 3.4.16.5 (e.g., carboxypeptidase C), EC 3.4.19.3 (e.g., pyroglutamyl-peptidase I), EC 3.4.21.X (e.g., serine endopeptidase), EC 3.4.21.1 (e.g., chymotrypsin), EC 3.4.21.19 (e.g., glutamyl endopeptidase), EC 3.4.21.26 (e.g., prolyl oligopeptidase), EC 3.4.21.4 (e.g., trypsin), EC 3.4.21.5 (e.g., thrombin), EC 3.4.21.63 (e.g., oryzen), EC 3.4.21.65 (e.g., thermomycolin), EC 3.4.21.80 (e.g., streptoglycin A), EC 3.4.22.X (e.g., cysteine ​​endopeptidase), EC 3.4.22.14 (e.g., actinidain), EC 3.4.22.2 (e.g., papain), EC 3.4. 22.3 (e.g., ficain), EC 3.4.22.32 (e.g., stem bromelain), EC 3.4.22.33 (e.g., fruit bromelain), EC 3.4.22.6 (e.g., chymopapain), EC 3.4.23.1 (e.g., pepsin A), EC 3.4.23.2 (e.g., pepsin B), EC 3.4.23.22 (e.g., endothiapepsin), EC 3.4.23.23 (e.g., mucorpepsin), EC 3.4.23.3 (e.g., gastricsin), EC 3.4.24.X (e.g., metalloendopeptidases), EC 3.4.24.39 (e.g., deuterolysin), EC 3.4.24.40 (e.g., serralysin), EC 3.5.1.1 (e.g., asparaginase), EC 3.5.1.11 (e.g., penicillin amidase), EC 3.5.1.14 (e.g., N-acyl-aliphatic-L-amino acid amidohydrolase), EC 3.5.1.2 (e.g., L-glutamine amidohydrolase), EC 3.5.1.28 (e.g., N-acetylmuramoyl-L-alanine amidase), EC 3.5.1.4 (e.g., amidase), EC 3.5.1. and hydrolase enzymes, including, but not limited to, EC 3 (hydrolase) enzymes selected from EC 3.5.44 (e.g., protein-L-glutamine amidohydrolase), EC 3.5.1.5 (e.g., urease), EC 3.5.1.52 (e.g., peptide-N(4)-(N-acetyl-β-glucosaminyl)asparagine amidase), EC 3.5.1.81 (e.g., N-acyl-D-amino acid deacylases), EC 3.5.4.6 (e.g., AMP deaminase), and EC 3.5.5.1 (e.g., nitrilases).

[0295] In other embodiments, the POI is a lyase enzyme, including but not limited to, an EC4 (lyase) enzyme selected from EC 4.1.2.10 (e.g., mandelonitrile lyase), EC 4.1.3.3 (e.g., N-acetylneuraminic acid lyase), EC 4.2.1.1 (e.g., carbonic anhydrase), EC 4.2.2.- (e.g., rhamnogalacturonan lyase), EC 4.2.2.10 (e.g., pectin lyase), EC 4.2.2.22 (e.g., pectate trisaccharide lyase), EC 4.2.2.23 (e.g., rhamnogalacturonan endolyase), and EC 4.2.2.3 (e.g., mannuronic acid-specific alginate lyase).

[0296] In certain other embodiments, the POI is an isomerase enzyme, including, but not limited to, an EC5 (isomerase) enzyme selected from EC 5.1.3.3 (e.g., aldose 1-epimerase), EC 5.1.3.30 (e.g., D-psicose 3-epimerase), EC 5.4.99.11 (e.g., isomaltulose synthase), and EC 5.4.99.15 (e.g., (1→4)-α-D-glucan 1-α-D-glucosylmutase).

[0297] In yet other embodiments, the POI is a ligase enzyme, including but not limited to, an EC6 (ligase) enzyme selected from EC 6.2.1.12 (e.g., 4-coumarinate:coenzyme A ligase) and EC 6.3.2.28 (e.g., L-amino acid α-ligase).

[0298] Thus, in certain embodiments, Bacillus host cells that produce industrial proteases provide particularly preferred expression hosts. Similarly, in certain other embodiments, Bacillus host cells that produce industrial amylases provide particularly preferred expression hosts.

[0299] For example, proteases typically secreted by Bacillus spp. are of two general types: neutral (or "metalloproteases") and alkaline (or "serine") proteases. For example, the Bacillus subtilisin protein (enzyme) is an exemplary serine protease for use in the present disclosure. A wide variety of Bacillus subtilisins have been identified and sequenced, including subtilisin 168, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 147, and subtilisin 309 (e.g., WO 1989 / 06279; and Stahl et al., 1984). In some embodiments of the present disclosure, the engineered Bacillus cells produce mutant (i.e., variant) proteases. Numerous references, e.g., WO 1999 / 20770; WO 1999 / 20726; WO 1999 / 20769; WO 1989 / 06279; U.S. Reissue Patent No. 34,606; U.S. Patent Nos. 4,914,031; 4,980,288; 5,208,158; 5,310,675; and 5,336,611, are incorporated herein by reference. Nos. 5,399,283; 5,441,882; 5,482,849; 5,631,217; 5,665,587; 5,700,676; 5,741,694; 5,858,757; 5,880,080; 6,197,567, and 6,218,165 provide examples of mutant proteases. Accordingly, in certain embodiments, the modified Bacillus cells of the present disclosure comprise an expression construct encoding a protease.

[0300] 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 their variants 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 encoding Bacillus species. No. 1999 / 43794 discloses maltogenic α-amylase variants, No. 1990 / 11352 discloses extremely thermostable α-amylase variants, and No. 2006 / 089107 discloses α-amylase variants with granular starch hydrolysis activity.

[0301] In other embodiments, the POI or mutant POI expressed and produced in the engineered cells of the present disclosure is a peptide, peptide hormone, growth factor, clotting factor, chemokine, cytokine, lymphokine, antibody, receptor, adhesion molecule, microbial antigen (e.g., HBV surface antigen, HPV E7, etc.), variants thereof, fragments thereof, etc. Other types of proteins (or variants) of interest can be proteins that can provide nutritional value to food or crops. Non-limiting examples include plant proteins that can inhibit the formation of anti-nutritional factors and plant proteins that have a more desirable amino acid composition (e.g., a higher lysine content than non-transgenic plants).

[0302] There are various methods known to those skilled in the art for detecting and measuring the activity of intracellularly and extracellularly expressed proteins. In particular, for proteases, there are assays based on the release of acid-soluble peptides from casein or hemoglobin, measured as absorbance at 280 nm or colorimetrically using the Folin method (Bergmeyer et al., 1984). Other assays involve the solubilization of chromogenic substrates (see, e.g., Ward, 1983). Other exemplary assays include the succinyl-Ala-Ala-Pro-Phe-para-nitroanilide assay (SAAPFpNA) and the 2,4,6-trinitrobenzenesulfonic acid sodium salt assay (TNBS assay). Numerous additional references known to those skilled in the art provide suitable methods (see, e.g., Wells et al., 1983; Christianson et al., 1994; and Hsia et al., 1999).

[0303] WO 2014 / 164777 discloses the Ceralpha α-amylase activity assay useful for measuring amylase activity as described herein.

[0304] Means for determining the secretion level of a protein of interest in a host cell and for detecting the expressed protein include the use of immunoassays using either polyclonal or monoclonal antibodies specific for the protein, examples of which include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescence-activated cell sorting (FACS).

[0305] VI. Exemplary Embodiments Non-limiting embodiments of the present disclosure include, but are not limited to, the following.

[0306] 1. A method for producing increased amounts of a protein of interest (POI) in an engineered Bacillus licheniformis cell, comprising: (a) modifying a parent B. licheniformis cell to express the POI by introducing therein a polynucleotide comprising a native prsA promoter operably linked to a native prsA open reading frame (ORF); and (b) fermenting the engineered cell under conditions suitable for production of the POI, wherein the engineered cell produces increased amounts of the POI compared to the parent cell when fermented under the same conditions.

[0307] 2. A method for producing increased amounts of a heterologous protein of interest (POI) in an engineered Bacillus licheniformis cell, comprising: (a) modifying a parent B. licheniformis cell by introducing therein a polynucleotide comprising (i) an expression cassette encoding the POI and (ii) a native prsA promoter sequence operably linked to a native prsA open reading frame (ORF) sequence; and (b) fermenting the engineered cell of step (a) under conditions suitable for production of the POI, wherein the engineered cell produces increased amounts of the POI compared to the parent cell when fermented under the same conditions.

[0308] 3. The method of embodiment 1 or embodiment 2, wherein the introduced polynucleotide comprises a native prsA promoter sequence comprising at least 95% sequence identity to SEQ ID NO: 100.

[0309] 4. The method of embodiment 1 or embodiment 2, wherein the introduced polynucleotide comprises a native prsA ORF that comprises at least 90% sequence identity to SEQ ID NO:101.

[0310] 5. The method of embodiment 1 or embodiment 2, wherein the parent cell comprises an endogenous prsA gene encoding a native prsA protein.

[0311] 6. The method of embodiment 5, wherein the endogenous prsA gene encodes a native prsA protein comprising about 90% sequence identity to SEQ ID NO: 155.

[0312] 7. The method of embodiment 1 or embodiment 2, wherein the introduced polynucleotide is integrated into the genome of the modified B. licheniformis cell.

[0313] 8. The method of embodiment 1 or embodiment 2, wherein the modified cell further comprises a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122.

[0314] 9. The method of embodiment 1 or embodiment 2, wherein the modified cell further comprises a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.

[0315] 10. The method of embodiment 1 or embodiment 2, wherein the modified cell comprises a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122 and a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.

[0316] 11. The method of embodiment 1 or embodiment 2, wherein the POI is an enzyme.

[0317] 12. The method of embodiment 11, wherein the enzyme is a protease or amylase.

[0318] 13. A modified B. licheniformis cell derived from a parent Bacillus licheniformis cell, the modified B. licheniformis cell comprising an introduced polynucleotide comprising a native prsA promoter sequence operably linked to a native prsA open reading frame (ORF) sequence.

[0319] 14. A modified B. licheniformis cell derived from a parent Bacillus licheniformis that contains an endogenous prsA gene encoding a native prsA protein, wherein the modified B. licheniformis cell contains an introduced polynucleotide that includes a native prsA promoter sequence operably linked to a native prsA open reading frame (ORF) sequence.

[0320] 15. The modified cell of embodiment 13 or embodiment 14, wherein the introduced polynucleotide comprises a native prsA promoter comprising at least 95% sequence identity to SEQ ID NO: 100.

[0321] 16. The modified cell of embodiment 13 or embodiment 14, wherein the introduced polynucleotide comprises a native prsA ORF comprising at least 90% sequence identity to SEQ ID NO: 101.

[0322] 17. The modified cell of embodiment 13 or embodiment 14, wherein the introduced polynucleotide encodes a native prsA protein that comprises about 90% sequence identity to SEQ ID NO: 155.

[0323] 18. The modified cell of embodiment 13 or embodiment 14, wherein the introduced polynucleotide is integrated into the genome of the modified B. licheniformis cell.

[0324] 19. The modified cell of embodiment 13 or embodiment 14, comprising a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122.

[0325] 20. The modified cell of embodiment 13 or embodiment 14, comprising a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.

[0326] 21. The modified cell of embodiment 13 or embodiment 14, comprising a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122 and a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.

[0327] 22. The modified cell of embodiment 13 or embodiment 14, comprising an introduced expression cassette encoding a heterologous protein of interest (POI).

[0328] 23. The modified cell of embodiment 22, wherein the POI is an enzyme.

[0329] 24. The modified cell of embodiment 13 or embodiment 14, wherein the parent cell expresses an endogenous POI.

[0330] 25. A protein of interest produced by the modified cell of embodiment 22 or embodiment 24.

[0331] 26. An engineered Bacillus licheniformis cell that produces an increased amount of a protein of interest (POI) relative to a parent B. licheniformis cell, wherein the engineered cell is derived from a parent B. licheniformis that expresses the POI, the engineered cell comprises an introduced polynucleotide comprising a native prsA promoter sequence operably linked to a native prsA open reading frame (ORF) sequence, and comprises a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158, and the engineered cell produces an increased amount of the POI relative to the parent strain when fermented under identical conditions.

[0332] 27. The modified cell of embodiment 26, comprising a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122.

[0333] 28. A modified B. licheniformis cell that produces an increased amount of a protein of interest (POI) relative to a parent Bacillus licheniformis cell, wherein the modified cell is derived from a parent B. licheniformis cell that expresses the POI, the modified cell comprises an introduced polynucleotide comprising a native prsA promoter operably linked to a native prsA open reading frame (ORF), and comprises a deleted or disrupted dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122, and the modified cell produces an increased amount of the POI relative to the parent strain when fermented under identical conditions.

[0334] 29. The modified cell of embodiment 28, further comprising a deleted or disrupted rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO: 158.

[0335] 30. The modified cell of embodiment 26 or embodiment 28, wherein the native prsA promoter comprises at least 95% sequence identity with SEQ ID NO: 100.

[0336] 31. The modified cell of embodiment 26 or embodiment 28, wherein the native prsA ORF comprises at least 90% sequence identity with SEQ ID NO: 101.

[0337] 32. The modified cell of embodiment 26 or embodiment 28, wherein the native prsA protein comprises about 90% sequence identity with SEQ ID NO: 155.

[0338] 33. The modified cell of embodiment 26 or embodiment 28, wherein the POI is an enzyme.

[0339] 34. The modified cell of embodiment 33, wherein the enzyme is a protease or amylase.

[0340] 35. A protein of interest produced by the modified cell of embodiment 26 or embodiment 28. [Example]

[0341] Certain aspects of the present invention 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. [Example]

[0342] Construction of CAS9 vectors targeting RGHR2 pathway genes The Cas9 protein from S. pyogenes (SEQ ID NO: 1) was codon-optimized for Bacillus (SEQ ID NO: 2) with the addition of an N-terminal nuclear localization sequence (NLS, "APKKKRKV"; SEQ ID NO: 3), a C-terminal NLS ("KKKKLK"; SEQ ID NO: 4), and a deca-histidine tag ("HHHHHHHHHH"; SEQ ID NO: 5), the aprE promoter (SEQ ID NO: 6) and terminator sequence (SEQ ID NO: 7) from B. subtilis, and amplified using Q5 DNA polymerase (NEB) with the forward (SEQ ID NO: 8) and reverse (SEQ ID NO: 9) primer pair shown in Table 1 below, according to the manufacturer's instructions.

[0343] [Table 1] The backbone (SEQ ID NO: 10) of plasmid pKB320 (SEQ ID NO: 11) was amplified using Q5 DNA polymerase (NEB) with the forward (SEQ ID NO: 12) and reverse (SEQ ID NO: 13) primer pairs shown in Table 2 below, according to the manufacturer's instructions.

[0344] [Table 2] The PCR product was purified using Zymo clean and concentrate 5 columns according to the manufacturer's instructions. Subsequently, the PCR product was assembled using extended overlap extension PCR (POE-PCR) with Q5 polymerase (NEB) by mixing the two fragments in an equimolar ratio. The following POE-PCR reaction cycle was performed: 98°C for 5 seconds, 64°C for 10 seconds, and 72°C for 4 minutes and 15 seconds (30 cycles). Five microliters of POE-PCR DNA was transformed into Top10 E. coli (Invitrogen) according to the manufacturer's instructions and selected on lysogeny (L) medium (Miller's formula; 1% w / v tryptone, 0.5% w / v yeast extract, 1% w / v NaCl) solidified with 1.5% agar and containing 50 μg / ml kanamycin sulfate. Colonies were grown at 37°C for 18 hours. Colonies were picked and plasmid DNA was prepared using a Qiaprep DNA miniprep kit according to the manufacturer's instructions and eluted in 55 μl of ddH O. The plasmid DNA was Sanger sequenced to verify correct assembly using the sequencing primers shown in Table 3 below. [Table 3]

[0345] The correctly assembled plasmid pRF694 (SEQ ID NO: 25) was used to construct plasmids pRF801 (SEQ ID NO: 26) and pRF806 (SEQ ID NO: 27) for editing the B. licheniformis genome at target site 1 (TS1; SEQ ID NO: 28) and target site 2 (TS2; SEQ ID NO: 29), as described below.

[0346] The serA1 open reading frame of B. licheniformis (SEQ ID NO: 30) contains a unique target site (TS), target site 1 (TS1; SEQ ID NO: 28), in the reverse orientation. The target site is adjacent to a proto-spacer adjacent motif (SEQ ID NO: 31) in the reverse orientation. The target site can be converted to DNA encoding a variable targeting (VT) domain (SEQ ID NO: 32).

[0347] A DNA sequence encoding the VT domain (SEQ ID NO: 32) is operably fused to a DNA sequence encoding a Cas9 endonuclease recognition domain (CER; SEQ ID NO: 33) so that, when transcribed by a bacterial cell's RNA polymerase, it produces a functional gRNA (gRNA) (SEQ ID NO: 34) that targets target site 1. The DNA encoding the gRNA was operably linked to a promoter operable in Bacillus sp. cells (e.g., the spac promoter; SEQ ID NO: 35) and a terminator operable in Bacillus sp. cells (e.g., the t0 terminator sequence of phage lambda; SEQ ID NO: 36) such that the promoter was located 5' to the DNA encoding the gRNA (SEQ ID NO: 33) and the terminator was located 3' downstream of the DNA encoding the gRNA (SEQ ID NO: 33).

[0348] An editing template for deleting the serA1 gene in response to Cas9 / gRNA cleavage was generated by amplification of two homology arms from B. licheniformis genomic DNA (gDNA). The first fragment corresponds to 500 bp immediately upstream of the serA1 open reading frame (SEQ ID NO: 37). This fragment was amplified using Q5 DNA polymerase according to the manufacturer's instructions and the forward (SEQ ID NO: 38) and reverse (SEQ ID NO: 39) primers listed in Table 4 below. These primers incorporate 18 bp of homology to the 5' end of the second fragment on the 3' end of the first fragment and 20 bp of homology to pRF694 at the 5' end of the first fragment. [Table 4]

[0349] The second fragment corresponds to 500 bp immediately downstream of the 3' end of the serA1 open reading frame (SEQ ID NO:40). This fragment was amplified using Q5 DNA polymerase according to the manufacturer's instructions and the forward (SEQ ID NO:41) and reverse (SEQ ID NO:42) primers listed in Table 5 below. These primers incorporate 28 bp of homology to the 3' end of the first fragment on the 5' end of the second fragment and 21 bp of homology to pRF694 on the 3' end of the second fragment. [Table 5]

[0350] DNA encoding the gRNA expression cassette for target site 1 (SEQ ID NO:43), the first (SEQ ID NO:37) and second (SEQ ID NO:40) homology arms was assembled into pRF801 (SEQ ID NO:26), an E. coli-B. licheniformis shuttle plasmid containing a Cas9 expression cassette (SEQ ID NO:2), using standard molecular biology techniques to generate an editing template (SEQ ID NO:44) comprised of the gRNA expression cassette (SEQ ID NO:43) encoding a gRNA targeting target site 1 within the serA1 open reading frame, and the first (SEQ ID NO:37) and second (SEQ ID NO:40) homology arms. The plasmid was verified by Sanger sequencing using the oligos listed in Table 3.

[0351] The B. licheniformis rghR1 open reading frame (SEQ ID NO: 45) contains a unique target site, target site 2 (TS2; SEQ ID NO: 29), on the reverse strand. This target site is adjacent to a proto-spacer adjacent motif (SEQ ID NO: 46) on the reverse strand. The DNA sequence encoding the target site (SEQ ID NO: 29) is operably fused to a DNA sequence encoding a Cas9 endonuclease recognition domain (CER; SEQ ID NO: 33) such that, when transcribed by a bacterial cell's RNA polymerase, it produces a functional gRNA (gRNA) (SEQ ID NO: 47) that targets target site 2. The DNA encoding the gRNA was operably linked to a promoter operable in a Bacillus sp. cell (e.g., the spac promoter derived from B. subtilis; SEQ ID NO: 35) and a terminator operable in a Bacillus sp. cell (e.g., the t0 terminator sequence of lambda phage; SEQ ID NO: 36) such that the promoter was located 5' of the DNA encoding the gRNA (SEQ ID NO: 47) and the terminator was located 3' of the DNA encoding the gRNA (SEQ ID NO: 47).

[0352] An editing template for modifying the rghR1 gene in response to Cas9 / gRNA cleavage was generated by amplification of two homology arms from B. licheniformis genomic DNA (gDNA). The first fragment corresponds to 500 bp immediately upstream of the rghR1 open reading frame (SEQ ID NO:48). This fragment was amplified using Q5 DNA polymerase and the primers listed in Table 6 below, according to the manufacturer's instructions. These primers incorporate 23 bp of homology to the 5' end of the second fragment on the 3' end of the first fragment and 20 bp of homology to pRF694 at the 5' end of the first fragment. [Table 6]

[0353] The second fragment corresponds to 500 bp immediately downstream of the 3' end of the rghR1 open reading frame (SEQ ID NO:51). This fragment was amplified using Q5 DNA polymerase and the primers listed in Table 7 below, according to the manufacturer's instructions. These primers incorporate 20 bp of homology to the 3' end of the first fragment on the 5' end of the second fragment and 21 bp of homology to pRF694 on the 3' end of the second fragment. [Table 7]

[0354] DNA encoding the target site 2 gRNA expression cassette (SEQ ID NO: 54), first (SEQ ID NO: 48) and second (SEQ ID NO: 51) homology arms was assembled into pRF694 (SEQ ID NO: 25) using standard molecular biology techniques to generate an editing template (SEQ ID NO: 55) comprised of pRF806 (SEQ ID NO: 27), an E. coli-B. licheniformis shuttle plasmid containing a Cas9 expression cassette (SEQ ID NO: 2), a gRNA expression cassette (SEQ ID NO: 54) encoding a gRNA targeting target site 2 within the rghR1 open reading frame, and the first (SEQ ID NO: 48) and second (SEQ ID NO: 51) homology arms. The plasmid was verified by Sanger sequencing using the oligos listed in Table 3. [Example]

[0355] Construction of CAS9 Y155H mutant and related targeting plasmids In this example, the Y155H mutant of S. pyogenes Cas9 (SEQ ID NO:56) was constructed in the pRF801 (SEQ ID NO:26) and pRF806 (SEQ ID NO:27) plasmids. To introduce the Y155H mutant into the pRF801 (SEQ ID NO:26) or pRF806 (SEQ ID NO:27) plasmids, site-directed mutagenesis was performed using the oligos shown in Table 8 below with the Quikchange mutagenesis kit and pRF801 (SEQ ID NO:26) or pRF806 (SEQ ID NO:27) as template DNA according to the manufacturer's instructions. [Table 8]

[0356] The resulting reaction product, pRF827 (SEQ ID NO:59), contained either the (Cas9) Y155H mutant expression cassette (SEQ ID NO:60), a gRNA expression cassette (SEQ ID NO:43) encoding gRNA targeting target site 1 within the serA1 open reading frame, and an editing template (SEQ ID NO:44) composed of the first (SEQ ID NO:37) and second (SEQ ID NO:40) homology arms; or pRF856 (SEQ ID NO:61), containing the (Cas9) Y155H mutant expression cassette (SEQ ID NO:60), a gRNA expression cassette targeting target site 2 within the rghR1 open reading frame (SEQ ID NO:54), and an editing template (SEQ ID NO:55) composed of the first (SEQ ID NO:48) and second (SEQ ID NO:51) homology arms. These plasmid DNAs were Sanger sequenced to verify correct assembly using the sequencing primers listed in Table 3 above.

[0357] Construction of plasmid pRF862

[0358] Plasmid pRF862 (SEQ ID NO: 62) was constructed by transferring a fragment of the Cas9 open reading frame containing the Y155H substitution (SEQ ID NO: 63) from pRF827 (SEQ ID NO: 59) that was amplified using the primers shown in Table 9. [Table 9]

[0359] The second fragment (SEQ ID NO:66) was amplified from pRF694 (SEQ ID NO:25) such that it contained the entire plasmid except for the fragment contained on the pRF827 fragment (SEQ ID NO:63) above. This fragment shares homology with the 5' and 3' ends of the pRF827 fragment (SEQ ID NO:60) for assembly and was amplified using the primers listed in Table 10 below. [Table 10]

[0360] The two fragments were assembled using NEBuilder according to the manufacturer's instructions and transformed into competent E. coli cells. The plasmid sequence was verified by Sanger sequencing as shown in Table 3. The sequence-verified isolate was saved as plasmid pRF862 (SEQ ID NO: 62).

[0361] pRF869 (SEQ ID NO:69), a plasmid targeting the rghR2 ORF (SEQ ID NO:70) and inserting three in-frame stop codons, was constructed using two parts: The first part (SEQ ID NO:71), containing an editing template (SEQ ID NO:72) for modifying the rghR2 ORF (SEQ ID NO:70) and a gRNA expression cassette (SEQ ID NO:73) targeting the rghR2 ORF (SEQ ID NO:70), was synthesized by IDT and amplified for assembly using the primer set shown in Table 11 below. [Table 11]

[0362] The synthetic fragment was inserted into pRF862 (SEQ ID NO: 62) by amplifying pRF862 using the primers shown in Table 12. [Table 12]

[0363] These two parts were assembled using NEBuilder according to the manufacturer's instructions and transformed into E. coli. The plasmid sequence was verified by Sanger sequencing as shown in Table 3. The sequence-verified isolate was saved as plasmid pRF869 (SEQ ID NO: 69).

[0364] Several additional Cas9 plasmids were constructed as described above in Examples 1 and 2. These plasmids, along with their target site sequences and editing template functions, are listed below in Table 13. [Table 13]

[0365] For all plasmids, rolling-circle amplification (RCA) was used to amplify the plasmids and generate suitable substrates for transformation using the TruPrime RCA kit (Sygnis). [Example]

[0366] Construction of engineered host strains In this example, a series of host modifications were introduced into a parent B. licheniformis strain, which contains deletions of the serAl gene (SEQ ID NO: 30) and the lysA gene (SEQ ID NO: 87), and is designated BF140.

[0367] One version of BF140, containing the pBl.comK plasmid (SEQ ID NO: 88) (Liu and Zuber, 1998; Hamoen et al., 1998), which contains a spectinomycin marker (SEQ ID NO: 89), DNA encoding the B. subtilis XylR repressor (SEQ ID NO: 90) and xylA promoter (SEQ ID NO: 91) operably linked to DNA encoding the B. licheniformis ComK protein (SEQ ID NO: 92), was transformed with a linear PCR product targeting the catH locus (SEQ ID NO: 93) for integration of a second copy of the B. licheniformis prsA gene. This construct contains an upstream homology arm to the catH locus (SEQ ID NO: 94) operably linked to the catH promoter (SEQ ID NO: 95), which is DNA encoding the CatH protein (SEQ ID NO: 96) operably linked to a dual terminator (SEQ ID NO: 97) composed of the catH terminator (SEQ ID NO: 98) operably linked to the spoVG terminator (SEQ ID NO: 99) of B. subtilis.

[0368] This construct contains the B. licheniformis prsA promoter (SEQ ID NO:100) operably linked to the prsA coding sequence (SEQ ID NO:101), which is in turn operably linked to a terminator from the B. licheniformis amyL gene (SEQ ID NO:102), which is operably linked to a downstream homology arm to the catH locus (SEQ ID NO:103). Briefly, BF140 / pBl.comK competent cells were generated. The BF140 / pBl.comK strain was grown overnight in L broth containing 100 ppm spectinomycin at 37°C with shaking at 250 RPM. The culture reached an OD of 0.7 the next day in fresh L broth containing 100 ppm spectinomycin. 600The cells were diluted to 0.05%. This new culture was grown for 1 hour at 37°C with shaking at 250 RPM. D-xylose was added to 0.1% w / v. The culture was grown for an additional 4 hours at 37°C with shaking at 250 RPM. The cells were harvested at 1700 g for 7 minutes. The cells were resuspended in 1 / 4 volume of spent culture medium containing 10% v / v DMSO. 100 μl of cells was mixed with 10 μL of catH::[catH prsAp-prsA] integration fragment (SEQ ID NO: 94). The cell / DNA mixture was incubated for 1.5 hours at 1400 RPM at 37°C. This mixture was then plated on L agar plates containing 10 ppm chloramphenicol. The inoculated plates were incubated for 48 hours at 37°C.

[0369] Colonies formed on L agar containing 10 ppm chloramphenicol were screened using colony PCR to confirm modification of the catH locus using the primers listed in Table 14 and standard PCR techniques. [Table 14]

[0370] This PCR product, a 2676 bp fragment (SEQ ID NO: 106), was sequenced using the Sanger method and the primers listed in Table 15. [Table 15]

[0371] An isolate with the correct catH::[catH prsAp-prsA] integration (SEQ ID NO: 93) was stored as strain BF547.

[0372] A version of BF547 containing the pBl.comK plasmid (SEQ ID NO: 88) was made competent as described above. 100 μl of competent cells was mixed with 5 μl of the RCA of pRF946 (SEQ ID NO: 81) and incubated at 1400 RPM and 37°C for 1.5 hours. The mixture was plated on L agar plates containing 20 ppm kanamycin to select for plasmid transformation. Plates were incubated at 37°C for 48 hours.

[0373] Colonies that formed on L agar containing 20 ppm kanamycin were meanwhile screened by colony PCR to confirm the deletion of the DNA encoding the 3' end of the catH promoter and the DNA encoding the CatH protein (SEQ ID NO:110), while retaining the catH::[prsAp-prsA] cassette (SEQ ID NO:111) using standard PCR techniques and the primers listed in Table 14 above.

[0374] The correct colony containing the catH::[prsAp-prsA] cassette (SEQ ID NO:111) produced a 1990 bp PCR product (SEQ ID NO:112), as opposed to the parent colony containing the catH::[catH prsAp-prsA] cassette (SEQ ID NO:93, which produced a PCR product 2676 bp in length) (SEQ ID NO:106). The difference was assessed visually using standard gel electrophoresis techniques. Isolates with PCR products of the correct size were sequenced using primers 1915 (SEQ ID NO:107) and 1916 (SEQ ID NO:108) shown in Table 15 above.

[0375] A sequence-verified isolate containing the catH::[prsAp-prsA] cassette (SEQ ID NO: 111) and that was phenotypically sensitive to chloramphenicol (10 ppm) was banked as BF561.

[0376] A version of BF561 containing the pBl.comK plasmid (SEQ ID NO:88) was made competent as described above. 100 μl of competent cells was mixed with 5 μl of the RCA of pZM221 (SEQ ID NO:84) or pRF879 (SEQ ID NO:78) and incubated at 1400 RPM and 37°C for 1.5 hours. The mixture was plated on L agar plates containing 20 ppm kanamycin to select for cells transformed with the plasmid.

[0377] For cells transformed with pZM221 (SEQ ID NO: 84) that formed colonies on L agar plates containing 20 ppm kanamycin, colonies were screened for the ΔdltA-2 allele (SEQ ID NO: 86), a deletion of 700 bp of the dltA coding sequence, using standard PCR techniques and the primers shown in Table 16. [Table 16]

[0378] Colonies with the ΔdltA-2 allele produce a 2067 bp (SEQ ID NO:115) PCR product using the primers shown in Table 16, while parental cells containing an intact dltA gene produce a 2767 bp (SEQ ID NO:116) PCR product, which could be differentiated using standard electrophoresis techniques. A colony containing a 700 bp internal deletion of dltA (SEQ ID NO:86) was stocked as BF598.

[0379] For cells transformed with pRF879 (SEQ ID NO: 78) that formed colonies on L agar plates containing 20 ppm kanamycin, colonies were screened for the ΔrghR2 allele (SEQ ID NO: 80), which is a deletion of the rghR2 coding sequence except for the first and last 9 bp, using standard PCR techniques and the primers shown in Table 17. [Table 17]

[0380] Colonies with the ΔrghR2 allele (SEQ ID NO:80) produce a 1523 bp (SEQ ID NO:119) PCR product using the primers shown in Table 17, while parental cells containing an intact rghR2 gene produce a 1922 bp (SEQ ID NO:120) PCR product. The difference between these two products can be distinguished using standard electrophoresis techniques. The colony containing the deletion of the rghR2 gene (SEQ ID NO:84) was stocked as BF602.

[0381] A version of BF598 containing the pBl.comK plasmid (SEQ ID NO:88) was made competent as described above. 100 μl of competent cells was mixed with 5 μl of the RCA of pRF879 (SEQ ID NO:78) and incubated at 1400 RPM and 37°C for 1.5 hours. The mixture was plated on L agar plates containing 20 ppm kanamycin to select for cells transformed with the plasmid.

[0382] For cells transformed with pRF879 (SEQ ID NO: 78) that formed colonies on L agar plates containing 20 ppm kanamycin, colonies were screened for the ΔrghR2 allele (SEQ ID NO: 80), which is a deletion of the rghR2 coding sequence except for the first and last 9 bp, using standard PCR techniques and the primers shown in Table 17 above.

[0383] Colonies with the ΔrghR2 allele (SEQ ID NO:80) produce a 1523 bp (SEQ ID NO:119) PCR product using the primers shown in Table 17, while parental cells containing an intact rghR2 gene produce a 1922 bp (SEQ ID NO:120) PCR product. The difference between these two products can be distinguished using standard electrophoresis techniques. The colony containing a deletion of the rghR2 gene (SEQ ID NO:80) was stocked as BF613. Table 18 below shows the modified host strains created in this example, along with the SEQ ID NOs for the three modified loci in this example. [Table 18] [Example]

[0384] Construction of amylase-expressing strains in engineered host strains In this example, a series of amylase and amylase variant expression cassettes were introduced into the strain family listed in Table 18 of Example 2 above.

[0385] Amylase 1

[0386] Amylase 1 (SEQ ID NO:126) is a native α-amylase from B. licheniformis, commonly referred to as AmyL. The first cassette for amylase 1 (SEQ ID NO:127) is integrated into the serA1 locus (SEQ ID NO:44) and contains a synthetic p3 promoter (SEQ ID NO:128) operably linked to DNA encoding a modified B. subtilis aprE 5'UTR (SEQ ID NO:129) operably linked to DNA encoding the B. licheniformis AmyL signal sequence (SEQ ID NO:130) operably linked to DNA encoding amylase 1 (SEQ ID NO:131) operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:102). The second amylase 1 cassette (SEQ ID NO:132), integrated within the lysA locus (SEQ ID NO:133), contains DNA encoding LysA (SEQ ID NO:134) and a synthetic p2 promoter (SEQ ID NO:135) operably linked to DNA encoding a modified B. subtilis aprE 5'UTR (SEQ ID NO:129), which is operably linked to DNA encoding a B. licheniformis AmyL signal sequence (SEQ ID NO:130), which is operably linked to DNA encoding amylase 1 (SEQ ID NO:131), which is operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:102).

[0387] Amylase 2

[0388] Amylase 2 (SEQ ID NO: 136) is a variant Bacillus sp. α-amylase generally described in WO 2018 / 184004, which is incorporated by reference in its entirety. The first cassette for amylase 2 (SEQ ID NO:137) is integrated within the serA1 locus (SEQ ID NO:44) and contains the B. subtilis rrnI promoter (SEQ ID NO:138) operably linked to DNA encoding a modified B. subtilis aprE 5'UTR (SEQ ID NO:139) operably linked to DNA encoding the B. licheniformis AmyL signal sequence (SEQ ID NO:130) operably linked to DNA encoding amylase 2 (SEQ ID NO:140) operably linked to the serA1 ORF (SEQ ID NO:30) and the B. licheniformis amyL transcription terminator (SEQ ID NO:102). The second amylase 2 cassette (SEQ ID NO:141), integrated within the lysA locus (SEQ ID NO:133) or the amyL locus (SEQ ID NO:142), contains DNA encoding LysA (SEQ ID NO:134) and a synthetic p3 promoter (SEQ ID NO:128) operably linked to DNA encoding a modified B. subtilis aprE 5'UTR (SEQ ID NO:139), which is operably linked to DNA encoding the B. licheniformis AmyL signal sequence (SEQ ID NO:130), which is operably linked to DNA encoding amylase 2 (SEQ ID NO:140), which is operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:102).

[0389] Amylase 3

[0390] Amylase 3 (SEQ ID NO: 143) is a mutant Cytophaga sp. α-amylase (see, e.g., WO 2014 / 164777; WO 2012 / 164800; and WO 2014 / 16483, each of which is incorporated by reference in its entirety). The first amylase 3 cassette (SEQ ID NO:144) is integrated into the serA1 locus (SEQ ID NO:44) and contains the synthetic p3 promoter (SEQ ID NO:128) operably linked to DNA encoding a modified B. subtilis aprE 5'UTR (SEQ ID NO:129) operably linked to DNA encoding the B. licheniformis amyL signal sequence (SEQ ID NO:130) operably linked to DNA encoding the serA1 ORF (SEQ ID NO:30) and amylase 3 (SEQ ID NO:145) operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:102). The second amylase 3 cassette (SEQ ID NO:146), integrated within the lysA locus (SEQ ID NO:133), contains DNA encoding LysA (SEQ ID NO:134) and a synthetic p2 promoter (SEQ ID NO:135) operably linked to DNA encoding a modified B. subtilis aprE 5'UTR (SEQ ID NO:129), which is operably linked to DNA encoding a B. licheniformis AmyL signal sequence (SEQ ID NO:130), which is operably linked to DNA encoding amylase 3 (SEQ ID NO:145), which is operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:102).

[0391] Amylase 4

[0392] Amylase 4 (SEQ ID NO: 147) is a mutant Cytophaga sp. α-amylase (see, e.g., WO 2014 / 164777; WO 2012 / 164800; and WO 2014 / 16483, each of which is incorporated by reference in its entirety). The first amylase 4 cassette (SEQ ID NO:148) is integrated into the serA1 locus (SEQ ID NO:44) and contains the synthetic p3 promoter (SEQ ID NO:128) operably linked to DNA encoding the B. subtilis aprE 5'UTR (SEQ ID NO:139), which is operably linked to DNA encoding the B. licheniformis amyL signal sequence (SEQ ID NO:130), which is operably linked to DNA encoding amylase 4 (SEQ ID NO:149), which is operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:129). The second amylase 4 cassette (SEQ ID NO:150), integrated within the lysA locus (SEQ ID NO:133), contains DNA encoding LysA (SEQ ID NO:134) and a synthetic p2 promoter (SEQ ID NO:135) operably linked to DNA encoding the B. subtilis aprE 5'UTR (SEQ ID NO:139), which is operably linked to DNA encoding the B. licheniformis AmyL signal sequence (SEQ ID NO:130), which is operably linked to DNA encoding amylase 4 (SEQ ID NO:149), which is operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:102).

[0393] Amylase 5

[0394] Amylase 5 (SEQ ID NO: 151) is an α-amylase from a mutant Bacillus sp. 707 (see, e.g., WO 2008 / 153805 and U.S. Patent Application Publication No. 2014 / 0057324). The first cassette for amylase 5 (SEQ ID NO:152) is integrated into the serA1 locus (SEQ ID NO:44) and contains the synthetic p3 promoter (SEQ ID NO:128) operably linked to DNA encoding the B. subtilis aprE 5'UTR (SEQ ID NO:139), which is operably linked to DNA encoding the B. licheniformis amyL signal sequence (SEQ ID NO:130), which is operably linked to DNA encoding amylase 5 (SEQ ID NO:153), which is operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:102). The second amylase 5 cassette (SEQ ID NO:154), integrated within the lysA locus (SEQ ID NO:133), contains DNA encoding LysA (SEQ ID NO:134) and a synthetic p2 promoter (SEQ ID NO:135) operably linked to DNA encoding the B. subtilis aprE 5'UTR (SEQ ID NO:139), which is operably linked to DNA encoding the B. licheniformis amyL signal sequence (SEQ ID NO:130), which is operably linked to DNA encoding amylase 5 (SEQ ID NO:153), which is operably linked to the B. licheniformis amyL transcription terminator (SEQ ID NO:102).

[0395] All amylase expression cassettes were transformed into the engineered host strains using the methods described in WO 2019 / 040412, which is incorporated herein by reference in its entirety. [Example]

[0396] Effect of engineered host background on amylase production In this example, modified host strains (i.e., Table 19; BF140, BF561, BF598, BF602, and BF613) containing two copies of the expression cassettes for Amylases 1-5 (Example 4) were assayed for α-amylase production using standard small-scale or laboratory-scale fermentation conditions (see WO 2018 / 156705 and WO 2019 / 055261, each incorporated herein by reference). α-Amylase production was quantified using Bradford or Ceralpha assay methods. The relative improvement in amylase production is comparable to an unmodified host containing the same α-amylase expression cassettes, as presented in Table 19 below. [Table 19]

[0397] Thus, all five amylases tested from a diverse group of α-amylases show an improvement in α-amylase production compared to the unmodified parent host BF140 in the modified background of native BF613 containing a deleted dltA-2 (ΔdltA-2) allele (SEQ ID NO: 125), a deleted rghR2 (ΔrghR2) allele (SEQ ID NO: 80), and the insertion of a second copy of the native prsA gene controlled by the prsA promoter (SEQ ID NO: 124).

[0398] For Amylase 2 and Amylase 3, the improvement in α-amylase production in the engineered background of BF602, which contains a deleted rghR2 (ΔrghR2) allele (SEQ ID NO: 80) and a second copy of the native prsA gene controlled by the native prsA promoter (SEQ ID NO: 124), was nearly as good as the improvement seen in the BF613 engineered host, suggesting that not only are the presence of these two alleles necessary for improvement for some amylases, but that the presence of the ΔdltA-2 allele is not detrimental to this improvement.

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Claims

1. 1. A method for producing increased amounts of a protein of interest (POI) in an engineered Bacillus licheniformis cell, comprising: (a) modifying a parent B. licheniformis cell to express a POI by introducing into the parent B. licheniformis cell a polynucleotide comprising a native B. licheniformis prsA promoter operably linked to a native B. licheniformis prsA open reading frame (ORF); and (b) fermenting the modified cells under conditions suitable for said production of the POI. wherein the modified cells produce increased amounts of the POI compared to the parent cells when fermented under the same conditions.

2. 1. A method for producing increased amounts of a protein of interest (POI) in an engineered Bacillus licheniformis cell, comprising: (a) introducing into a parent B. licheniformis cell a polynucleotide comprising (i) an expression cassette encoding a POI, and (ii) a native B. licheniformis prsA promoter operably linked to a native B. licheniformis prsA open reading frame (ORF); and (b) fermenting the modified cells of step (a) under conditions suitable for said production of said POI. wherein the modified cells produce increased amounts of the POI compared to the parent cells when fermented under the same conditions.

3. 3. The method of claim 1 or claim 2, wherein the introduced polynucleotide comprises a native B. licheniformis prsA promoter sequence that comprises at least 95% sequence identity to SEQ ID NO:

100.

4. 3. The method of claim 1 or claim 2, wherein the introduced polynucleotide comprises a native B. licheniformis prsA ORF sequence that comprises at least 90% sequence identity to SEQ ID NO:

101.

5. 3. The method of claim 1 or claim 2, wherein the parent cell comprises an endogenous B. licheniformis prsA gene encoding a native B. licheniformis prsA protein.

6. 3. The method of claim 1 or claim 2, wherein the introduced polynucleotide is integrated into the genome of the modified cell.

7. The method of claim 1 or claim 2, wherein the modified cell does not contain a functional dltA gene comprising at least 90% sequence identity to SEQ ID NO: 122 and / or a functional rghR2 gene comprising at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO:

158.

8. The method of claim 1 or claim 2, wherein the POI is an enzyme.

9. 1. A modified B. licheniformis cell derived from a parent Bacillus licheniformis cell, the modified B. licheniformis cell comprising an endogenous B. licheniformis prsA gene encoding a native B. licheniformis prsA protein, and further comprising an introduced polynucleotide comprising a native B. licheniformis prsA promoter operably linked to the native B. licheniformis prsA open reading frame (ORF).

10. 10. The modified cell of claim 9, wherein the introduced polynucleotide comprises a native B. licheniformis prsA promoter that comprises at least 95% sequence identity to SEQ ID NO:

100.

11. 10. The modified cell of claim 9, wherein the introduced polynucleotide comprises a native B. licheniformis prsA ORF that contains at least 90% sequence identity to SEQ ID NO:

101.

12. 10. The engineered cell of claim 9, wherein the introduced polynucleotide encodes a native B. licheniformis prsA protein that comprises 90% or greater sequence identity to SEQ ID NO:

155.

13. The modified cell of claim 9, which does not contain a functional dltA gene having at least 90% sequence identity to SEQ ID NO: 122 and / or a functional rghR2 gene having at least 90% sequence identity to SEQ ID NO: 121 or SEQ ID NO:

158.

14. 10. The modified cell of claim 9, comprising an introduced expression construct encoding a heterologous protein of interest (POI).

15. The modified cell of claim 14, wherein the POI is an enzyme.

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