Compositions and methods for enhancing protein production in Gram-positive bacterial cells

Abstract

This disclosure, in a broad sense, relates to Gram-positive bacterial strains containing phenotypes with enhanced protein production capacity. Accordingly, certain embodiments relate to compositions and methods for constructing recombinant (modified) Gram-positive bacterial strains for enhanced production of the protein of interest.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 518,482, filed Aug. 9, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure generally relates to the fields of bacteriology, microbiology, genetics, molecular biology, enzymology, industrial protein production, etc. Certain embodiments of the present disclosure relate to Gram - positive bacterial cells having a phenotype with enhanced protein production ability, and compositions and methods for constructing recombinant Gram - positive bacterial cells, etc.

[0003] Reference to Sequence Listing The content of the electronic submission of the text file of the sequence listing named "NB42147USPSP_SequenceListing.xml", created on Jul. 25, 2023, and having a size of 48 KB, is incorporated herein by reference in its entirety.

Background Art

[0004] Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens are frequently used as microbial factories for producing industrial-related proteins due to their excellent fermentation properties and high yields. For example, Bacillus species host cells are well known to produce enzymes (e.g., amylase, cellulase, mannanase, pectin lysase, protease, pullulanase, etc.) required for food, textiles, laundry, medical device cleaning, and the pharmaceutical industry. These non-pathogenic Gram-positive bacteria produce proteins that contain no harmful by-products (e.g., lipopolysaccharide (LPS), also known as endotoxin), and have therefore received a "Qualified Presumption of Safety" (QPS) rating from the European Food Safety Authority (EFSA). Many of their products have also received a "Generally Recognized As Safe" (GRAS) rating from the U.S. Food and Drug Administration.

[0005] Therefore, the production of proteins (e.g., enzymes, antibodies, receptors, etc.) via microbial host cells is of particular interest in the field of biotechnology. Similarly, the optimization of Bacillus host cells that produce and secrete one or more proteins of interest is highly relevant, especially in the context of industrial biotechnology, where even small improvements in protein yield can be extremely important when proteins are produced on a large scale industrially. For example, the expression of many secreted proteins can still be difficult and unpredictable in terms of yield, etc. As described below in this specification, this disclosure relates to a highly desirable and unmet need for obtaining and constructing Gram-positive cells (e.g., protein-producing hosts) with enhanced protein-producing capacity. [Overview of the project] [Means for solving the problem]

[0006] As described herein, certain embodiments of this disclosure relate to methods and compositions for enhancing protein production in Gram-positive bacterial (host) cells. Accordingly, certain one or more embodiments provide, among other things, Gram-positive bacterial cells / strains expressing heterologous proteins, Gram-positive bacterial cells secreting heterologous proteins into fermentation broth, and Gram-positive bacterial cells comprising introduced (synthesized) secGEY operons expressing SecG, SecE, and SecY translocon proteins. Therefore, certain embodiments relate to nucleic acid (DNA) sequences encoding non-natural (synthetic) secGEY operons, SecG, SecE, and SecY translocon proteins (e.g., secG ORF encoding a functional SecG protein, secE ORF encoding a functional SecE protein, 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 signaling (secreted) peptide sequences, DNA sequences encoding protease PRO region sequences, DNA sequences encoding one or more ribosome-binding sites (RBS), promoter region (DNA) sequences, and 5' untranslated region (5'-UTR) sequences, etc.

[0007] In certain embodiments, the disclosure provides recombinant Gram-positive bacterial cells comprising an introduced (synthetic) secGEY operon and one or more introduced expression cassettes encoding heterologous subtilisin proteins. In certain embodiments, the introduced secGEY operon comprises a secG open reading frame (ORF) sequence having at least about 80% to 100% identity to SEQ ID NO: 2, a secE ORF sequence having at least about 80% to 100% identity to SEQ ID NO: 31, and a secY ORF sequence having at least about 80% to 100% identity to SEQ ID NO: 32. In certain other embodiments, the introduced (synthetic) secGEY operon comprises an upstream (5') wild-type secG promoter and a secG 5'-UTR sequence, with a downstream secGEY open reading frame (ORF) operably ligated thereto. In other embodiments, the introduced secGEY operon comprises an upstream (5') heterologous promoter and a 5'-UTR sequence, with a downstream secGEY ORF operably ligated thereto. In further embodiments, the recombinant cells include at least two transexpression cassettes encoding the same or different subtilisins, or at least three transexpression cassettes encoding the same or different subtilisins. In certain embodiments, the cassette encodes an alkaline subtilisin. In other embodiments, the cassette encodes an alkaline subtilisin having an isoelectric point (pI) of about 8.5 to about 10. In certain other embodiments, the heterologous subtilisin has at least about 80% amino acid identity to the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23. For example, in certain other embodiments, the heterologous subtilisin contains 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 with respect to the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23.

[0008] As described herein, the non-natural (synthetic) secGEY operon of this disclosure is constructed, in which the secG, secE, and secY open reading frame (ORF) polynucleotide sequences (i.e., encoding the secG, secE, and secY translocon proteins, respectively) are operably linked in any order. For example, in a particular embodiment, a non-natural (synthetic) secGEY operon comprises an upstream secE ORF, a downstream secY ORF operably coupled thereto, and a downstream secG ORF operably coupled thereto (e.g., 5'-[secE]-[secY]-[secG]-3'), and a non-natural secGEY operon comprises an upstream secE ORF, a downstream secG ORF operably coupled thereto, and a downstream secY ORF operably coupled thereto (e.g., 5'-[secE]-[secG]-[secY]-3'), and a non-natural secGEY operon comprises an upstream secY ORF, a downstream secE ORF operably coupled thereto, and a downstream secG ORF operably coupled thereto An ORF (e.g., 5'-[secY]-[secG]-[secE]-3') is included, and a non-natural secGEY operon includes an upstream secG ORF, a downstream secY ORF operably coupled to it, and a downstream secE ORF (e.g., 5'-[secG]-[secY]-[secE]-3') operably coupled to it.

[0009] Accordingly, certain embodiments relate to polynucleotides encoding the synthetic secGEY operon of the present disclosure. For example, in certain embodiments, the present disclosure provides a polynucleotide construct encoding the synthetic secGEY operon, the polynucleotide comprising at least an upstream (5') promoter region sequence, a downstream nucleic acid operably ligated thereto that encodes a secG protein having at least about 80% to 100% identity to SEQ ID NO: 28, a downstream nucleic acid operably ligated thereto that encodes a secE protein having at least about 80% to 100% identity to SEQ ID NO: 29, and a downstream (3') nucleic acid operably ligated thereto that encodes a secY protein having at least about 80% to 100% identity to SEQ ID NO: 30.

[0010] In other embodiments, the Disclosure provides a method for producing heterologous subtilisin proteins in recombinant (modified) bacterial cells. Thus, certain embodiments of the Disclosure provide a method for producing heterologous subtilisin in modified Gram-positive bacterial cells, comprising (a) obtaining Gram-positive bacterial cells that express / produce heterologous subtilisin and introducing a synthetic secGEY operon into the cells, and (b) fermenting the modified cells under conditions suitable for subtilisin production. In other embodiments, the Disclosure provides a method for producing heterologous subtilisin in modified Gram-positive bacterial cells, comprising (a) obtaining Gram-positive bacterial cells and introducing (i) a synthetic secGEY operon and (ii) an expression cassette encoding heterologous subtilisin into the cells, and (b) fermenting the modified cells under conditions suitable for subtilisin production.

[0011] In a particular embodiment of this method, subtilisin is secreted into the fermentation broth when fermented under conditions suitable for subtilisin production. In another embodiment, modified cells produce an increased amount of subtilisin compared to control cells fermented under the same conditions, the control cells containing the same transexpression cassette encoding the same heterologous subtilisin but without the transsecGEY operon. In yet another embodiment of this method, the transsecGEY operon contains a secG ORF sequence with at least about 80-100% identity to SEQ ID NO: 2. In yet another embodiment, the transsecGEY operon contains a secE ORF sequence with at least about 80-100% identity to SEQ ID NO: 31. In yet another embodiment, the transsecGEY operon contains a secY ORF sequence with at least about 80-100% identity to SEQ ID NO: 32. In certain other embodiments of this method, the introduced secGEY operon comprises an upstream secG promoter and secG 5'-UTR sequence having at least about 95% to 100% identity with respect to SEQ ID NO: 1, and a downstream secGEY ORF operably linked thereto. In other embodiments, the introduced secGEY operon comprises an upstream (5') heterologous promoter and 5'-UTR sequence, and a downstream secGEY ORF operably linked thereto. In yet another embodiment, the cell comprises at least two introduced cassettes encoding the same or different heterologous subtilisins, or at least three introduced expression cassettes encoding the same or different heterologous subtilisins. In certain other embodiments, the cassette encodes an alkaline subtilisin. In other embodiments, the cassette encodes an alkaline subtilisin having an isoelectric point (pI) of about 8.5 to about 10. In yet another embodiment of this method, the cassette encodes an alkaline subtilisin of subgroup I-S2. In certain other embodiments, the heterologous subtilisin contains at least about 80% to 100% amino acid identity with respect to the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23.In certain relevant embodiments, the heterologous subtilisin contains 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 with the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23. In other embodiments, the cassette encoding the subtilisin contains an upstream promoter region sequence, a downstream nucleic acid encoding a protein signal sequence operably ligated thereto, a downstream nucleic acid encoding a pro region sequence operably ligated thereto, and a downstream nucleic acid encoding a mature subtilisin operably ligated thereto. In certain embodiments of the method, the increased amount of subtilisin produced is at least about 5% greater compared to control cells fermented under the same conditions. [Brief explanation of the drawing]

[0012] [Figure 1]Figure 1 shows the nucleic acid (DNA) sequences of the non-natural (synthetic) secGEY operons constructed and described herein. More specifically, various non-natural 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 "PhbsS-secGEY"; SEQ ID NO: 13), as described in the following examples. As shown in Figure 1, synthetic operon 1 (PsecG-secGEY) includes an upstream (5') secG promoter region containing a secG promoter (PsecG) and a secG 5'-UTR sequence (Figure 1A; SEQ ID NO: 1), a downstream DNA sequence operably ligated thereto that encodes a secG transloconprotein (SEQ ID NO: 28) (Figure 1B; secG ORF, SEQ ID NO: 2), a downstream DNA sequence operably ligated thereto that includes a secEORF (Figure 1C; SEQ ID NO: 3) encoding a secE ribosome binding site (RBS) and a secE transloconprotein (SEQ ID NO: 29), and a downstream DNA sequence operably ligated thereto that includes a secY RBS and a secY ORF (Figure 1D; SEQ ID NO: 4) encoding a secE transloconprotein (SEQ ID NO: 30).Similarly, synthetic operon 2 (Figure 1; PspoVG-secGEY, SEQ ID NO: 11) comprises an upstream (5') spoVG promoter region containing the spoVG promoter (PspoVG) and the spoVG 5'-UTR sequence (Figure 1E; SEQ ID NO: 10), a downstream DNA sequence operably linked thereto that encodes the secG transloconprotein (SEQ ID NO: 28) (Figure 1F; secG ORF, SEQ ID NO: 2), a downstream DNA sequence operably linked thereto that encodes the secE ribosome binding site (RBS) and the secE transloconprotein (SEQ ID NO: 29) (Figure 1G; SEQ ID NO: 3), and a downstream DNA sequence operably linked thereto that encodes the secY RBS and the secY transloconprotein (SEQ ID NO: 30) (Figure 1H; SEQ ID NO: 4). Synthetic operon 3 (Phbs-secGEY; SEQ ID NO: 13) comprises the hbs promoter (Phbs) and spoVG The DNA sequence includes an upstream (5)hbs promoter region containing a 5'-UTR sequence (Figure 1I; SEQ ID NO: 12), a downstream DNA sequence operably ligated thereto that encodes a secG transloconprotein (SEQ ID NO: 28) (Figure 1J; secG ORF, SEQ ID NO: 2), a downstream DNA sequence operably ligated thereto that includes a secE ribosome binding site (RBS) and a secE ORF (Figure 1K; SEQ ID NO: 3) encoding a secE transloconprotein (SEQ ID NO: 29), and a downstream DNA sequence operably ligated thereto that includes a secY RBS and a secY ORF (Figure 1L; SEQ ID NO: 4) encoding a secY transloconprotein (SEQ ID NO: 30). In addition, as shown in Figures 1C, 1D, 1G, 1H, 1K, and 1L, the secE and secY ribosome binding sites are indicated as underlined nucleotides. [Figure 2]Figure 2 shows the mature amino acid sequences of natural B. clausii subtilisin (Figure 2A; Reporter 1, Sequence ID 21), natural B. amyloliquefaciens subtilisin (Figure 2B; Reporter 2, Sequence ID 22), and natural B. gibsonii subtilisin (Figure 2C; Reporter 3, Sequence ID 23). More specifically, as shown in Figure 2, the natural B. clausii subtilisin reporter contains 269 amino acid residues with a theoretical isoelectric point (pI) of approximately 9.30 and a molecular weight (Mw) of approximately 26.725 Da (Figure 2A), the natural B. amyloliquefaciens subtilisin reporter contains 275 amino acid residues with a theoretical pI of approximately 6.30 and an Mw of approximately 27,533 Da (Figure 2B), and the natural B. gibsonii subtilisin reporter contains 269 amino acid residues with a theoretical pI of approximately 9.57 and an Mw of approximately 27,498 Da (Figure 2C). [Figure 3] Figure 3 shows the BLAST-P alignment of the natural B. clausii subtilisin (mature sequence; SEQ ID NO: 21) to the natural B. amyloliquefaciens subtilisin (mature sequence; SEQ ID NO: 22). As shown in Figure 3, SEQ ID NOs. 21 and 22 contain approximately 60% amino acid identity. [Figure 4] Figure 4 shows the BLAST-P alignment of the natural B. gibsonii subtilisin (mature sequence; SEQ ID NO: 23) to the natural B. amyloliquefaciens subtilisin (mature sequence; SEQ ID NO: 22). As shown in Figure 4, SEQ ID NO: 23 and SEQ ID NO: 22 contain approximately 57% amino acid identity. [Figure 5]Figure 5 shows the BLAST-P alignment of the natural B. clausii subtilisin (mature sequence; SEQ ID NO: 21) to the natural B. gibsonii subtilisin (mature sequence; SEQ ID NO: 23). As shown in Figure 5, SEQ ID NOs. 21 and 23 contain approximately 81% amino acid identity. [Modes for carrying out the invention]

[0013] A brief explanation of biological sequences Sequence ID 1 is a nucleic acid (DNA) promoter and a 5'-UTR sequence named "PsecG," which contains a functional combination of the Bacillus subtilis secG promoter and secG 5'-UTR sequence. Sequence ID 2 is a DNA sequence containing a Bacillus subtilis secG open reading frame (ORF) that encodes the natural SecG protein. Sequence ID 3 is a DNA sequence containing a Bacillus subtilis ribosome-binding sequence (RBS) and a secE ORF encoding a native SecE protein. Sequence ID 4 is a DNA sequence containing the Bacillus subtilis RBS and a secY ORF encoding the natural SecY protein. Sequence ID 5 is a DNA sequence containing the BPN' terminator region of Bacillus amyloliquefaciens. Sequence ID 6 is the downstream (3') Bacillus subtilis (B. subtilis) pksR embedded cassette homologous region (HR). Sequence ID 7 is a synthetic DNA sequence containing a non-natural secGEY operon named "PsecG-secGEY". Sequence ID 8 is a DNA sequence containing the Bacillus subtilis (B. subtilis) alanine racemase (alrA) gene. Sequence ID 9 is an upstream (5') Bacillus subtilis (B. subtilis) pksR embedded cassette HR. SEQ ID NO: 10 is a synthetic DNA promoter region named "PspoVG", which contains the Bacillus subtilis spoVG promoter and the spoVG 5'-UTR sequence in an operable combination. SEQ ID NO: 11 is a synthetic DNA sequence containing a non-natural secGEY operon named "PspoVG-secGEY". SEQ ID NO: 12 is a synthetic DNA promoter region named "Phbs", which contains the Bacillus subtilis hbs promoter and the Bacillus subtilis spoVG 5'-UTR sequence in an operable combination. SEQ ID NO: 13 is a synthetic DNA sequence containing a non-natural secGEY operon named "Phbs-secGEY". SEQ ID NO: 14 is the upstream (5') Bacillus subtilis aprE integration cassette homologous region (HR). SEQ ID NO: 15 is a synthetic DNA sequence containing the B. licheniformis citZ promoter and the kanamycin resistance gene. SEQ ID NO: 16 is the downstream (3') Bacillus subtilis aprE integration cassette HR. SEQ ID NO: 17 is the Bacillus subtilis AprE signal (secretion) peptide amino acid sequence. SEQ ID NO: 18 is the native pro-region amino acid sequence of the B. lentus subtilisin protease. SEQ ID NO: 19 is the signal peptide amino acid sequence of the B. amyloliquefaciens subtilisin (BPN') protease. SEQ ID NO: 20 is the native pro-region amino acid sequence of the B. amyloliquefaciens subtilisin (BPN') protease. SEQ ID NO: 21 is the mature amino acid sequence of the native B. clausii subtilisin protease. Sequence number 22 is the mature amino acid sequence of a native B. amyloliquefaciens subtilisin protease. Sequence number 23 is the mature amino acid sequence of a native B. gibsonii subtilisin protease. Sequence number 24 is an artificial nucleic acid promoter sequence named the "P2" promoter. Sequence number 25 is an artificial nucleic acid promoter sequence named the "P2-00788" promoter. Sequence number 26 is an artificial nucleic acid promoter sequence named the "P4" promoter. Sequence number 27 is a DNA sequence containing the B. subtilis spoVG terminator (term) sequence. Sequence number 28 is the amino acid sequence of the native B. subtilis secG protein. Sequence number 29 is the amino acid sequence of the native B. subtilis secE protein. Sequence number 30 is the amino acid sequence of the native B. subtilis secY protein. Sequence number 31 is the DNA sequence of the wild-type B. subtilis secE open reading frame encoding the native secE protein of sequence number 29. Sequence number 32 is the DNA sequence of the wild-type B. subtilis secY open reading frame encoding the native secY protein of sequence number 30.

[0014] Detailed description As described herein, certain embodiments of this disclosure relate to compositions and methods for enhancing protein production in Gram-positive bacterial (host) cells. Accordingly, certain one or more embodiments of this disclosure provide, among other things, Gram-positive bacterial cells / strains expressing heterologous proteins, compositions and methods thereof; Gram-positive bacterial cells secreting heterologous proteins into fermentation broth, compositions and methods thereof; Gram-positive bacterial cells comprising introduced (non-natural) secGEY operons expressing SecG, SecE, and SecY translocon proteins, compositions and methods thereof; and / or combinations thereof. In one or more other specific embodiments, the Disclosure relates to nucleic acid (DNA) sequences encoding non-natural secGEY operons, SecG, SecE, and SecY translocon proteins (e.g., secG ORFs encoding a functional SecG protein, secE ORFs encoding a functional SecE protein, secY ORFs 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 signaling (secreted) peptide sequences, DNA sequences encoding protease PRO region sequences, DNA sequences encoding one or more ribosome-binding sites (RBS), promoter region (DNA) sequences, and 5' untranslated region (5'-UTR) sequences, etc.

[0015] I. Definition In consideration of the compositions and methods described herein, which are further described herein, the following terms and phrases are defined. Terms not defined herein shall have the common meanings used in the art.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the compositions and methods of the present invention belong. Any methods and materials similar to or equivalent to those described herein may be used to carry out or test the compositions and methods of the present invention, but representative methods and materials are described below. All publications and patents cited herein are incorporated herein by reference in their entirety.

[0017] It should be further noted that claims may be written in a manner that excludes optional elements. Therefore, this statement is intended to serve as a precedent for the use of exclusive terms such as “solely,” “only,” “excluding,” and “not including,” or for the use of “negative” limitations or conditions, in relation to the enumeration of elements in the claims. For example, in a particular embodiment, a control (isogenetic) Gram-positive bacterial cell is constructed, and this control cell “does not contain” (i.e., excludes) the introduced (synthesized) secGEY operon.

[0018] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described herein has individual components and features that can be readily separated from or combined with any of the features of some of the other embodiments without departing from the scope or spirit of the compositions and methods described herein. Any of the described methods may be carried out in the order of events described, or in any other logically conceivable order.

[0019] As generally described herein and as shown in the examples, certain embodiments of this disclosure provide, among other things, recombinant Gram-positive bacterial cells comprising a transduced (non-natural) secGEY operon expressing a protein of interest and expressing SecG, SecE, and SecY translocon proteins, and methods for producing one or more proteins of interest in Gram-positive bacterial cells comprising a transduced (non-natural) secGEY operon expressing SecG, SecE, and SecY proteins.

[0020] The phrases “Gram-positive bacteria,” “Gram-positive cells,” “Gram-positive strains,” and / or “Gram-positive bacterial cells” as used herein have the same meanings as they do in the art. For example, Gram-positive bacterial cells include all strains of the phyla Actinobacteria and Firmicutes. In certain embodiments, such Gram-positive bacteria are of the classes Bacilli, Clostridia, and Mollicutes.

[0021] As used herein, the genus "Bacillus" includes all species within the genus "Bacillus" known to those skilled in the art, including Bacillus subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, Examples include, but are not limited to, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulars, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus is constantly undergoing taxonomic reorganization. Therefore, the genus includes reclassified species, and is not limited to the following, but includes organisms such as B. stearothermophilus, now called Geobacillus stearothermophilus.

[0022] In this specification, a particular range is indicated by a number preceded by the term “approximately”. In this specification, the term “approximately” is used to provide literal support for the exact number preceded by the term, and for numbers that are close to or approximate to the number preceded by the term. In determining whether a number is close to or approximate to a specifically enumerated number, a close or approximate number that is not enumerated may be a number that, in the context in which it is shown, provides a substantial equivalent of the specifically enumerated number. For example, with respect to a number, the term “approximately” refers to a range of -10% to +10% of that number, unless the term is otherwise explicitly defined in the context.

[0023] The term “derived from” includes the terms “resulting from,” “obtained from,” “available from,” and “created from,” and generally indicates that one particular material or composition has its origins in another particular material or composition, or has features that can be described by reference to another particular material or composition. For example, the recombinant Gram-positive bacterial cells of this disclosure may be derived from / obtained from any known Gram-positive bacterial strain.

[0024] 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 manipulated genetic modification or is modified by the introduction of a heterologous nucleic acid molecule, or a cell (e.g., a microbial cell) that has been modified so that the expression of heterologous or endogenous nucleic acid molecules or genes can be controlled. Recombinant also refers to a cell that originates from or is a progeny of a non-natural cell having one or more such modifications. Examples of genetic modifications include modifications that introduce an expressible nucleic acid molecule encoding a protein, or the addition, deletion, replacement, and / or other functional modifications of other nucleic acid molecules in the cell’s genetic material. For example, recombinant cells may express genes or other nucleic acid molecules (e.g., fusion or chimeric proteins) that are not found in the same or homologous form in natural (wild-type) cells, or they may be overexpressed, underexpressed, minimally expressed, or not expressed at all, thus providing an altered expression pattern of endogenous genes. "Recombination," "recombination," or the formation of "recombinant" nucleic acids generally refers to an aggregate of two or more nucleic acid fragments, which generates a chimeric gene.

[0025] As used herein, “nucleic acid” refers to nucleotides or polynucleotide sequences, which may represent either sense strands or antisense strands, and which may be double-stranded or single-stranded, as well as fragments or parts thereof, and DNA, cDNA, and RNA of genomic or synthetic origin. As a result of the degeneracy of the genetic code, it will be understood that a large number of nucleotide sequences may encode a given protein.

[0026] It is understood that the polynucleotides (or nucleic acid molecules) described herein include “genes,” “vectors,” and “plasmids.”

[0027] Therefore, the term "gene" refers to a polynucleotide that codes for a specific sequence of amino acids, including all or part of the coding sequence of a protein, and may include regulatory (non-transcribed) DNA sequences such as promoter sequences that determine the conditions under which a gene is expressed. The transcription region of a gene may include introns, 5'-untranslated regions (UTRs), and untranslated regions (UTRs) including the 3'-UTR, as well as the coding sequence.

[0028] As used herein, “endogenous gene” refers to a gene located in its natural position within the genome of an organism.

[0029] As used herein, the terms “heterogeneous,” “non-endogenous,” or “foreign” genes refer to genes that are not normally found in a host organism but are introduced into the host organism through gene transfer. The term “foreign” gene includes native genes inserted into non-natural organisms and / or chimeric genes inserted into natural or non-natural organisms.

[0030] As used herein, “heterogeneous regulatory sequences” refer to gene expression regulatory sequences (e.g., promoters, enhancers, terminators, etc.) that do not function in nature to regulate (control) the expression of the gene of interest. Generally, heterogeneous nucleic acids are not endogenous (natural) to the cell or part of the genome in which they are present, but are added to the cell by infection, transfection, transformation, transduction, microinjection, and electroporation, etc. A “heterogeneous” nucleic acid construct may contain the same or different combinations of regulatory sequences and DNA coding sequences (ORFs) as those found in natural host cells.

[0031] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecules of this disclosure. Expression may also refer to the translation of mRNA into polypeptides. Therefore, the term "expression" includes, but is not limited to, all processes involved in polypeptide production, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0032] As used herein, the term "coding sequence" refers to a nucleotide sequence that directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of a coding sequence are generally determined by an open reading frame (hereinafter "ORF"), which typically begins with an ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0033] As used herein, terms such as “promoter,” “promoter region,” “promoter element,” and “promoter sequence” refer to nucleic acid (DNA) sequences that can control the transcription of a gene coding sequence (CDS / ORF) to messenger RNA (mRNA) when the promoter region sequence is located upstream (5') and operably ligated to a downstream (3') gene (ORF). As will be generally understood by those skilled in the art, a promoter typically provides a site for specific binding by RNA polymerase and transcription initiation. In certain embodiments, the term “promoter” refers to the minimum portion of the promoter nucleic acid sequence required for transcription initiation (i.e., including the RNA polymerase binding site). For example, a promoter generally includes a “-10” (consensus sequence) element and a “-35” (consensus sequence) element located upstream (5') of the target gene CDS and relative to the +1 transcription initiation site (TSS). The core promoter elements, -10 and -35, are commonly referred to in the art as the "TATAAT" (Privnowbox) consensus region and the "TTGACA" consensus region, respectively. The core promoter (-10 and -35) regions are generally separated by approximately 15 to 20 intervening base pairs (nucleotides).

[0034] A promoter may be entirely derived from a native gene, or composed of various elements derived from various naturally occurring promoters, or may further include synthetic nucleic acid segments. Those skilled in the art will understand that various promoters can direct gene expression in various cell types, at various developmental stages, or in response to various environmental or physiological conditions. Promoters may include constitutive promoters, inductive promoters, tunable promoters, hybrid promoters, synthetic promoters, tandem promoters, etc. The promoter that elicits the most gene expression in most cell types is generally referred to as a "constitutive promoter." Furthermore, since the precise boundaries of regulatory sequences are often not fully understood, it is recognized that DNA fragments of various lengths may possess identical promoter activity.

[0035] As used herein, a “functional promoter sequence” that controls the expression of a gene of interest, ligated to the protein-coding sequence of the gene of interest, refers to a promoter sequence that controls the transcription and translation of the coding sequence in a desired Gram-positive host cell. For example, in a particular embodiment, the disclosure provides a polynucleotide comprising an upstream (5') promoter that functions in Gram-positive cells, wherein the functional promoter region is operably ligated to a nucleic acid sequence encoding a protein of interest.

[0036] As used herein, the term “precursor protein” refers to an inactive protein. In certain embodiments, a full-length protein is synthesized as a pro-sequence precursor to a mature protein (abbreviated as “preprotein”). In other embodiments, a full-length protein is synthesized as a signal peptide sequence, a pro-sequence, and a precursor to a mature protein (abbreviated as “preproprotein”). For example, a pre-sequence typically acts as a signal peptide for transport, and a pro-sequence is typically essential for the correct folding of the associated (mature) protein.

[0037] As used herein, the term "mature protein" refers to an active protein, as opposed to an inactive precursor (full-length) protein.

[0038] As used herein, the terms “signal sequence,” “secretory signal,” and “signal peptide” may be used interchangeably and refer to sequences of amino acid residues involved in the secretion or direct transport of precursor proteins. Signal (pre) sequences are typically cleaved from precursor proteins by signal peptidases during translocation. Signal (pre) sequences are typically positioned at the N-terminus of a mature protein sequence or at the N-terminus of a pro-region (PRO) sequence when a signal (pre) sequence and a pro-region (PRO) sequence are used upstream (5') of a mature POI sequence in a responsive combination.

[0039] The terms “pro sequence,” “pro-sequence,” and “pro region sequence” as used herein may be used interchangeably and may be abbreviated as “PRO” sequence. The term “pro sequence” as used herein has the same meaning as understood in the art. For example, “subtilisin,” an alkaline serine protease from Bacillus subtilis, is first produced as preprosubtilisin, which consists of a signal (pre) sequence for protein secretion, followed by a 77-amino acid pro region (PRO) sequence, and then an amino acid sequence encoding mature subtilisin (e.g., preprosubtilisin). The pro sequence is often essential for the correct folding of the associated (mature) protein and acts as an intramolecular chaperone (e.g., directly catalyzing the protein folding reaction). Similarly, the pro sequence may be required for both the folding and intracellular transport (or secretion) of the mature protein of interest, suggesting that these two functions are closely related.

[0040] The phrases “five prime(5')untranslated region,” “5' untranslated region,” and / or “5' transcript reader” as used herein may be used interchangeably and may be abbreviated as “5'-UTR.” As is generally understood in the art, the 5'-UTR is known to be a region of messenger RNA (mRNA) located immediately upstream (5') of the start codon.

[0041] When a nucleic acid is functionally related to another nucleic acid sequence, the nucleic acids are "operably ligated." For example, DNA encoding a secretory leader (i.e., a signal sequence) is operably ligated to the DNA encoding the polypeptide if it is expressed as a preprotein involved in polypeptide secretion; a promoter or enhancer is operably ligated to a coding sequence (CDS, ORF) if it affects the transcription of the sequence; or a ribosome binding site (RBS) is operably ligated to a coding sequence if it is positioned to facilitate translation. Generally, "operably ligated" means that the ligated DNA sequences are adjacent and, in the case of a secretory leader, are adjacent to the reading phase. However, enhancers do not need to be adjacent. Ligation is achieved by ligation at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or linker is used according to conventional practice. For this reason, the term "operably ligated" generally refers to the association (juxtaposition) of nucleic acid sequences on a single nucleic acid fragment such that the function of one is influenced by the function of the other. For example, if a promoter (pro) controls the transcription of the gene CDS, it is operablely ligated to the gene coding sequence (gene CDS) (e.g., 5'-[pro]-[gene CDS]-3').

[0042] As used herein, “preferred regulatory sequence” refers to a nucleotide sequence located upstream (5' non-coding sequence), within the sequence, or downstream (3' non-coding sequence) of a coding sequence, and which affects the transcription, RNA processing, stability, or translation of the associated coding sequence. Examples of regulatory sequences include promoters, translational leader sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0043] In a particular embodiment, an upstream (5') promoter (pro) region sequence, a downstream DNA sequence (ss) operably ligated thereto encoding a signal peptide (secretion) sequence, and a downstream (3') DNA sequence (ORF) operably ligated thereto encoding the mature protein of interest may be schematically represented as 5'-[pro]-[ss]-[ORF]-3'. In a particular other embodiment, an upstream promoter (pro) region sequence, a downstream DNA sequence (ss) operably ligated thereto encoding a signal peptide (secretion) sequence, a downstream DNA sequence (PRO) operably ligated thereto encoding the pro region amino acid sequence, and a downstream DNA sequence (ORF) operably ligated thereto encoding the mature protein of interest may be schematically represented as 5'-[pro]-[ss]-[PRO]-[ORF]-3'. In a particular other embodiment, an upstream promoter (pro) region sequence, operably ligated thereto, a downstream DNA sequence (ss) encoding a signal peptide (secretion) sequence, operably ligated thereto, a downstream DNA sequence (PRO) encoding a pro region amino acid sequence, operably ligated thereto, a downstream DNA sequence (ORF) encoding the mature protein of interest, and operably ligated thereto, a downstream terminator (term) sequence may be schematically represented as 5'-[pro]-[ss]-[PRO]-[ORF]-[term]-3'.

[0044] In this specification, the promoter region sequence containing the Bacillus subtilis secG promoter and secG 5'-UTR in an operable combination will be abbreviated as "PsecG" (SEQ ID NO: 1), the promoter region sequence containing the Bacillus subtilis spoVG promoter and spoVG 5'-UTR sequence in an operable combination will be abbreviated as "PspoVG" (SEQ ID NO: 10), and the promoter region sequence containing the Bacillus subtilis hbs promoter and spoVG 5'-UTR sequence in an operable combination will be abbreviated as "Phbs" (SEQ ID NO: 12).

[0045] As used herein, the “secG” open reading frame (ORF; SEQ ID NO: 2) encodes the natural Bacillus subtilis SecG transloconprotein (SEQ ID NO: 28), the “secE” ORF (SEQ ID NO: 31) encodes the natural Bacillus subtilis SecE transloconprotein (SEQ ID NO: 29), and the “secY” ORF (SEQ ID NO: 32) encodes the natural Bacillus subtilis SecY transloconprotein (SEQ ID NO: 30).

[0046] As used herein, the phrase “non-natural (artificial) secGEY operon” refers to one or more synthetic expression vectors / cassettes of this disclosure that express extra copies of Bacillus subtilis (B. subtilis) secG, secE, and secY genes (ORFs) encoding the natural SecG, SecE, and SecY (translocon) proteins. In certain one or more embodiments or aspects, recombinant Gram-positive cells containing an introduced (i.e., non-natural) secGEY operon may be described as recombinant (modified) cells that “overexpress” the natural SecG, SecE, and SecY translocon proteins.

[0047] The phrases “non-natural secGEY operon” and / or “recombinant cells overexpressing SecG, SecE, and SecY transloconproteins” as used herein do not imply any restriction on the order of the Sec transloconproteins encoded by the non-natural secGEY operon. For example, a person skilled in the art can easily construct a non-natural secGEY operon in which the ORFs encoding the Sec transloconproteins are in any order, for example, a non-natural secGEY operon comprising an upstream secE ORF, a downstream secG ORF operably linked thereto, a downstream secYORF operably linked thereto, or a non-natural secGEY operon comprising an upstream secE ORF, a downstream secY ORF operably linked thereto, a downstream secG ORF operably linked thereto, or a non-natural secGEY operon comprising an upstream secY ORF, a downstream secG ORF operably linked thereto, a downstream secE ORF operably linked thereto.

[0048] Similarly, as shown in Figure 1, the ORFs at the second (e.g., Figures 1C and 1G) and / or third positions (e.g., Figures 1D and 1H) of the non-natural secGEY operon may further contain a ribosome binding site immediately upstream (5') of the ORF encoding the Sec translocon protein. For example, the 20 nucleotides containing the ribosome binding site (RBS) immediately upstream of the secE ORF are underlined in Figure 1C (SEQ ID NO: 3) and Figure 1G (SEQ ID NO: 3), and the 20 nucleotides containing the ribosome binding site (RBS) immediately upstream of the secY ORF are underlined in Figure 1D (SEQ ID NO: 4) and Figure 1H (SEQ ID NO: 4).

[0049] As used herein, the unnatural secGEY operon named "PsecG-secGEY" (operon 1; SEQ ID NO: 7) comprises an upstream (5') PsecG promoter region sequence (Figure 1A; SEQ ID NO: 1), an operably coupled downstream secG ORF (Figure 1B; SEQ ID NO: 2), an operably coupled downstream secE ORF (Figure 1C; SEQ ID NO: 3), an operably coupled downstream secY ORF (Figure 1D; SEQ ID NO: 4), and an operably coupled downstream (3) BPN terminator sequence (SEQ ID NO: 5). The unnatural secGEY operon named "PspoVG-secGEY" (operon 2; SEQ ID NO: 11) comprises an upstream PspoVG promoter region sequence (Figure 1E; SEQ ID NO: 10), an operably coupled downstream secG ORF (Figure 1F; SEQ ID NO: 2), an operably coupled secE ORF (Figure 1G; SEQ ID NO: 3), and an operably coupled downstream secY An unnatural secGEY operon named "Phbs-secGEY" (operon 3; sequence number 13) includes an ORF (Figure 1H; sequence number 4), an operably coupled downstream (3)BPN terminator sequence (sequence number 5) thereto, an upstream Phbs promoter region sequence (Figure 1I; sequence number 12), an operably coupled downstream secG ORF (Figure 1J; sequence number 2) thereto, an operably coupled downstream secE ORF (Figure 1K; sequence number 3) thereto, an operably coupled downstream secY ORF (Figure 1L; sequence number 4) thereto, and an operably coupled downstream (3)BPN terminator sequence (sequence number 5) thereto.

[0050] Exemplary proteases as used herein may be referred to as “reporter proteins.” In certain embodiments of this disclosure, the exemplary reporter proteins are expressed / produced by one or more recombinant (modified) cells of this disclosure. In certain embodiments, the reporter proteins include, but are not limited to, natural and variant Bacillus species subtilisins. In one or more specific embodiments, exemplary subtilisin reporters include, but are not limited to, natural B. clausii subtilisin and its functional variant, natural B. gibsonii subtilisin and its functional variant, natural B. lentus subtilisin and its functional variant, natural B. licheniformis subtilisin (AprL) and its functional variant, natural Bacillus subtilis subtilis (AprE) and its functional variant, and natural B. amyloliquefaciens subtilis (BPN') and its functional variant. In certain embodiments, the subtilisin reporters of exemplary B. clausii, B. gibsonii, and / or B. lentus may be referred to as alkaline proteases. For example, alkaline subtilisins generally have an isoelectric point (pI) of about 9.5, while the subtilisins of B. licheniformis, B. subtilis, and B. amyloliquefaciens have a pI of about 6.5.

[0051] As used herein, the term "subtilisin" refers to all members of the S8 serine protease family as described in MEROPS - The Peptidase Database (Rawlings et al., 2006). The term subtilisin includes a wide variety of identified and sequenced Bacillus subtilisins, such as subtilisin 168, subtilisin BPN', subtilisin Carlsberg, and variant proteases derived therefrom.

[0052] The phrases “subtilisin-1,” “subtilisin-1 reporter,” “reporter 1,” and “reporter 1 protease,” as used herein, may be used interchangeably and in particular refer to the natural B. clausii subtilisin shown in Sequence ID No. 21, or its functional variants.

[0053] The phrases “subtilisin-2,” “subtilisin-2 reporter,” “reporter 2,” and “reporter 2 protease,” as used herein, may be used interchangeably and in particular refer to the natural B. amyloliquefaciens subtilisin shown in SEQ ID NO: 22, or its functional variants.

[0054] The phrases “subtilisin-3,” “subtilisin-3 reporter,” “reporter 3,” and “reporter 3 protease,” as used herein, may be used interchangeably and in particular refer to the natural B. gibsonii subtilisin shown in SEQ ID NO: 23, or its functional variants.

[0055] In certain embodiments, the disclosure relates to one or more variant subtilisins derived from parent (natural) subtilis sequences, such as natural Bacillus subtilis subtilis (e.g., 168), natural B. amyloliquefaciens (e.g., BPN'), natural B. licheniformis subtilis (e.g., Carlsberg), natural B. lentus subtilis (e.g., 309), and B. alcalophilus subtilis (e.g., PB92). For example, in certain embodiments, the Disclosure provides recombinant expression cassettes encoding functional subtilisin variants derived from natural B. clausii subtilisin (SEQ ID NO: 21), natural B. amyloliquefaciens subtilisin (SEQ ID NO: 22), and natural B. gibsonii subtilisin (SEQ ID NO: 23), etc. More specifically, those skilled in the art can easily design, construct, screen, and identify functional subtilisin variants using routine methods known in the art. In particular, International Publication Nos. 2010 / 056634, 2011 / 130222, 2015 / 089447, 2016 / 202839, 2017 / 207762, and 2023 / 114936 (each incorporated herein by reference in its entirety) describe suitable methods and compositions for constructing functional subtilisin variants derived from natural B. clausii subtilisin (SEQ ID NO: 21), functional subtilisin variants derived from natural B. amyloliquefaciens subtilisin (SEQ ID NO: 22), and functional subtilisin variants derived from natural B. gibsonii subtilisin (SEQ ID NO: 23), etc.

[0056] As used herein, the Bacillus subtilis strain named “CZ437” comprises two transfected (integrated) expression cassettes encoding reporter 1 protease, the cassette comprising, in a 5'-to-3' direction and in an operable combination, an artificial “P2” promoter sequence (SEQ ID NO: 24), linked thereto, a DNA sequence (ss) encoding an aprE signal peptide sequence (SEQ ID NO: 17), linked thereto, a DNA sequence encoding a pro-region sequence (SEQ ID NO: 18), and linked thereto, a DNA sequence encoding reporter 1 (SEQ ID NO: 21). In certain embodiments, strain CZ437 may be referred to as a control (isogenetic) cell, in particular, when compared to one or more modified strains (e.g., strains BPC0123, BPC0182, BPC0184) that overexpress SecG, SecE, and SecY transloconproteins.

[0057] As used herein, the Bacillus subtilis strain named "BPC0178" comprises three transfected (integrated) expression cassettes encoding reporter 2 protease, the cassette comprising, in a 5'-to-3' direction and in an operable combination, an artificial "P4" promoter sequence (SEQ ID NO: 26), a DNA sequence (ss) (SEQ ID NO: 19) linked thereto, encoding a BPN' signal peptide sequence, a DNA sequence (SEQ ID NO: 20) linked thereto, encoding a BPN' pro-region sequence, and a DNA sequence (SEQ ID NO: 22) linked thereto. In certain embodiments, the BPC0178 strain may be referred to as a control cell, in particular, when compared to one or more modified strains (e.g., BPC0166, BPC0167, BPC0168) that overexpress SecG, SecE, and SecY transloconproteins.

[0058] As used herein, the Bacillus subtilis strain named "BPC0229" comprises two transfected (integrated) expression cassettes encoding reporter 3 protease, the cassette comprising, in a 5'-to-3' direction and in an operable combination, an artificial "P2-0078" promoter sequence (SEQ ID NO: 25), a DNA sequence (ss) (SEQ ID NO: 17) linked thereto, encoding an aprE signal peptide sequence, a DNA sequence (SEQ ID NO: 18) linked thereto, and a DNA sequence (SEQ ID NO: 23) linked thereto, encoding a pro-region sequence. In certain embodiments, the BPC0229 strain may be referred to as a control cell, in particular, when compared to one or more modified strains (e.g., AL394, AL395, AL396) that overexpress SecG, SecE, and SecY transloconproteins.

[0059] As used herein, “host cell” refers to a cell capable of acting as a host or expression vehicle for a newly introduced DNA sequence. Thus, in certain embodiments of this disclosure, the host cell is a Gram-positive (e.g., Bacillus) and / or Gram-negative (e.g., Escherichia coli) cell.

[0060] As used herein, “modified cell” refers to a recombinant cell that contains at least one genetic modification that is not present in the parent cell, reference cell, or control cell from which the modified cell is derived.

[0061] In this specification, when the expression and / or production of a protein of interest (POI) in recombinant (modified) cells is compared with the expression and / or production of the same POI in unmodified (control) cells, it will be understood that the modified and unmodified cells are grown / cultured / fermented under the same conditions (e.g., the same conditions such as medium, temperature, and pH).

[0062] As used herein, "increased quantity" refers specifically to an "increased quantity" of the protein of interest (POI) expressed / produced by recombinant cells, when used in phrases such as "recombinant cells express / produce an increased quantity of the protein of interest compared to unmodified (control) cells," but this "increased quantity" is always compared to unmodified (control) cells expressing / producing the same POI, and modified and unmodified cells are grown / cultured / fermented under the same conditions.

[0063] As used herein, “increasing” or “enhancing” protein production means that an increased amount of protein (e.g., the protein of interest) is produced. The protein may be produced within 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 levels of protein or enzyme activity (e.g., amylase activity) compared to the parental host cell, or as total extracellular protein produced.

[0064] The terms “modification” and “genetic modification” as used herein are interchangeable and include: (a) introduction, replacement, or removal of one or more nucleotides in any of the genes disclosed herein (or its ORF), or introduction, substitution, or removal of one or more nucleotides in a regulatory element necessary for the transcription or translation of the gene or its ORF; (b) gene disruption; (c) gene transformation; (d) gene deletion; (e) gene downregulation; (f) specific mutagenesis; and / or (g) random mutagenesis.

[0065] In certain embodiments, genetic modification refers in particular to the introduction, replacement, or removal of one or more nucleotides in a nucleic acid (DNA) sequence encoding a pro-region (amino acid) sequence of the present disclosure. For example, in certain embodiments, the DNA sequence encoding the natural pro-region amino acid sequence described in Sequence ID No. 18 is genetically modified as described herein.

[0066] The term "introduce" as used in phrases such as "introducing 'genes,' 'polynucleotides,' 'open reading frames' (ORFs), 'genetic coding sequences,' 'vectors,' and 'expression cassettes,' etc., into Gram-positive bacterial cells" includes, but is not limited to, methods known in the art for introducing polynucleotides (DNA) into cells, including, protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, and conjugation.

[0067] As used herein, “transformed” or “transformed” refers to cells that have been transformed by the use of recombinant DNA technology. Transformation typically occurs by inserting one or more nucleotide sequences (e.g., polynucleotides, ORFs, or genes) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence not naturally present in the cell being transformed). Thus, transformation generally refers to the introduction of exogenous DNA into a host cell such that the DNA is maintained as a chromosomal integration body or a self-replicating extrachromosomal vector.

[0068] As used herein, “transformed DNA,” “transformed sequence,” and “DNA construct” refer to DNA used to introduce a sequence into a host cell or organism. Transformed DNA is DNA used to introduce a sequence into a host cell or organism. Such DNA may be produced in vitro by PCR or any other suitable technique. In some embodiments, the transformed DNA includes an incoming sequence, while in other embodiments, the transformed DNA further includes an incoming sequence flanked by a homology box. In yet another embodiment, the transformed DNA includes other non-homologous sequences (i.e., stuffer sequences or flanks) appended to its ends. The ends can be closed so that the transformed DNA forms a ring, for example, by insertion into a vector.

[0069] As used herein, “gene disruption” or “gene disruption” is used interchangeably and broadly refers to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., a protein). Therefore, as used herein, gene disruption includes, but is not limited to, frameshift mutations, immature stop codons (i.e., those that prevent the production of a functional protein), replacements that eliminate or reduce the activity of an internal protein deletion (that prevents the production of a functional protein), and insertions that disrupt coding sequences, as well as mutations that remove the operable link between the native promoter and the open reading frame necessary for transcription.

[0070] As used herein, “introduced sequence” refers to a DNA sequence introduced into a bacterial cell chromosome. In some embodiments, the introduced sequence is part of a DNA construct. In other embodiments, the introduced sequence encodes one or more proteins of interest. In some embodiments, the introduced sequence includes sequences that may or may not already be present in the genome of the cell to be transformed (i.e., homologous or heterologous sequences). In some embodiments, the introduced sequence encodes one or more proteins, genes, and / or mutated or modified genes of interest. In alternative embodiments, the introduced sequence encodes a functional wild-type gene or operon, a functional mutated gene or operon, or a non-functional gene or operon. In some embodiments, the non-functional sequence may be inserted into a gene to disrupt its function. In another embodiment, the introduced sequence includes a selection marker. In further embodiments, the introduced sequence includes two homology boxes.

[0071] As used herein, “homology box” refers to a nucleic acid sequence homologous to a sequence within a bacterial cell chromosome. More specifically, a homology box is an upstream or downstream region having about 80–100% sequence identity, about 90–100% sequence identity, or about 95–100% sequence identity to a direct flanking coding region of a gene or part of a gene that is deleted, disrupted, inactivated, or downregulated according to the present invention. These sequences indicate where a DNA construct is incorporated into the bacterial cell chromosome and which portion of the chromosome is replaced by the incoming sequence. Not intended to limit the disclosure, homology boxes may range from about 1 base pair (bp) to 200 kilobases (kb). Preferably, homology boxes include about 1 bp–10.0 kb; 1 bp–5.0 kb; 1 bp–2.5 kb; 1 bp–1.0 kb; and 0.25 kb–2.5 kb. Furthermore, homology boxes may include approximately 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb, and 0.1 kb. In some embodiments, the 5' and 3' ends of the selection marker are flanked by homology boxes, where the homology boxes contain nucleic acid sequences that directly flank the coding region of the gene.

[0072] As used herein, the terms "host cell genome," "bacterial (host) cell genome," or "Bacillus species (host) cell genome" include chromosomal genes and extrachromosomal genes.

[0073] As used herein, the terms “plasmid,” “vector,” and “cassette” often refer to extrachromosomal elements that carry genes typically not part of the cell’s central metabolism and are usually in the form of circular double-stranded DNA molecules. Such elements may be linear or circular, single-stranded or double-stranded DNA or RNA self-replicating sequences, genomic integration sequences, phages, or nucleotide sequences derived from any source in which a number of nucleotide sequences are spliced ​​into a unique structure that can introduce a promoter fragment and DNA sequence for a selected gene product into the cell along with a suitable 3' untranslated sequence.

[0074] As used herein, the term "plasmid" refers to a circular double-stranded (ds)DNA construct used as a cloning vector and forming an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes. In some embodiments, the plasmid becomes integrated into the genome of a host cell. In some embodiments, the plasmid is present in the parent cell and lost in the daughter cell.

[0075] As used herein, "transformation cassette" refers to a specific vector that contains a gene (or its ORF) and, in addition to the exogenous gene, has an element that promotes the transformation of a particular host cell.

[0076] As used herein, the term "vector" refers to any nucleic acid that can replicate (proliferate) within a cell and deliver a new gene or DNA segment into the cell. Thus, the term refers to a nucleic acid construct designed for transport between various host cells. Examples of vectors include "epicosomes" (i.e., those that can replicate autonomously or be incorporated into the chromosomes of a host organism), such as viruses, bacteriophages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes such as YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), and PLAC (plant artificial chromosome).

[0077] An "expression vector" refers to a vector that has the ability to incorporate and express foreign DNA within a cell. Many expression vectors for prokaryotes and eukaryotes are commercially available and are known to those skilled in the art. The selection of an appropriate expression vector is within the scope of the knowledge of those skilled in the art.

[0078] As used herein, the terms “expression cassette” and “expression vector” refer to a nucleic acid construct resulting recombinantly or synthetically from a set of specific nucleic acid elements (i.e., these are the vectors or vector elements described herein) that enable the transcription of a particular nucleic acid within a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the DNA construct also includes a set of specific nucleic acid elements that enable the transcription of a particular nucleic acid within a target cell. In certain embodiments, the DNA construct of this disclosure includes a selection marker and an inactivated chromosomal segment or gene segment or DNA segment as defined herein.

[0079] As used herein, a “targeted vector” is a vector that contains a polynucleotide sequence homologous to a region in the chromosome of a host cell being transformed, and that can drive homologous recombination in that region. For example, a targeted vector is used to introduce mutations into the chromosome of a host cell by homologous recombination. In some embodiments, a targeted vector contains other non-homologous sequences, which are appended to the terminals, for example (i.e., stuffer sequences or flanking sequences). The terminals can be closed, for example, by insertion into the vector, so that the targeted vector forms a ring. For example, in a particular embodiment, parent B. licheniformis (host) cells are modified (e.g., transformed) by introducing one or more “targeted vectors” into them.

[0080] As used herein, the term “protein of interest” or “POI” refers to a polypeptide of interest that is desired to be expressed in modified (recombinant) Gram-positive host cells, and the POI is preferably expressed at an increased level (i.e., compared to “unmodified” (parent, control, isogeneic cells)). Thus, as used herein, the POI may be an enzyme, substrate-binding protein, surfactant protein, structural protein, and receptor protein, etc. In certain embodiments, the modified cells of this disclosure produce an increased amount of the heterologous protein of interest compared to control cells. In certain embodiments, the increase in the amount of the protein of interest produced by the modified cells of this disclosure is an increase of at least 0.5%, at least 1.0%, at least 5.0%, or greater than 5.0% compared to control cells.

[0081] Similarly, as defined herein, “gene of interest” or “GOI” refers to a nucleic acid sequence (e.g., polynucleotide, gene, or ORF) that encodes a “protein of interest.” A “gene of interest” encoding a “protein of interest” may be a naturally occurring gene, a mutant gene, or a synthetic gene.

[0082] As used herein, the terms “polypeptide” and “protein” are interchangeable and refer to polymers of any length containing amino acid residues linked by peptide bonds. Hereinafter, conventional single-letter or three-letter codes are used for amino acid residues. Polypeptides may be linear or branched, may contain modified amino acids, and may be cleaved by non-amino acids. Furthermore, the term polypeptide encompasses amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeling components. Also included within this definition are, for example, polypeptides containing one or more analogues of amino acids (including, for example, non-natural amino acids), and other modifications known in the art.

[0083] As used herein, the term “variant” polypeptide typically refers to a polypeptide derived from a parent (or reference) polypeptide by the replacement, addition, or deletion of one or more amino acids using recombinant DNA technology. A variant polypeptide may differ from the parent polypeptide by a small number of amino acid residues and may be defined by the level of homology / identity of its primary amino acid sequence with the parent (reference) polypeptide. Preferably, the variant polypeptide has amino acid sequence identity with the parent (reference) polypeptide sequence of 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%.

[0084] As used herein, “variant” polynucleotide means a polynucleotide that has a certain degree of sequence homology / identity with a parent polynucleotide or that hybridizes with a parent polynucleotide (or its complement) under stringent hybridization conditions. Preferably, the variant polynucleotide has nucleotide sequence identity of 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% with the parent (reference) polynucleotide sequence.

[0085] As used herein, “mutation” refers to any change or alteration within a nucleic acid sequence. Several types of mutations exist, including point mutations, deletion mutations, silent mutations, frameshift mutations, and splicing mutations. Mutations can be performed specifically (e.g., via site-directed mutagenesis) or randomly (e.g., via chemical agents, or through passaging through repair-negative bacterial strains).

[0086] In this specification, the term “substitution” as used in the context of polypeptides or their sequences means the substitution (i.e., replacement) of one amino acid with another.

[0087] As used herein, the term “homologousness” refers to homologous polynucleotides or homologous polypeptides. If two or more polynucleotides or two or more polypeptides are homologous, this means that the homologous polynucleotides or homologous polypeptides have a “degree of identity” of at least 60%, more preferably at least 70%, even more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, and most preferably at least 98%. Whether two polynucleotide sequences or polypeptide sequences have a sufficiently high degree of identity to be homologous as defined herein can be suitably examined by aligning the two sequences using a computer program known in the art, for example, "GAP" provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman and Wunsch, (1970). For DNA sequence comparison, use GAP with the following settings: GAP creation penalty 5.0 and GAP extension penalty 0.3).

[0088] As used herein, the term “identity percentage (%)” refers to the level of identity between nucleic acid sequences encoding polypeptides or between amino acid sequences of polypeptides when aligned using a sequence alignment program.

[0089] As used herein, "specific productivity" refers to the total amount of protein produced per unit time per cell over a given period of time.

[0090] As used herein, the terms “purified,” “isolated,” and “enriched” mean that a biomolecule (e.g., a polypeptide or polynucleotide) is modified from its native state by separating it from some or all of the native components from which it naturally associates. Such isolation or purification may be achieved by separation techniques known in the art, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation or other protein salting-out, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation, to remove undesirable whole cells, cell debris, impurities, foreign proteins, or enzymes from the final composition. The purified or isolated biomolecular composition may then be further enriched with components that provide additional benefits, such as activators, inhibitors, desirable ions, pH-controlling compounds, or other enzymes or chemicals.

[0091] II. Overexpression of the synthetic SecGEY operon in Gram-positive bacterial cells enhances protein production. As briefly shown above, the production of many heterologous proteins remains difficult and unpredictable in terms of protein titer and yield. In particular, bottlenecks in secretory protein production can vary depending on the host strain and protein sequence. In the context of Gram-positive bacterial hosts, the initial steps for protein secretion include the recognition of signal peptides and the targeting of preproteins to translocation mechanisms. For example, as outlined by Pohl and Harwood (2010), Bacillus secretory pathways include the Sec-dependent (Sec) pathway and the twin-arginine translocation (Tat) pathway, where the Sec pathway is responsible for the secretion of most proteins. As described by Pohl and Harwood (2010), preproteins are targeted to membrane-embedded Sec translocons, which are heterotrimeric protein complexes that form a main channel named SecYEG that facilitate the translocation of secretory proteins across the cytoplasmic membrane. Protein secretion via the Sec pathway is generally a complex system involving substrate recognition, intracellular chaperonening, piloting to Sec translocases, and post-translocation folding events.

[0092] In certain embodiments, Freudl (2018) describes the secretion of recombinant proteins into the culture supernatant of bacterial (host) expression systems, focusing particularly on protein export systems that require the fusion of a Sec-specific or Tat-specific signal peptide to the amino-terminus (N) of a desired target protein. For example, as concluded in this publication, the most promising method for finding the optimal signal peptide for a desired protein is to screen for the greatest possible diversity of signal peptides resulting from mutations of the signal peptide using a large signal peptide library, or alternatively, from the optimization of a given signal peptide using site-directed or random mutagenesis strategies. International Publication No. 1999 / 04006 describes relevant methods for secreting proteins in Gram-positive hosts, including expression vectors encoding Gram-positive microbial SecG proteins and transvestments encoding SecG proteins. International Publication No. 2008 / 141281 describes a modified secretory system for Gram-positive microbial hosts comprising a transvestment vector encoding a heterologous truncated SecG protein that can promote the secretion of a desired protein, the transvestment vector being able to replace or complement an endogenous (secG) gene encoding a native SecG protein. U.S. Patent Application Publication No. 2009 / 0029417 describes a recombinant microorganism that produces cellulase and overexpresses the Bacillus subtilis (B. subtilis) secY gene. International Publication No. 2008 / 126929 describes a recombinant microorganism that produces cellulase and overexpresses the Bacillus subtilis (B. subtilis) secY gene, which further requires the deletion of one or more spore-forming related genes.

[0093] In another embodiment, Mulder et al. (2013) described IPTG induction (P) for the expression of the α-amylase (amyQ) gene. gracThis publication describes recombinant Gram-positive bacterial host systems for secreting B. amyloliquefaciens α-amylase using a promoter. For example, as described in this publication, recombinant host cells are P grac amyQ gene cassette under promoter control, and xylose induction (P xyl ) This includes a secYEG gene cassette (for the expression of SecG, SecE, and SecY proteins) under the control of a promoter, where the synthesis of SecYEG and AmyQ was induced by 0.5% xylose and 100 μg of IPTG, respectively. Chen et al. (2015) describe a study aimed at identifying bottlenecks in the Sec pathway and improving the secretion of heterologous proteins by so-called molecular genetic techniques. For example, Chen et al. (2015) describes a study using strong constitutive P HpaII Promoter and subsequent natural signal peptide (SP) amyl and SP amyS We described recombinant cells expressing both α-amylases (AmyL and AmyS) under the control of [the gene], in which 23 major genes or gene operons involved in or closely related to the Sec pathway were overexpressed. From this, we concluded that deficiencies in the DnaK series PrsA lipoprotein and chaperone were the major rate-limiting factors for heterologous protein secretion.

[0094] More recently, studies from various laboratories have described the essential and non-essential contributions of various Sec pathway components to protein secretion in Bacillus subtilis (B. subtilis). In a particular aspect, Neef et al. (2020) compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases to the secretion efficiency of three highly expressed proteins (i.e., α-amylase AmyE from Bacillus subtilis, AmyL from B. licheniformis, and serine protease BPN from B. amyloliquefaciens). For example, as described in Neef et al. (2020), isogenic Bacillus subtilis strains lacking the chaperone DnaK, the translocase subunit SecDF or SecG, or the genes for the signal peptidases Sip, SipT, SipU, SipV, or SipW, or the genes for the cell envelope-associated proteases SppA, tepA, PrsW, WprA, YqeZ, HtrA, or HtrB were constructed. As summarized in this publication, the results show that mutations in secDF, secG, or rasP have a significant impact on the secretion of AmyE, AmyL, and BPN', although the actual effect size depends on the protein investigated. Furthermore, as concluded by Neef et al. (2020), the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion is unaffected by dnaK deletion, and deletion of certain sip genes revealed a strong differential effect of specific signal peptidases on the scale of the secretory stress response. Therefore, recombinant protein production in Bacillus subtilis cells remains challenging due to a bottleneck in the general Sec pathway and the bacterium's inherent ability to secrete a highly potent cocktail of proteases (Neef et al., 2021).In particular, Neef et al. (2021) examined the advantages and disadvantages of Bacillus subtilis and L. lactis, which are Gram-positive bacterial cell factories, and concluded that Bacillus subtilis and related Bacillus species are generally the most suitable for the mass production of recombinant proteins. However, they noted that Bacillus subtilis has high proteolytic activity, which represents a serious disadvantage. This can lead to product loss and / or accumulation of degraded product derivatives (which can be overcome by deleting protease genes), and the resulting strains are more susceptible to autodegradation, often resulting in an increased amount of contaminated cytoplasmic proteins in the fermentation broth.

[0095] Based on the foregoing, certain embodiments of this disclosure relate to surprising and unexpected results, which are described and illustrated below. More specifically, as described herein, the applicant has contemplated, designed and constructed recombinant Gram-positive (bacterial) cell / strains that overexpress SecG, SecE, and SecY transloconproteins from an introduced non-natural (synthetic) secGEY operon and express one or more introduced cassettes encoding subtilisin reporter proteins. In particular, as shown in Examples 1–6 of this disclosure, the applicant has surprisingly observed that overexpression of the complete complement of SecG, SecE, and SecY transloconproteins results in enhanced production of certain reporter proteins. Control and modified Bacillus subtilis cell / strains were constructed as further described in Examples 1, 3, and 5, and are summarized in Table 1 below.

[0096] [Table 1]

[0097] As presented and described in Example 2, the expression of reporter 1 protein in the presence of SecGEY overexpression was compared with the expression of the same reporter 1 protein without SecGEY overexpression. More specifically, three SecGEY overexpression strains (BPC0123, BPC0182, BPC0184) and the control strain CZ437 were constructed, and reporter 1 production was evaluated. For example, in the first experiment, the CZ437 control strain and the SecGEY overexpression strain BPC0123 were cultured, and protease activity was sampled after 24 and 40 hours of growth, as shown in Tables 2 and 3 (Example 2) below. Surprisingly, as shown in Table 3, compared to the CZ437 control strain after 40 hours of culture, it was observed that reporter 1 protein production was significantly increased (i.e., by approximately 60%) when the SecGEY protein was overexpressed from the secG promoter in strain BPC0123. Similarly, in the second experiment (Tables 4 and 5), the control strain CZ437 and the SecGEY overexpressing strains BPC0123, BPC0182, and BPC0184 were cultured, and protease activity was sampled after 22 hours of growth (Example 2). In particular, as shown in Table 5, surprisingly, when considering protease production per OD600, reporter 1 protein production was observed to be significantly increased (i.e., approximately 30-40%) in all three SecGEY overexpressing strains compared to the CZ437 control strain. Similarly, as shown in the data presented in Example 2, suitable promoter region sequences for expressing non-natural secGEY operons generally include promoter region sequences that are functional in host Gram-positive bacterial cells (e.g., secG promoter sequence, spoVG promoter sequence, and hbs promoter sequence, etc.).

[0098] As presented and described in Examples 3 and 4, the applicant further constructed Bacillus cells overexpressing SecG, SecE, and SecY in a host background expressing heterologous reporter 2 proteins. More specifically, three SecGEY overexpressing strains (BPC0166, BPC0167, and BPC0168) and a control strain BPC0178 were constructed and their reporter 2 production was evaluated. For example, as shown in Tables 6 and 7 (Example 4), the BPC0178 control strain and the SecGEY overexpressing strains (BPC0166, BPC0167, and BPC0168) were cultured and sampled for reporter 2 activity after 20 and 40 hours of growth. Here, reporter 2 protein production was similar or less for all three strains overexpressing SecGEY.

[0099] As presented and described in Examples 5 and 6, the applicant further constructed Bacillus cells overexpressing SecG, SecE, and SecY in a host background expressing heterologous reporter 3 proteins. More specifically, three SecGEY overexpressing strains (AL394, AL395, and AL396) and a control strain BPC0229 were constructed and their reporter 3 production was evaluated. For example, as shown in Tables 8 and 9 (Example 6), the BPC0229 control strain and the SecGEY overexpressing strains (AL394, AL395, and AL396) were cultured and reporter 3 activity was sampled after 16, 24, and 40 hours of growth. More specifically, as shown in Table 9 (Example 6), when considering protease production per OD600, reporter 3 protein production was significantly increased (i.e., by approximately 10–25%) in all three SecGEY overexpressing strains.

[0100] Therefore, as generally summarized above and described in the following examples, recombinant Gram-positive bacterial cells (e.g., Bacillus species cells) overexpressing SecG, SecE, and SecY proteins derived from the introduced non-natural secGEY operon can express / produce specific reporter proteases (e.g., reporter 1 and reporter 3) at significantly higher levels compared to other reporter proteases (e.g., reporter 2). For example, without intending to be bound by theory, mechanism of action, or mode of action, the applicant has surprisingly found that recombinant Gram-positive bacterial cells overexpressing SecG, SecE, and SecY transloconproteins (i.e., including the introduced secGEY operon) can produce increased amounts of alkaline subtilisin (e.g., B. clausii subtilisin SEQ ID NO: 21 and B. gibsonii subtilisin SEQ ID NO: 23) compared to control cells expressing the same alkaline subtilisin without the introduced secGEY operon. In contrast, recombinant cells overexpressing SecG, SecE, and SecY transloconproteins (i.e., including the introduced secGEY operon) produced equal amounts of B. amyloliquefaciens subtilisin (BPN'; SEQ ID NO: 22) compared to control cells expressing the same BPN' subtilisin without the introduced secGEY operon.

[0101] Based on the above, certain embodiments relate to recombinant Gram-positive bacterial cells expressing a non-natural secGEY operon and alkaline subtilisin protease. Thus, certain embodiments relate to recombinant Gram-positive bacterial cells comprising an introduced (synthesized) secGEY operon, wherein the recombinant cells express / produce / secrete an alkaline subtilisin protease having at least about 80% to 100% sequence identity with respect to the natural subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0102] In certain embodiments, natural subtilisins (and their functional variants) that exhibit 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 to so-called “true” subtilisins (subgroup I-S1). For example, a subgroup of serine proteases referred to as “subtilases” has been proposed, as generally described by Siezen and Leunissen (1997), and subtilases have been defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. In particular, the publications of Siezen and Leunissen provide an overview of the serine protease subtilase family (Table 1, Gram-positive bacteria), mainly including enzymes from the genus Bacillus, which have subgroups of true subtilisins (>64% identity), hyperalkaline proteases (>55% identity), and intracellular proteases (>37% identity), and show numerous minor variants of true subtilisins and hyperalkaline proteases (Table 2). More specifically, a subgroup of subtilases named "I-S1" (or "true" subtilisins) includes classic subtilisins such as Bacillus subtilis 168 subtilisin (aprA), Bacillus amyloliquefaciens subtilisin (BPN'), and Bacillus licheniformis subtilisin (Carlsberg). The second subgroup of subtilases named "I-S2" (or alkaline subtilisin) includes subtilisins such as B. alcalophilus PB92 alkaline subtilisin (PB92), B. lentus 309 alkaline subtilisin (309; Savinase®), and B. lentus 147 alkaline subtilisin (147; Esperase®).For example, as shown in Figure 2, the alkaline subtilisin (mature) protein sequence of the natural B. clausii (Figure 2A, SEQ ID NO: 21) contains 269 amino acid residues with an isoelectric point (pI) of approximately 9.30, the alkaline subtilisin (mature) protein sequence of the natural B. gibsonii (Figure 2C, SEQ ID NO: 23) contains 269 amino acid residues with a pI of approximately 9.57, while the "true" subtilisin mature protein sequence of the natural B. amyloliquefaciens (Figure 2B, SEQ ID NO: 22) contains 275 amino acid residues with a pI of approximately 6.30. More specifically, the isoelectric points described are theoretical values ​​calculated at average resolution using the Expasy (Swiss Bioinformatics Resource Portal) "Compute pI / MW tool".

[0103] In certain embodiments, one or more preferred pro-region sequences of this disclosure are derived from natural (wild-type, reference) subtilisin proteases. For example, in certain embodiments, preferred pro-region sequences are derived from natural pro-region amino acid sequences positioned upstream (5') of a mature subtilisin ORF. In other embodiments, particularly preferred pro-region sequences are the natural B. lentus subtilisin pro-region sequence (and its functional variants) of Sequence ID No. 18. For example, International Publication No. 2008 / 112258 generally describes recombinant Gram-positive bacterial cells and methods for producing serine proteases therefrom, such as modified (variant) pro-region sequences derived from full-length natural B. clausii alkaliserine (Maxacal) protease. Similarly, International Publication No. 2010 / 123754 describes compositions and methods for generating serine proteases using one or more modified pro-region sequences derived from full-length natural B. clausii serine protease or full-length natural B. lentus serine (GG36) protease. International Publication No. 2011 / 014278 further describes modified (variant) pro-region sequences suitable for generating serine proteases such as natural (or variant) B. amyloliquefaciens serine (BPN') protease.

[0104] Therefore, in one or more specific embodiments, the present disclosure provides recombinant Gram-positive bacterial cells comprising an introduced secGEY operon and one or more introduced expression cassettes encoding a subtilisin reporter protein. For example, as briefly summarized above and further described in the following examples, an artificial secGEY operon and / or expression cassette encoding a subtilisin reporter protein is generally constructed by operably linking one or more nucleic acid (DNA) sequence elements, including but not limited to promoter region sequences, 5'-UTR sequences, 3'-UTR sequences, ribosome-binding site (RBS) / Shine-Dalgano (SD) sequences, signal peptide (secretion) sequences, pro-region sequences, open reading frame (ORF) sequences, and terminator sequences.

[0105] In one or more specific embodiments, an expression cassette encoding an exemplary reporter protein is constructed and introduced into the cells of the Disclosure. In one or more specific embodiments, the exemplary reporter protein of the Disclosure is a protease, but is not limited to, natural B. clausii subtilisin (and its functional variants), natural B. gibsonii subtilisin (and its functional variants), and natural B. lentus subtilisin (and its functional variants). In particular, DNA sequences encoding natural subtilisin proteases (and therefore functional variants) are generally available and suitable for use according to one or more embodiments of the Disclosure.

[0106] In certain embodiments, modified cells produce an increased amount of subtilisin (protease) compared to control (or parent) cells, the increase being at least about 0.01%, at least about 0.10%, at least about 0.50%, at least about 1.0%, at least about 2.0%, at least about 3.0%, at least about 4.0%, at least about 5.0%, or greater than 5.0%. In certain embodiments, the increased amount of subtilisin is determined by assaying enzyme activity, assaying protein function, and assaying / quantifying specific productivity (Qp), etc. For example, those skilled in the art can utilize routine methods and techniques known in the art for detection, assay, and measurement of protein expression, production, and secretion, etc.

[0107] III. Recombinant Polynucleotides and Molecular Biology Certain embodiments of this disclosure provide, among other things, recombinant Gram-positive bacterial cells comprising a transduced (non-natural) secGEY operon that expresses a subtilisin protease and overexpresses SecG, SecE, and SecY proteins, and a method for generating a subtilisin protease in Gram-positive bacterial cells comprising the transduced (non-natural) secGEY operon and nucleic acid (DNA) sequences encoding natural SecG, SecE, and SecY proteins (e.g., a secG ORF encoding a natural or functional variant SecG protein, a SecE ORF encoding a natural or functional variant SecE protein, and a secY ORF encoding a natural or functional variant SecY protein).

[0108] Generally, as described herein and presented in the following examples, recombinant polynucleotides (vectors, expression cassettes, etc.) and recombinant (modified) Bacillus strains can be readily constructed using routine molecular biology and microbiology techniques, as well as methods known to those skilled in the art. Therefore, this disclosure generally relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods used in this disclosure include Sambrook et al., (2nd Edition, 1989); Kriegler (1990); and Ausubel et al., (1994). Similarly, those skilled in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., Escherichia coli, Bacillus species, etc.).

[0109] Therefore, in certain embodiments, the polynucleotides (genes, vectors, plasmids, DNA elements, etc.) of the Disclosure may be genetically modified, including, but not limited to, (a) introduction, replacement, or removal of one or more nucleotides within a gene (or its ORF) or within a regulatory element required for the transcription or translation of a gene or its ORF, (b) gene disruption, (c) gene transformation, (d) gene deletion, (e) gene downregulation (e.g., interfering RNA), (f) specific mutagenesis, and / or (g) random mutagenesis. In certain embodiments, the modified Bacillus cells of the Disclosure are constructed by increasing and / or decreasing (or eliminating) gene expression using methods well known in the Art, such as insertion, disruption, substitution, or deletion. The portion of the gene to be modified or inactivated may be, for example, a coding region (CDS, ORF) or a regulatory (DNA) element required for the expression of a coding region. Examples of such regulatory or control sequences may include promoter sequences or their functional portions (i.e., portions sufficient to affect the expression of a nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcriptional terminator sequences, and transcriptional activator sequences.

[0110] Gene deletion techniques eliminate gene expression or induce the expression of a non-functional (or reducedly active) protein product by enabling partial or complete removal of a gene. In such methods, gene deletion can be achieved by homologous recombination using a plasmid constructed to contain adjacent 5' and 3' regions that flank the gene. The adjacent 5' and 3' regions can be introduced into Bacillus cells on a temperature-sensitive plasmid, such as pE194, by associating with a second selection marker at an acceptable temperature established within the cell. Subsequently, cells are shifted to an unacceptable temperature to select cells with the plasmid incorporated into the chromosome at one of the homologous flanking regions. Selection for plasmid integration is influenced by selection for the second selection marker. After integration, the recombination event at the second homologous flanking region is stimulated by shifting the cells to an acceptable temperature for several generations without selecting cells. Cells are plated to obtain single colonies, which are then tested for the disappearance of both selection markers. Therefore, those skilled in the art can easily identify nucleotide regions in the coding and / or non-coding sequences of genes that are suitable for complete or partial deletion. In other embodiments, the modified Bacillus cells of the Disclosure are constructed by introducing, replacing, or removing one or more nucleotides in a gene or regulatory element required for its transcription or translation.

[0111] In certain embodiments, modified Bacillus cells are constructed via CRISPR-Cas9 editing. For example, a wild-type gene encoding a native protein of interest (or a functional variant protein of interest) may be modified via CRISPR-Cas9 editing with a nucleic acid-inducible endonuclease that finds the target DNA by binding to a guide RNA (e.g., Cas9) and Cpfl, or guide DNA (e.g., NgAgo), which recruits an endonuclease to a target sequence on the DNA, where the endonuclease can cause single-strand or double-strand breaks in the DNA. These targeted DNA breaks serve as substrates for DNA repair and can be recombined with a provided editing template (e.g., an editing template for replacing a native gene promoter sequence with a heterologous promoter). For example, a gene encoding a nucleic acid-induced endonuclease (for this purpose, Cas9 derived from S. pyogenes), or a codon-optimized gene encoding a Cas9 nuclease, generates a Bacillus Cas9 expression cassette by being operably linked to a promoter and a terminator active in Bacillus cells. Similarly, one or more target sites specific to the gene of interest can be readily identified by those skilled in the art. For example, to construct a DNA construct encoding a gRNA directed to a target site within a gene of interest using Streptococcus pyogenes Cas9, the variable targeting domain (VT) would contain a nucleotide at the target site, which is the 5' position of the (PAM) protospacer adjacent motif (NGG). This nucleotide is fused to the DNA encoding the Cas9 endonuclease recognition domain (CER) for S. pyogenes Cas9. The combination of the DNA encoding the VT domain and the DNA encoding the CER domain results in the gRNA-encoding DNA.Therefore, Bacillus expression cassettes for gRNA are created by operably linking the DNA encoding the gRNA to a promoter and a terminator that are active in Bacillus cells.

[0112] In certain embodiments, endonuclease-induced DNA breaks are repaired / replaced by incoming sequences. For example, nucleotide editing templates are provided so that the cell's DNA repair mechanisms can utilize them to precisely repair DNA breaks resulting from the Cas9 expression cassette and gRNA expression cassette described above. For instance, the 5' end of approximately 500 bp of the target gene can be fused to the 3' end of approximately 500 bp of the target gene to generate an editing template, which is then used by the mechanisms of Bacillus host cells to repair DNA breaks resulting from RGEN. The Cas9 expression cassette, gRNA expression cassette, and editing template can be delivered to cells simultaneously using a variety of methods. Transformed cells are screened for the locus of the target gene by PCR amplification, which amplifies the locus using forward and reverse primers. These primers can amplify wild-type loci or modified loci edited by RGEN. These fragments are then sequenced using sequencing primers to identify edited colonies.

[0113] In other embodiments, modified Bacillus cells are constructed by random or specific mutagenesis, including but not limited to chemical mutagenesis and translocation, using methods well known in the art. Gene modification can be performed by subjecting parental cells to mutagenesis and screening for mutant cells in which gene expression has been altered. Mutagenesis, which may be specific or random, can be performed, for example, by using a suitable physical or chemical mutagenerator, by using a suitable oligonucleotide, or by subjecting a DNA sequence to PCR-generated mutagenesis. Furthermore, mutagenesis can be performed by using any combination of these mutagenesis methods. Examples of physical or chemical mutagenerators suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrite, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When using such agents, mutagenesis is typically performed by incubating the parent cells to be mutageneised in the presence of the optimal mutagenesis agent under favorable conditions, and then selecting mutant cells that exhibit reduced gene expression or no expression at all.

[0114] International Publication No. 2003 / 083125 discloses methods for modifying Bacillus cells, such as creating Bacillus deletion strains and DNA constructs, using PCR fusion to bypass E. coli. International Publication No. 2002 / 14490 discloses methods for modifying Bacillus cells, including (1) construction and transformation of embedded plasmids (pComK), (2) random mutagenesis of coding, signaling, and propeptide sequences, (3) homologous recombination, (4) increasing transformation efficiency by adding non-homologous flanks to transformed DNA, (5) optimizing double crossover integration, (6) site-directed mutagenesis, and (7) markerless deletion. Those skilled in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli and Bacillus). In fact, transformation methods such as protoplast transformation and assembly, transduction, and protoplast fusion are known and suitable for use in this disclosure. Transformation methods are particularly preferred for introducing the DNA constructs of this disclosure into host cells.

[0115] In addition to commonly used methods, some embodiments directly transform host cells (i.e., without using intermediate cells to amplify the DNA construct or in any other process before introducing it into host cells). Methods for introducing the DNA construct into host cells include physical and chemical methods known in the art for introducing DNA into host cells without insertion into a plasmid or vector. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In additional embodiments, the DNA construct is co-transformed with the plasmid without insertion into the plasmid. In further embodiments, the selection marker is deleted or substantially excised from the modified Bacillus strain by methods known in the art. In some embodiments, the isolation of the vector from the host chromosome removes the intrinsic chromosomal region while leaving a flanking region within the chromosome.

[0116] Promoters and promoter sequences used for the expression of genes, their open reading frames (ORFs), and / or variant sequences within Bacillus cells are generally known to those skilled in the art. The promoter sequences of this disclosure are generally selected to be functional in Bacillus cells and include, but are not limited to, naturally occurring promoter sequences, synthetic promoter sequences, and / or combinations thereof, such promoters (sequences) are operable / functional in Bacillus cells. Examples of synthetic (manipulated) 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 species promoter sequences include, but are not limited to, the Bacillus subtilis alkaline protease (aprE) promoter, the Bacillus subtilis α-amylase promoter, the B. amyloliquefaciens α-amylase promoter, the Bacillus subtilis-derived neutral protease (nprE) promoter, the mutant aprE promoter (e.g., International Publication No. 2001 / 51643), the B. licheniformis tuf promoter, the B. licheniformis citZ promoter, or any other functional promoter derived from Bacillus species cells. A method for screening and generating a promoter library with a range of activity (promoter intensities) in Bacillus cells is described in International Publication No. 2003 / 089604.

[0117] IV. Fermentation of Gram-positive cells to produce the protein of interest In certain embodiments, this disclosure provides recombinant microbial cells that produce subtilisin proteases. More specifically, certain embodiments relate to genetically modified (recombinant) Gram-positive bacterial cells that express heterologous polynucleotides encoding subtilisin proteases. Thus, certain embodiments relate to the growth, culture, and fermentation of microbial cells for protein production. Generally, fermentation methods well known in the art are used to ferment microbial cells.

[0118] In some embodiments, cells are grown under batch fermentation conditions or continuous fermentation conditions. Classical batch fermentation is a closed system in which the composition of the culture medium is set at the start of fermentation and is not changed during fermentation. At the start of fermentation, the desired organism is inoculated into the medium. This method allows fermentation to be carried out without adding any components to the system. Typically, batch fermentation is considered "batch" in terms of the addition of a carbon source, and attempts are often made to control factors such as pH and oxygen concentration. The metabolites and biomass composition of the batch system are constantly changing until fermentation is stopped. In batch culture, cells progress through a static induction phase to a high-growth logarithmic phase, and finally to a quiescent phase in which the growth rate decreases or stops. If left untreated, cells in the quiescent phase will eventually die. Generally, cells in the logarithmic phase are responsible for the production of the majority of the product.

[0119] A suitable variation of the standard batch system is the "fed-batch fermentation" system. In this variation of the typical batch system, the substrate is gradually added as fermentation progresses. The fed-batch system is useful when catabolite inhibition is likely to inhibit cellular metabolism, and when a limited amount of substrate in the culture medium is desirable. In the fed-batch system, it is difficult to measure the actual substrate concentration, so it is estimated based on changes in measurable factors such as pH, dissolved oxygen, and the partial pressure of exhaust gases such as CO2. Batch fermentation and fed-batch fermentation are common and well-known in the art.

[0120] Continuous fermentation is an open system in which a specified fermentation medium is continuously added to a bioreactor for processing, while an equal amount of acclimatized medium is simultaneously removed. Continuous fermentation generally maintains the culture at a constant density where the cells are primarily in the logarithmic growth phase. Continuous fermentation allows for the regulation of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, limiting nutrients such as a carbon or nitrogen source can be maintained at a constant ratio while all other parameters can be regulated. In other systems, numerous factors affecting growth can be continuously changed while maintaining a constant cell concentration, as measured by the turbidity of the medium. Continuous systems attempt to maintain steady-state growth conditions. Therefore, cell loss resulting from the removal of medium should be balanced with respect to the cell growth rate during fermentation. Methods for regulating nutrients and growth factors in the continuous fermentation process, as well as techniques for maximizing the rate of product formation, are well known in the field of industrial microbiology.

[0121] Cultivation / fermentation is generally achieved in a growth medium containing aqueous inorganic salt medium, organic growth factors, carbon and energy source substances, molecular oxygen, and, of course, the starting inoculum of the microbial host used.

[0122] To ensure proper microbial growth, maximize the absorption of carbon and energy sources by cells during the microbial transformation process, and achieve maximum cell yield at maximum cell density in the fermentation medium, it is necessary to supply suitable amounts of inorganic nutrients in appropriate proportions, in addition to carbon and energy sources, oxygen, assimilated nitrogen, and microbial inoculant.

[0123] The composition of aqueous inorganic media can vary widely, in part, depending on the microorganisms and substrates used, as is known in the art. In addition to nitrogen, the inorganic media contains appropriate amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium in appropriate soluble and assimilated ionic composite forms, and also preferably certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, also in appropriate soluble and assimilated forms, all of which are known in the art.

[0124] Fermentation is an aerobic process in which the necessary molecular oxygen is supplied by a molecular oxygen-containing gas, such as air, oxygen-enriched air, or even substantially pure molecular oxygen, which is provided to maintain the contents of the fermentation vessel at an appropriate partial pressure of oxygen that is effective in helping microbial species to grow vigorously.

[0125] Fermentation temperatures can vary somewhat, but for most microbial cells, the temperature will generally be in the range of approximately 20°C to 40°C.

[0126] Furthermore, microorganisms require an assimilated nitrogen source. This assimilated nitrogen source can be any nitrogen-containing compound, or a compound capable of releasing nitrogen in a form suitable for metabolic utilization by microorganisms. Various organic nitrogen source compounds, such as protein hydrolysates, can be used, but typically, inexpensive 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 used by bubbling aqueous fermenters (fermentation media) in suitable amounts. Simultaneously, such ammonia can also be used to assist in pH control.

[0127] The pH range in aqueous microbial fermentation products (fermentation mixtures) should be within the exemplary range of approximately 2.0 to 8.0. The preferred pH range for microorganisms is, to some extent, dependent on the culture medium used and the specific microorganism, and can be easily determined by those skilled in the art, but it will change somewhat with changes in the culture medium.

[0128] It is preferable to carry out fermentation in a manner that allows control of the carbon-containing substrate as a limiting factor, thereby ensuring good conversion of the carbon-containing substrate to cells and avoiding contamination of cells with a considerable amount of unconverted substrate. The latter is not a problem with water-soluble substrates, as any trace amounts of residue can be easily washed away. However, it may be a problem with non-water-soluble substrates, requiring additional product treatment steps such as appropriate washing.

[0129] As mentioned above, the time it takes to reach this level is not critical and can vary depending on the specific microorganisms and the fermentation process being carried out. However, methods for determining the carbon source concentration in the fermentation medium and for determining whether the desired carbon source level has been achieved are well known in the art.

[0130] If necessary, some or all of the carbon and energy source material, and / or some of the assimilated nitrogen source such as ammonia, may be added to the aqueous inorganic medium before supplying it to the fermenter.

[0131] It is preferable that each flow introduced into the reactor be controlled at a predetermined rate, or as needed, as determined by monitoring the concentration of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the fermenter exhaust gas, cell density (measurable by dry cell weight), or light transmittance. The supply rates of various materials can be varied to obtain the fastest possible cell growth rate and the highest possible yield of microbial cells relative to the substrate charge, in line with the efficient utilization of carbon and energy sources.

[0132] In batch or preferred fed-batch operations, all equipment, reactors, or fermentation means, vessels or containers, piping, and associated circulation or cooling systems are first sterilized, for example, at about 121°C for at least about 15 minutes, usually using steam. Next, the culture of the selected microorganisms is inoculated into the sterilized reactor in the presence of all necessary nutrients, including oxygen, and a carbon-containing substrate. The type of fermenter used is not important.

[0133] V. Exemplary Embodiments Non-limiting embodiments of the compositions and methods disclosed herein are as follows:

[0134] 1. Recombinant Gram-positive (host) cells containing an introduced secGEY operon and an expression cassette encoding a heterologous subtilisin.

[0135] 2. The introduced secGEY operon comprises a secG open reading frame (ORF) sequence having at least about 80% to 100% identity with SEQ ID NO: 2, wherein the secG ORF encodes a functional SecG protein, in the recombinant cell of Embodiment 1.

[0136] 3. The introduced secGEY operon comprises a secE ORF sequence having at least about 80% to 100% identity with SEQ ID NO: 31, and the secE ORF encodes a functional SecE protein, in the recombinant cell of Embodiment 1.

[0137] 4. The introduced secGEY operon comprises a secY ORF sequence having at least approximately 80% to 100% identity with SEQ ID NO: 32, and the secY ORF encodes a functional SecY protein, in the recombinant cell of Embodiment 1.

[0138] 5. Recombinant cells of Embodiment 1, wherein the introduced secGEY operon encodes a functional secG protein containing at least approximately 80-100% amino acid identity with respect to SEQ ID NO: 28.

[0139] 6. Recombinant cells of Embodiment 1, wherein the introduced secGEY operon encodes a functional secE protein containing at least about 80-100% amino acid identity with respect to SEQ ID NO: 29.

[0140] 7. Recombinant cells of Embodiment 1, wherein the introduced secGEY operon encodes a functional secY protein containing at least about 80-100% amino acid identity with respect to SEQ ID NO: 30.

[0141] 8. The introduced secGEY operon comprises an upstream (5')secG promoter and a secG 5'-UTR sequence having at least about 80% to 100% identity with respect to SEQ ID NO: 1, and a downstream secGEY ORF operably ligated thereto, in a recombinant cell of Embodiment 1.

[0142] 9. The introduced secGEY operon comprises a recombinant cell of Embodiment 1, including a heterogeneous upstream (5') promoter and a 5'-UTR sequence, and a downstream secGEY ORF operably ligated thereto.

[0143] 10. Recombinant cells of Embodiment 9, wherein the heterologous upstream promoter and 5'-UTR sequence have at least about 90% to 100% identity with the spoVG promoter and spoVG-5'-UTR of SEQ ID NO: 10.

[0144] 11. Recombinant cells of Embodiment 9, wherein the heterologous upstream promoter and 5'-UTR sequence have at least about 90% to 100% identity with the hbs promoter and spoVG-5'-UTR of SEQ ID NO: 12.

[0145] 12. Recombinant cells of Embodiment 1, wherein the introduced secGEY operon has at least about 80% to 100% identity with SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 13.

[0146] 13. Recombinant cells of Embodiment 1, comprising at least two introduction cassettes encoding the same or different heterologous subtilisins, or at least three introduction expression cassettes encoding the same or different heterologous subtilisins.

[0147] 14. The cassette is a recombinant cell according to Embodiment 1 or Embodiment 13, encoding alkaline subtilisin. 15. Recombinant cells of Embodiment 1 or Embodiment 13, wherein the cassette encoding subtilisin comprises an upstream promoter region sequence, a downstream nucleic acid encoding a protein signal sequence operably ligated thereto, a downstream nucleic acid encoding a pro region sequence operably ligated thereto, and a downstream nucleic acid encoding a mature subtilisin operably ligated thereto.

[0148] 16. Recombinant cells of Embodiment 15, in which the promoter sequence is a functionally potent promoter in Gram-positive cells.

[0149] 17. Recombinant cells of Embodiment 15, wherein the signal sequence is a native subtilisin signal sequence or a functional variant thereof.

[0150] 18. Recombinant cells of Embodiment 15, wherein the signal sequence has at least approximately 95% to 100% amino acid identity to the aprE signal sequence of SEQ ID NO: 17, or at least approximately 95% to 100% amino acid identity to the BPN' signal sequence of SEQ ID NO: 19.

[0151] 19. Recombinant cells of Embodiment 15, wherein the pro region sequence is a native subtilisin pro region sequence or a functional variant thereof.

[0152] 20. Recombinant cells of Embodiment 15, wherein the pro-region sequence contains at least about 95% to 100% amino acid identity with respect to the pro-region of SEQ ID NO: 18.

[0153] 21. Recombinant cells of Embodiment 15, wherein the mature subtilisin contains at least about 80% to 100% amino acid identity with respect to the mature subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0154] 22. A polynucleotide construct encoding a synthetic secGEY operon, wherein the polynucleotide comprises at least an upstream promoter sequence, a downstream nucleic acid encoding a secG protein operably linked thereto having at least about 80% to 100% identity with SEQ ID NO: 28, a downstream nucleic acid encoding a secE protein operably linked thereto having at least about 80% to 100% identity with SEQ ID NO: 29, and a downstream nucleic acid encoding a secY protein operably linked thereto having at least about 80% to 100% identity with SEQ ID NO: 30.

[0155] 23. The nucleic acid encoding the secG protein is a polynucleotide of Embodiment 22, which contains at least about 80% to 100% identity with respect to the secG ORF of Sequence ID No. 2.

[0156] 24. The nucleic acid encoding the secE protein is a polynucleotide of Embodiment 22, which contains at least about 80% to 100% identity with respect to the secE ORF of Sequence ID No. 31.

[0157] 25. The nucleic acid encoding the secY protein is a polynucleotide of Embodiment 22, which contains at least about 80% to 100% identity with respect to the secY ORF of Sequence ID No. 32.

[0158] 26. The nucleic acid encoding the secE protein is a polynucleotide of Embodiment 24, comprising a secE ribosome-binding site (RBS), a secE ORF positioned upstream of the RBS and operably ligated thereto.

[0159] 27. A polynucleotide of Embodiment 26, having at least about 80% to 100% identity with respect to the secE RBS and secE ORF of Sequence ID No. 3.

[0160] 28. The nucleic acid encoding the secY protein is a polynucleotide of Embodiment 25, comprising a secY ribosome-binding site (RBS), a secY ORF positioned upstream of the RBS and operably ligated thereto.

[0161] 29. A polynucleotide of Embodiment 28, having at least about 80% to 100% identity with respect to the secY RBS and secY ORF of Sequence ID No. 4.

[0162] 30. A method for producing heterologous subtilisin in modified Gram-positive bacterial cells, comprising: (a) obtaining Gram-positive bacterial cells that produce heterologous subtilisin and introducing a synthetic secGEY operon into the cells; and (b) fermenting the modified cells under conditions suitable for subtilisin production.

[0163] 31. A method for producing heterologous subtilisin in modified Gram-positive bacterial cells, comprising: (a) obtaining Gram-positive bacterial cells and introducing into the cells (i) an expression cassette encoding heterologous subtilisin and (ii) an expression cassette encoding a synthetic secGEY operon; and (b) fermenting the modified cells under conditions suitable for subtilisin production.

[0164] 32. Subtilisin is secreted into the fermentation broth when fermentation is carried out under conditions suitable for subtilisin production, according to the method of Embodiment 30 or Embodiment 31.

[0165] 33. The method of Embodiment 30, wherein modified cells, when fermented under the same conditions, produce an increased amount of subtilisin compared to control cells, and the control cells do not contain the introduced synthetic secGEY operon.

[0166] 34. The method of Embodiment 31, wherein modified cells, when fermented under the same conditions, produce an increased amount of subtilisin compared to control cells, the control cells contain the same transexpression cassette encoding the same heterologous subtilisin, and the control cells do not contain the transsynthetic secGEY operon.

[0167] 35. The introduced secGEY operon comprises a secG open reading frame (ORF) sequence having at least about 80% to 100% identity with SEQ ID NO: 2, wherein the secG ORF encodes a functional SecG protein, according to the method of Embodiment 30 or Embodiment 31.

[0168] 36. The introduced secGEY operon comprises a secE open reading frame (ORF) sequence having at least about 80% to 100% identity with SEQ ID NO: 31, wherein the secE ORF encodes a functional secE protein, according to the method of Embodiment 30 or Embodiment 31.

[0169] 37. The introduced secGEY operon comprises a secY open reading frame (ORF) sequence having at least about 80% to 100% identity with SEQ ID NO: 32, wherein the secY ORF encodes a functional SecY protein, according to the method of Embodiment 30 or Embodiment 31.

[0170] 38. The method of Embodiment 30 or Embodiment 31, wherein the introduced secGEY operon encodes a functional secG protein having at least about 80% to 100% amino acid identity with respect to SEQ ID NO: 28.

[0171] 39. The method of Embodiment 30 or Embodiment 31, wherein the introduced secGEY operon encodes a functional secE protein having at least about 80% to 100% amino acid identity with respect to SEQ ID NO: 29.

[0172] 40. The method of Embodiment 30 or Embodiment 31, wherein the introduced secGEY operon encodes a functional secY protein having at least about 80% to 100% amino acid identity with respect to SEQ ID NO: 30.

[0173] 41. The introduced secGEY operon comprises an upstream (5')secG promoter and a secG 5'-UTR sequence having at least about 95% to 100% identity with respect to SEQ ID NO: 1, and a downstream secGEY ORF operably coupled thereto, according to the method of Embodiment 30 or Embodiment 31.

[0174] 42. The introduced secGEY operon comprises a heterogeneous upstream (5') promoter and a 5'-UTR array, and a downstream secGEY ORF operably coupled thereto, as described in Embodiment 30 or Embodiment 31.

[0175] 43. The method of Embodiment 42, wherein the heterogeneous promoter and 5'-UTR sequence have at least about 95% to 100% identity with respect to the spoVG promoter and spoVG-5'-UTR of Sequence ID No. 10.

[0176] 44. The method of Embodiment 42, wherein the heterogeneous promoter and 5'-UTR sequence have at least about 95% to 100% identity with respect to the hbs promoter and spoVG-5'-UTR of Sequence ID No. 12.

[0177] 45. The method of Embodiment 30 or Embodiment 31, wherein the introduced secGEY operon has at least about 80% to 100% identity with SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 13.

[0178] 46. ​​The method of Embodiment 31, wherein the cells include at least two transexpression cassettes encoding the same or different heterologous subtilisins, or at least three transexpression cassettes encoding the same or different heterologous subtilisins.

[0179] 47. The cassette encodes an alkaline subtilisin, as per the method of Embodiment 31 or Embodiment 46.

[0180] 48. The method of Embodiment 31 or Embodiment 46, wherein the cassette encoding the subtilisin comprises an upstream promoter region sequence, a downstream nucleic acid encoding a protein signal sequence operably ligated thereto, a downstream nucleic acid encoding a pro region sequence operably ligated thereto, and a downstream nucleic acid encoding a mature subtilisin operably ligated thereto.

[0181] 49. The method of Embodiment 48, wherein the promoter sequence is a functionally potent promoter in Gram-positive cells.

[0182] 50. The method of Embodiment 48, wherein the signal sequence is a natural subtilisin signal sequence or a functional variant thereof.

[0183] 51. The method of Embodiment 50, wherein the signal sequence has at least about 95% to 100% amino acid identity with respect to the aprE signal sequence of SEQ ID NO: 17, or at least about 95% to 100% amino acid identity with respect to the BPN' signal sequence of SEQ ID NO: 19.

[0184] 52. The method of Embodiment 48, wherein the pro region sequence is a natural subtilisin pro region sequence or a functional variant thereof.

[0185] 53. The method of Embodiment 48, wherein the pro-region sequence contains at least about 90% to 100% amino acid identity with respect to the pro-region of SEQ ID NO: 18.

[0186] 54. The method of Embodiment 48, wherein the mature subtilisin has at least about 80% to 100% amino acid identity with respect to the mature subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0187] 55. The method of Embodiment 33 or Embodiment 34, wherein the increased amount of subtilisin is at least about 5% greater compared to control cells when fermented under the same conditions.

[0188] 56. The increase in subtilisin is determined by the suc-AAPF-pNA assay, as in the method of Embodiment 33 or Embodiment 34. [Examples]

[0189] Certain aspects of the present invention may be further understood in light of the following examples, but these examples should not be construed as limiting. Modifications of materials and methods will be obvious to those skilled in the art. The standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).

[0190] Example 1 Construction of Bacillus subtilis cells that express heterologous proteins and overexpress SecG, SecE, and SecY. This embodiment describes the construction of Bacillus subtilis cells that overexpress SecG, SecE, and SecY in a host background expressing heterologous reporter 1 proteins. More specifically, a control Bacillus subtilis strain named CZ437 includes two introduced reporter 1 expression cassettes, each containing a promoter sequence (P2), an open reading frame (ORF) encoding the reporter 1 protein, positioned upstream (5') and operably ligated thereto, the cassettes being incorporated into the Bacillus subtilis skf locus (skf::P2-reporter 1) and the Bacillus subtilis aprE locus (aprE::P2-reporter 1). The design and construction of a modified Bacillus subtilis strain expressing subtilisin reporter 1 and overexpressing the artificial (non-natural) secGEY operon of this disclosure was carried out as follows.

[0191] We constructed a first modified Bacillus subtilis strain expressing SecG, SecE, and SecY proteins as a non-natural operon (PsecG-secGEY) initiated by a secG promoter derived from those native gene loci and incorporated into the Bacillus subtilis (B. subtilis) pksR locus. More specifically, the non-natural operon PsecG-secGEY was constructed by overlap extension PCR and includes the Bacillus subtilis (B. subtilis) secG promoter and secG 5-UTR (SEQ ID NO: 1), a secG ORF (SEQ ID NO: 2) positioned upstream (5') and operably ligated thereto, a secE ribosome-binding sequence (RBS) and ORF (SEQ ID NO: 3) operably ligated thereto, a secY RBS and ORF (SEQ ID NO: 4) operably ligated thereto, and a BPN' terminator derived from Bacillus amyloliquefaciens (SEQ ID NO: 5) operably ligated thereto. An embedded cassette containing the homologous region downstream (3') of Bacillus subtilis (B. subtilis) pksR (SEQ ID NO: 6), the non-natural operon PsecG-secGEY (SEQ ID NO: 7) operably linked to it, the Bacillus subtilis (B. subtilis) alrA gene (SEQ ID NO: 8) operably linked to it, and the upstream (5') homologous region of Bacillus subtilis (B. subtilis) pksR (SEQ ID NO: 9) operably linked to it was constructed by overlap extension PCR and transformed into a Bacillus subtilis (B. subtilis) strain having an alrA deletion and containing one copy of the =reporter 1 expression cassette (skf:P2 reporter 1) integrated at the skf locus. The resulting strain was transformed with a second copy of the reporter 1 cassette (aprE::P2 reporter 1) integrated at the aprE locus to create the Bacillus subtilis (B. subtilis) strain BPC0123.

[0192] A second modified strain was constructed expressing SecG, SecE, and SecY as a non-natural operon (PspoVG-secGEY) initiated by a spoVG promoter (PspoVG) derived from those natural loci and incorporated into the pksR locus. More specifically, the non-natural operon PspoVG-secGEY was constructed by overlap extension PCR and contains the Bacillus subtilis (B. subtilis) spoVG promoter and spoVG 5-UTR (SEQ ID NO: 10), a secG ORF (SEQ ID NO: 2) positioned upstream (5') and operably ligated to it, secE RBS and ORF (SEQ ID NO: 3) operably ligated to it, secY RBS and ORF (SEQ ID NO: 4) operably ligated to it, and a BPN' terminator (SEQ ID NO: 5) derived from B. amyloliquefaciens operably ligated to it. An embedded cassette containing the Bacillus subtilis (B. subtilis) pksR downstream (3') homologous region (SEQ ID NO: 6), the non-natural operon PspoVG-secGEY (SEQ ID NO: 11) operably linked to it, the Bacillus subtilis (B. subtilis) alrA gene (SEQ ID NO: 8) operably linked to it, and the Bacillus subtilis (B. subtilis) pksR upstream (5') homologous region (SEQ ID NO: 9) operably linked to it was constructed by overlap extension PCR and transformed into a Bacillus subtilis (B. subtilis) strain having an alrA deletion and containing one copy of the -reporter 1 expression cassette (skf:P2-reporter 1) integrated at the skf locus. The resulting strain was transformed with a second copy of the subtilis sine variant 1 expression cassette (aprE::P2-reporter 1) integrated at the aprE locus to create the Bacillus subtilis (B. subtilis) strain BPC0182.

[0193] A third modified strain was constructed expressing SecG, SecE, and SecY as a non-natural operon (Phbs-secGEY) initiated by the hbs promoter (Phbs) derived from those native gene loci and incorporated into the pksR locus. More specifically, the non-natural operon Phbs-secGEY was constructed by overlap extension PCR and contains the Bacillus subtilis (B. subtilis) hbs promoter and Bacillus subtilis (B. subtilis) spoVG5-UTR (SEQ ID NO: 12), a secG ORF (SEQ ID NO: 2) positioned upstream (5') and operably ligated to it, secE RBS and ORF (SEQ ID NO: 3) operably ligated to it, secY RBS and ORF (SEQ ID NO: 4) operably ligated to it, and a BPN' terminator (SEQ ID NO: 5) derived from B. amyloliquefaciens operably ligated to it. An embedded cassette containing the Bacillus subtilis (B. subtilis) pksR downstream (3') homologous region (SEQ ID NO: 6), the non-natural operon Phbs-secGEY (SEQ ID NO: 13) operably linked to it, the Bacillus subtilis (B. subtilis) alrA gene (SEQ ID NO: 8) operably linked to it, and the Bacillus subtilis (B. subtilis) pksR upstream (5') homologous region (SEQ ID NO: 9) operably linked to it was constructed by overlap extension PCR and transformed into a Bacillus subtilis (B. subtilis) strain having an alrA deletion and containing one copy of the reporter 1 expression cassette (skf:P2-reporter 1) integrated at the skf locus. The resulting strain was transformed with a second copy of the subtilis sine variant 1 expression cassette (aprE::P2-reporter 1) integrated at the aprE locus to create the Bacillus subtilis (B. subtilis) strain BPC0184.

[0194] Example 2 Expression of reporter 1 proteases in Bacillus subtilis cells overexpressing SecG, SecE, and SecY. In this example, the expression of heterologous reporter 1 protein in the presence of SecGEY overexpression was compared with the expression of the same reporter 1 protein without SecGEY overexpression. More specifically, reporter 1 production under small-scale conditions was evaluated for three SecGEY overexpression strains (BPC0123, BPC0182, BPC0184) and the control strain CZ437 as follows: Single colonies were inoculated into a pre-culture in trypsin-soybean broth (1.7% tryptone, 0.3% soytone, 0.25% glucose, 0.5% sodium chloride, 0.25% dibasic potassium phosphate) and grown at 37°C and 250 RPM. Using the pre-culture, the cells were inoculated at a 1:1000 dilution into 20 ml of 0.5 × MPS2 medium supplemented with 80 mM MOPS adjusted to pH 7.3, and the cultures were grown in a flask at 37°C and 250 RPM. MPS2 medium consists of: 10v / v% 10×MOPS-based medium (8.4w / v% MOPS, 2.9w / v% sodium chloride, 1.5w / v% potassium hydroxide, 0.05w / v% potassium sulfate, 0.05w / v% magnesium chloride, 0.7w / v% tricine, 10v / v% micronutrients), 10w / v% maltrin M150, 6w / v% soyton, 0.78w / v% dipotassium phosphate, 0.36w / v% urea, 0.2w / v% monopotassium phosphate, 0.06w / v% trisodium citrate dihydrate, and pH adjusted with potassium hydroxide. The micronutrients were composed of 100x stock solution per liter, 1.47g sodium citrate 2H2O, 1.47g CaCl22H2O, 400mg FeSO47H2O, 100mg MnSO4H2O, 100mg ZnSO4H2O, 50mg CuCl22H2O, 100mg CoCl26H2O, and 100mg Na2MoO42H2O.

[0195] As essentially described in International Publication No. 2020112609 (incorporated herein by reference), the amount of reporter 1 protease in the culture supernatant was determined using the suc-AAPF-pNA assay. The substrate is N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA). During hydrolysis of the peptide substrate by the protease, 4-nitroanilide is cleaved to produce 4-nitroaniline, a yellow chromophore. Therefore, the absorbance at 405 nm is measured, and the slope of the absorbance change (mOD / min) directly correlates with the amount of protease in the analyte. The 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 sample was added to a microtiter plate containing 190 μL of AAPF stock diluted in Tris buffer (100 mg / ml AAPF stock in DMSO diluted 100× in 100 mM Tris, 0.005% Tween 80, pH 8.6). The absorbance of the solution was measured at 405 nm using a SpectraMax spectrophotometer.

[0196] In the first experiment, control strain CZ437 and SecGEY-overexpressing strain BPC0123 were cultured, and protease activity was sampled after 24 and 40 hours of growth. For example, Table 2 below shows the reporter 1 protein production of strain BPC0123 (and the associated coefficient of variation (CV; n≧4)) compared to the reporter 1 protein production of control strain CZ437. Similarly, Table 3 below shows the relative reporter 1 production of strains CZ437 and BPC0123, normalized to the cell aggregate volume in the culture, as measured by OD600. Thus, as generally shown in Tables 2 and 3, reporter 1 protein production increased with SecGEY overexpressed from the secG promoter.

[0197] [Table 2]

[0198] [Table 3]

[0199] In the second experiment, control strain CZ437 and SecGEY-overexpressing strains BPC0123, BPC0182, and BPC0184 were cultured, and protease activity was sampled after 22 hours of growth. More specifically, the reporter 1 production of SecGEY-overexpressing strains BPC0123, BPC0182, and BPC0184 compared to control strain CZ437, along with the associated coefficient of variation (CV; n=6), is shown in Table 4 below. Similarly, the relative reporter 1 production normalized to the cell aggregate volume in the culture, as measured by OD600, is shown in Table 5 below. As shown in Tables 4 and 5, when considering protease production by OD600, reporter 1 protein production increased in all three SecGEY-overexpressing strains (see Table 5).

[0200] [Table 4]

[0201] [Table 5]

[0202] Example 3 Construction of Bacillus subtilis cells that express heterologous proteins and overexpress SecG, SecE, and SecY. This embodiment describes the construction of Bacillus subtilis cells overexpressing SecG, SecE, and SecY in a host background expressing heterologous reporter 2 proteins. More specifically, a control Bacillus subtilis strain named BPC0178 contains three introduced reporter 2 expression cassettes, each containing a promoter sequence (P4), an open reading frame (ORF) encoding the subtilisin reporter 2 protein, positioned upstream (5') and operably ligated thereto, the three cassettes being incorporated into the Bacillus subtilis skf locus (skf::P4--reporter 2), the Bacillus subtilis nprE locus (nprE::P4-reporter 2), and the Bacillus subtilis ppsC locus (ppsC::P4-reporter 2). The design and construction of a modified Bacillus subtilis strain expressing reporter 2 and overexpressing the artificial (non-natural) secGEY operon of this disclosure was carried out as follows.

[0203] We constructed a first modified Bacillus subtilis strain expressing SecG, SecE, and SecY proteins as a non-natural operon (PsecG-secGEY) initiated by a secG promoter derived from those native gene loci and incorporated into the Bacillus subtilis (B. subtilis) aprE locus. More specifically, the non-natural operon PsecG-secGEY was constructed by overlap extension PCR and includes the Bacillus subtilis (B. subtilis) secG promoter and secG 5-UTR (SEQ ID NO: 1), a secG ORF (SEQ ID NO: 2) positioned upstream (5') and operably ligated thereto, secE RBS and ORF (SEQ ID NO: 3) operably ligated thereto, secY RBS and ORF (SEQ ID NO: 4) operably ligated thereto, and a BPN' terminator (SEQ ID NO: 5) derived from Bacillus amyloliquefaciens. An embedded cassette containing the upstream (5') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 14), the non-natural operon PsecG-secGEY (SEQ ID NO: 7) operably linked thereto, the promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked thereto, and the downstream (3') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 16) operably linked thereto was constructed by overlap extension PCR. This cassette was then transformed into a Bacillus subtilis (B. subtilis) strain containing three copies of the reporter 2 expression cassette embedded at the skf locus (skf::P4-reporter 2), nprE locus (nprE::P4-reporter 2), and ppsC locus (ppsC:P4-reporter 2), creating the Bacillus subtilis (B. subtilis) strain BPC0166.

[0204] A second modified strain was constructed expressing SecG, SecE, and SecY as a non-natural operon (PspoVG-secGEY) initiated by a spoVG promoter derived from those native gene loci and incorporated into the aprE locus. More specifically, the non-natural operon PspoVG-secGEY was constructed by overlap extension PCR and contains the Bacillus subtilis (B. subtilis) spoVG promoter and spoVG 5-UTR (SEQ ID NO: 10), a secG ORF (SEQ ID NO: 2) positioned upstream (5') and operably ligated to it, secE RBS and ORF (SEQ ID NO: 3) operably ligated to it, secY RBS and ORF (SEQ ID NO: 4) operably ligated to it, and a BPN' terminator (SEQ ID NO: 5) derived from B. amyloliquefaciens operably ligated to it. An embedded cassette containing the upstream (5') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 14), the non-natural operon PspoVG-secGEY (SEQ ID NO: 11) operably linked thereto, the promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked thereto, and the downstream (3') homologous region of Bacillus subtilis (B. subtilis) aprE (HR; SEQ ID NO: 16) operably linked thereto was constructed by overlap extension PCR. This was then transformed into a Bacillus subtilis (B. subtilis) strain containing three copies of the reporter 2 expression cassette embedded at the skf locus (skf::P4-reporter 2), nprE locus (nprE::P4-reporter 2), and ppsC locus (ppsC:P4-reporter 2), creating the Bacillus subtilis (B. subtilis) strain BPC0167.

[0205] A third modified strain was constructed expressing SecG, SecE, and SecY as a non-natural operon (Phbs-secGEY) initiated by an hbs promoter derived from those native gene loci and incorporated into the aprE locus. More specifically, the non-natural operon Phbs-secGEY was constructed by overlap extension PCR and includes the Bacillus subtilis (B. subtilis) hbs promoter and Bacillus subtilis (B. subtilis) spoVG5-UTR (SEQ ID NO: 12), positioned upstream (5') and operably ligated to it secG ORF (SEQ ID NO: 2), operably ligated to secERBS and ORF (SEQ ID NO: 3), operably ligated to secY RBS and ORF (SEQ ID NO: 4), and operably ligated to the BPN' terminator derived from B. amyloliquefaciens (B. amyloliquefaciens) (SEQ ID NO: 5). An embedded cassette containing the upstream (5') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 14), the non-natural operon Phbs-secGEY (SEQ ID NO: 13) operably linked thereto, the promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked thereto, and the downstream (3') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 16) operably linked thereto was constructed by overlap extension PCR. This cassette was then transformed into a Bacillus subtilis (B. subtilis) strain containing three copies of the reporter 2 expression cassette embedded at the skf locus (skf::P4-reporter 2), nprE locus (nprE::P4-reporter 2), and ppsC locus (ppsC:P4-reporter 2), creating the Bacillus subtilis (B. subtilis) strain BPC0168.

[0206] Example 4 Expression of reporter 2 proteases in Bacillus subtilis cells overexpressing SecG, SecE, and SecY. In this example, the expression of heterologous reporter 2 protein in the presence of SecGEY overexpression was compared with the expression of the same reporter 2 protein without SecGEY overexpression. More specifically, reporter 2 production was evaluated for three SecGEY overexpression strains (BPC0166, BPC0167, BPC0168) and the control strain BPC0178 under small-scale conditions as follows: Single colonies were inoculated into a pre-culture in trypsin-soybean broth (1.7% tryptone, 0.3% soytone, 0.25% glucose, 0.5% sodium chloride, 0.25% dibasic potassium phosphate) and grown at 37°C and 250 RPM. Using the pre-culture, the cells were inoculated at a 1:1000 dilution into 20 ml of 0.5 × MPS2 medium supplemented with 80 mM MOPS adjusted to pH 7.3, and the cultures were grown in a flask at 37°C and 250 RPM. MPS2 medium consists of: 10v / v% 10×MOPS-based medium (8.4w / v% MOPS, 2.9w / v% sodium chloride, 1.5w / v% potassium hydroxide, 0.05w / v% potassium sulfate, 0.05w / v% magnesium chloride, 0.7w / v% tricine, 10v / v% micronutrients), 10w / v% maltrin M150, 6w / v% soyton, 0.78w / v% dipotassium phosphate, 0.36w / v% urea, 0.2w / v% monopotassium phosphate, 0.06w / v% trisodium citrate dihydrate, and pH adjusted with potassium hydroxide. The micronutrients were composed of 100x stock solution per liter, 1.47g sodium citrate 2H2O, 1.47g CaCl22H2O, 400mg FeSO47H2O, 100mg MnSO4H2O, 100mg ZnSO4H2O, 50mg CuCl22H2O, 100mg CoCl26H2O, and 100mg Na2MoO42H2O.

[0207] As essentially described in International Publication No. 2020112609, the amount of reporter 2 protease in the culture supernatant was determined using the suc-AAPF-pNA assay. The substrate is N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA). During the hydrolysis of the peptide substrate by the protease, 4-nitroanilide is cleaved to produce 4-nitroaniline, a yellow chromophore. Therefore, the absorbance at 405 nm is measured, and the slope of the absorbance change (mOD / min) directly correlates with the amount of protease in the analyte sample. The 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 sample was added to a microtiter plate containing 190 μL of AAPF stock diluted in Tris buffer (100 mg / ml AAPF stock in DMSO diluted 100× in 100 mM Tris, 0.005% Tween 80, pH 8.6). The absorbance of the solution was measured at 405 nm using a SpectraMax spectrophotometer.

[0208] The control strain BPC0178, along with SecGEY overexpressing strains BPC0166, BPC0167, and BPC0168, were cultured, and protease activity was sampled after 20 and 40 hours of growth. More specifically, the reporter 2 production of SecGEY overexpressing strains BPC0166, BPC0167, and BPC0168 compared to the control strain BPC0178, and the associated coefficient of variation (CV; n=3) are shown in Table 6 below, and the relative reporter 2 production normalized to the cell aggregate volume in the culture, measured by OD600, is shown in Table 7 below. As shown in Tables 6 and 7, reporter 2 protein production is equivalent or less for all three strains overexpressing SecGEY.

[0209] [Table 6]

[0210] [Table 7]

[0211] Example 5 Construction of Bacillus subtilis cells that express heterologous proteins and overexpress SecG, SecE, and SecY. This embodiment describes the construction of Bacillus subtilis cells overexpressing SecG, SecE, and SecY in a host background expressing heterologous reporter 3 proteins. More specifically, a control Bacillus subtilis strain named BPC0229 contains two introduced reporter 3 expression cassettes, each containing a promoter sequence (P2-00788), an open reading frame (ORF) encoding a subtilis sine variant 3 protein, positioned upstream (5') and operably ligated thereto, the cassettes being incorporated into the Bacillus subtilis skf locus (skf::P2-00788-reporter3) and the Bacillus subtilis pksR locus (pksR::P2-00788-reporter3). The design and construction of a modified Bacillus subtilis strain expressing reporter 3 protein and overexpressing the artificial (non-natural) secGEY operon of this disclosure was carried out as follows.

[0212] We constructed a first modified Bacillus subtilis strain expressing SecG, SecE, and SecY proteins as a non-natural operon (PsecG-secGEY) initiated by a secG promoter derived from those native gene loci and incorporated into the Bacillus subtilis (B. subtilis) aprE gene locus. More specifically, the non-natural operon PsecG-secGEY was constructed by overlap extension PCR and includes the Bacillus subtilis (B. subtilis) secG promoter and secG 5-UTR (SEQ ID NO: 1), a secG ORF (SEQ ID NO: 2) positioned upstream (5') and operably ligated thereto, secERBS and ORF (SEQ ID NO: 3), secY RBS and ORF (SEQ ID NO: 4) operably ligated thereto, and a BPN' terminator derived from Bacillus amyloliquefaciens (SEQ ID NO: 5) operably ligated thereto. An embedded cassette containing the upstream (5') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 14), the non-natural operon PsecG-secGEY (SEQ ID NO: 7) operably linked thereto, the promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked thereto, and the downstream (3') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 16) operably linked thereto was constructed by overlap extension PCR. This cassette was then transformed into a Bacillus subtilis (B. subtilis) strain containing two copies of the reporter 3 expression cassette embedded at the skf locus (skf::P2-00788-reporter 3) and the pksR locus (pksR::P2-00788-reporter 3) to create Bacillus subtilis (B. subtilis) strain AL394.

[0213] A second modified strain was constructed expressing SecG, SecE, and SecY as a non-natural operon (PspoVG-secGEY) initiated by a spoVG promoter derived from those native gene loci and incorporated into the aprE locus. More specifically, the non-natural operon PspoVG-secGEY was constructed by overlap extension PCR and contains the Bacillus subtilis (B. subtilis) spoVG promoter and spoVG 5-UTR (SEQ ID NO: 10), a secG ORF (SEQ ID NO: 2) positioned upstream (5') and operably ligated to it, secE RBS and ORF (SEQ ID NO: 3) operably ligated to it, secY RBS and ORF (SEQ ID NO: 4) operably ligated to it, and a BPN' terminator (SEQ ID NO: 5) derived from B. amyloliquefaciens operably ligated to it. An embedded cassette containing the upstream (5') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 14), the non-natural operon PspoVG-secGEY (SEQ ID NO: 11) operably linked thereto, the promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked thereto, and the downstream (3') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 16) operably linked thereto was constructed by overlap extension PCR. This cassette was then transformed into a Bacillus subtilis (B. subtilis) strain containing two copies of the reporter 3 expression cassette embedded at the skf locus (skf::P2-00788-reporter 3) and the pksR locus (pksR::P2-00788-reporter 3) to create Bacillus subtilis (B. subtilis) strain AL395.

[0214] A third modified strain was constructed expressing SecG, SecE, and SecY as a non-natural operon (Phbs-secGEY) initiated by an hbs promoter derived from those native gene loci and incorporated into the aprE locus. More specifically, the non-natural operon Phbs-secGEY was constructed by overlap extension PCR and contains the Bacillus subtilis hbs promoter and Bacillus subtilis spoVG 5-UTR (SEQ ID NO: 12), a secG ORF (SEQ ID NO: 2) positioned upstream (5') and operably ligated to it, secE RBS and ORF (SEQ ID NO: 3) operably ligated to it, secY RBS and ORF (SEQ ID NO: 4) operably ligated to it, and a BPN' terminator derived from B. amyloliquefaciens (SEQ ID NO: 5) operably ligated to it. An embedded cassette containing the upstream (5') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 14), the non-natural operon Phbs-secGEY (SEQ ID NO: 13) operably linked thereto, the promoter and kanamycin resistance gene (SEQ ID NO: 15) operably linked thereto, and the downstream (3') homologous region of Bacillus subtilis (B. subtilis) aprE (SEQ ID NO: 16) operably linked thereto was constructed by overlap extension PCR. This cassette was then transformed into a Bacillus subtilis (B. subtilis) strain containing two copies of the reporter 3 expression cassette embedded at the skf locus (skf::P2-00788-reporter 3) and the pksR locus (pksR::P2-00788-reporter 3) to create Bacillus subtilis (B. subtilis) strain AL396.

[0215] Example 6 Expression of reporter 3 proteases in Bacillus subtilis cells overexpressing SecG, SecE, and SecY. In this example, the expression of heterologous reporter 3 protein in the presence of SecGEY overexpression was compared with the expression of the same reporter 3 protein without SecGEY overexpression. More specifically, reporter 3 production was evaluated for three SecGEY overexpression strains (AL394, AL395, AL396) and the control strain BPC0229 under small-scale conditions as follows: Single colonies were inoculated into a pre-culture in trypsin-soybean broth (1.7% tryptone, 0.3% soytone, 0.25% glucose, 0.5% sodium chloride, 0.25% dibasic potassium phosphate) and grown at 37°C and 250 RPM. Using the pre-culture, the cells were inoculated at a 1:1000 dilution into 20 ml of 0.5 × MPS2 medium supplemented with 80 mM MOPS and 5 mM CaCl2 adjusted to pH 7.3, and the cultures were grown in a flask at 37°C and 250 RPM. MPS2 medium consists of: 10v / v% 10×MOPS-based medium (8.4w / v% MOPS, 2.9w / v% sodium chloride, 1.5w / v% potassium hydroxide, 0.05w / v% potassium sulfate, 0.05w / v% magnesium chloride, 0.7w / v% tricine, 10v / v% micronutrients), 10w / v% maltrin M150, 6w / v% soyton, 0.78w / v% dipotassium phosphate, 0.36w / v% urea, 0.2w / v% monopotassium phosphate, 0.06w / v% trisodium citrate dihydrate, and pH adjusted with potassium hydroxide. The micronutrients were composed of 100x stock solution per liter, 1.47g sodium citrate 2H2O, 1.47g CaCl22H2O, 400mg FeSO47H2O, 100mg MnSO4H2O, 100mg ZnSO4H2O, 50mg CuCl22H2O, 100mg CoCl26H2O, and 100mg Na2MoO42H2O.

[0216] As essentially described in International Publication No. 2020112609, the amount of reporter 3 protease in the culture supernatant was determined using the suc-AAPF-pNA assay. The substrate is N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA). During the hydrolysis of the peptide substrate by the protease, 4-nitroanilide is cleaved to produce 4-nitroaniline, a yellow chromophore. Therefore, the absorbance at 405 nm is measured, and the slope of the absorbance change (mOD / min) directly correlates with the amount of protease in the analyte sample. The 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 sample was added to a microtiter plate containing 190 μL of AAPF stock diluted in Tris buffer (100 mg / ml AAPF stock in DMSO diluted 100× in 100 mM Tris, 0.005% Tween 80, pH 8.6). The absorbance of the solution was measured at 405 nm using a SpectraMax spectrophotometer.

[0217] The control strain BPC0229, as well as SecGEY-overexpressing strains AL394, AL395, and AL396, were cultured, and reporter 3 protease activity was sampled after 16, 24, and 40 hours of growth. More specifically, the reporter 3 production of SecGEY-overexpressing strains AL394, AL395, and AL396 compared to the control strain BPC0229, and the associated coefficient of variation (CV; n=3) are shown in Table 7 below, and the relative reporter 3 protease production normalized to the cell aggregate volume in the culture, as measured by OD600, is shown in Table 8 below. As shown in Tables 7 and 8, when considering protease production by OD600, reporter 3 protein production increased in all three SecGEY-overexpressing strains.

[0218] [Table 8]

[0219]

Table 9

[0220] References 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, 17:52 2018. 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, 19:52 2020. 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

1. Recombinant Gram-positive bacterial cells containing an introduced secGEY operon and an expression cassette encoding a heterologous subtilisin.

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

2.

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

31.

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

32.

5. The recombinant cell according to claim 1, wherein the introduced secGEY operon comprises an upstream (5') wild-type secG promoter and a secG 5'-UTR sequence or a functional variant thereof, and a downstream secGEY open reading frame operably linked thereto, or the introduced secGEY operon comprises a heterogeneous upstream (5') promoter and a 5'-UTR sequence, and a downstream secGEY open reading frame (ORF) operably linked thereto.

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

13.

7. Recombinant cell according to claim 1, comprising at least two introduction cassettes encoding the same or different subtilisins, or at least three introduction expression cassettes encoding the same or different subtilisins.

8. The cassette encodes alkaline subtilisin, according to claim 1 or 7, recombinant cell.

9. The recombinant cell according to claim 1 or 7, wherein the subtilisin has at least 80% amino acid identity with respect 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 (5') promoter sequence, a downstream nucleic acid operably linked thereto that encodes a secG protein having at least 80% identity with SEQ ID NO: 28, a downstream nucleic acid operably linked thereto that encodes a secE protein having at least 80% identity with SEQ ID NO: 29, and a downstream (3') nucleic acid operably linked thereto that encodes a secY protein having at least 80% identity with SEQ ID NO:

30.

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

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

13. A method for generating heterologous subtilisins in modified Gram-positive bacterial cells, comprising: (a) Obtain Gram-positive bacterial cells that produce heterologous subtilisin, and introduce a synthetic secGEY operon into the cells, (b) Fermenting the modified cells under conditions suitable for the production of the subtilisin A method that includes this.

14. A method for generating heterologous subtilisins in modified Gram-positive bacterial cells, comprising: (a) Obtain Gram-positive bacterial cells, and introduce (i) an expression cassette encoding the heterologous subtilisin and (ii) a synthetic secGEY operon into the cells, (b) Fermenting the modified cells under conditions suitable for the production of the subtilisin A method that includes this.

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

16. The method according to claim 13 or 14, wherein the modified cells produce an increased amount of the subtilisin compared to control cells fermented under the same conditions, the control cells contain the same introduction cassette encoding the same subtilisin, and the control cells do not contain the introduction secGEY operon.

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

2.

18. The method according to claim 13 or 14, wherein the introduced secGEY operon includes a secE open reading frame (ORF) sequence having at least 80% identity with respect to sequence number 31.

19. The method according to claim 13 or 14, wherein the introduced secGEY operon includes a secY open reading frame (ORF) sequence having at least 80% identity with sequence number 32.

20. The method according to claim 13 or 14, wherein the introduced secGEY operon comprises an upstream secG promoter and a secG 5'-UTR array having at least about 95% identity with respect to SEQ ID NO: 1, and a downstream secGEY ORF operably coupled thereto.

21. The method according to claim 13 or 14, wherein the introduced secGEY operon includes a different upstream (5') promoter and a 5'-UTR array, and a downstream secGEY ORF operably coupled thereto.

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

13.

23. The method according to claim 14, wherein the cells include at least two introduction cassettes encoding the same or different subtilisins, or at least three introduction expression cassettes encoding the same or different subtilisins.

24. The method according to claim 14 or 23, wherein the cassette encodes an alkaline subtilisin.

25. The method according to claim 14 or 23, wherein the cassette encoding the subtilisin comprises an upstream promoter region sequence, a downstream nucleic acid encoding a protein signal sequence operably linked thereto, a downstream nucleic acid encoding a pro region sequence operably linked thereto, and a downstream nucleic acid encoding the mature subtilisin operably linked thereto.

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

23.

27. The method according to claim 15, wherein the increased amount of subtilisin is at least about 5% greater than that of the control cells when fermented under the same conditions.

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