Compositions and methods for enhanced protein production in gram-positive bacterial cells

Abstract

The present disclosure is generally related to Gram-positive bacterial strains comprising enhanced protein productivity phenotypes. Certain aspects are therefore related to compositions and methods for constructing recombinant (modified) Gram-positive bacterial strains for the enhanced production of proteins of interest.

Description

COMPOSITIONS AND METHODS FOR ENHANCED PROTEIN PRODUCTION IN GRAM-POSITIVE BACTERIAL CELLS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit to U.S. Provisional Patent Application No.63 / 518,482, filed August 9, 2023, which is incorporated herein by referenced in its entirety. FIELD

[0002] The present disclosure is generally related to the fields of bacteriology, microbiology, genetics, molecular biology, enzymology, industrial protein production the like. Certain embodiments of the disclosure are related to Gram-positive bacterial cells comprising enhanced protein productivity phenotypes, compositions, and methods for constructing recombinant Gram-positive bacterial cells, and the like. REFERENCE TO A SEQUENCE LISTING

[0003] The contents of the electronic submission of the text file Sequence Listing, named “NB42147USPSP_SequenceListing.xml” was created on July 25, 2023 and is 48 KB in size, which is hereby incorporated by reference in its entirety. BACKGROUND

[0004] Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and the like are frequently used as microbial factories for the production of industrial relevant proteins, due to their excellent fermentation properties and high yields. For example, Bacillus sp. host cells are well known for their production of enzymes (e.g., amylases, cellulases, mannanases, pectate lysases, proteases, pullulanases, etc.) necessary for food, textile, laundry, medical instrument cleaning, pharmaceutical industries and the like. Because these non-pathogenic Gram-positive bacteria produce proteins that completely lack toxic by-products (e.g., lipopolysaccharides; LPS, also known as endotoxins) they have obtained the “Qualified Presumption of Safety” (QPS) status of the European Food Safety Authority (EFSA), and many of their products gained a “Generally Recognized As Safe” (GRAS) status from the US Food and Drug Administration.

[0005] Thus, the production of proteins (e.g., enzymes, antibodies, receptors, etc.) via microbial host cells is of particular interest in the biotechnological arts. Likewise, the optimization of Bacillus host cells for the production and secretion of one or more protein(s) of interest is of high relevance, particularly in the industrial biotechnology setting, wherein small improvements in protein yield are quite significant when the protein is produced in large industrial quantities. For example, the expression of many secreted proteinscan still be challenging and unpredictable with respect to yield and the like. As described hereinafter, the present disclosure is related to the highly desirable and unmet needs for obtaining and constructing Gram- positive cells (e.g., protein production hosts) having enhanced protein production capabilities. SUMMARY OF INVENTION

[0006] As described herein, certain embodiments of the disclosure are directed to methods and compositions for enhanced protein production in Gram-positive bacterial (host) cells. Certain one or more embodiments therefore provide, inter alia, Gram-positive bacterial cells / strains expressing heterologous proteins, Gram-positive bacterial cells secreting heterologous proteins into the fermentation broth, Gram- positive bacterial cells comprising an introduced (synthetic) secGEY operon expressing SecG, SecE and SecY translocon proteins, and the like. Certain embodiments are therefore related to non-native (synthetic) secGEY operons, nucleic acid (DNA) sequences encoding SecG, SecE and SecY translocon proteins (e.g., a secG ORF encoding a functional SecG protein, a secE ORF encoding a functional SecE protein, a secY ORF encoding a functional SecY protein), DNA sequences encoding heterologous proteins of interest, DNA sequences encoding (heterologous) precursor proteases, DNA sequences encoding (heterologous) mature proteases, DNA sequences encoding protease signal (secretion) peptide sequences, DNA sequences encoding protease PRO region sequences, DNA sequences encoding one or more ribosome binding sites (RBS), promoter region (DNA) sequences, 5ʹ-untranslated region (5ʹ-UTR) sequences, and the like.

[0007] In certain embodiments, the disclosure provides recombinant Gram-positive bacterial cells comprising an introduced (synthetic) secGEY operon and comprising one or more introduced expression cassettes encoding a heterologous subtilisin protein. In certain embodiments, the introduced secGEY operon comprises a secG open reading frame (ORF) sequence comprising at least about 80% to 100% identity to SEQ ID NO: 2, a secE ORF sequence comprising at least about 80% to 100% identity to SEQ ID NO: 31 and a secY ORF sequence comprising at least about 80% to 100% identity to SEQ ID NO: 32. In certain other embodiments, the introduced (synthetic) secGEY operon comprises an upstream (5ʹ) wild- type secG promoter and secG 5ʹ-UTR sequence operably linked to the downstream secGEY open reading frames (ORFs). In other embodiments, the introduced secGEY operon comprises an upstream (5ʹ) heterologous promoter and 5ʹ-UTR sequence operably linked to the downstream secGEY ORFs. In yet other embodiments, the recombinant cell comprises at least two introduced cassettes encoding the same subtilisin or different subtilisins, or comprises at least three introduced expression cassettes encoding the same subtilisin or different subtilisins. In certain embodiments, the cassette or cassettes encode an alkaline subtilisin. In another embodiment, the cassette or cassettes encode an alkaline subtilisin comprising an isoelectric point (pI) between about 8.5 to about 10. In certain other embodiments, the heterologous subtilisin comprises at least about 80% amino acid identity to the mature amino acid sequence of SEQ IDNO: 21 or SEQ ID NO: 23. For instance, in certain embodiments, the heterologous subtilisin comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23.

[0008] As described herein, a non-native (synthetic) secGEY operon of the disclosure is constructed wherein the secG, secE and secY open reading frame (ORF) polynucleotide sequences (i.e., encoding secG, secE and secY translocon proteins respectively) are operably linked in any order. For example, in certain embodiments, a non-native (synthetic) secGEY operon comprises an upstream secE ORF operably linked to a downstream secY ORF operably linked to a downstream secG ORF (e.g., 5ʹ-[secE]-[secY]-[secG]-3ʹ), a non-native secGEY operon comprises an upstream secE ORF operably linked to a downstream secG ORF operably linked to a downstream secY ORF (e.g., 5ʹ-[secE]-[secG]-[secY]-3ʹ), a non-native secGEY operon comprises an upstream secY ORF operably linked to a downstream secE ORF operably linked to a downstream secG ORF (e.g., 5ʹ-[secY]-[secE]-[secG]-3ʹ), a non-native secGEY operon comprises an upstream secY ORF operably linked to a downstream secG ORF operably linked to a downstream secE ORF (e.g., 5ʹ-[secY]-[secG]-[secE]-3ʹ), a non-native secGEY operon comprises an upstream secG ORF operably linked to a downstream secY ORF operably linked to a downstream secE ORF (e.g., 5ʹ-[secG]- [secY]-[secE]-3ʹ), and the like.

[0009] Certain embodiments are therefore related to polynucleotides encoding synthetic secGEY operons of the disclosure. For instance, in certain embodiments the disclosure provides a polynucleotide construct encoding a synthetic secGEY operon, wherein the polynucleotide comprises at least an upstream (5ʹ) promoter region sequence operably linked to a downstream nucleic acid encoding a secG protein comprising at least about 80% to 100% identity to SEQ ID NO: 28 operably linked to a downstream nucleic acid encoding a secE protein comprising at least about 80% to 100% identity to SEQ ID NO: 29 operably linked to a downstream (3ʹ) nucleic acid encoding a secY protein comprising at least about 80% to 100% identity to SEQ ID NO: 30.

[0010] In other embodiments, the disclosure provides methods for producing heterologous subtilisin proteins in recombinant (modified) bacterial cells. Certain embodiments are therefore directed to a method for producing a heterologous subtilisin in a modified Gram-positive bacterial cell comprising (a) obtaining a Gram-positive bacterial cell expressing / producing a heterologous subtilisin and introducing into the cell a synthetic secGEY operon and (b) fermenting the modified cell under suitable conditions for the production of the subtilisin. In other embodiments, the disclosure provides a method for producing a heterologous subtilisin in a modified Gram-positive bacterial cell comprising (a) obtaining a Gram-positive bacterial cell and introducing into the cell (i) a synthetic secGEY operon and (ii) an expression cassette encoding aheterologous subtilisin and (b) fermenting the modified cell under suitable conditions for the production of the subtilisin.

[0011] In certain embodiments of the methods, the subtilisin is secreted into the fermentation broth when fermented under suitable conditions for the production of the subtilisin. In other embodiments, the modified cell produces an increased amount of the subtilisin relative to a control cell fermented under the same conditions, wherein the control cell comprises the same introduced expression cassette encoding the same heterologous subtilisin, but does not comprise an introduced secGEY operon. In other embodiments of the methods, the introduced secGEY operon comprises a secG ORF sequence comprising at least about 80 to 100% identity to SEQ ID NO: 2. In another embodiment, the introduced secGEY operon comprises a secE ORF sequence comprising at least about 80% to 100% identity to SEQ ID NO: 31. In yet other embodiments, the introduced secGEY operon comprises a secY ORF sequence comprising at least about 80% to 100% identity to SEQ ID NO: 32. In certain other embodiments of the methods, the introduced secGEY operon comprises an upstream secG promoter and secG 5ʹ-UTR sequence comprising at least about 95% to 100% identity to SEQ ID NO: 1 operably linked to the downstream secGEY ORFs. In other embodiments, the introduced secGEY operon comprises an upstream (5ʹ) heterologous promoter and 5ʹ-UTR sequence operably linked to the downstream secGEY ORFs. In another embodiment, the cell comprises at least two introduced cassettes encoding the same subtilisin or different heterologous subtilisins, or comprises at least three introduced expression cassettes encoding the same subtilisin or different heterologous subtilisins. In certain other embodiments, the cassette or cassettes encode an alkaline subtilisin. In other embodiments, the cassette or cassettes encode an alkaline subtilisin comprising an isoelectric point (pI) between about 8.5 to about 10. In yet other embodiments of the methods, the cassette or cassettes encode an alkaline subtilisin of subgroup I-S2. In certain other embodiments, the heterologous subtilisin comprises at least about 80% to 100% amino acid identity to the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23. In certain related embodiments, the heterologous subtilisin comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23. In other embodiments, the cassette or cassettes encoding the subtilisin comprise an upstream promoter region sequence operably linked to a downstream nucleic acid encoding a protein signal sequence operably linked to a downstream nucleic acid encoding a pro-region sequence operably linked to a downstream nucleic acid encoding the mature subtilisin. In particular embodiments of the methods, the increased amount of the subtilisin produced is at least about 5% increased relative to the control cell when fermented under the same conditions.BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 shows the nucleic acid (DNA) sequences of the non-native (synthetic) secGEY operons constructed and described herein. More particularly, as set forth below in the Examples, various non-native secGEY operons were constructed, such as synthetic operon 1 (named “PsecG-secGEY”; SEQ ID NO: 7), synthetic operon 2 (named “PspoVG-secGEY”; SEQ ID NO: 11) and synthetic operon 3 (named “Phbs- secGEY”; SEQ ID NO: 13). As shown in FIG.1, synthetic operon 1 (PsecG-secGEY) includes an upstream (5ʹ) secG promoter region comprising a secG promoter (PsecG) and secG 5ʹ-UTR sequence (FIG.1A; SEQ ID NO: 1) operably linked to a downstream DNA sequence (FIG.1B; secG ORF, SEQ ID NO: 2) encoding a secG translocon protein (SEQ ID NO: 28) operably linked to a downstream DNA sequence comprising a secE ribosomal binding site (RBS) and secE ORF (FIG. 1C; SEQ ID NO: 3) encoding a secE translocon protein (SEQ ID NO: 29) operably linked to a downstream DNA sequence comprising a secY RBS and secY ORF (FIG. 1D; SEQ ID NO: 4) encoding a secY translocon protein (SEQ ID NO: 30). Likewise, synthetic operon 2 (FIG.1; PspoVG-secGEY, SEQ ID NO: 11) includes an upstream (5ʹ) spoVG promoter region comprising a spoVG promoter (PspoVG) and spoVG 5ʹ-UTR sequence (FIG. 1E; SEQ ID NO: 10) operably linked to a downstream DNA sequence (FIG. 1F; secG ORF, SEQ ID NO: 2) encoding a secG translocon protein (SEQ ID NO: 28) operably linked to a downstream DNA sequence comprising a secE ribosomal binding site (RBS) and secE ORF (FIG.1G; SEQ ID NO: 3) encoding a secE translocon protein (SEQ ID NO: 29) operably linked to a downstream DNA sequence comprising a secY RBS and secY ORF (FIG. 1H; SEQ ID NO: 4) encoding a secY translocon protein (SEQ ID NO: 30), and synthetic operon 3 (Phbs-secGEY; SEQ ID NO: 13) includes an upstream (5ʹ) hbs promoter region comprising a hbs promoter (Phbs) and spoVG 5ʹ-UTR sequence (FIG. 1I; SEQ ID NO: 12) operably linked to a downstream DNA sequence (FIG. 1J; secG ORF, SEQ ID NO: 2) encoding a secG translocon protein (SEQ ID NO: 28) operably linked to a downstream DNA sequence comprising a secE ribosomal binding site (RBS) and secE ORF (FIG.1K; SEQ ID NO: 3) encoding a secE translocon protein (SEQ ID NO: 29) operably linked to a downstream DNA sequence comprising a secY RBS and secY ORF (FIG.1L; SEQ ID NO: 4) encoding a secY translocon protein (SEQ ID NO: 30). In addition, as shown in FIG.1C, FIG.1D, FIG.1G, FIG.1H, FIG.1K and FIG.1L, the secE and secY ribosome binding sites are presented as underlined nucleotides.

[0013] Figure 2 presents the mature amino acid sequences of the native B. clausii subtilisin (FIG.2A; Reporter-1, SEQ ID NO: 21), the native B. amyloliquefaciens subtilisin (FIG. 2B; Reporter-2, SEQ ID NO: 22) and the native B. gibsonii subtilisin (FIG.2C; Reporter-3, SEQ ID NO: 23). More specifically, as shown in FIG. 2, the native B. clausii subtilisin reporter comprises 269 amino acid residues with a theoretical isoelectric point (pI) of about 9.30 and a molecular weight (Mw) of about 26,725 Da (FIG.2A), the native B. amyloliquefaciens subtilisin reporter comprises 275 amino acid residues with a theoretical pIof about 6.30 and Mw of about 27,533 Da (FIG.2B), and the native B. gibsonii subtilisin reporter comprises 269 amino acid residues with a theoretical pI of about 9.57 and a Mw of about 27,498 Da (FIG.2C).

[0014] Figure 3 presents a BLAST-P alignment of the native B. clausii subtilisin (mature sequence; SEQ ID NO: 21) versus the native B. amyloliquefaciens subtilisin (mature sequence; SEQ ID NO: 22). As shown in FIG.3, SEQ ID NO: 21 and SEQ ID NO: 22 comprise approximately 60% amino acid identity.

[0015] Figure 4 presents a BLAST-P alignment of the native B. gibsonii subtilisin (mature sequence; SEQ ID NO: 23) versus the native B. amyloliquefaciens subtilisin (mature sequence; SEQ ID NO: 22). As shown in FIG.4, SEQ ID NO: 23 and SEQ ID NO: 22 comprise approximately 57% amino acid identity.

[0016] Figure 5 presents a BLAST-P alignment of the native B. clausii subtilisin (mature sequence; SEQ ID NO: 21) versus the native B. gibsonii subtilisin (mature sequence; SEQ ID NO: 23). As shown in FIG.5, SEQ ID NO: 21 and SEQ ID NO: 23 comprise approximately 81% amino acid identity. BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0017] SEQ ID NO: 1 is a nucleic acid (DNA) promoter and 5ʹ-UTR sequence named “PsecG”, comprising a Bacillus subtilis secG promoter and secG 5ʹ-UTR sequence in operable combination.

[0018] SEQ ID NO: 2 is a DNA sequence comprising a B. subtilis secG open reading frame (ORF) encoding a native SecG protein.

[0019] SEQ ID NO: 3 is a DNA sequence comprising a B. subtilis ribosome binding sequence (RBS) and secE ORF encoding a native SecE protein.

[0020] SEQ ID NO: 4 is a DNA sequence comprising a B. subtilis RBS and secY ORF encoding a native SecY protein.

[0021] SEQ ID NO: 5 is a DNA sequence comprising a Bacillus amyloliquefaciens BPNʹ terminator region.

[0022] SEQ ID NO: 6 is a downstream (3ʹ) B. subtilis pksR integration cassette homology region (HR)

[0023] SEQ ID NO: 7 is a synthetic DNA sequence comprising a non-native secGEY operon named “PsecG-secGEY”.

[0024] SEQ ID NO: 8 is a DNA sequence comprising a B. subtilis alanine racemase (alrA) gene.

[0025] SEQ ID NO: 9 is an upstream (5ʹ) B. subtilis pksR integration cassette HR.

[0026] SEQ ID NO: 10 is a synthetic DNA promoter region named “PspoVG” comprising a B. subtilis spoVG promoter and spoVG 5ʹ-UTR sequence in operable combination.

[0027] SEQ ID NO: 11 is a synthetic DNA sequence comprising a non-native secGEY operon named “PspoVG-secGEY”.

[0028] SEQ ID NO: 12 is a synthetic DNA promoter region named “Phbs” comprising a B. subtilis hbs promoter and B. subtilis spoVG 5ʹ-UTR sequence in operable combination.

[0029] SEQ ID NO: 13 is a synthetic DNA sequence comprising a non-native secGEY operon named “Phbs-secGEY”.

[0030] SEQ ID NO: 14 is an upstream (5ʹ) B. subtilis aprE integration cassette homology region (HR).

[0031] SEQ ID NO: 15 is a synthetic DNA sequence comprising a B. licheniformis citZ promoter and a kanamycin resistance gene.

[0032] SEQ ID NO: 16 is a downstream (3ʹ) B. subtilis aprE integration cassette HR.

[0033] SEQ ID NO: 17 is a B. subtilis AprE signal (secretion) peptide amino acid sequence.

[0034] SEQ ID NO: 18 is the native pro-region amino acid sequence of the B. lentus subtilisin protease.

[0035] SEQ ID NO: 19 is a signal peptide amino acid sequence of the B. amyloliquefaciens subtilisin (BPNʹ) protease.

[0036] SEQ ID NO: 20 is the native pro-region amino acid sequence of the B. amyloliquefaciens subtilisin (BPNʹ) protease.

[0037] SEQ ID NO: 21 is the mature amino acid sequence of the native B. clausii subtilisin protease.

[0038] SEQ ID NO: 22 is the mature amino acid sequence of the native B. amyloliquefaciens subtilisin protease.

[0039] SEQ ID NO: 23 is the mature amino acid sequence of the native B. gibsonii subtilisin protease.

[0040] SEQ ID NO: 24 is an artificial nucleic acid promoter sequence named “P2” promoter.

[0041] SEQ ID NO: 25 is an artificial nucleic acid promoter sequence named “P2-00788” promoter.

[0042] SEQ ID NO: 26 is an artificial nucleic acid promoter sequence named “P4” promoter.

[0043] SEQ ID NO: 27 is a DNA sequence comprising a B. subtilis spoVG terminator (term) sequence.

[0044] SEQ ID NO: 28 is the amino acid sequence of the native B. subtilis secG protein.

[0045] SEQ ID NO: 29 is the amino sequence of the native B. subtilis secE protein.

[0046] SEQ ID NO: 30 is the amino sequence of the native B. subtilis secY protein.

[0047] SEQ ID NO: 31 is the DNA sequence of the wild-type B. subtilis secE open reading frame encoding the native secE protein of SEQ ID NO: 29.

[0048] SEQ ID NO: 32 is the DNA sequence of the wild-type B. subtilis secY open reading frame encoding the native secY protein of SEQ ID NO: 30. DETAILED DESCRIPTION

[0049] As described herein, certain embodiments of the disclosure are related to compositions and methods for enhanced protein production in Gram-positive bacterial (host) cells. Certain one or more embodiments of the disclosure therefore provide, inter alia, Gram-positive bacterial cells / strains expressing heterologous proteins, compositions thereof and methods thereof, Gram-positive bacterial cells secreting heterologous proteins into the fermentation broth, compositions thereof and methods thereof, Gram-positive bacterial cells comprising an introduced (non-native) secGEY operon expressing SecG, SecE and SecY transloconproteins, compositions thereof and methods thereof, and / or combinations of the foregoing. In certain other one or more embodiments, the disclosure relates to non-native secGEY operons, nucleic acid (DNA) sequences encoding SecG, SecE and SecY translocon proteins (e.g., a secG ORF encoding a functional SecG protein, a secE ORF encoding a functional SecE protein, a secY ORF encoding a functional SecY protein), DNA sequences encoding heterologous proteins of interest, DNA sequences encoding (heterologous) precursor proteases, DNA sequences encoding (heterologous) mature proteases, DNA sequences encoding protease signal (secretion) peptide sequences, DNA sequences encoding protease PRO region sequences, DNA sequences encoding one or more ribosome binding sites (RBS), promoter region (DNA) sequences, 5ʹ-untranslated region (5ʹ-UTR) sequences, and the like. I. DEFINITIONS

[0050] In view of the aforementioned compositions and methods of the disclosure, as further described herein, the following terms and phrases are defined. Terms not defined herein should be accorded their ordinary meaning as used in the art.

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present compositions and methods apply. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present compositions and methods, representative illustrative methods and materials are now described. All publications and patents cited herein are incorporated by reference in their entirety.

[0052] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only”, “excluding”, “not including” and the like, in connection with the recitation of claim elements, or use of a “negative” limitation or proviso thereof. For example, in certain embodiments control (isogenic) Gram- positive bacterial cells are constructed, wherein the control cells “do not include” (i.e., excludes) the introduced (synthetic) secGEY operon.

[0053] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present 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.

[0054] As generally described herein and set forth in the Examples, certain one or more embodiments of the disclosure provide, inter alia, recombinant Gram-positive bacterial cells expressing proteins of interest and comprising an introduced (non-native) secGEY operon expressing SecG, SecE and SecY transloconproteins, methods for producing one or more proteins of interest in Gram-positive bacterial cells comprising an introduced (non-native) secGEY operon expressing SecG, SecE and SecY proteins, and the like.

[0055] As used herein, the phrases “Gram-positive bacteria”, Gram-positive cells” “Gram-positive bacterial strains”, and / or “Gram positive bacterial cells” have the same meaning as used in the art. For example, Gram-positive bacterial cells include all strains of Actinobacteria and Firmicutes. In certain embodiments, such Gram-positive bacteria are of the classes Bacilli, Clostridia and Mollicutes.

[0056] As used herein, the genus “Bacillus” includes all species within the genus “Bacillus”’ as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus”.

[0057] Certain ranges are presented herein with numerical values being preceded by the term “about”. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.

[0058] The term “derived” encompasses the terms “originated” “obtained,” “obtainable,” and “created,” and generally indicates that one specified material or composition finds its origin in another specified material or composition, or has features that can be described with reference to another specified material or composition. For example, recombinant Gram-positive bacterial cells of the disclosure may be derived / obtained from any known Gram-positive bacterial strains.

[0059] As used herein, the terms “recombinant” or “non-natural” refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has at least one engineered genetic alteration, or has been modified by the introduction of a heterologous nucleic acid molecule, or refer to a cell (e.g., a microbial cell) that has been altered such that the expression of a heterologous or endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from a non-natural cell or is progeny of a non-natural cell having one or more such modifications. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, or other nucleic acid molecule additions, deletions, substitutions and / or other functional alteration of a cell’s genetic material.For example, recombinant cells may express genes or other nucleic acid molecules that are not found in identical or homologous form within a native (wild-type) cell (e.g., a fusion or chimeric protein), or may provide an altered expression pattern of endogenous genes, such as being over-expressed, under-expressed, minimally expressed, or not expressed at all. “Recombination”, “recombining” or generating a “recombined” nucleic acid is generally the assembly of two or more nucleic acid fragments wherein the assembly gives rise to a chimeric gene.

[0060] As used herein, “nucleic acid” refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, as well as to DNA, cDNA, and RNA of genomic or synthetic origin, which may be double- stranded or single-stranded, whether representing the sense or antisense strand. It will be understood that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences may encode a given protein.

[0061] It is understood that the polynucleotides (or nucleic acid molecules) described herein include “genes”, “vectors” and “plasmids”.

[0062] Accordingly, the term “gene”, refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all, or part of a protein coding sequence, and may include regulatory (non- transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions (UTRs), including introns, 5′-untranslated regions (UTRs), and 3′-UTRs, as well as the coding sequence.

[0063] As used herein, an “endogenous gene” refers to a gene in its natural location in the genome of an organism.

[0064] As used herein, a “heterologous” gene, a “non-endogenous” gene, or a “foreign” gene refer to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. The term “foreign” gene(s) comprises native genes inserted into a non-native organism and / or chimeric genes inserted into a native or non-native organism.

[0065] As used herein, a “heterologous control sequence”, refers to a gene expression control sequence (e.g., promoters, enhancers, terminators, etc.) which does not function in nature to regulate (control) the expression of the gene of interest. Generally, heterologous nucleic acids are not endogenous (native) to the cell, or a part of the genome in which they are present, and have been added to the cell, by infection, transfection, transduction, transformation, microinjection, electroporation, and the like. A “heterologous” nucleic acid construct may contain a control sequence and DNA coding sequence (ORF) combination that is the same as, or different, from a control sequence / DNA coding sequence combination found in the native host cell.

[0066] As used herein, the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA, derived from a nucleic acid molecule of the disclosure. Expression may alsorefer to translation of mRNA into a polypeptide. Thus, the term “expression” includes any steps involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, secretion and the like.

[0067] 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 an ATG start codon. The coding sequence typically includes DNA, cDNA, and recombinant nucleotide sequences.

[0068] As used herein, the terms “promoter”, “promoter region”, “promoter element”, “promoter sequence” and the like, refer to a nucleic acid (DNA) sequence capable of controlling the transcription of a gene coding sequence (CDS / ORF) into messenger RNA (mRNA) when the promoter region sequence is placed upstream (5′) and operably linked to the downstream (3′) gene (ORF). As generally understood by of skilled in the art, promoters typically provide a site for specific binding by RNA polymerase and the initiation of transcription. In certain aspects, the term “promoter” refers to the minimal portion of the promoter nucleic acid sequence required to initiate transcription (i.e., comprising RNA polymerase binding sites). For example, a promoter generally comprises a “-10” (consensus sequence) element and a “-35” (consensus sequence) element, which are upstream (5′) and relative to the +1 transcription start site (TSS) of the gene CDS to be transcribed. The core promoter -10 and -35 elements are generally referred to in the art as the “TATAAT” (Pribnow box) consensus region and the “TTGACA” consensus region, respectively. The spacing of the core promoter (-10 and -35) regions are generally separated (spaced) by about fifteen- twenty (15-20) intervening base pairs (nucleotides).

[0069] Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters can be constitutive promoters, inducible promoters, tunable promoters, hybrid promoters, synthetic promoters, tandem promoters, etc. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0070] As used herein, a “functional promoter sequence controlling the expression of a gene of interest linked to the gene of interest’s protein coding sequence” refers to a promoter sequence which controls the transcription and translation of the coding sequence in a desired Gram-positive host cell. For example, in certain embodiments, the present disclosure provides polynucleotides comprising an upstream (5′) promoterfunctional in a Gram-positive cell, wherein the functional promoter region is operably linked to a nucleic acid sequence encoding a protein of interest.

[0071] As used herein, the term “precursor protein” refers to an inactive form of a protein. In certain aspects, a full-length protein is synthesized as precursor, in the form of a pro-sequence and mature protein (abbreviated, “pre-protein”). In other aspects, a full-length protein is synthesized as precursor, in the form of a signal peptide sequence, a pro-sequence and mature protein (abbreviated, “pre-pro-protein”). For example, pre-sequences usually act as signal peptides for transport, and pro-sequences are typically essential for the correct folding of the associated (mature) protein.

[0072] As used herein, the term “mature protein” refers to an active form of a protein, in contrast to the inactive precursor (full-length) protein.

[0073] As used herein, the terms “signal sequence”, “secretion signal” and “signal peptide” may be used interchangeably and refer to a sequence of amino acid residues that participate in the secretion or direct transport of a precursor protein. The signal (pre) sequence is typically cleaved from the precursor protein by a signal peptidase during translocation. The signal (pre) sequence is typically located N-terminal to the mature protein sequence, or located N-terminal to the pro-region (PRO) sequence when a signal (pre) sequence and a pro-region (PRO) sequence are used in operable combination and upstream (5′) of the mature POI sequence.

[0074] As used herein, the terms “pro sequence”, “pro-sequence” and “pro region sequence” may be used interchangeably and abbreviated as “PRO” sequence. The term pro-sequence as used herein has the same meaning as understood in the art. For example, the B. subtilis alkaline serine protease “subtilisin” is first produced as a pre-pro-subtilisin, which consists of a signal (pre) sequence for protein secretion followed by a seventy-seven (77) amino acid pro-region (PRO) sequence followed by the amino acid sequence encoding the mature subtilisin (e.g., pre-pro-subtilisin). Pro-sequences are often essential for the correct folding of the associated (mature) protein, acting as an intra-molecular chaperone (e.g., catalyzing the protein-folding reaction directly). Likewise, pro-sequences may be required for both folding and intracellular transport (or secretion) of the mature protein of interest, suggesting that these two functionalities are intimately related.

[0075] As used herein, the phrases “five prime (5′) untranslated region”, “5′ untranslated region” and / or “5′ transcript leader” may be used interchangeably and abbreviated as “5′-UTR”. As generally understood in the art, the 5′-UTR is known to be the region of a messenger RNA (mRNA) that is directly upstream (5′) from the initiation codon.

[0076] 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., a signal sequence) is operably linked to DNA encoding a polypeptide if it is expressed as a pre-protein that participates in the secretion ofthe polypeptide; a promoter or enhancer is operably linked to a coding sequence (CDS, ORF) if it affects the transcription of the sequence; or a ribosome binding site (RBS) 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. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Thus, the term operably linked generally refers to the association (juxtaposition) 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 (pro) sequence is operably linked to a gene coding sequence (gene CDS) if it controls the transcription of the gene CDS (e.g., 5′-[pro]-[gene CDS]-3′).

[0077] 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, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing site, effector binding site and stem-loop structure.

[0078] In certain aspects, an upstream (5′) promoter (pro) region sequence operably linked to a downstream DNA sequence (ss) encoding a signal peptide (secretion) sequence operably linked to a downstream (3′) DNA sequence (ORF) encoding a mature protein of interest may be schematically presented as 5′-[pro]-[ss]-[ORF]-3′. In certain other embodiments, an upstream promoter (pro) region sequence operably linked to a downstream DNA sequence (ss) encoding a signal peptide (secretion) sequence operably linked to a downstream DNA sequence (PRO) encoding a pro-region amino acid sequence operably linked to a downstream DNA sequence (ORF) encoding a mature protein of interest may be schematically presented as 5′-[pro]-[ss]-[PRO]-[ORF]-3′. In certain other embodiments, an upstream promoter (pro) region sequence operably linked to a downstream DNA sequence (ss) encoding a signal peptide (secretion) sequence operably linked to a downstream DNA sequence (PRO) encoding a pro-region amino acid sequence operably linked to a downstream DNA sequence (ORF) encoding a mature protein of interest operably linked to a downstream terminator (term) sequence may be schematically presented as 5′- [pro]-[ss]-[PRO]-[ORF]-[term]-3′.

[0079] As used herein, a promoter region sequence comprising a B. subtilis secG promoter and secG 5ʹ-UTR in operable combination is abbreviated hereinafter “PsecG” (SEQ ID NO: 1), a promoter region sequence comprising a B. subtilis spoVG promoter and spoVG 5ʹ-UTR sequence in operable combination is abbreviated hereinafter “PspoVG” (SEQ ID NO: 10), and a promoter region sequence comprising a B. subtilis hbs promoter and spoVG 5ʹ-UTR sequence in operable combination is abbreviated hereinafter “Phbs” (SEQ ID NO: 12).

[0080] As used herein, a “secG” open reading frame (ORF; SEQ ID NO: 2) encodes a native B. subtilis SecG translocon protein (SEQ ID NO: 28), a “secE” ORF (SEQ ID NO: 31) encodes a native B. subtilis SecE translocon protein (SEQ ID NO: 29), and a “secY” ORF (SEQ ID NO: 32) encodes a native B. subtilis SecY translocon protein (SEQ ID NO: 30).

[0081] As used herein, the phrase “non-native (artificial) secGEY operon” refers to one or more synthetic expression vectors / cassettes of the disclosure expressing extra copies of the B. subtilis secG, secE and secY genes (ORF) encoding native SecG, SecE and SecY (translocon) proteins. In certain one or more embodiments or aspects, recombinant Gram-positive cells comprising an introduced (i.e., non-native) secGEY operon may be described as recombinant (modified) cells “overexpressing” native SecG, SecE and SecY translocon proteins.

[0082] As used herein, phrases such as “non-native secGEY operon” and / or “recombinant cells overexpressing SecG, SecE and SecY translocon proteins” are not meant to limit the order of Sec translocon proteins which are encoded by the non-native secGEY operon. For example, one of skill in the art may readily construct non-native secGEY operons wherein the ORFs encoding the Sec translocon proteins are in any order, such as a non-native secGEY operon comprising an upstream secE ORF operably linked to a downstream secG ORF operably linked to a downstream secY ORF, or a non-native secGEY operon comprising an upstream secE ORF operably linked to a downstream secY ORF operably linked to a downstream secG ORF, or a non-native secGEY operon comprising an upstream secY ORF operably linked to a downstream secG ORF operably linked to a downstream secE ORF and the like.

[0083] Likewise, as presented in FIG.1, the ORFs in the second (e.g., FIG.1C and FIG.1G) and / or third positions (e.g., FIG. 1D and FIG. 1H) of the non-native secGEY operon may further include ribosomal binding sites immediately upstream (5ʹ) of the ORF encoding the Sec translocon protein. For instance, the twenty (20) nucleotides comprising the ribosomal binding site (RBS) immediately upstream of the secE ORF are underlined in FIG. 1C (SEQ ID NO: 3) and FIG. 1G (SEQ ID NO: 3), and the twenty (20) nucleotides comprising the ribosomal binding site (RBS) immediately upstream of the secY ORF are underlined in FIG.1D (SEQ ID NO: 4) and FIG.1H (SEQ ID NO: 4).

[0084] As used herein, the non-native secGEY operon named “PsecG-secGEY” (operon 1; SEQ ID NO: 7) comprises an upstream (5ʹ) PsecG promoter region sequence (FIG.1A; SEQ ID NO: 1) operably linked to a downstream secG ORF (FIG.1B; SEQ ID NO: 2) operably linked to a downstream secE ORF (FIG.1C; SEQ ID NO: 3) operably linked to a downstream secY ORF (FIG.1D; SEQ ID NO: 4) operably linked to a downstream (3ʹ) BPNʹ terminator sequence (SEQ ID NO: 5), the non-native secGEY operon named “PspoVG-secGEY” (operon 2; SEQ ID NO: 11) comprises an upstream PspoVG promoter region sequence (FIG.1E; SEQ ID NO: 10) operably linked to a downstream secG ORF (FIG.1F; SEQ ID NO: 2) operably linked to a downstream secE ORF (FIG. 1G; SEQ ID NO: 3) operably linked to a downstream secY ORF(FIG. 1H; SEQ ID NO: 4) operably linked to a downstream (3ʹ) BPNʹ terminator sequence (SEQ ID NO: 5), and the non-native secGEY operon named “Phbs-secGEY” (operon 3; SEQ ID NO: 13) comprises an upstream Phbs promoter region sequence (FIG.1I; SEQ ID NO: 12) operably linked to a downstream secG ORF (FIG. 1J; SEQ ID NO: 2) operably linked to a downstream secE ORF (FIG. 1K; SEQ ID NO: 3) operably linked to a downstream secY ORF (FIG. 1L; SEQ ID NO: 4) operably linked to a downstream (3ʹ) BPNʹ terminator sequence (SEQ ID NO: 5).

[0085] As used herein, exemplary proteases may be referred to as “reporter proteins”. In certain one or more embodiments of the disclosure, exemplary reporter proteins are expressed / produced by one or more recombinant (modified) cells of the disclosure. In certain embodiments, reporter proteins include, but are not limited to, native and variant Bacillus sp. subtilisins. In certain one or more embodiments, exemplary subtilisin reporters include, but are not limited to, the native B. clausii subtilisin and functional variants thereof, the native B. gibsonii subtilisin and functional variants thereof, the native B. lentus subtilisin and functional variants thereof, the native B. licheniformis subtilisin (AprL) and functional variants thereof, the native B. subtilis subtilisin (AprE) and functional variants thereof, the native B. amyloliquefaciens subtilisin (BPNʹ) and functional variants thereof, and the like. In certain aspects, exemplary B. clausii, B. gibsonii and / or B. lentus subtilisin reporters may be referred to as alkaline proteases. For instance, alkaline subtilisins generally have an isoelectric point (pI) of about 9.5, whereas the B. licheniformis, B. subtilis and B. amyloliquefaciens subtilisins have a pI of about 6.5.

[0086] As used herein, the term “subtilisin” refers to any member of the S8 serine protease family as described in MEROPS—The Peptidase Data base (Rawlings et al., 2006). The term subtilisin includes a wide variety of Bacillus subtilisins which have been identified and sequenced e.g., subtilisin 168, subtilisin BPNʹ, subtilisin Carlsberg, etc., and includes mutant (variant) proteases derived therefrom and the like.

[0087] As used herein, phrases such as “subtilisin-1”, “subtilisin-1 reporter”, “reporter-1”, “reporter-1 protease” and the like may be used interchangeably, and particularly refer to the native B. clausii subtilisin set forth in SEQ ID NO: 21, or a functional variant thereof.

[0088] As used herein, phrases such as “subtilisin-2”, “subtilisin-2 reporter”, “reporter-2”, “reporter-2 protease”, and the like may be used interchangeably, and particularly refer to the native B. amyloliquefaciens subtilisin set forth in SEQ ID NO: 22, or a functional variant thereof.

[0089] As used herein, phrases such as “subtilisin-3”, “subtilisin-3 reporter”, “reporter-3”, “reporter-3 protease” and the like may be used interchangeably, and particularly refer to the native B. gibsonii subtilisin set forth in SEQ ID NO: 23, or a functional variant thereof.

[0090] In certain embodiments, the disclosure is related to one or more variant subtilisins derived from a parent (native) subtilisin sequence, such as the native B. subtilis subtilisin (e.g., 168), the native B. amyloliquefaciens (e.g., BPNʹ), the native B. licheniformis subtilisin (e.g., Carlsberg), the native B. lentussubtilisin (e.g., 309), the B. alcalophilus subtilisin (e.g., PB92) and the like. For instance, in certain embodiments, the disclosure provides recombinant expression cassettes encoding functional subtilisin variants derived from a native B. clausii subtilisin (SEQ ID NO: 21), a native B. amyloliquefaciens subtilisin (SEQ ID NO: 22), a native B. gibsonii subtilisin (SEQ ID NO: 23) and the like. More particularly, one of skill may readily design, construct, screen, and identity functional subtilisin variants using routine methods known in the art. In particular, PCT Publication Nos. WO2010 / 056634, WO2011 / 130222, WO2015 / 089447, WO2016 / 202839, WO2017 / 207762 and WO2023 / 114936 (each incorporated herein by reference in its entirety) describe suitable methods and compositions for constructing functional subtilisin variants derived from a native B. clausii subtilisin (SEQ ID NO: 21), functional subtilisin variants derived from a native B. amyloliquefaciens subtilisin (SEQ ID NO: 22), functional subtilisin variants derived from a native B. gibsonii subtilisin (SEQ ID NO: 23) and the like.

[0091] As used herein, the B. subtilis strain named “CZ437” comprises two (2) introduced (integrated) expression cassettes encoding the reporter-1 protease, wherein the cassettes comprises in the 5ʹ to 3ʹ direction and in operable combination, an artificial “P2” promoter sequence (SEQ ID NO: 24) linked to a DNA sequence (ss) encoding an aprE signal peptide sequence (SEQ ID NO: 17) linked to a DNA sequence encoding a pro-region sequence (SEQ ID NO: 18) linked to a DNA sequence encoding reporter-1 (SEQ ID NO: 21). In certain embodiments, strain CZ437 may be referred to as a control (isogenic) cell, particularly when being compared with one or more modified strains overexpressing SecG, SecE and SecY translocon proteins (e.g., strains BPC0123, BPC0182, BPC0184).

[0092] As used herein, the B. subtilis strain named “BPC0178” comprises three (3) introduced (integrated) expression cassettes encoding the reporter-2 protease, wherein the cassettes comprises in the 5ʹ to 3ʹ direction and in operable combination, an artificial “P4” promoter sequence (SEQ ID NO: 26) linked to a DNA sequence (ss) encoding a BPNʹ signal peptide sequence (SEQ ID NO: 19) linked to a DNA sequence encoding a BPNʹ pro-region sequence (SEQ ID NO: 20) linked to a DNA sequence encoding reporter-2 (SEQ ID NO: 22). In certain embodiments, strain BPC0178 may be referred to as a control cell, particularly when being compared with one or more modified strains overexpressing SecG, SecE and SecY translocon proteins (e.g., strains BPC0166, BPC0167, BPC0168).

[0093] As used herein, the B. subtilis strain named “BPC0229” comprises two (2) introduced (integrated) expression cassettes encoding the reporter-3 protease, wherein the cassettes comprises in the 5ʹ to 3ʹ direction and in operable combination, an artificial “P2-0078” promoter sequence (SEQ ID NO: 25) linked to a DNA sequence (ss) encoding an aprE signal peptide sequence (SEQ ID NO: 17) linked to a DNA sequence encoding a pro-region sequence (SEQ ID NO: 18) linked to a DNA sequence encoding reporter- 3 (SEQ ID NO: 23). In certain embodiments, strain BPC0229 may be referred to as a control cell,particularly when being compared with one or more modified strains overexpressing SecG, SecE and SecY translocon proteins (e.g., strains AL394, AL395, AL396).

[0094] As used herein, a “host cell” refers to a cell that has the capacity to act as a host or expression vehicle for a newly introduced DNA sequence. Thus, in certain embodiments of the disclosure, the host cells are Gram-positive (e.g., Bacilli) and / or Gram-negative (e.g., E. coli) cells.

[0095] As used herein, a “modified cell” refers to a recombinant cell that comprises at least one genetic modification which is not present in the parental, reference, or control cell from which the modified cell is derived.

[0096] As used herein, when the expression and / or production of a protein of interest (POI) in a recombinant (modified) cell is being compared to the expression and / or production of the same POI in an unmodified (control) cell, it will be understood that the modified and unmodified cells are grown / cultivated / fermented under the same conditions (e.g., the same conditions such as media, temperature, pH and the like).

[0097] As used herein, an “increased amount”, when used in phrases such as a “recombinant cell ‘expresses / produces an increased amount’ of a protein of interest relative to the unmodified (control) cell”, particularly refers to an “increased amount” of a protein of interest (POI) expressed / produced in by the recombinant cell, which “increased amount” is always relative to the unmodified (control) cells expressing / producing the same POI, wherein the modified and unmodified cells are grown / cultured / fermented under the same conditions.

[0098] As used herein, “increasing” protein production or “increased” protein production is meant an increased amount of protein produced (e.g., a protein of interest). The protein may be produced inside the host cell, or secreted (or transported) into the culture medium. In certain embodiments, the protein of interest is produced (secreted) into the culture medium. Increased protein production may be detected for example, as higher maximal level of protein or enzymatic activity (e.g., such as protease activity), or total extracellular protein produced as compared to the parental host cell.

[0099] As used herein, the terms “modification” and “genetic modification” are used interchangeably and include: (a) the introduction, substitution, or removal of one or more nucleotides in a gene (or an ORF thereof), 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 ORF thereof, (b) a gene disruption, (c) a gene conversion, (d) a gene deletion, (e) the down-regulation of a gene, (f) specific mutagenesis and / or (g) random mutagenesis of any one or more the genes disclosed herein.

[0100] In certain aspects, genetic modifications particularly refer to the introduction, substitution, or removal of one or more nucleotides in a nucleic acid (DNA) sequence encoding pro-region (amino acid)sequence of the disclosure. For example, in certain embodiments, a DNA sequence encoding a native pro- region amino acid sequence set forth in SEQ ID NO: 18 is genetically modified as described herein.

[0101] As used herein, the term “introducing”, as used in phrases such as “introducing into a Gram-positive bacterial cell a ‘gene’, a ‘polynucleotide’, an ‘open reading frame’ (ORF), a ‘gene coding sequence, a ‘vector’, an ‘expression cassette’”, and the like, includes methods known in the art for introducing polynucleotides (DNA) into a cell, including, but not limited to protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation and the like.

[0102] As used herein, “transformed” or “transformation” mean a cell has been transformed by use of recombinant DNA techniques. Transformation typically occurs by insertion of one or more nucleotide sequences (e.g., a polynucleotide, an ORF or gene) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence that is not naturally occurring in cell that is to be transformed). Transformation therefore generally refers to introducing an exogenous DNA into a host cell so that the DNA is maintained as a chromosomal integrant or a self-replicating extra-chromosomal vector.

[0103] As used herein, “transforming DNA”, “transforming sequence”, and “DNA construct” refer to DNA that is used to introduce sequences into a host cell or organism. Transforming DNA is DNA used to introduce sequences into a host cell or organism. The DNA may be generated in vitro by PCR or any other suitable techniques. In some embodiments, the transforming DNA comprises an incoming sequence, while in other embodiments it further comprises an incoming sequence flanked by homology boxes. In yet a further embodiment, the transforming DNA comprises other non-homologous sequences, added to the ends (i.e., stuffer sequences or flanks). The ends can be closed such that the transforming DNA forms a closed circle, such as, for example, insertion into a vector.

[0104] As used herein, “disruption of a gene” or a “gene disruption”, are used interchangeably and refer broadly to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., a protein). Thus, as used herein, a gene disruption includes, but is not limited to, frameshift mutations, premature stop codons (i.e., such that a functional protein is not made), substitutions eliminating or reducing activity of the protein, internal deletions (such that a functional protein is not made), insertions disrupting the coding sequence, mutations removing the operable link between a native promoter required for transcription and the open reading frame, and the like.

[0105] As used herein “an incoming sequence” refers to a DNA sequence that is introduced into the bacterial cell chromosome. In some embodiments, the incoming sequence is part of a DNA construct. In other embodiments, the incoming sequence encodes one or more proteins of interest. In some embodiments, the incoming sequence comprises a sequence that may or may not already be present in the genome of the cell to be transformed (i.e., it may be either a homologous or heterologous sequence). In some embodiments, the incoming sequence encodes one or more proteins of interest, a gene, and / or amutated or modified gene. In alternative embodiments, the incoming sequence encodes a functional wild- type gene or operon, a functional mutant gene or operon, or a nonfunctional gene or operon. In some embodiments, the non-functional sequence may be inserted into a gene to disrupt function of the gene. In another embodiment, the incoming sequence includes a selective marker. In a further embodiment the incoming sequence includes two homology boxes.

[0106] As used herein, “homology box” refers to a nucleic acid sequence, which is homologous to a sequence in the bacterial cell chromosome. More specifically, a homology box is an upstream or downstream region having between about 80 and 100% sequence identity, between about 90 and 100% sequence identity, or between about 95 and 100% sequence identity with the immediate flanking coding region of a gene or part of a gene to be deleted, disrupted, inactivated, down-regulated and the like, according to the invention. These sequences direct where in the bacterial cell chromosome a DNA construct is integrated and directs what part of the chromosome is replaced by the incoming sequence. While not meant to limit the present disclosure, a homology box may include about between 1 base pair (bp) to 200 kilobases (kb). Preferably, a homology box includes about between 1 bp and 10.0 kb; between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb, and between 0.25 kb and 2.5 kb. A homology box may also include 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 a selective marker are flanked by a homology box wherein the homology box comprises nucleic acid sequences immediately flanking the coding region of the gene.

[0107] As used herein, a host cell “genome”, a bacterial (host) cell “genome”, or a Bacillus sp. (host) cell “genome” includes chromosomal and extrachromosomal genes.

[0108] As used herein, the terms “plasmid”, “vector” and “cassette” refer to extrachromosomal elements, often carrying genes which are typically not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- stranded or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0109] As used herein, the term “plasmid” refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes. In some embodiments, plasmids become incorporated into the genome of the host cell. in some embodiments plasmids exist in a parental cell and are lost in the daughter cell.

[0110] A used herein, a “transformation cassette” refers to a specific vector comprising a gene (or ORF thereof), and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.

[0111] As used herein, the term “vector” refers to any nucleic acid that can be replicated (propagated) in cells and can carry new genes or DNA segments into cells. Thus, the term refers to a nucleic acid construct designed for transfer between different host cells. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), PLACs (plant artificial chromosomes), and the like, that are “episomes” (i.e., replicate autonomously or can integrate into a chromosome of a host organism).

[0112] An “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA in a cell. Many prokaryotic and eukaryotic expression vectors are commercially available and know to one skilled in the art. Selection of appropriate expression vectors is within the knowledge of one skilled in the art.

[0113] As used herein, the terms “expression cassette” and “expression vector” refer to a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell (i.e., these are vectors or vector elements, as described above). The recombinant expression cassette 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, DNA constructs also include a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. In certain embodiments, a DNA construct of the disclosure comprises a selective marker and an inactivating chromosomal or gene or DNA segment as defined herein.

[0114] As used herein, a “targeting vector” is a vector that includes polynucleotide sequences that are homologous to a region in the chromosome of a host cell into which the targeting vector is transformed and that can drive homologous recombination at that region. For example, targeting vectors find use in introducing mutations into the chromosome of a host cell through homologous recombination. In some embodiments, the targeting vector comprises other non-homologous sequences, e.g., added to the ends (i.e., stuffer sequences or flanking sequences). The ends can be closed such that the targeting vector forms a closed circle, such as, for example, insertion into a vector. For example, in certain embodiments, a parental B. licheniformis (host) cell is modified (e.g., transformed) by introducing therein one or more “targeting vectors”.

[0115] As used herein, the term “protein of interest” or “POI” refers to a polypeptide of interest that is desired to be expressed in a modified (recombinant) Gram-positive host cell, wherein the POI is preferably expressed at increased levels (i.e., relative to the “unmodified” (parental, control, isogenic cell). Thus, as used herein, a POI may be an enzyme, a substrate-binding protein, a surface-active protein, a structural protein, a receptor protein, and the like. In certain embodiments, a modified cell of the disclosure produces an increased amount of a heterologous protein of interest relative to the control cell. In particular embodiments, an increased amount of a protein of interest produced by a modified cell of the disclosure is at least a 0.5% increase, at least a 1.0% increase, at least a 5.0% increase, or a greater than 5.0% increase, relative to the control cell.

[0116] Similarly, as defined herein, a “gene of interest” or “GOI” refers a nucleic acid sequence (e.g., a polynucleotide, a gene or an ORF) which encodes a POI. A “gene of interest” encoding a “protein of interest” may be a naturally occurring gene, a mutated gene or a synthetic gene.

[0117] 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. The conventional one (1) letter or three (3) letter codes for amino acid residues are used herein. The polypeptide may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term polypeptide also encompasses an amino acid polymer that has been 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 the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0118] As used herein, a “variant” polypeptide refers to a polypeptide that is derived from a parent (or reference) polypeptide by the substitution, addition, or deletion of one or more amino acids, typically by recombinant DNA techniques. Variant polypeptides may differ from a parent polypeptide by a small number of amino acid residues and may be defined by their level of primary amino acid sequence homology / identity with a parent (reference) polypeptide. Preferably, variant polypeptides have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity with a parent (reference) polypeptide sequence.

[0119] As used herein, a “variant” polynucleotide refers to a polynucleotide having a specified degree of sequence homology / identity with a parent polynucleotide, or hybridizes with a parent polynucleotide (or a complement thereof) under stringent hybridization conditions. Preferably, a variant polynucleotide 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%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% nucleotide sequence identity with a parent (reference) polynucleotide sequence.

[0120] As used herein, a “mutation” refers to any change or alteration in a nucleic acid sequence. Several types of mutations exist, including point mutations, deletion mutations, silent mutations, frame shift mutations, splicing mutations and the like. Mutations may be performed specifically (e.g., via site directed mutagenesis) or randomly (e.g., via chemical agents, passage through repair minus bacterial strains).

[0121] As used herein, in the context of a polypeptide or a sequence thereof, the term “substitution” means the replacement (i.e., substitution) of one amino acid with another amino acid.

[0122] As used herein, the term “homology” relates to homologous polynucleotides or polypeptides. If 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%, still more preferably at least 90%, more preferably at least 95%, and most preferably at least 98%. Whether two polynucleotide or polypeptide sequences have a sufficiently high degree of identity to be homologous as defined herein, can suitably be investigated 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). Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0123] As used herein, the term “percent (%) identity” refers to the level of nucleic acid or amino acid sequence identity between the nucleic acid sequences that encode a polypeptide or the polypeptide's amino acid sequences, when aligned using a sequence alignment program.

[0124] As used herein, “specific productivity” is total amount of protein produced per cell per time over a given time period.

[0125] As used herein, the terms “purified”, “isolated” or “enriched” are meant that a biomolecule (e.g., a polypeptide or polynucleotide) is altered from its natural state by virtue of separating it from some, or all of, the naturally occurring constituents with which it is associated in nature. Such isolation or purification may be accomplished by art-recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulphate precipitation or other protein salt precipitation, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis or separation on a gradient to remove whole cells, cell debris, impurities, extraneous proteins, or enzymes undesired in the final composition. It is further possible to then add constituents to a purified or isolated biomolecule composition which provide additional benefits, forexample, activating agents, anti-inhibition agents, desirable ions, compounds to control pH or other enzymes or chemicals. II. OVEREXPRESSION OF A SYNTHETIC SecGEY OPERON IN GRAM-POSITIVE BACTERIAL CELLS ENHANCES PROTEIN PRODUCTION

[0126] As briefly set forth above, the production of many heterologous proteins remains challenging and unpredictable with respect to protein titers, yields and the like. In particular, bottlenecks for secreted protein production can vary dependent on host strain and protein sequence, among other things. In the context of Gram-positive bacterial hosts, the initial steps for protein secretion involve the recognition of the signal peptide and targeting of the pre-protein to the translocation machinery. For instance, as generally reviewed in Pohl and Harwood (2010), Bacillus secretion pathways include the Sec-dependent (Sec) and twin- arginine translocation (Tat) pathways, wherein the Sec pathway is responsible for secretion of the majority of proteins. As described by Pohl and Harwood (2010), pre-proteins are targeted to the membrane- embedded Sec translocon, a heterotrimeric protein complex which forms the main channel named SecYEG that facilitates translocation of secreted proteins across the cytoplasmic membrane, wherein the secretion of proteins via the Sec pathway is a complex system generally involving substrate recognition, intracellular chaperoning and piloting to the Sec translocase, post-translocation folding events and the like.

[0127] In certain aspects, Freudl (2018) has generally described the secretion of recombinant proteins into the culture supernatant of bacterial (host) expression systems, particularly focusing on protein export systems requiring the fusion of a Sec-specific signal peptide, or a Tat-specific signal peptide to the amino- terminal (N) end of the desired target protein. For instance, as concluded in this publication, the most promising way to find the optimal signal peptide for a desired protein is to screen the largest possible diversity of signal peptides, either generated by signal peptide variation using large signal peptide libraries or, alternatively, by optimization of a given signal peptide using site-directed or random mutagenesis strategies. PCT Publication No. WO1999 / 04006 describes an expression vector encoding a gram-positive microorganism SecG protein, and related methods for secreting a protein in the gram-positive host comprising the introduced vector encoding the SecG protein. PCT Publication No. WO2008 / 141281 describes a modified secretion system for Gram-positive microbial hosts comprising an introduced vector encoding a heterologous truncated SecG protein capable of facilitating secretion of a desired protein, wherein the introduced vector can replace the endogenous (secG) gene encoding the native SecG protein or complement the endogenous (secG) gene encoding the native SecG protein. US Patent Publication No. US2009 / 0029417 describes a recombinant microorganism producing a cellulase and overexpressing a B. subtilis secY gene. PCT Publication No. WO2008 / 126929 describes a recombinant microorganism producing a cellulase and overexpressing a B. subtilis secY gene, wherein the microorganism further requires a deletion of one or more sporulation-associated genes.

[0128] In other aspects, Mulder et al. (2013) describe a recombinant Gram-positive bacterial host system for secretion of a B. amyloliquefaciens α-amylase using the IPTG-inducible (Pgrac) promoter for expression of the α-amylase (amyQ) gene. For instance, as described in this publication, the recombinant host cell includes an amyQ gene cassette under control of the Pgrac promoter, and a secYEG gene cassette under the control of a xylose-inducible (Pxyl) promoter (for expression of SecG, SecE and SecY proteins), wherein the synthesis of SecYEG and AmyQ was induced by 0.5% xylose and 100 μg IPTG, respectively. Chen et al. (2015) describe a study aimed at identifying bottlenecks of the Sec pathway and improving secretion of heterologous proteins by so called molecular genetic techniques. For instance, Chen et al. (2015) describes a recombinant cell expressing two α-amylases (AmyL and AmyS) both under the control of the strong constitutive PHpaIIpromoter followed by their native signal peptides (SPamyLand SPamyS), wherein twenty- three (23) main genes or gene operons involved in or closely related to the Sec pathway were overexpressed, concluding that deficiency of PrsA lipoprotein and chaperones of the DnaK series were main rate-limiting factors for heterologous proteins secretion.

[0129] More recently, studies from different laboratories have described the essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. In certain aspects, Neef et al. (2020) compared the contributions of non-essential Sec pathway components and cell envelope- associated proteases on the secretion efficiency of three proteins expressed at high level (i.e., α-amylases AmyE from B. subtilis and AmyL from B. licheniformis, and serine protease BPNʹ from B. amyloliquefaciens). For instance, as described in Neef et al. (2020), isogenic B. subtilis strains lacking the genes for chaperone DnaK, translocase subunits SecDF, or SecG, or signal peptidases SipS, SipT, SipU, SipV or SipW, the genes for the cell envelope-associated proteases SppA, TepA, PrsW, WprA, YqeZ, HtrA or HtrB were constructed. As summarized in this publication, results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPNʹ secretion, but the actual effect size depends on the investigated protein. Additionally, as concluded in Neef et al. (2020), the chaperone DnaK is important for BPNʹ secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion, and the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. Thus, recombinant protein production in B. subtilis cells remains challenging due to bottlenecks in the general Sec pathway, as well as this bacterium’s intrinsic capability to secrete a cocktail of highly potent proteases (Neef et al., 2021). In particular, Neef et al. (2021) consider and summarize the pros and cons of the Gram-positive bacterial cell factories B. subtilis and L. lactis, generally concluding that B. subtilis and closely related bacilli are most suitable for the bulk production of recombinant proteins, noting however that B. subtilis is highly proteolytic which represents a serious drawback as this may lead to a loss of product and / or the accumulation of cleaved product derivatives, which may be overcome by the deletion of protease genes, wherein the resulting strains are oftentimes moresensitive to autolysis, leading to increased amounts of contaminating cytoplasmic proteins in the fermentation broth.

[0130] Based on the foregoing, certain embodiments of the disclosure are related to surprising and unexpected results set forth and exemplified hereinafter. More particularly, as described herein, Applicant has contemplated, designed, and constructed recombinant Gram-positive (bacterial) cells / strains overexpressing SecG, SecE and SecY translocon proteins from an introduced non-native (synthetic) secGEY operon and expressing one or more introduced cassettes encoding subtilisin reporter proteins. In particular, as shown in Examples 1-6 of the instant disclosure, Applicant has surprisingly observed that overexpression of the full complement of SecG, SecE and SecY translocon proteins results in the enhanced production of certain reporter proteins. As further described in Examples 1, 3 and 5, control and modified B. subtilis cells / strains were constructed and are summarized below in TABLE 1. TABLE 1 STRAIN NAMES AND GENETIC MODIFICATIONS N m Str in M difi ti n

[0131] As presented and described in Example 2, the expression of the reporter-1 protein in the presence of SecGEY overexpression was compared to the expression of the same reporter-1 protein without SecGEY overexpression. More particularly, three (3) SecGEY overexpression strains (BPC0123, BPC0182, BPC0184) and a control strain CZ437 were constructed and assessed for reporter-1 production. For example, in a first experiment the CZ437 control strain and SecGEY overexpression strain BPC0123 were cultured and sampled for protease activity after twenty-hour (24) hours and forty (40) hours of growth, as shown below in TABLES 2 and 3 (Example 2). As presented in TABLE 3, it was surprisingly observedthat the production of the reporter-1 protein was significantly increased (i.e., about 60%) when SecGEY proteins are overexpressed from the secG promoter in strain BPC0123, as compared to the CZ437 control strain after 40 hours of cultivation. Similarly, in a second experiment (TABLES 4 and 5), control strain CZ437 and SecGEY overexpression strains BPC0123, BPC0182, and BPC0184 were cultured and sampled for protease activity after 22 hours of growth (Example 2). In particular, as shown in TABLE 5, it was surprisingly observed that the production of the reporter-1 protein was significantly increased for all three strains overexpressing SecGEY (i.e., about 30-40%) when considering protease production per OD600, as compared to the CZ437 control strain. Likewise, as indicated from the data presented in Example 2, suitable promoter region sequences for expressing the non-native secGEY operon generally include promoter region sequences functional in the host Gram-positive bacterial cell (e.g., a secG promoter sequence, a spoVG promoter sequence, a hbs promoter sequence, and the like).

[0132] As presented and described in Examples 3 and 4, Applicant further constructed Bacillus cells overexpressing SecG, SecE, and SecY in a host background expressing a heterologous reporter-2 protein. More particularly, three (3) SecGEY overexpression strains (BPC0166, BPC0167 and BPC0168) and a control strain BPC0178 were constructed and assessed for reporter-2 production. For example, as presented in TABLES 6 and 7 (Example 4), the BPC0178 control strain and SecGEY overexpression strains (BPC0166, BPC0167 and BPC0168) were cultured and sampled for reporter-2 activity after 20 and 40 hours of growth, wherein production of the reporter-2 protein was equivalent or lower for all three strains overexpressing SecGEY.

[0133] As presented and described in Examples 5 and 6, Applicant further constructed Bacillus cells overexpressing SecG, SecE, and SecY in a host background expressing a heterologous reporter-3 protein. More particularly, three (3) SecGEY overexpression strains (AL394, AL395 and AL396) and a control strain BPC0229 were constructed and assessed for reporter-3 production. For example, as presented in TABLES 8 and 9 (Example 6), the BPC0229 control strain and SecGEY overexpression strains (AL394, AL395 and AL396) were cultured and sampled for reporter-3 activity after 16, 24 and 40 hours of growth. More particularly, as shown in TABLE 9 (Example 6), production of the reporter-3 protein was significantly increased (i.e., about 10-25%) for all three strains overexpressing SecGEY when considering protease production per OD600.

[0134] Thus, as generally summarized above, and set forth below in the Examples, recombinant Gram- positive bacterial cells (e.g., Bacillus sp. cells) overexpressing SecG, SecE and SecY proteins from the introduced non-native secGEY operon are capable expressing / producing certain reporter proteases (e.g., reporter-1 and reporter-3) at significantly increased levels as compared to other reporter proteases (e.g., reporter-2). For example, without wishing to be bound by theory, mechanism or mode of operation, Applicant has surprisingly observed herein that recombinant Gram-positive bacterial cells overexpressingthe SecG, SecE and SecY translocon proteins (i.e., comprising an introduced secGEY operon) are capable of producing increased amounts of alkaline subtilisins (e.g., B. clausii subtilisin SEQ ID NO: 21 and B. gibsonii subtilisin SEQ ID NO: 23) as compared to control cells expressing the same alkaline subtilisin, wherein the control cells do not comprise the introduced secGEY operon. In contrast, recombinant cells overexpressing the SecG, SecE and SecY translocon proteins (i.e., comprising an introduced secGEY operon) produced equivalent amounts of the B. amyloliquefaciens subtilisin (BPNʹ; SEQ ID NO: 22) as compared to control cells expressing the same BPNʹ subtilisin, wherein the control cells do not comprise the introduced secGEY operon.

[0135] Based on the foregoing, certain embodiments are related to recombinant Gram-positive bacterial cells expressing a non-native secGEY operon and an alkaline subtilisin protease. Certain embodiments are therefore related to recombinant Gram-positive bacterial cells comprising an introduced (synthetic) secGEY operon, wherein the recombinant cells express / produce / secrete an alkaline subtilisin protease comprising at least about 80% to 100% sequence identity to the native subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0136] In certain embodiments, native subtilisins (and functional variants thereof) comprising at least about 80% identity to SEQ ID NO: 21 or SEQ ID NO: 23 are referred to as alkaline subtilisins of subgroup I-S2, in contrast the so called “true” subtilisins (subgroup I-S1). For instance, as generally described in Siezen and Leunissen (1997), a sub-group of the serine proteases designated “subtilases” has been proposed, wherein the subtilases were defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. In particular, the Siezen and Leunissen publication presents an overview of the subtilase family of serine proteases (Table 1, Gram- positive bacteria), which includes mainly enzymes from Bacillus, with subgroups of true subtilisins (>64% identity), high-alkaline proteases (>55% identity), and intracellular proteases (>37% identity), wherein numerous minor variants of true subtilisins and high-alkaline proteases are shown (Table 2). More particularly, one subgroup of the subtilases named “I-S1” (or “true” subtilisins) comprise the classical subtilisins, such as the B. subtilis 168 subtilisin (aprA), the B. amyloliquefaciens subtilisin (BPNʹ), the B. licheniformis subtilisin (Carlsberg), and the like. A second subgroup of the subtilases, named “I-S2” (or alkaline subtilisins) comprise subtilisins such as the B. alcalophilus PB92 alkaline subtilisin (PB92), the B. lentus 309 alkaline subtilisin (309; Savinase™), the B. lentus 147 alkaline subtilisin (147; Esperase™), and the like. For example, as shown in FIG.2, the native B. clausii alkaline subtilisin (mature) protein sequence (FIG. 2A, SEQ ID NO: 21) comprises 269 amino acid residues and with an isoelectric point (pI) of about 9.30 and the native B. gibsonii alkaline subtilisin (mature) protein sequence (FIG. 2C, SEQ ID NO: 23) comprises 269 amino acid residues with a pI of about 9.57, whereas the native B. amyloliquefaciens “true” subtilisin mature protein sequence (FIG. 2B, SEQ ID NO: 22) comprises 275 amino acid residues with apI of about 6.30. More particularly, the isoelectric points described are the theoretical values computed using the Expasy (Swiss Bioinformatics Resource Portal) “Compute pI / Mw tool”, with average resolution.

[0137] In certain embodiments, one or more suitable pro-region sequences of the disclosure are derived from native (wild-type, reference) subtilisin proteases. For instance, in certain embodiments, suitable pro- region sequences are derived from a native pro-region amino acid sequence located upstream (5ʹ) of the mature subtilisin ORF. In other embodiments, a particularly suitable pro-region sequence is the native B. lentus subtilisin pro-region sequence of SEQ ID NO: 18 (and functional variants thereof). For example, PCT Publication No WO2008 / 112258 generally describes recombinant Gram-positive bacterial cells and methods thereof for producing serine proteases, such as modified (variant) pro-region sequences derived from the full-length native B. clausii alkaline serine (Maxacal) protease. Likewise, PCT Publication No. WO2010 / 123754 describes compositions and methods for producing serine proteases using one or more modified pro-region sequences derived from full-length native B. clausii serine protease or full-length native B. lentus serine (GG36) protease. PCT Publication No. WO2011 / 014278 further describes modified (variant) pro-region sequences suitable for producing serine proteases, such as the native (or variant) B. amyloliquefaciens serine (BPNʹ) protease.

[0138] Thus, in certain one or more embodiments, the disclosure provides recombinant Gram-positive bacterial cells comprising an introduced secGEY operon and one or more introduced expression cassettes encoding subtilisin reporter proteins. For instance, as briefly summarized above, and further described below in the Examples, artificial secGEY operon and / or expression cassettes encoding subtilisin reporter proteins are generally constructed by operably linking one or more nucleic acid (DNA) sequence elements, including, but not limited to, promoter region sequences, 5ʹ-UTR sequences, 3ʹ-UTR sequences, ribosomal binding site (RBS) / Shine-Dalgarno (SD) sequences, signal peptide (secretion) sequences, pro-region sequences, open reading frame (ORF) sequences, terminator sequences, and the like.

[0139] In certain one or more embodiments, expression cassettes encoding exemplary reporter proteins are constructed and introduced into cells of the disclosure. In certain one or more embodiments, exemplary reporter proteins of the disclosure are proteases, including, but not limited to, a native B. clausii subtilisin (and functional variants thereof), a native B. gibsonii subtilisin (and functional variants thereof), a native B. lentus subtilisin (and functional variants thereof), and the like. In particular, DNA sequences encoding native subtilisin proteases (and functional variants therefore) are generally available and suitable for use according to one or more embodiments of the discourse.

[0140] In certain embodiments, a modified cell produces an increased amount of a subtilisin (protease) relative to a control (or parental) cell, wherein the increased amount is at least about a 0.01% increase, at least about a 0.10% increase, at least about a 0.50% increase, at least about a 1.0% increase, at least about a 2.0% increase, at least about a 3.0% increase, at least about a 4.0% increase, at least about a 5.0% increase,or an increase greater than 5.0%. In certain aspects, an increased amount of a subtilisin is determined by assaying enzymatic activity, assaying protein function, assaying / quantifying specific productivity (Qp) and the like. For example, one skilled in the art may utilize routine methods and techniques known in the art for detecting, assaying, measuring, etc. protein expression, production, secretion and the like. III. RECOMBINANT POLYNUCLEOTIDES AND MOLECULAR BIOLOGY

[0141] Certain one or more embodiments of the disclosure provide, inter alia, recombinant Gram-positive bacterial cells expressing subtilisin proteases and comprising an introduced (non-native) secGEY operon overexpressing SecG, SecE and SecY proteins, methods for producing subtilisin proteases in Gram-positive bacterial cells comprising introduced (non-native) secGEY operons, nucleic acid (DNA) sequences encoding native SecG, SecE and SecY proteins (e.g., a secG ORF encoding a native or functional variant SecG protein, a secE ORF encoding a native or functional variant SecE protein, a secY ORF encoding a native or functional variant SecY protein), and the like.

[0142] As generally described herein and presented below in the Examples, recombinant polynucleotides (vectors, expression cassettes, etc.), recombinant (modified) Bacillus strains and the like are readily constructed using routine molecular biology and microbiology techniques and methods know to one skilled in the art. Therefore, the instant disclosure generally relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in present disclosure include Sambrook et al., (2nd Edition, 1989); Kriegler (1990) and Ausubel et al., (1994). Likewise, those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli, Bacilli, etc.).

[0143] Thus, in certain embodiments, a polynucleotide (genes, vectors, plasmids, DNA elements, etc.) of the disclosure may be genetically modified, wherein genetic modifications include, but are not limited to, (a) the introduction, substitution, or removal of one or more nucleotides in a gene (or an ORF thereof), 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 ORF thereof, (b) a gene disruption, (c) a gene conversion, (d) a gene deletion, (e) the down-regulation of a gene (e.g., interfering RNA), (f) specific mutagenesis and / or (g) random mutagenesis of any one or more the genes disclosed herein. In certain embodiments, a modified Bacillus cell of the disclosure is constructed by increasing the expression of a gene and / or by reducing (or eliminating) the expression of a gene, using methods well known in the art, for example, insertions, disruptions, replacements, or deletions. The portion of the gene to be modified or inactivated may be, for example, the coding region (CDS, ORF) or a regulatory (DNA) element required for expression of the coding region. An example of such a regulatory or control sequences may be a promoter sequence or a functional part thereof, (i.e., a part which is sufficient for affecting expression of the nucleic acid sequence). Other control sequences for modification include, but are not limited to, a leader sequence, a pro-peptidesequence, a signal sequence, a transcription terminator sequence, a transcriptional activator sequence and the like.

[0144] Gene deletion techniques enable the partial or complete removal of gene(s), thereby eliminating their expression, or expressing a non-functional (or reduced activity) protein product. In such methods, the deletion of the gene(s) may be accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5′ and 3′ regions flanking the gene. The contiguous 5′ and 3′ regions may be introduced into a Bacillus cell, for example, on a temperature-sensitive plasmid, such as pE194, in association with a second selectable marker at a permissive temperature to allow the plasmid to become established in the cell. The cell is then shifted to a non-permissive temperature to select for cells that have the plasmid integrated into the chromosome at one of the homologous flanking regions. Selection for integration of the plasmid is affected by selection for the second selectable marker. After integration, a recombination event at the second homologous flanking region is stimulated by shifting the cells to the permissive temperature for several generations without selection. The cells are plated to obtain single colonies and the colonies are examined for loss of both selectable markers. Thus, a person of skill in the art may readily identify nucleotide regions in the gene's coding sequence and / or the gene's non-coding sequence suitable for complete or partial deletion. In other embodiments, a modified Bacillus cell of the disclosure is constructed by introducing, substituting, or removing one or more nucleotides in the gene or a regulatory element required for the transcription or translation thereof.

[0145] In certain embodiments, a modified Bacillus cell is constructed via CRISPR-Cas9 editing. For example, a wild-type gene encoding a native protein of interest (or functional variant protein of interest thereof) may be modified via CRISPR-Cas9 editing, by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g., Cas9) and Cpfl or a guide DNA (e.g., NgAgo), which recruits the endonuclease to the target sequence on the DNA, wherein the endonuclease can generate a single or double stranded break in the DNA. This targeted DNA break becomes a substrate for DNA repair, and can recombine with a provided editing template (e.g., an editing template to replace the native gene promoter sequence with a heterologous promoter). For example, the gene encoding the nucleic acid guided endonuclease (for this purpose Cas9 from S. pyogenes) or a codon optimized gene encoding the Cas9 nuclease is operably linked to a promoter active in the Bacillus cell and a terminator active in Bacillus cell, thereby creating a Bacillus Cas9 expression cassette. Likewise, one or more target sites unique to the gene of interest are readily identified by a person skilled in the art. For example, to build a DNA construct encoding a gRNA-directed to a target site within the gene of interest using Streptococcus pyogenes Cas9, the variable targeting domain (VT) will comprise nucleotides of the target site which are 5' of the (PAM) proto-spacer adjacent motif (NGG), which nucleotides are fused to DNA encoding the Cas9 endonuclease recognition domain for S. pyogenes Cas9 (CER). The combination of the DNA encoding a VT domain andthe DNA encoding the CER domain thereby generate a DNA encoding a gRNA. Thus, a Bacillus expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in Bacillus cells and a terminator active in Bacillus cells.

[0146] In certain embodiments, the DNA break induced by the endonuclease is repaired / replaced with an incoming sequence. For example, to precisely repair the DNA break generated by the Cas9 expression cassette and the gRNA expression cassette described above, a nucleotide editing template is provided, such that the DNA repair machinery of the cell can utilize the editing template. For example, about 500-bp 5' of targeted gene can be fused to about 500-bp 3' of the targeted gene to generate an editing template, which template is used by the Bacillus host's machinery to repair the DNA break generated by the RGEN. The Cas9 expression cassette, the gRNA expression cassette and the editing template can be co-delivered to the cells using many different methods. The transformed cells are screened by PCR amplifying the target gene locus, by amplifying the locus with a forward and reverse primer. These primers can amplify the wild-type locus or the modified locus that has been edited by the RGEN. These fragments are then sequenced using a sequencing primer to identify edited colonies.

[0147] In other embodiments, a modified Bacillus cell is constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis and transposition. Modification of the gene may be performed by subjecting the parental cell to mutagenesis and screening for mutant cells in which expression of the gene has been altered. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods. Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'- nitrosoguanidine (NTG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the parental cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutant cells exhibiting reduced or no expression of the gene.

[0148] International PCT Publication No. WO2003 / 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. PCT Publication No. WO2002 / 14490 discloses methods for modifying Bacillus cells including (1) the construction and transformation of an integrative plasmid (pComK), (2) random mutagenesis of coding sequences, signal sequences and pro-peptide sequences, (3) homologous recombination, (4) increasing transformation efficiency by adding non-homologous flanks to the transformation DNA, (5) optimizingdouble cross-over integrations, (6) site directed mutagenesis and (7) marker-less deletion. Those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli and Bacillus). Indeed, such methods as transformation including protoplast transformation and congression, transduction, and protoplast fusion are known and suited for use in the present disclosure. Methods of transformation are particularly preferred to introduce a DNA construct of the present disclosure into a host cell.

[0149] In addition to commonly used methods, in some embodiments, host cells are directly transformed (i.e., an intermediate cell is not used to amplify, or otherwise process, the DNA construct prior to introduction into the host cell). Introduction of the DNA construct into the host cell includes those physical and chemical methods known in the art to introduce 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, DNA constructs are co-transformed with a plasmid without being inserted into the plasmid. In further embodiments, a selective marker is deleted or substantially excised from the modified Bacillus strain by methods known in the art. In some embodiments, resolution of the vector from a host chromosome leaves the flanking regions in the chromosome, while removing the indigenous chromosomal region.

[0150] Promoters and promoter sequence regions for use in the expression of genes, open reading frames (ORFs) thereof and / or variant sequences thereof in Bacillus cells are generally known on one of skill in the art. Promoter sequences of the disclosure are generally chosen so that they are functional in the Bacillus cells, and include, but are not limited to, naturally occurring promoter sequences, synthetic promoter sequences, and / or promoter sequence combinations thereof and the like, which promoter (sequences) are operable / functional in Bacillus cells. Examples of synthetic (engineered) promoters capable of producing heterologous (foreign) proteins in Bacillus cells include, but are not limited to, the promoter systems described by Zhou et al. (2019), Wang et al. (2019) and Castillo-Hair et al. (2019). Certain other exemplary Bacillus promoter sequences include, but are not limited to, the B. subtilis alkaline protease (aprE) promoter, the α-amylase promoter of B. subtilis, the α-amylase promoter of B. amyloliquefaciens, the neutral protease (nprE) promoter from B. subtilis, a mutant aprE promoter (e.g., PCT Publication No. WO2001 / 51643), a B licheniformis tuf promoter, a B licheniformis citZ promoter, or any other functional promoter from Bacillus sp. cells. Methods for screening and creating promoter libraries with a range of activities (promoter strength) in Bacillus cells is describe in PCT Publication No. WO2003 / 089604. IV. FERMENTING GRAM-POSITIVE CELLS FOR PRODUCTION OF PROTEINS OF INTEREST

[0151] In certain embodiments, the disclosure provides recombinant microbial cells producing subtilisin proteases. More particularly, certain embodiments are related genetically modified (recombinant) Gram-positive bacterial cells expressing heterologous polynucleotides encoding subtilisin proteases. Thus, particular embodiments are related to growing, cultivating, fermenting and the like, microbial cells for the production of proteins. In general, fermentation methods well known in the art are used to ferment the microbial cells.

[0152] In some embodiments, the cells are grown under batch or continuous fermentation conditions. A classical batch fermentation is a closed system, where the composition of the medium is set at the beginning of the fermentation and is not altered during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism(s). In this method, fermentation is permitted to occur without the addition of any components to the system. Typically, a batch fermentation qualifies as a “batch” with respect to the addition of the carbon source, and attempts are often made to control factors such as pH and oxygen concentration. The metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped. Within batch cultures, cells progress through a static lag phase to a high growth log phase and finally to a stationary phase, where growth rate is diminished or halted. If untreated, cells in the stationary phase eventually die. In general, cells in log phase are responsible for the bulk of production of product.

[0153] A suitable variation on the standard batch system is the “fed-batch fermentation” system. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression likely inhibits the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors, such as pH, dissolved oxygen and the partial pressure of waste gases, such as CO2. Batch and fed-batch fermentations are common and well known in the art.

[0154] Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density, where cells are primarily in log phase growth. Continuous fermentation allows for the modulation 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 source or nitrogen source, is maintained at a fixed rate and all other parameters are allowed to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[0155] Culturing / fermenting is generally accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, a carbon and energy source material, molecular oxygen, and, of course, a starting inoculum of the microbial host to be employed.

[0156] In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the fermentation media.

[0157] The composition of the aqueous mineral medium can vary over a wide range, depending in part on the microorganism and substrate employed, as is known in the art. The mineral media should include, in addition to nitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium, in suitable soluble assimilable ionic and combined forms, and also present preferably should be certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art.

[0158] The fermentation reaction is an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion.

[0159] The fermentation temperature can vary somewhat, but for most microbial cells the temperature generally will be within the range of about 20°C to 40°C.

[0160] The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself is convenient for large scale operations, and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control.

[0161] The pH range in the aqueous microbial ferment (fermentation admixture) should be in the exemplary range of about 2.0 to 8.0. Preferences for pH range of microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus change somewhat with change in media as can be readily determined by those skilled in the art.

[0162] Preferably, the fermentation is conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon-containing substrate to cells and avoiding contamination of the cells with a substantial amount of unconverted substrate. The latter is not a problem with water-soluble substrates, since any remaining traces are readily washed off. It may be a problem, however, in the case of non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps.

[0163] As described above, the time to reach this level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved.

[0164] If desired, part or all of the carbon and energy source material and / or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermenter.

[0165] Each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in response to a need determinable by monitoring such as concentration of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gases from the fermenter, cell density measurable by dry cell weights, light transmittancy, or the like. The feed rates of the various materials can be varied so as to obtain as rapid a cell growth rate as possible, consistent with efficient utilization of the carbon and energy source, to obtain as high a yield of microorganism cells relative to substrate charge as possible.

[0166] In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling devices, and the like, are initially sterilized, usually by employing steam such as at about 121°C for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermenter employed is not critical. V. EXEMPLARY EMBODIMENTS

[0167] Non-limiting embodiments of compositions and methods disclosed herein are as follows:

[0168] 1. A recombinant Gram-positive (host) cell comprising an introduced secGEY operon and an expression cassette encoding a heterologous subtilisin.

[0169] 2. The recombinant cell of embodiment 1, wherein the introduced secGEY operon comprises a secG open reading frame (ORF) sequence comprising at least about 80% to 100% identity to SEQ ID NO: 2, wherein the secG ORF encodes a functional SecG protein.

[0170] 3. The recombinant cell of embodiment 1, wherein the introduced secGEY operon comprises a secE ORF sequence comprising at least about 80% to 100% identity to SEQ ID NO: 31, wherein the secE ORF encodes a functional SecE protein.

[0171] 4. The recombinant cell of embodiment 1, wherein the introduced secGEY operon comprises a secY ORF sequence comprising at least about 80% to 100% identity to SEQ ID NO: 32, wherein the secY ORF encodes a functional SecY protein.

[0172] 5. The recombinant cell of embodiment 1, wherein the introduced secGEY operon encodes a functional secG protein comprising at least about 80% to 100% amino acid identity to SEQ ID NO: 28.

[0173] 6. The recombinant cell of embodiment 1, wherein the introduced secGEY operon encodes a functional secE protein comprising at least about 80% to 100% amino acid identity to SEQ ID NO: 29.

[0174] 7. The recombinant cell of embodiment 1, wherein the introduced secGEY operon encodes a functional secY protein comprising at least about 80% to 100% amino acid identity to SEQ ID NO: 30.

[0175] 8. The recombinant cell of embodiment 1, wherein the introduced secGEY operon comprises an upstream (5ʹ) secG promoter and secG 5ʹ-UTR sequence comprising at least about 80% to 100% identity to SEQ ID NO: 1 operably linked to the downstream secGEY ORFs.

[0176] 9. The recombinant cell of embodiment 1, wherein the introduced secGEY operon comprises a heterologous upstream (5ʹ) promoter and 5ʹ-UTR sequence operably linked to the downstream secGEY ORFs.

[0177] 10. The recombinant cell of embodiment 9, wherein heterologous upstream promoter and 5ʹ-UTR sequence comprise at least about 90% to 100% identity to the spoVG promoter and spoVG-5'-UTR of SEQ ID NO: 10.

[0178] 11. The recombinant cell of embodiment 9, wherein heterologous upstream promoter and 5ʹ-UTR sequence comprise at least about 90% to 100% identity to the hbs promoter and spoVG-5'-UTR of SEQ ID NO: 12.

[0179] 12. The recombinant cell of embodiment 1, wherein the introduced secGEY operon comprises at least about 80% to 100% identity to SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 13.

[0180] 13. The recombinant cell of embodiment 1, comprising at least two introduced cassettes encoding the same subtilisin or different heterologous subtilisins, or comprising at least three introduced expression cassettes encoding the same subtilisin or different heterologous subtilisins.

[0181] 14. The recombinant cell of embodiment 1 or embodiment 13, wherein the cassette or cassettes encode an alkaline subtilisin.

[0182] 15. The recombinant cell of embodiment 1 or embodiment 13, wherein the cassette encoding the subtilisin comprises an upstream promoter region sequence operably linked to a downstream nucleic acidencoding a protein signal sequence operably linked to a downstream nucleic acid encoding a pro-region sequence operably linked to a downstream nucleic acid encoding the mature subtilisin.

[0183] 16. The recombinant cell of embodiment 15, wherein the promoter sequence is a strong promoter functional in the Gram-positive cell.

[0184] 17. The recombinant cell of embodiment 15, wherein the signal sequence is a native subtilisin signal sequence or a functional variant thereof.

[0185] 18. The recombinant cell of embodiment 15, wherein the signal sequence comprises at least about 95% to 100% amino acid identity to the aprE signal sequence of SEQ ID NO: 17, or at least about 95% to 100% amino acid identity to the BPNʹ signal sequence of SEQ ID NO: 19.

[0186] 19. The recombinant cell of embodiment 15, wherein the pro-region sequence is a native subtilisin pro-region sequence or a functional variant thereof.

[0187] 20. The recombinant cell of embodiment 15, wherein the pro-region sequence comprises at least about 95% to 100% amino acid identity to the pro-region of SEQ ID NO: 18.

[0188] 21. The recombinant cell of embodiment 15, wherein the mature subtilisin comprises at least about 80% to 100% amino acid identity to the mature subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0189] 22. A polynucleotide construct encoding a synthetic secGEY operon, wherein the polynucleotide comprises at least an upstream promoter sequence operably linked to a downstream nucleic acid encoding a secG protein comprising at least about 80% to 100% identity to SEQ ID NO: 28 operably linked to a downstream nucleic acid encoding a secE protein comprising at least about 80% to 100% identity to SEQ ID NO: 29 operably linked to a downstream nucleic acid encoding a secY protein comprising at least about 80% to 100% identity to SEQ ID NO: 30.

[0190] 23. The polynucleotide of embodiment 22, wherein the nucleic acid encoding the secG protein comprises at least about 80% to 100% identity to the secG ORF of SEQ ID NO: 2.

[0191] 24. The polynucleotide of embodiment 22, wherein the nucleic acid encoding the secE protein comprises at least about 80% to 100% identity to the secE ORF of SEQ ID NO: 31.

[0192] 25. The polynucleotide of embodiment 22, wherein the nucleic acid encoding the secY protein comprises at least about 80% to 100% identity to the secY ORF of SEQ ID NO: 32.

[0193] 26. The polynucleotide of embodiment 24, wherein the nucleic acid encoding the secE protein comprises a secE ribosomal binding site (RBS) positioned upstream and operably linked to the secE ORF.

[0194] 27. The polynucleotide of embodiment 26, comprising at least about 80% to 100% identity to the secE RBS and secE ORF of SEQ ID NO: 3.

[0195] 28. The polynucleotide of embodiment 25, wherein the nucleic acid encoding the secY protein comprises a secY ribosomal binding site (RBS) positioned upstream and operably linked to the secY ORF.

[0196] 29. The polynucleotide of embodiment 28, comprising at least about 80% to 100% identity to the secY RBS and secY ORF of SEQ ID NO: 4.

[0197] 30. A method for producing a heterologous subtilisin in a modified Gram-positive bacterial cell comprising (a) obtaining a Gram-positive bacterial cell producing a heterologous subtilisin and introducing into the cell a synthetic secGEY operon, and (b) fermenting the modified cell under suitable conditions for the production of the subtilisin.

[0198] 31. A method for producing a heterologous subtilisin in a modified Gram-positive bacterial cell comprising (a) obtaining a Gram-positive bacterial cell and introducing into the cell (i) an expression cassette encoding the heterologous subtilisin and (ii) an expression cassette encoding a synthetic secGEY operon, and (b) fermenting the modified cell under suitable conditions for the production of the subtilisin.

[0199] 32. The method of embodiment 30 or embodiment 31, wherein the subtilisin is secreted into the fermentation broth when fermented under suitable conditions for the production of the subtilisin.

[0200] 33. The method of embodiment 30, wherein the modified cell produces an increased amount of the subtilisin relative to (vis-à-vis) a control cell when fermented under the same conditions, wherein the control cell does not comprise an introduced synthetic secGEY operon.

[0201] 34. The method of embodiment 31, wherein the modified cell produces an increased amount of the subtilisin relative to (vis-à-vis) a control cell when fermented under the same conditions, wherein the control cell comprises the same introduced expression cassette encoding the same heterologous subtilisin, wherein the control cell does not comprise an introduced synthetic secGEY operon.

[0202] 35. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon comprises a secG open reading frame (ORF) sequence comprising at least about 80% to 100% identity to SEQ ID NO: 2, wherein the secG ORF encodes a functional SecG protein.

[0203] 36. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon comprises a secE open reading frame (ORF) sequence comprising at least about 80% to 100% identity to SEQ ID NO: 31, wherein the secE ORF encodes a functional SecE protein.

[0204] 37. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon comprises a secY open reading frame (ORF) sequence comprising at least about 80% to 100% identity to SEQ ID NO: 32, wherein the secY ORF encodes a functional SecY protein.

[0205] 38. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon encodes a functional secG protein comprising at least about 80% to 100% amino acid identity to SEQ ID NO: 28.

[0206] 39. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon encodes a functional secE protein comprising at least about 80% to 100% amino acid identity to SEQ ID NO: 29.

[0207] 40. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon encodes a functional secY protein comprising at least about 80% to 100% amino acid identity to SEQ ID NO: 30.

[0208] 41. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon comprises an upstream (5ʹ) secG promoter and secG 5ʹ-UTR sequence comprising at least about 95% to 100% identity to SEQ ID NO: 1 operably linked to the downstream secGEY ORFs.

[0209] 42. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon comprises a heterologous upstream (5ʹ) promoter and 5ʹ-UTR sequence operably linked to the downstream secGEY ORFs.

[0210] 43. The method of embodiment 42, wherein heterologous promoter and 5ʹ-UTR sequence comprise at least about 95% to 100% identity to the spoVG promoter and spoVG-5'-UTR of SEQ ID NO: 10.

[0211] 44. The method of embodiment 42, wherein heterologous promoter and 5ʹ-UTR sequence comprise at least about 95% to 100% identity to the hbs promoter and spoVG-5'-UTR of SEQ ID NO: 12.

[0212] 45. The method of embodiment 30 or embodiment 31, wherein the introduced secGEY operon comprises at least about 80% to 100% identity to SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 13.

[0213] 46. The method of embodiment 31, wherein the cell comprises at least two introduced cassettes encoding the same subtilisin or different heterologous subtilisins, or comprises at least three introduced expression cassettes encoding the same subtilisin or different heterologous subtilisins.

[0214] 47. The method of embodiment 31 or embodiment 46, wherein the cassette or cassettes encode an alkaline subtilisin.

[0215] 48. The method of embodiment 31 or embodiment 46, wherein the cassette or cassettes encoding the subtilisin comprise an upstream promoter region sequence operably linked to a downstream nucleic acid encoding a protein signal sequence operably linked to a downstream nucleic acid encoding a pro-region sequence operably linked to a downstream nucleic acid encoding the mature subtilisin.

[0216] 49. The method of embodiment 48, wherein the promoter sequence is a strong promoter functional in the Gram-positive cell.

[0217] 50. The method of embodiment 48, wherein the signal sequence is a native subtilisin signal sequence or a functional variant thereof.

[0218] 51. The method of embodiment 50, wherein the signal sequence comprises at least about 95% to 100% amino acid identity to the aprE signal sequence of SEQ ID NO: 17, or at least about 95% to 100% amino acid identity to the BPNʹ signal sequence of SEQ ID NO: 19.

[0219] 52. The method of embodiment 48 wherein the pro-region sequence is a native subtilisin pro- region sequence or a functional variant thereof.

[0220] 53. The method of embodiment 48, wherein the pro-region sequence comprises at least about 90% to 100% amino acid identity to the pro-region of SEQ ID NO: 18.

[0221] 54. The method of embodiment 48, wherein the mature subtilisin comprises at least about 80% to 100% amino acid identity to the mature subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0222] 55. The method of embodiment 33 or embodiment 34, wherein the increased amount of the subtilisin is at least about 5% increased relative to the control cell when fermented under the same conditions.

[0223] 56. The method of embodiment 33 or embodiment 34, wherein the increased amount of the subtilisin is determined by suc-AAPF-pNA assay. EXAMPLES

[0224] Certain aspects of the present invention may be further understood in light of the following examples, which should not be construed as limiting. Modifications to materials and methods will be apparent to those skilled in the art. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989). EXAMPLE 1 CONSTRUCTION OF BACILLUS SUBTILIS CELLS EXPRESSING A HETEROLOGOUS PROTEIN AND OVEREXPRESSING SECG, SECE AND SECY

[0225] The present example describes the construction of Bacillus subtilis cells overexpressing SecG, SecE, and SecY in a host background expressing a heterologous reporter-1 protein. More particularly, a control B. subtilis strain named CZ437 comprises two (2) introduced reporter-1 expression cassettes comprising a promoter sequence (P2) positioned upstream (5ʹ) and operably linked to an open reading frame (ORF) encoding the reporter-1 protein, wherein the cassettes are integrated into the B. subtilis skf locus (skf::P2-reporter-1) and the B. subtilis aprE locus (aprE::P2-reporter-1). The design and construction of modified B. subtilis strains expressing the subtilisin reporter-1 and overexpressing an artificial (non- native) secGEY operon of the disclosure were performed as follows.

[0226] A first modified B. subtilis strain expressing SecG, SecE, and SecY proteins from their native loci and as a non-native operon (PsecG-secGEY) initiated by the secG promoter integrated into the B. subtilis pksR locus was constructed. More particularly, the non-native operon PsecG-secGEY was constructed by overlap extension PCR and comprises the B. subtilis secG promoter and secG 5ʹ-UTR (SEQ ID NO: 1) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO:2) operably linked to the secE ribosome binding sequence (RBS) and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a B. subtilis pksR downstream (3ʹ) homology region (SEQ ID NO: 6)operably linked to the non-native operon PsecG-secGEY (SEQ ID NO: 7) operably linked to the B. subtilis alrA gene (SEQ ID NO: 8) operably linked to a B. subtilis pksR upstream (5ʹ) homology region (SEQ ID NO: 9) was constructed by overlap extension PCR and transformed into a B. subtilis strain with an alrA deletion and containing one copy of the =reporter-1 expression cassette integrated at the skf locus (skf::P2- reporter-1). The resulting strain was transformed with a second copy of the reporter-1 cassette integrated at the aprE locus (aprE::P2-reporter-1) to create B. subtilis strain BPC0123.

[0227] A second modified strain expressing SecG, SecE, and SecY from their native loci and as a non- native operon (PspoVG-secGEY) initiated by the spoVG promoter (PspoVG) integrated into the pksR locus was constructed. More particularly, the non-native operon PspoVG-secGEY was constructed by overlap extension PCR and comprises the B. subtilis spoVG promoter and spoVG 5ʹ-UTR (SEQ ID NO: 10) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO: 2) operably linked to the secE RBS and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from B. amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a B. subtilis pksR downstream (3ʹ) homology region (SEQ ID NO: 6) operably linked to the non-native operon PspoVG-secGEY (SEQ ID NO: 11) operably linked to the B. subtilis alrA gene (SEQ ID NO: 8) operably linked to a B. subtilis pksR upstream (5ʹ) homology region (SEQ ID NO: 9) was constructed by overlap extension PCR and transformed into a B. subtilis strain with an alrA deletion and containing one copy of the -reporter-1 expression cassette integrated at the skf locus (skf::P2-reporter-1). The resulting strain was transformed with a second copy of the subtilisin variant-1 expression cassette integrated at the aprE locus (aprE::P2-reporter-1) to create B. subtilis strain BPC0182.

[0228] A third modified strain expressing SecG, SecE, and SecY from their native loci and as a non-native operon (Phbs-secGEY) initiated by the hbs promoter (Phbs) integrated into the pksR locus was constructed. More particularly, the non-native operon Phbs-secGEY was constructed by overlap extension PCR and comprises a B. subtilis hbs promoter and B. subtilis spoVG 5ʹ-UTR (SEQ ID NO: 12) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO: 2) operably linked to the secE RBS and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from B. amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing B. subtilis pksR downstream (3ʹ) homology region (SEQ ID NO: 6) operably linked to the non-native operon Phbs-secGEY (SEQ ID NO: 13) operably linked to the B. subtilis alrA gene (SEQ ID NO: 8) operably linked to a B. subtilis pksR upstream (5ʹ) homology region (SEQ ID NO: 9) was constructed by overlap extension PCR and transformed into a B. subtilis strain with an alrA deletion and containing one copy of the reporter-1 expression cassette integrated at the skf locus (skf::P2-reporter-1). The resulting strain was transformed with a second copy of the subtilisin variant-1 expression cassette integrated at the aprE locus (aprE::P2- reporter-1) to create B. subtilis strain BPC0184.EXAMPLE 2 EXPRESSION OF REPORTER-1 PROTEASE IN BACILLUS SUBTILIS CELLS OVEREXPRESSING SECG, SECE AND SECY

[0229] In the present example, expression of the heterologous reporter-1 protein in the presence of SecGEY overexpression was compared to expression of the same reporter-1 protein without SecGEY overexpression. More particularly, the three (3) SecGEY overexpression strains (BPC0123, BPC0182, BPC0184) and the control strain CZ437 were assessed for reporter-1 production under small scale conditions as follows. Pre-cultures in Tryptic Soy Broth (1.7% tryptone, 0.3% soytone, 0.25% glucose, 0.5% sodium chloride, 0.25% potassium phosphate dibasic) were inoculated with a single colony and grown at 37°C 250 RPM. The pre-culture was used to inoculate at a 1:1000 dilution 20 ml of 0.5X MPS2 medium, supplemented with 80 mM MOPS adjusted to pH7.3, and cultures grown in flasks at 37°C 250 RPM. MPS2 medium consists of: 10% v / v 10xMOPS based medium (8.4% w / v MOPS, 2.9% w / v Sodium Chloride, 1.5% w / v Potassium Hydroxide, 0.05% w / v Potassium Sulfate, 0.05% w / v Magnesium Chloride, 0.7% w / v Tricine, 10% v / v micronutrients), 10% w / v Maltrin M150, 6% w / v soytone, 0.78% w / v Dipotassium phosphate, 0.36% w / v urea, 0.2% w / v Monopotassium phosphate, 0.06% w / v tri-Sodium citrate dihydrate, adjust pH with Potassium hydroxide. The micronutrients were made up as a 100X stock solution in one (1) liter, 1.47 g Sodium citrate 2H2O, 1.47 g CaCl22H2O, 400 mg FeSO47H20, 100 mg MnSO4H2O, 100 mg ZnSO4H20, 50 mg CuCl22H20, 100 mg CoCl26H20, 100 mg Na2MoO42H20.

[0230] The amount reporter-1 protease in the culture supernatant was determined using the suc-AAPF- pNA assay essentially as described in WO2020112609 (incorporated herein by reference). The substrate is N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA). Upon hydrolysis of the peptide substrate by the protease, the 4-nitroanilide is cleaved and yields 4-nitroaniline, which is a yellow chromophore. Thus, the absorbance at 405 nm is measured and the slope of the absorbance change (mOD / min) directly correlates to the amount of protease in the analyzed sample. Filtered culture supernatant was diluted in dilution buffer (100 mM Tris, 0.005% Tween 80, 10 mM CaCl2, pH 8.6) and 10 µl of the diluted samples were added to microtiter plates containing 190 µl of AAPF stock diluted in Tris buffer (100 mg / ml AAPF stock in DMSO diluted 100X in 100 mM Tris, 0.005% Tween 80, pH 8.6). Absorbance of the solution was measured at 405 nm using a SpectraMax spectrophotometer.

[0231] In a first experiment, control strain CZ437 and SecGEY overexpression strain BPC0123 were cultured and sampled for protease activity after twenty-four (24) hours and forty (40) hours of growth. For example, reporter-1 protein production of strain BPC0123 relative to reporter-1 protein production of the control strain CZ437 (and associated coefficient of variation (CV; n≥4)), are presented below in TABLE 2. Likewise, TABLE 3 below shows the relative reporter-1 production of strains CZ437 and BPC0123 normalized to the amount of cell mass in the culture, as measured by OD600,. Thus, as generally shown inTABLES 2 and 3, production of the reporter-1 protein was increased with SecGEY overexpressed from the secG promoter. TABLE 2 SMALL-SCALE PRODUCTION OF PROTEASETABLE 3 SMALL-SCALE PRODUCTION OF PROTEASE NORMALIZED TO CULTURE OD600 i EY 24 h R l i 4 h R l i

[0232] In a second experiment, control strain CZ437 and SecGEY overexpression strains BPC0123, BPC0182, and BPC0184 were cultured and sampled for protease activity after twenty-two (22) hours of growth. More particularly, reporter-1 production of SecGEY overexpression strains BPC0123, BPC0182 and BPC0184, relative to the control strain CZ437, and associated coefficient of variation (CV; n=6), are presented below in TABLE 4. Likewise, the relative reporter-1 production normalized to the amount of cell mass in the culture, as measured by OD600, is presented below in TABLE 5. As shown in TABLES 4 and 5, production of reporter-1 protein was increased for all three strains overexpressing SecGEY when considering protease production per OD600 (see, TABLE 5). TABLE 4 SMALL-SCALE PRODUCTION OF PROTEASE Strain secGEY 22-hour RelativeTABLE 5 SMALL-SCALE PRODUCTION OF PROTEASE NORMALIZED TO CULTURE OD600EXAMPLE 3 CONSTRUCTION OF BACILLUS SUBTILIS CELLS EXPRESSING A HETEROLOGOUS PROTEIN AND OVEREXPRESSING SECG, SECE AND SECY

[0233] The present example describes the construction of Bacillus subtilis cells overexpressing SecG, SecE, and SecY in a host background expressing a heterologous reporter-2 protein. More particularly, a control B. subtilis strain named BPC0178 comprises three (3) introduced reporter-2 expression cassettes comprising a promoter sequence (P4) positioned upstream (5ʹ) and operably linked to an open reading frame (ORF) encoding the subtilisin reporter-2 protein, wherein the three cassettes are integrated into the B. subtilis skf locus (skf::P4-reporter-2), the B. subtilis nprE locus (nprE::P4-reporter-2) and the B. subtilis ppsC locus (ppsC::P4-reporter-2). The design and construction of modified B. subtilis strains expressing the reporter-2 and overexpressing an artificial (non-native) secGEY operon of the disclosure were performed as follows.

[0234] A first modified B. subtilis strain expressing SecG, SecE, and SecY proteins from their native loci and as a non-native operon (PsecG-secGEY) initiated by the secG promoter integrated into the B. subtilis aprE locus was constructed. More particularly, the non-native operon PsecG-secGEY was constructed by overlap extension PCR and comprises the B. subtilis secG promoter and secG 5ʹ-UTR (SEQ ID NO: 1) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO: 2) operably linked to the secE RBS and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a B. subtilis aprE upstream (5ʹ) homology region (SEQ ID NO: 14) operably linked to the non- native operon PsecG-secGEY (SEQ ID NO: 7) operably linked to a promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked to a B. subtilis aprE downstream (3ʹ) homology region (SEQ ID NO: 16) was constructed by overlap extension PCR and transformed into a B. subtilis strain containing three copies of the reporter-2 expression cassette integrated at the skf locus (skf::P4-reporter-2), nprE locus (nprE::P4- reporter-2) and ppsC locus (ppsC::P4- reporter-2) to create B. subtilis strain BPC0166.

[0235] A second modified strain expressing SecG, SecE, and SecY from their native loci and as a non- native operon (PspoVG-secGEY) initiated by the spoVG promoter integrated into the aprE locus was constructed. More particularly, the non-native operon PspoVG-secGEY was constructed by overlap extension PCR and comprises the B. subtilis spoVG promoter and spoVG 5ʹ-UTR (SEQ ID NO: 10) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO: 2) operably linked to the secE RBS and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from B. amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a B. subtilis aprE upstream (5ʹ) homology region (SEQ ID NO: 14) operably linked to the non-native operon PspoVG-secGEY (SEQ ID NO: 11) operably linked to a promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked to a B. subtilis aprE downstream (3ʹ) homology region (HR; SEQ ID NO: 16) was constructed by overlap extension PCR and transformed into a B. subtilis strain containing three copies of the reporter-2 expression cassette integrated at the skf locus (skf::P4-reporter-2), nprE locus (nprE::P4- reporter-2) and ppsC locus (ppsC::P4-reporter-2) to create B. subtilis strain BPC0167.

[0236] A third modified strain expressing SecG, SecE, and SecY from their native loci and as a non-native operon (Phbs-secGEY) initiated by the hbs promoter integrated into the aprE locus was constructed. More particularly, the non-native operon Phbs-secGEY was constructed by overlap extension PCR and comprises a B. subtilis hbs promoter and B. subtilis spoVG 5ʹ-UTR (SEQ ID NO: 12) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO: 2) operably linked to the secE RBS and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from B. amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a B. subtilis aprE upstream (5ʹ) homology region (SEQ ID NO: 14) operably linked to the non-native operon Phbs-secGEY (SEQ ID NO: 13) operably linked to a promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked to a B. subtilis aprE downstream (3ʹ) homology region (SEQ ID NO: 16) was constructed by overlap extension PCR and transformed into a B. subtilis strain containing three copies of the reporter-2 expression cassette integrated at the skf locus (skf::P4- reporter-2), nprE locus (nprE::P4-reporter2) and ppsC locus (ppsC::P4- reporter-2) to create B. subtilis strain BPC0168. EXAMPLE 4 EXPRESSION OF REPORTER-2 PROTEASE IN BACILLUS SUBTILIS CELLS OVEREXPRESSING SECG, SECE AND SECY

[0237] In the present example, expression of the heterologous reporter-2 protein in the presence of SecGEY overexpression was compared to expression of the same reporter-2 protein without SecGEY overexpression. More particularly, the three (3) SecGEY overexpression strains (BPC0166, BPC0167, BPC0168) and the control strain BPC0178 were assessed for reporter-2 production under small scale conditions as follows. Pre-cultures in Tryptic Soy Broth (1.7% tryptone, 0.3% soytone, 0.25% glucose,0.5% sodium chloride, 0.25% potassium phosphate dibasic) were inoculated with a single colony and grown at 37°C 250 RPM. The pre-culture was used to inoculate at a 1:1000 dilution 20 ml of 0.5X MPS2 medium, supplemented with 80 mM MOPS adjusted to pH7.3, and cultures grown in flasks at 37°C 250 RPM. MPS2 medium consists of: 10% v / v 10xMOPS based medium (8.4% w / v MOPS, 2.9% w / v Sodium Chloride, 1.5% w / v Potassium Hydroxide, 0.05% w / v Potassium Sulfate, 0.05% w / v Magnesium Chloride, 0.7% w / v Tricine, 10% v / v micronutrients), 10% w / v Maltrin M150, 6% w / v soytone, 0.78% w / v Dipotassium phosphate, 0.36% w / v urea, 0.2% w / v Monopotassium phosphate, 0.06% w / v tri-Sodium citrate dihydrate, adjust pH with Potassium hydroxide. The micronutrients were made up as a 100X stock solution in one (1) liter, 1.47 g Sodium citrate 2H2O, 1.47 g CaCl22H2O, 400 mg FeSO47H20, 100 mg MnSO4H2O, 100 mg ZnSO4H20, 50 mg CuCl22H20, 100 mg CoCl26H20, 100 mg Na2MoO42H20.

[0238] The amount reporter-2 protease in the culture supernatant was determined using the suc-AAPF- pNA assay essentially as described in WO2020112609. The substrate is N-succinyl-L-Ala-L-Ala-L-Pro-L- Phe-p-nitroanilide (suc-AAPF-pNA). Upon hydrolysis of the peptide substrate by the protease, the 4- nitroanilide is cleaved and yields 4-nitroaniline, which is a yellow chromophore. Thus, the absorbance at 405 nm is measured and the slope of the absorbance change (mOD / min) directly correlates to the amount of protease in the analyzed sample. Filtered culture supernatant was diluted in dilution buffer (100 mM Tris, 0.005% Tween 80, 10 mM CaCl2, pH 8.6) and 10 µl of the diluted samples were added to microtiter plates containing 190 µl of AAPF stock diluted in Tris buffer (100 mg / ml AAPF stock in DMSO diluted 100X in 100 mM Tris, 0.005% Tween 80, pH 8.6). Absorbance of the solution was measured at 405 nm using a SpectraMax spectrophotometer.

[0239] Control strain BPC0178 and SecGEY overexpression strains BPC0166, BPC0167, and BPC0168 were cultured and sampled for protease activity after twenty (20) and forty (40) hours of growth. More particularly, reporter-2 production of SecGEY overexpression strains BPC0166, BPC0167 and BPC0168, relative to the control strain BPC0178, and associated coefficient of variation (CV; n=3), are presented below in TABLE 6, and the relative reporter-2 production normalized to the amount of cell mass in the culture, as measured by OD600, is presented below in TABLE 7. As shown in TABLES 6 and 7, production of reoporter-2 protein is equivalent or lower for all three strains overexpressing SecGEY.TABLE 6 SMALL-SCALE PRODUCTION OF PROTEASETABLE 7 SMALL-SCALE PRODUCTION OF PROTEASE NORMALIZED TO CULTURE OD600 i EY 2 h R l i 4 h R l iEXAMPLE 5 CONSTRUCTION OF BACILLUS SUBTILIS CELLS EXPRESSING A HETEROLOGOUS PROTEIN AND OVEREXPRESSING SECG, SECE AND SECY

[0240] The present example describes the construction of Bacillus subtilis cells overexpressing SecG, SecE, and SecY in a host background expressing a heterologous reporter-3 protein. More particularly, a control B. subtilis strain named BPC0229 comprises two (2) introduced reporter-3 expression cassettes comprising a promoter sequence (P2-00788) positioned upstream (5ʹ) and operably linked to an open reading frame (ORF) encoding the subtilisin variant-3 protein, wherein the cassettes are integrated into the B. subtilis skf locus (skf::P2-00788- reporter-3) and the B. subtilis pksR locus (pksR::P2-00788- reporter- 3). The design and construction of modified B. subtilis strains expressing the reporter-3 protein and overexpressing an artificial (non-native) secGEY operon of the disclosure were performed as follows.

[0241] A first modified B. subtilis strain expressing SecG, SecE, and SecY proteins from their native loci and as a non-native operon (PsecG-secGEY) initiated by the secG promoter integrated into the B. subtilis aprE locus was constructed. More particularly, the non-native operon PsecG-secGEY was constructed by overlap extension PCR and comprises the B. subtilis secG promoter and secG 5ʹ-UTR (SEQ ID NO: 1) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO: 2) operably linked to the secERBS and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a B. subtilis aprE upstream (5ʹ) homology region (SEQ ID NO: 14) operably linked to the non- native operon PsecG-secGEY (SEQ ID NO: 7) operably linked to a promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked to a B. subtilis aprE downstream (3ʹ) homology region (SEQ ID NO: 16) was constructed by overlap extension PCR and transformed into a B. subtilis strain containing two copies of the reporter-3 expression cassette integrated at the skf locus (skf::P2-00788-reporter-3) and pksR locus (pksR::P2-00788- reporter-3) to create B. subtilis strain AL394.

[0242] A second modified strain expressing SecG, SecE, and SecY from their native loci and as a non- native operon (PspoVG-secGEY) initiated by the spoVG promoter integrated into the aprE locus was constructed. More particularly, the non-native operon PspoVG-secGEY was constructed by overlap extension PCR and comprises the B. subtilis spoVG promoter and spoVG 5ʹ-UTR (SEQ ID NO: 10) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO: 2) operably linked to the secE RBS and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from B. amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a B. subtilis aprE upstream (5ʹ) homology region (SEQ ID NO: 14) operably linked to the non-native operon PspoVG-secGEY (SEQ ID NO: 11) operably linked to a promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked to a B. subtilis aprE downstream (3ʹ) homology region (SEQ ID NO: 16) was constructed by overlap extension PCR and transformed into a B. subtilis strain containing two copies of the reporter-3 expression cassette integrated at the skf locus (skf::P2-00788- reporter-3) and pksR locus (pksR::P2-00788-reporter-3) to create B. subtilis strain AL395.

[0243] A third modified strain expressing SecG, SecE, and SecY from their native loci and as a non-native operon (Phbs-secGEY) initiated by the hbs promoter integrated into the aprE locus was constructed. More particularly, the non-native operon Phbs-secGEY was constructed by overlap extension PCR and comprises a B. subtilis hbs promoter and B. subtilis spoVG 5ʹ-UTR (SEQ ID NO: 12) positioned upstream (5ʹ) and operably linked to the secG ORF (SEQ ID NO: 2) operably linked to the secE RBS and ORF (SEQ ID NO: 3) operably linked to the secY RBS and ORF (SEQ ID NO: 4) operably linked to the BPN’ terminator from B. amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a B. subtilis aprE upstream (5ʹ) homology region (SEQ ID NO: 14) operably linked to the non-native operon Phbs-secGEY (SEQ ID NO: 13) operably linked to a promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked to a B. subtilis aprE downstream (3ʹ) homology region (SEQ ID NO: 16) was constructed by overlap extension PCR and transformed into a B. subtilis strain containing two copies of the reporter-3 expression cassette integrated at the skf locus (skf::P2-00788- reporter-3) and pksR locus (pksR::P2-00788- reporter-3) to create B. subtilis strain AL396.EXAMPLE 6 EXPRESSION OF REPORTER-3 PROTEASE IN BACILLUS SUBTILIS CELLS OVEREXPRESSING SECG, SECE AND SECY

[0244] In the present example, expression of the heterologous reporter-3 protein in the presence of SecGEY overexpression was compared to expression of the same reporter-3 protein without SecGEY overexpression. More particularly, the three (3) SecGEY overexpression strains (AL394, AL395, AL396) and the control strain BPC0229 were assessed for reporter-3 production under small scale conditions as follows. Pre-cultures in Tryptic Soy Broth (1.7% tryptone, 0.3% soytone, 0.25% glucose, 0.5% sodium chloride, 0.25% potassium phosphate dibasic) were inoculated with a single colony and grown at 37°C 250 RPM. The pre-culture was used to inoculate at a 1:1000 dilution 20 ml of 0.5X MPS2 medium, supplemented with 80 mM MOPS and 5mM CaCl2adjusted to pH7.3, and cultures grown in flasks at 37°C 250 RPM. MPS2 medium consists of: 10% v / v 10xMOPS based medium (8.4% w / v MOPS, 2.9% w / v Sodium Chloride, 1.5% w / v Potassium Hydroxide, 0.05% w / v Potassium Sulfate, 0.05% w / v Magnesium Chloride, 0.7% w / v Tricine, 10% v / v micronutrients), 10% w / v Maltrin M150, 6% w / v soytone, 0.78% w / v Dipotassium phosphate, 0.36% w / v urea, 0.2% w / v Monopotassium phosphate, 0.06% w / v tri-Sodium citrate dihydrate, adjust pH with Potassium hydroxide. The micronutrients were made up as a 100X stock solution in one (1) liter, 1.47 g Sodium citrate 2H2O, 1.47 g CaCl22H2O, 400 mg FeSO47H20, 100 mg MnSO4H2O, 100 mg ZnSO4H20, 50 mg CuCl22H20, 100 mg CoCl26H20, 100 mg Na2MoO42H20.

[0245] The amount reporter-3 protease in the culture supernatant was determined using the suc-AAPF- pNA assay essentially as described in WO2020112609. The substrate is N-succinyl-L-Ala-L-Ala-L-Pro-L- Phe-p-nitroanilide (suc-AAPF-pNA). Upon hydrolysis of the peptide substrate by the protease, the 4- nitroanilide is cleaved and yields 4-nitroaniline, which is a yellow chromophore. Thus, the absorbance at 405 nm is measured and the slope of the absorbance change (mOD / min) directly correlates to the amount of protease in the analyzed sample. Filtered culture supernatant was diluted in dilution buffer (100 mM Tris, 0.005% Tween 80, 10 mM CaCl2, pH 8.6) and 10 µl of the diluted samples were added to microtiter plates containing 190 µl of AAPF stock diluted in Tris buffer (100 mg / ml AAPF stock in DMSO diluted 100X in 100 mM Tris, 0.005% Tween 80, pH 8.6). Absorbance of the solution was measured at 405 nm using a SpectraMax spectrophotometer.

[0246] Control strain BPC0229 and SecGEY overexpression strains AL394, AL395, and AL396 were cultured and sampled for reporter-3 protease activity after sixteen (16), twenty-four (24) and forty (40) hours of growth. More particularly, reporter-3 production of SecGEY overexpression strains AL394, AL395 and AL396, relative to the control strain BPC0229, and associated coefficient of variation (CV; n=3), are presented below in TABLE 7, and the relative reporter-3 protease production normalized to the amount of cell mass in the culture, as measured by OD600, is presented below in TABLE 8. As shown inTABLES 7 and 8, production of the reporter-3 protein was increased for all three strains overexpressing SecGEY when considering protease production per OD600. TABLE 8 SMALL-SCALE PRODUCTION OF PROTEASETABLE 9 SMALL-SCALE PRODUCTION OF PROTEASE NORMALIZED TO CULTURE OD600 Strain secGEY 16-hour 24-hour 40-hourREFERENCES PCT Publication No. WO1999 / 04006 PCT Publication No. WO2008 / 126929 PCT Publication No. WO2010 / 056634 PCT Publication No. WO2011 / 130222 PCT Publication No. WO2015 / 089447 PCT Publication No. WO2016 / 202839 PCT Publication No. WO2017 / 207762 PCT Publication No. WO2020 / 112609 PCT Publication No. WO2023 / 114936 PCT Publication No. WO2008 / 141281 US Patent Publication No. US2009 / 0029417 Chen et al., “Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression”, Microbial Cell Factories, 14:92 2015. Freudl, “Signal peptides for recombinant protein secretion in bacterial expression systems”, Microbial Cell Factories, 17:522018. Mulder et al., “Construction of an artificial secYEG operon allowing high level secretion of α-amylase”, Protein Expression and Purification, 89, pages 92-96, 2013. Neef et al., “Relative contributions of non‑essential Sec pathway components and cell envelope‑associated proteases to high‑level enzyme secretion by Bacillus subtilis”, Microbial Cell Factories, 19:522020. Neef et al., “Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential”, Essays in Biochemistry, 65: 187–195, 2021. Pohl and Harwood, “Heterologous Protein Secretion by Bacillus Species: From the Cradle to the Grave”, Advances in Applied Microbiology, Vol.73, Chapter I, 2010. Rawlings et al., MEROPS: the peptidase database, Nucleic Acids Res, 34 Database issue, D270-272, 2006. Siezen and Leunissen, “Subtilases: The superfamily of subtilisin-like serine proteases”, Protein Science, 6, pages 501-523, 1997.

Claims

CLAIMS 1. A recombinant Gram-positive bacterial cell comprising an introduced secGEY operon and an expression cassette encoding a heterologous subtilisin.

2. The recombinant cell of claim 1, wherein the introduced secGEY operon comprises a secG open reading frame (ORF) sequence comprising at least 80% identity to SEQ ID NO:

2.

3. The recombinant cell of claim 1, wherein the introduced secGEY operon comprises a secE ORF sequence comprising at least 80% identity to SEQ ID NO:

31.

4. The recombinant cell of claim 1, wherein the introduced secGEY operon comprises a secY ORF sequence comprising at least 80% identity to SEQ ID NO:

32.

5. The recombinant cell of claim 1, wherein the introduced secGEY operon comprises an upstream (5ʹ) wild-type secG promoter and secG 5ʹ-UTR sequence or a functional variant thereof operably linked to the downstream secGEY open reading frames, or wherein the introduced secGEY operon comprises a heterologous upstream (5ʹ) promoter and 5ʹ-UTR sequence operably linked to the downstream secGEY open reading frames (ORFs).

6. The recombinant cell of claim 1, wherein the introduced secGEY operon comprises at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO:

13.

7. The recombinant cell of claim 1, comprising at least two introduced cassettes encoding the same subtilisin or different subtilisins, or comprising at least three introduced expression cassettes encoding the same subtilisin or different subtilisins.

8. The recombinant cell of claim 1 or claim 7, wherein the cassette or cassettes encode an alkaline subtilisin.

9. The recombinant cell of claim 1 or claim 7, wherein the subtilisin comprises at least 80% amino acid identity to the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO:

23.

10. A polynucleotide construct encoding a synthetic secGEY operon, wherein the polynucleotide comprises at least an upstream (5ʹ) promoter sequence operably linked to a downstream nucleic acid encoding a secG protein comprising at least 80% identity to SEQ ID NO: 28 operably linked to a downstream nucleic acid encoding a secE protein comprising at least 80% identity to SEQ ID NO: 29 operably linked to a downstream (3ʹ) nucleic acid encoding a secY protein comprising at least 80% identity to SEQ ID NO:

30.

11. The polynucleotide of claim 10, wherein the nucleic acid encoding the secE protein comprises a secE ribosomal binding site (RBS) positioned upstream and operably linked to the secE ORF.

12. The polynucleotide of claim 10, wherein the nucleic acid encoding the secY protein comprises a secY ribosomal binding site (RBS) positioned upstream and operably linked to the secY ORF.

13. A method for producing a heterologous subtilisin in a modified Gram-positive bacterial cell comprising: (a) obtaining a Gram-positive bacterial cell producing a heterologous subtilisin and introducing into the cell a synthetic secGEY operon, and (b) fermenting the modified cell under suitable conditions for the production of the subtilisin.

14. A method for producing a heterologous subtilisin in a modified Gram-positive bacterial cell comprising: (a) obtaining a Gram-positive bacterial cell and introducing into the cell (i) an expression cassette encoding the heterologous subtilisin and (ii) a synthetic secGEY operon, and (b) fermenting the modified cell under suitable conditions for the production of the subtilisin.

15. The method of claim 13 or claim 14, wherein the subtilisin is secreted into the fermentation broth when fermented under suitable conditions for the production of the subtilisin.

16. The method of claim 13 or claim 14, wherein the modified cell produces an increased amount of the subtilisin relative to a control cell fermented under the same conditions, wherein the control cell comprises same introduced cassette encoding the same subtilisin, wherein the control cell does not comprise an introduced secGEY operon.

17. The method of claim 13 or claim 14, wherein the introduced secGEY operon comprises a secG open reading frame (ORF) sequence comprising at least 80% identity to SEQ ID NO:

2.

18. The method of claim 13 or claim 14, wherein the introduced secGEY operon comprises a secE open reading frame (ORF) sequence comprising at least 80% identity to SEQ ID NO:

31.

19. The method of claim 13 or claim 14, wherein the introduced secGEY operon comprises a secY open reading frame (ORF) sequence comprising at least 80% identity to SEQ ID NO:

32.

20. The method of claim 13 or claim 14, wherein the introduced secGEY operon comprises an upstream secG promoter and secG 5ʹ-UTR sequence comprising at least about 95% identity to SEQ ID NO: 1 operably linked to the downstream secGEY ORFs.

21. The method of claim 13 or claim 14, wherein the introduced secGEY operon comprises a heterologous upstream (5ʹ) promoter and 5ʹ-UTR sequence operably linked to the downstream secGEY ORFs.

22. The method of claim 13 or claim 14, wherein the introduced secGEY operon comprises at least about 80% identity to SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO:

13.

23. The method of claim 14, wherein the cell comprises at least two introduced cassettes encoding the same subtilisin or different subtilisins, or comprises at least three introduced expression cassettes encoding the same subtilisin or different subtilisins.

24. The method of claim 14 or claim 23, wherein the cassette or cassettes encode an alkaline subtilisin.

25. The method of claim 14 or claim 23, wherein the cassette or cassettes encoding the subtilisin comprise an upstream promoter region sequence operably linked to a downstream nucleic acid encoding a protein signal sequence operably linked to a downstream nucleic acid encoding a pro- region sequence operably linked to a downstream nucleic acid encoding the mature subtilisin.

26. The method of claim 25, wherein the mature subtilisin comprises at least about 80% amino acid identity to the mature subtilisin of SEQ ID NO: 21 or SEQ ID NO:

23.

27. The method of claim 15, wherein the increased amount of the subtilisin is at least about 5% increased relative to the control cell when fermented under the same conditions.

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