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

Abstract

The present disclosure relates generally to Gram-positive bacterial strains having enhanced protein productivity phenotypes. Accordingly, certain aspects relate to compositions and methods for constructing recombinant (modified) Gram-positive bacterial strains for enhanced production of proteins of interest.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 518,482, filed August 9, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure generally relates to the fields of bacteriology, microbiology, genetics, molecular biology, enzymology, and industrial protein production. Some embodiments of this disclosure relate to Gram-positive bacterial cells with enhanced protein productivity phenotypes, compositions and methods for constructing recombinant Gram-positive bacterial cells, etc. References to sequence lists

[0003] The electronic submission of the text file sequence list named “NB42147USPSP_SequenceListing.xml” was created on July 25, 2023, and is 48 KB in size. It is hereby incorporated in its entirety by reference. Background Technology

[0004] Gram-positive bacteria such as *Bacillus subtilis*, *Bacillus licheniformis*, and *Bacillus amyloliquefaciens* are frequently used as microbial factories for producing industrially relevant proteins due to their excellent fermentation properties and high yields. For example, host cells of *Bacillus* species are well-known for producing enzymes (e.g., amylase, cellulase, mannanase, pectinase, protease, amylopectinase, etc.) required by the food, textile, laundry, medical device cleaning, and pharmaceutical industries. Because these non-pathogenic Gram-positive bacteria produce proteins completely free of toxic byproducts (e.g., lipopolysaccharide; LPS, also known as endotoxin), they have achieved Qualified for Safety (QPS) status from the European Food Safety Authority (EFSA), and many of their products have achieved Generally Recognized As Safe (GRAS) status 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 significance in the field of biotechnology. Similarly, the optimization of Bacillus host cells for the production and secretion of one or more target proteins is highly relevant, especially in industrial biotechnological environments where even small improvements in protein yield are significant when proteins are produced in large industrial quantities. For example, the expression of many secreted proteins can still be challenging and unpredictable in terms of yield and other aspects. As described below, this disclosure relates to a highly desired and unmet need for obtaining and constructing Gram-positive cells (e.g., protein-producing hosts) with enhanced protein-producing capabilities. Summary of the Invention

[0006] As described herein, certain embodiments of this disclosure relate to methods and compositions for enhancing protein production in Gram-positive bacterial (host) cells. Therefore, certain embodiments particularly provide Gram-positive bacterial cells / strains expressing heterologous proteins, Gram-positive bacterial cells secreting heterologous proteins into fermentation broth, Gram-positive bacterial cells containing an introduced (synthetic) secGEY operon expressing SecG, SecE, and SecY transloson proteins, etc. Therefore, some embodiments involve non-natural (synthetic) secGEY operons, nucleic acid (DNA) sequences encoding SecG, SecE, and SecY transloson 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 target proteins, DNA sequences encoding (heterologous) precursor proteases, DNA sequences encoding (heterologous) mature proteases, DNA sequences encoding protease signal (secretory) peptide sequences, DNA sequences encoding protease PRO region sequences, DNA sequences encoding one or more ribosome binding sites (RBS), promoter region (DNA) sequences, 5'-untranslated region (5'-UTR) sequences, etc.

[0007] In some embodiments, this disclosure provides recombinant Gram-positive bacterial cells comprising an introduced (synthetic) secGEY operon and one or more introduced expression cassettes encoding a heterologous subtilisin protein. In some embodiments, 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, a secE ORF sequence having at least about 80% to 100% identity with SEQ ID NO: 31, and a secY ORF sequence having at least about 80% to 100% identity with SEQ ID NO: 32. In some other embodiments, the introduced (synthetic) secGEY operon comprises an upstream (5') wild-type secG promoter and a secG 5'-UTR sequence operably linked to a downstream secGEY open reading frame (ORF). In other embodiments, the introduced secGEY operon comprises an upstream (5') heterologous promoter and a 5'-UTR sequence operably linked to a downstream secGEY ORF. In some other embodiments, the recombinant cell comprises at least two introduced cassettes encoding the same or different subtilisin, or at least three introduced expression cassettes encoding the same or different subtilisin. In some embodiments, the one or more cassettes encode alkaline subtilisin. In another embodiment, the one or more cassettes encode alkaline subtilisin, which contains an isoelectric point (pI) between about 8.5 and about 10. In some other embodiments, the heterologous subtilisin has at least about 80% amino acid identity with the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23. For example, in some embodiments, the heterologous subtilisin protease has 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.

[0008] As described herein, a non-natural (synthetic) secGEY operon of this disclosure was constructed in which the secG, secE, and secY open reading frame (ORF) polynucleotide sequences (i.e., encoding the secG, secE, and secY transloson proteins, respectively) are operatively linked in any order. For example, in some embodiments, the non-natural (synthetic) secGEY operon comprises an upstream secE ORF operably connected to a downstream secY ORF operably connected to a downstream secGO RRF (e.g., 5'-[secE]-[secY]-[secG]-3'), the non-natural secGEY operon comprises an upstream secE ORF operably connected to a downstream secG ORF operably connected to a downstream secY ORF (e.g., 5'-[secE]-[secG]-[secY]-3'), the non-natural secGEY operon comprises an upstream secY ORF operably connected to a downstream secE ORF operably connected to a downstream secG ORF (e.g., 5'-[secY]-[secE]-[secG]-3'), and the non-natural secGEY operon comprises an upstream secY ORF operably connected to a downstream secG ORF operably connected to a downstream secG ORF (e.g., 5'-[secY]-[secE]-[secG]-3'). The downstream secG ORF is operatively connected to the downstream secE ORF (e.g., 5'-[secY]-[secG]-[secE]-3'), and the non-natural secGEY operator contains an upstream secG ORF operatively connected to the downstream secY ORF operatively connected to the downstream secE ORF (e.g., 5'-[secG]-[secY]-[secE]-3'), etc.

[0009] Therefore, some embodiments relate to polynucleotides encoding the synthetic secGEY operon of this disclosure. For example, in some embodiments, this disclosure provides a polynucleotide construct encoding the synthetic secGEY operon, wherein the polynucleotide comprises at least an upstream (5') promoter region sequence operatively linked to a downstream nucleic acid encoding a secG protein having at least about 80% to 100% identity with SEQ ID NO: 28, the downstream nucleic acid encoding the secG protein being operatively linked to a downstream nucleic acid encoding a secE protein having at least about 80% to 100% identity with SEQ ID NO: 29, and the downstream nucleic acid encoding the secE protein being operatively linked to a downstream (3') nucleic acid encoding a secY protein having at least about 80% to 100% identity with SEQ ID NO: 30.

[0010] In other embodiments, this disclosure provides a method for producing heterologous subtilisin protein in recombinant (modified) bacterial cells. Therefore, some embodiments relate to a method for producing heterologous subtilisin in modified Gram-positive bacterial cells, the method comprising: (a) obtaining Gram-positive bacterial cells expressing / producing heterologous subtilisin and introducing a synthetic secGEY operon into the cells, and (b) fermenting the modified cells under conditions suitable for producing subtilisin. In other embodiments, this disclosure provides a method for producing heterologous subtilisin in modified Gram-positive bacterial cells, the method 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 producing subtilisin.

[0011] In some embodiments of these methods, the subtilisin is secreted into the fermentation broth during fermentation under conditions suitable for producing subtilisin. In other embodiments, the modified cells produce an increased amount of subtilisin relative to control cells fermenting under the same conditions, wherein the control cells contain the same introduced expression cassette encoding the same heterologous subtilisin but do not contain the introduced secGEY operon. In other embodiments of these methods, the introduced secGEY operon contains a secG ORF sequence having at least about 80% to 100% identity with SEQ ID NO: 2. In another embodiment, the introduced secGEY operon contains a secE ORF sequence having at least about 80% to 100% identity with SEQ ID NO: 31. In still other embodiments, the introduced secGEY operon contains a secY ORF sequence having at least about 80% to 100% identity with SEQ ID NO: 32. In some other embodiments of these methods, the introduced secGEY operon comprises an upstream secG promoter and a secG 5'-UTR sequence having at least about 95% to 100% identity with SEQ ID NO: 1, operably linked to a downstream secGEY ORF. In other embodiments, the introduced secGEY operon comprises an upstream (5') heterologous promoter and a 5'-UTR sequence operably linked to a downstream secGEY ORF. In yet another embodiment, the cell comprises at least two introduced cassettes encoding the same or different heterologous subtilisin, or at least three introduced expression cassettes encoding the same or different heterologous subtilisin. In some other embodiments, the one or more cassettes encode alkaline subtilisin. In other embodiments, the one or more cassettes encode alkaline subtilisin, which contains an isoelectric point (pI) between about 8.5 and about 10. In still other embodiments of these methods, the one or more cassettes encode alkaline subtilisin of subgroup I-S2. In some other embodiments, the heterologous subtilisin protease has at least about 80% to 100% amino acid identity with the mature amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 23. In some related embodiments, the heterologous subtilisin protease has 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 one or more cassettes encoding subtilisin include an upstream promoter region sequence operatively linked to a downstream nucleic acid encoding a protein signal sequence, the downstream nucleic acid encoding the protein signal sequence being operatively linked to a downstream nucleic acid encoding a pre-coding region sequence, and the downstream nucleic acid encoding the pre-coding region sequence being operatively linked to a downstream nucleic acid encoding mature subtilisin. In specific embodiments of these methods, when fermented under the same conditions, the amount of subtilisin produced is increased by at least about 5% relative to control cells. Attached Figure Description

[0012] Figure 1 The nucleic acid (DNA) sequences of the non-natural (synthetic) secGEY operons constructed and described herein are shown. 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 "Phbs-secGEY"; SEQ ID NO: 13), as described in the examples below. Figure 1 As shown, the synthesized operon 1 (PsecG-secGEY) comprises: an upstream (5') secG promoter region containing the secG promoter (PsecG) and the secG 5'-UTR sequence ( Figure 1 A; SEQ ID NO: 1), which is operatively linked to a downstream DNA sequence ( Figure 1 B; secG ORF, SEQ ID NO: 2), the downstream DNA sequence ( Figure 1 B; secG ORF, SEQ ID NO: 2) encodes the secG transloson protein (SEQ ID NO: 28), which is operatively linked to a downstream DNA sequence containing the secE ribosome binding site (RBS) and the secE ORF. Figure 1 C; SEQ ID NO: 3), the downstream DNA sequence ( Figure 1 C; SEQ ID NO: 3) encodes the secE transloson protein (SEQ ID NO: 29), which is operatively linked to a downstream DNA sequence containing secY RBS and secY ORF. Figure 1 D; SEQ ID NO: 4), the downstream DNA sequence ( Figure 1 D; SEQ ID NO: 4) encodes the secY transloson protein (SEQ ID NO: 30). Similarly, the synthetic operon 2 ( Figure 1PspoVG-secGEY, SEQ ID NO: 11) contains an upstream (5') spoVG promoter region, which contains the spoVG promoter (PspoVG) and the spoVG 5'-UTR sequence ( Figure 1 E; SEQ ID NO: 10), which is operatively linked to a downstream DNA sequence ( Figure 1 F; secG ORF, SEQ ID NO: 2), the downstream DNA sequence ( Figure 1 F; secG ORF, SEQ ID NO: 2) encodes the secG transloson protein (SEQ ID NO: 28), which is operatively linked to a downstream DNA sequence containing the secE ribosome binding site (RBS) and the secE ORF. Figure 1 G; SEQ ID NO:3), the downstream DNA sequence ( Figure 1 G; SEQ ID NO: 3) encodes the secE transloson protein (SEQ ID NO: 29), which is operatively linked to a downstream DNA sequence containing secY RBS and secY ORF. Figure 1 H; SEQ ID NO: 4), the downstream DNA sequence ( Figure 1 H; SEQ ID NO: 4) encodes the secY transloson protein (SEQ ID NO: 30), and the synthesized operon 3 (Phbs-secGEY; SEQ ID NO: 13) contains an upstream (5') hbs promoter region containing the hbs promoter (Phbs) and the spoVG 5'-UTR sequence ( Figure 1 I; SEQ ID NO: 12), which is operatively linked to a downstream DNA sequence ( Figure 1 J;secG ORF, SEQ ID NO: 2), the downstream DNA sequence ( Figure 1 J; secG ORF, SEQ ID NO: 2) encodes the secG transloson protein (SEQ ID NO: 28), which is operatively linked to a downstream DNA sequence containing the secE ribosome binding site (RBS) and the secE ORF. Figure 1 K; SEQ ID NO: 3), the downstream DNA sequence ( Figure 1 K; SEQ ID NO: 3) encodes the secE transloson protein (SEQ ID NO: 29), which is operatively linked to a downstream DNA sequence containing secY RBS and secY ORF. Figure 1 L; SEQ ID NO: 4), the downstream DNA sequence ( Figure 1L; SEQ ID NO: 4) encodes the secY transloson protein (SEQ ID NO: 30). Additionally, as... Figure 1 C Figure 1 D、 Figure 1 G, Figure 1 H, Figure 1 K and Figure 1 As shown in L, the secE and secY ribosome binding sites are represented by underlined nucleotides.

[0013] Figure 2 It presents the natural Bacillus clausii subtilis protease (B. clausii). Figure 2 A; Reporter-1, SEQ ID NO: 21), natural Bacillus amyloliquefaciens (B. amyloliquefaciens) subtilisin ( Figure 2 B; Reporter-2, SEQ ID NO: 22) and native Bacillus gibsonii subtilisin ( Figure 2 C; the mature amino acid sequence of reporter-3 (SEQ ID NO: 23). More specifically, as Figure 2 As shown, the natural Bacillus subtilis protease 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 2 A), the natural Bacillus amyloliquefaciens subtilis protease reporter contains 275 amino acid residues, with a theoretical pI of approximately 6.30 and an Mw of approximately 27,533 Da ( Figure 2 B), and the natural Bacillus girdrensis subtilis protease reporter contains 269 amino acid residues, with a theoretical pI of approximately 9.57 and a Mw of approximately 27,498 Da ( Figure 2 C).

[0014] Figure 3 The BLAST-P alignment of native Bacillus clausti subtilis protease (mature sequence; SEQ ID NO: 21) with native Bacillus amyloliquefaciens subtilis protease (mature sequence; SEQ ID NO: 22) is presented. Figure 3 As shown, SEQ ID NO: 21 and SEQ ID NO: 22 have approximately 60% amino acid identity.

[0015] Figure 4 The BLAST-P alignment of native Bacillus giganteus subtilisin (mature sequence; SEQ ID NO: 23) with native Bacillus amyloliquefaciens subtilisin (mature sequence; SEQ ID NO: 22) is presented. Figure 4As shown, SEQ ID NO: 23 and SEQ ID NO: 22 have approximately 57% amino acid identity.

[0016] Figure 5 The BLAST-P alignment of native Bacillus clausti subtilis protease (mature sequence; SEQ ID NO: 21) with native Bacillus giganteus subtilis protease (mature sequence; SEQ ID NO: 23) is presented. Figure 5 As shown, SEQ ID NO: 21 and SEQ ID NO: 23 have approximately 81% amino acid identity. Brief description of biological sequences

[0017] SEQ ID NO: 1 is a nucleic acid (DNA) promoter named “PsecG” and its 5'-UTR sequence, which contains the Bacillus subtilis secG promoter and secG 5'-UTR sequence that can be manipulated together.

[0018] SEQ ID NO: 2 is a DNA sequence containing a Bacillus subtilis secG read frame (ORF) encoding a natural SecG protein.

[0019] SEQ ID NO: 3 is a DNA sequence containing the Bacillus subtilis ribosome binding sequence (RBS) and the secE ORF encoding the natural SecE protein.

[0020] SEQ ID NO: 4 is a DNA sequence containing Bacillus subtilis RBS and secY ORF encoding the natural secY protein.

[0021] SEQ ID NO: 5 is a DNA sequence containing the BPN' terminator region of Bacillus amyloliquefaciens.

[0022] SEQ ID NO: 6 is the downstream (3') Bacillus subtilis pksR integration box homology region (HR).

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

[0024] SEQ ID NO: 8 is a DNA sequence containing the Bacillus subtilis alanine racemic enzyme (alrA) gene.

[0025] SEQ ID NO: 9 is the upstream (5') Bacillus subtilis pksR integration box HR.

[0026] SEQ ID NO: 10 is a synthetic DNA promoter region named “PspoVG”, which contains the operably combinable Bacillus subtilis spoVG promoter and spoVG 5'-UTR sequence.

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

[0028] 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 that can be manipulated together.

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

[0030] SEQ ID NO: 14 is the upstream (5') Bacillus subtilis aprE integration box homology region (HR).

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

[0032] SEQ ID NO: 16 is the downstream (3') Bacillus subtilis aprE integration box HR.

[0033] SEQ ID NO: 17 is the amino acid sequence of the Bacillus subtilis AprE signal (secreted) peptide.

[0034] SEQ ID NO: 18 is the natural pre-region amino acid sequence of Bacillus subtilis protease from B. lentus.

[0035] SEQ ID NO: 19 is the amino acid sequence of the signal peptide of Bacillus subtilis protease (BPN').

[0036] SEQ ID NO: 20 is the native pre-region amino acid sequence of Bacillus amyloliquefaciens subtilisin (BPN') protease.

[0037] SEQ ID NO: 21 is the mature amino acid sequence of natural Bacillus subtilis protease.

[0038] SEQ ID NO: 22 is the mature amino acid sequence of natural Bacillus amyloliquefaciens subtilis protease.

[0039] SEQ ID NO: 23 is the mature amino acid sequence of natural Bacillus subtilis protease.

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

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

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

[0043] SEQ ID NO: 27 is a DNA sequence containing the Bacillus subtilis spoVG term sequence.

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

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

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

[0047] SEQ ID NO: 31 is the DNA sequence of the wild-type Bacillus subtilis secE read frame encoding the natural secE protein of SEQ ID NO: 29.

[0048] SEQ ID NO: 32 is the DNA sequence of the wild-type Bacillus subtilis secY read frame encoding the natural secY protein of SEQ ID NO: 30. Detailed Implementation

[0049] As described herein, certain embodiments of this disclosure relate to compositions and methods for enhancing protein production in Gram-positive bacterial (host) cells. Therefore, certain embodiments of this disclosure particularly provide Gram-positive bacterial cells / strains expressing heterologous proteins, compositions thereof and methods thereof, Gram-positive bacterial cells secreting heterologous proteins into fermentation broth, compositions thereof and methods thereof, Gram-positive bacterial cells comprising an introduced (non-natural) secGEY operon expressing SecG, SecE, and SecY transloson proteins, compositions thereof and methods thereof, and / or combinations thereof. In some other embodiments, this disclosure relates to a non-natural secGEY operon, nucleic acid (DNA) sequences encoding SecG, SecE, and SecY transloson proteins (e.g., secG ORF encoding a functional SecG protein, secEORF encoding a functional SecE protein, secY ORF encoding a functional SecY protein), DNA sequences encoding a heterologous target protein, DNA sequences encoding a (heterologous) precursor protease, DNA sequences encoding a (heterologous) mature protease, DNA sequences encoding a protease signal (secretory) peptide sequence, DNA sequences encoding a protease PRO region sequence, DNA sequences encoding one or more ribosome binding sites (RBS), promoter region (DNA) sequences, 5'-untranslated region (5'-UTR) sequences, etc.

[0050] I. Definition

[0051] In view of the foregoing compositions and methods of this disclosure, as further described herein, the following terms and phrases are defined. Terms not defined herein shall be interpreted as having their ordinary meaning as used in the art.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the compositions and methods of the invention are applied. While any methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of the compositions and methods of the invention, representative illustrative methods and materials are described herein. All publications and patents cited herein are incorporated herein by reference in their entirety.

[0053] It should be further noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended as a basis (or condition) for the use of exclusive terms such as “alone,” “only,” “excluding,” “not including,” etc., or for the use of “negative” to define the elements of the claims. For example, in some embodiments, control (synthetic) Gram-positive bacterial cells were constructed, wherein the control cells “do not include” (i.e., exclude) the introduced (synthetic) secGEY operon.

[0054] Upon reading this disclosure, it will be apparent to those skilled in the art that each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the inventive compositions and methods described herein. Any method of description may be performed in the order of the events stated or in any other logically possible order.

[0055] As summarized herein and illustrated in the examples, certain embodiments of this disclosure particularly provide recombinant Gram-positive bacterial cells expressing a target protein and comprising an introduced (non-natural) secGEY operon expressing SecG, SecE, and SecY translocation proteins, and methods for producing one or more target proteins in Gram-positive bacterial cells comprising an introduced (non-natural) secGEY operon expressing SecG, SecE, and SecY proteins.

[0056] As used herein, the phrases “Gram-positive bacteria,” “Gram-positive cells,” “Gram-positive bacterial strains,” and / or “Gram-positive bacterial cells” have the same meaning as used in the art. For example, Gram-positive bacterial cells include all strains of the phyla Actinobacteria and Firmicutes. In some embodiments, such Gram-positive bacteria belong to the classes Bacillus, Clostridia, and Mollicutes.

[0057] As used herein, “Bacillus” includes all species within the genus “Bacillus” as known to those skilled in the art, including but not limited to Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus lautus, and Bacillus thuringiensis. It should be recognized that the genus Bacillus is undergoing continuous taxonomic recombination. Therefore, this genus aims to include species that have been reclassified, including but not limited to organisms such as *Geobacillus stearothermophilus* (now named *Geobacillus stearothermophilus*).

[0058] This document provides certain ranges in which numerical values ​​are preceded by the term "approximately". The term "approximately" is used herein to provide textual support for the exact number that follows it, as well as for numbers that are close to or approximate to the number following the term. In determining whether a number is close to or approximate to a particular stated number, the close to or approximate unstated number may be a number that is substantially equivalent to the number for which the particular statement is provided in the context in which it is presented. For example, with respect to numerical values, the term "approximately" refers to the range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context.

[0059] The term “derived” encompasses the terms “of origin,” “obtained,” “available,” and “generated,” and generally indicates that a specified material or composition finds its origin in another specified material or composition, or has features that can be described with reference to another specified material or composition. For example, the recombinant Gram-positive bacterial cells of this disclosure can be derived from / obtained from any known Gram-positive bacterial strain.

[0060] As used herein, the terms “recombinant” or “non-natural” refer to an organism, microorganism, cell, nucleic acid molecule, or vector having at least one engineered genetic alteration or having been modified by the introduction of a heterologous nucleic acid molecule, or to a cell (e.g., a microbial cell) that has been altered to control the expression of a heterologous or endogenous nucleic acid molecule or gene. Recombination also refers to a cell derived from a non-natural cell, or a descendant of a non-natural cell having one or more such modifications. Genetic alterations include, for example, the introduction of an expressible nucleic acid molecule encoding a protein, or the addition, deletion, substitution, and / or other functional alterations of the cell’s genetic material. For example, recombinant cells may express genes or other nucleic acid molecules (e.g., fusion proteins or chimeric proteins) not found in natural (wild-type) cells, or may provide altered endogenous gene expression patterns, such as overexpression, low expression, minimal expression, or no expression at all. “Recombination” or “recombining” of nucleic acids is typically an assembly of two or more nucleic acid fragments that produce a chimeric gene.

[0061] As used herein, “nucleic acid” refers to nucleotide or polynucleotide sequences and fragments or portions thereof, as well as DNA, cDNA, and RNA of genomic or synthetic origin, which may be double-stranded or single-stranded, whether representing a sense strand or an antisense strand. It should be understood that, due to the degeneracy of the genetic code, multiple nucleotide sequences can encode a given protein.

[0062] It should be understood that the polynucleotides (or nucleic acid molecules) mentioned in this article include “genes”, “vectors”, and “plasmids”.

[0063] Accordingly, the term "gene" refers to a specific sequence of polynucleotides that encodes amino acids, contains all or part of the protein-coding sequence, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine, for example, the conditions for gene expression. The transcribed region of a gene may include untranslated regions (UTRs) (including introns, 5'-untranslated regions (UTRs), and 3'-UTRs) as well as coding sequences.

[0064] As used in this article, "endogenous gene" refers to a gene located in its natural location within the genome of an organism.

[0065] As used herein, a "heterologous" gene, a "non-endogenous" gene, or a "foreign" gene refers to a gene that is not normally found in a host organism but is introduced into the host organism through gene transfer. The term "one or more foreign genes" includes natural genes inserted into a non-natural organism and / or chimeric genes inserted into a natural or non-natural organism.

[0066] As used herein, a "heterologous control sequence" refers to a gene expression control sequence (e.g., promoter, enhancer, terminator, etc.) that does not inherently function to regulate (control) the expression of a target gene. Typically, heterologous nucleic acids are not endogenous (natural) to the cells or part of the genome in which they are present and have been added to cells through infection, transfection, transduction, transformation, microinjection, electroporation, etc. "Heterologous" nucleic acid constructs may contain control sequence and DNA coding sequence (ORF) combinations that are the same as or different from those found in the natural host cell.

[0067] 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 can also refer to the translation of mRNA into a polypeptide. Therefore, the term "expression" includes any steps involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0068] As used herein, the term "coding sequence" refers to a nucleotide sequence that directly identifies the amino acid sequence of the protein product it encodes. The boundaries of a coding sequence are typically defined by a reading frame (hereinafter, "ORF") that usually begins with the ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0069] As used herein, the terms “promoter,” “promoter region,” “promoter element,” “promoter sequence,” etc., refer to a nucleic acid (DNA) sequence capable of controlling the transcription of a gene coding sequence (CDS / ORF) into messenger RNA (mRNA), wherein the promoter region sequence is positioned upstream (5′) and operatively linked to the downstream (3′) gene (ORF). As commonly understood by those skilled in the art, a promoter typically provides a site for the specific binding of RNA polymerase and the initiation of transcription. In some respects, the term “promoter” refers to the smallest portion of the promoter nucleic acid sequence required to initiate transcription (i.e., containing the RNA polymerase binding site). For example, a promoter typically contains a “-10” (shared sequence) element and a “-35” (shared sequence) element, both located upstream (5′) and opposite to the +1 transcription start site (TSS) of the CDS of the gene to be transcribed. The core promoter -10 and -35 elements are commonly referred to in the art as the “TATAAT” (Pribnow box) shared region and the “TTGACA” shared region, respectively. The spaces between the core promoter (-10 and -35) regions are typically separated by about fifteen to twenty (15-20) intercalation base pairs (nucleotides).

[0070] Promoters can be entirely derived from natural genes, or composed of different elements derived from different promoters found in nature, or even contain synthetic nucleic acid segments. Those skilled in the art will understand that different promoters can guide gene expression in different cell types, at different developmental stages, or in response to different environmental or physiological conditions. Promoters can be constitutive promoters, inducible promoters, tunable promoters, heterozygous promoters, synthetic promoters, tandem promoters, etc. Promoters that enable gene expression in most cell types most of the time are generally referred to as "constitutive promoters." It should be further recognized that, because the exact boundaries of regulatory sequences are not fully defined in most cases, DNA fragments of different lengths can possess the same promoter activity.

[0071] As used herein, a “functional promoter sequence that controls the expression of a target gene linked to a protein-coding sequence of the target gene” 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 some embodiments, this disclosure provides a polynucleotide comprising a functional upstream (5′) promoter in Gram-positive cells, wherein the functional promoter region is operatively linked to a nucleic acid sequence encoding a target protein.

[0072] As used herein, the term "pre-protein" refers to the inactive form of a protein. In some respects, full-length proteins are synthesized as precursors in the form of a pre-sequence and a mature protein (abbreviated as "pre-protein"). In other respects, full-length proteins are synthesized as precursors in the form of a signal peptide sequence, a pre-sequence, and a mature protein (abbreviated as "pre-pro-protein"). For example, the pre-sequence often serves as the signal peptide for transport, and the pre-sequence is typically essential for the proper folding of the associated (mature) protein.

[0073] As used in this article, the term "mature protein" refers to the active form of a protein as opposed to the inactive precursor (full-length) protein.

[0074] As used herein, the terms “signal sequence,” “secretion signal,” and “signal peptide” are used interchangeably and refer to the sequence of amino acid residues involved in the secretion or directed transport of a precursor protein. Typically, the signal (pre) sequence is cleaved from the precursor protein by a signal peptidase during translocation. The signal (pre) sequence is typically located at the N-terminus of the mature protein sequence or at the N-terminus of the pre-PRO sequence, in which case the signal (pre) sequence and the pre-PRO sequence are used in an operative combination and are located upstream (5′) of the mature POI sequence.

[0075] As used herein, the terms “pro sequence,” “pro-sequence,” and “pro region sequence” are used interchangeably and are abbreviated as “PRO sequence.” The term pro sequence, as used herein, has the same meaning as understood in the art. For example, the Bacillus subtilis basic serine protease “subtilisin” is first produced as pre-pro-subtilisin, which consists of a signal (pre) sequence for protein secretion, followed by a 77-amino acid pre-region (PRO) sequence, followed by an amino acid sequence encoding the mature subtilisin (e.g., pre-pro-subtilisin). The pro sequence acts as an intramolecular chaperone (e.g., directly catalyzing protein folding reactions) and is generally essential for the proper folding of the associated (mature) protein. Similarly, the folding of mature target proteins and intracellular transport (or secretion) may both require a pro sequence, suggesting a close relationship between these two functions.

[0076] As used herein, the phrases “5′ untranslated region,” “5′ untranslated region,” and / or “5′ leader sequence” are used interchangeably and are abbreviated as “5′-UTR.” As is generally understood in the art, the 5′-UTR is a region of messenger RNA (mRNA) located directly upstream (5′) of the start codon.

[0077] When a nucleic acid is functionally related to another nucleic acid sequence, that nucleic acid is "operably linked." For example, if DNA encoding a secretory leader sequence (i.e., a signal sequence) is expressed as a preprotein involved in polypeptide secretion, then the DNA encoding the secretory leader sequence is operably linked to the DNA encoding the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to that sequence (CDS, ORF); or if a ribosome binding site (RBS) is localized to promote translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, contiguous and in the reading phase. However, enhancers do not need to be contiguous. Linkage is achieved by joining at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to conventional practice. Therefore, the term "operably linked" generally refers to the association (juxtaposition) of nucleic acid sequences on a single nucleic acid fragment, such that the function of one nucleic acid fragment is influenced by the other. For example, if a promoter (pro) sequence controls the transcription of a gene coding sequence (gene CDS), then the promoter sequence is operatively linked to the gene CDS (e.g., 5′-[pro]-[gene CDS]-3′).

[0078] As used herein, a “suitable regulatory sequence” refers to a nucleotide sequence located upstream (5' non-coding sequence), inside, or downstream (3' non-coding sequence) of a coding sequence that affects the transcription, RNA processing, or stability or translation of the relevant coding sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0079] In some embodiments, the upstream (5′) promoter (pro) region sequence is operatively linked to a downstream DNA sequence (ss) encoding a signal peptide (secretion) sequence, which is operatively linked to a downstream (3′) DNA sequence (ORF) encoding a mature target protein, which can be schematically represented as 5′-[pro]-[ss]-[ORF]-3′. In some other embodiments, the upstream promoter (pro) region sequence is operatively linked to a downstream DNA sequence (ss) encoding a signal peptide (secretion) sequence, which is operatively linked to a downstream DNA sequence (PRO) encoding a pre-region amino acid sequence, which is operatively linked to a downstream DNA sequence (ORF) encoding a mature target protein, which can be schematically represented as 5′-[pro]-[ss]-[PRO]-[ORF]-3′. In some other embodiments, an upstream promoter (pro) region sequence is operatively linked to a downstream DNA sequence (ss) encoding a signal peptide (secretion) sequence, which is operatively linked to a downstream DNA sequence (PRO) encoding a pre-region amino acid sequence, which is operatively linked to a downstream DNA sequence (ORF) encoding a mature target protein, which is operatively linked to a downstream term sequence, which can be schematically represented as 5′-[pro]-[ss]-[PRO]-[ORF]-[term]-3′.

[0080] As used herein, the promoter region sequence comprising the operably combinable Bacillus subtilis secG promoter and secG 5'-UTR is hereinafter abbreviated as "PsecG" (SEQ ID NO: 1), the promoter region sequence comprising the operably combinable Bacillus subtilis spoVG promoter and spoVG 5'-UTR sequence is hereinafter abbreviated as "PspoVG" (SEQ ID NO: 10), and the promoter region sequence comprising the operably combinable Bacillus subtilis hbs promoter and spoVG 5'-UTR sequence is hereinafter abbreviated as "Phbs" (SEQ ID NO: 12).

[0081] As used herein, the “secG” read frame (ORF; SEQ ID NO: 2) encodes the native Bacillus subtilis SecG transloson protein (SEQ ID NO: 28), the “secE” ORF (SEQ ID NO: 31) encodes the native Bacillus subtilis SecE transloson protein (SEQ ID NO: 29), and the “secY” ORF (SEQ ID NO: 32) encodes the native Bacillus subtilis SecY transloson protein (SEQ ID NO: 30).

[0082] As used herein, the phrase "non-natural (artificial) secGEY operon" refers to one or more synthetic expression vectors / cassettes of this disclosure that express additional copies of the Bacillus subtilis secG, secE, and secY genes (ORF) encoding natural SecG, SecE, and SecY (translocation) proteins. In some embodiments or aspects, recombinant Gram-positive cells comprising the introduced (i.e., non-natural) secGEY operon can be described as recombinant (modified) cells that "overexpress" natural SecG, SecE, and SecY translocation proteins.

[0083] As used herein, phrases such as “non-natural secGEY operon” and / or “recombinant cells overexpressing SecG, SecE, and SecY transloson proteins” are not intended to limit the order of the Sec transloson proteins encoded by the non-natural secGEY operon. For example, those skilled in the art can readily construct non-natural secGEY operons in which the ORFs encoding the Sec transloson proteins are in any order, such as a non-natural secGEY operon containing an upstream secE ORF operatively linked to a downstream secG ORF operatively linked to a downstream secY ORF, or a non-natural secGEY promoter containing an upstream secE ORF operatively linked to a downstream secY ORF operatively linked to a downstream secG ORF, or a non-natural secGEY operon containing an upstream secY ORF operatively linked to a downstream secG ORF operatively linked to a downstream secEO RRF, etc.

[0084] Similarly, as in Figure 1 As presented in the second (e.g., of the non-natural secGEY operator) Figure 1 C and Figure 1 G) and / or a third position (e.g., Figure 1 D and Figure 1The ORF in H) may further include a ribosome binding site immediately upstream (5') of the ORF encoding the Sec translocation protein. For example, twenty (20) nucleotides containing a ribosome binding site (RBS) immediately upstream of the secE ORF. Figure 1 C (SEQ ID NO: 3) and Figure 1 G(SEQ ID NO: 3) Underline And contains twenty (20) nucleotides of ribosome binding site (RBS) immediately upstream of secYORF. Figure 1 D (SEQ ID NO: 4) and Figure 1 H (SEQ ID NO: 4) Underline .

[0085] As used herein, the non-natural secGEY operon (operon 1; SEQ ID NO: 7), named “PsecG-secGEY”, contains: an upstream (5') PsecG promoter region sequence ( Figure 1 A; SEQ ID NO: 1), the upstream (5') PsecG promoter subregion sequence is operatively linked to the downstream secG ORF ( Figure 1 B; SEQ ID NO: 2), this downstream secG ORF is operatively connected to the downstream secE ORF ( Figure 1 C; SEQ ID NO: 3), this downstream secE ORF is operatively connected to the downstream secY ORF ( Figure 1 D; SEQ ID NO: 4), the downstream secY ORF is operatively linked to the downstream (3') BPN' terminator sequence (SEQ ID NO: 5), the non-natural secGEY operon named "PspoVG-secGEY" (operon 2; SEQ ID NO: 11) containing the upstream PspoVG promoter region sequence ( Figure 1 E; SEQ ID NO: 10), this upstream PspoVG promoter subregion sequence is operatively linked to the downstream secG ORF ( Figure 1 F; SEQ ID NO: 2), this downstream secG ORF is operatively connected to the downstream secE ORF ( Figure 1 G; SEQ ID NO: 3), this downstream secE ORF is operatively connected to the downstream secY ORF ( Figure 1H; SEQ ID NO: 4), the downstream secY ORF is operatively linked to the downstream (3') BPN' terminator sequence (SEQ ID NO: 5), and the non-natural secGEY operator named "Phbs-secGEY" (operator 3; SEQ ID NO: 13) contains the upstream Phbs promoter region sequence ( Figure 1 I; SEQ ID NO: 12), this upstream Phbs promoter region sequence is operatively linked to the downstream secG ORF ( Figure 1 J; SEQ ID NO: 2), this downstream secG ORF is operatively connected to the downstream secE ORF ( Figure 1 K; SEQ ID NO: 3), the downstream secE ORF is operatively connected to the downstream secY ORF ( Figure 1 L; SEQ ID NO: 4), the downstream secY ORF is operatively connected to the downstream (3') BPN' terminating subsequence (SEQ ID NO: 5).

[0086] As used herein, exemplary proteases may be referred to as “reporter proteins.” In certain embodiments of this disclosure, exemplary reporter proteins are expressed / produced by one or more recombinant (modified) cells of this disclosure. In some embodiments, reporter proteins include, but are not limited to, native and variant Bacillus species subtilisin. In certain embodiments, exemplary subtilisin reporters include, but are not limited to, native Bacillus clausti subtilisin and its functional variants, native Bacillus giganteus subtilisin and its functional variants, native Bacillus retardans subtilisin and its functional variants, native Bacillus licheniformis subtilisin (AprL) and its functional variants, native Bacillus subtilisin (AprE) and its functional variants, native Bacillus amyloliquefaciens subtilisin (BPN') and its functional variants, etc. In some aspects, exemplary Bacillus clausti, Bacillus giganteus, and / or Bacillus retardans subtilisin reporters may be referred to as alkaline proteases. For example, alkaline subtilisin typically has an isoelectric point (pI) of about 9.5, while Bacillus licheniformis, Bacillus subtilis, and Bacillus amyloliquefaciens subtilisin have a pI of about 6.5.

[0087] As used herein, the term "subtilisin" refers to any member of the S8 serine protease family, as described in MEROPS—The Peptidase Data base (Rawlings et al., 2006). The term subtilisin includes a variety of identified and sequenced Bacillus subtilisin proteases, such as subtilisin 168, subtilisin BPN', subtilisin Carlsberg, etc., and includes mutant (variant) proteases derived therefrom.

[0088] As used herein, phrases such as “subtilisin-1”, “subtilisin-1 reporter”, “reporter-1”, and “reporter-1 protease” are used interchangeably and specifically refer to the natural Bacillus clausti subtilisin or a functional variant thereof listed in SEQ ID NO: 21.

[0089] As used herein, phrases such as “subtilisin-2”, “subtilisin-2 reporter”, “reporter-2”, “reporter-2 protease”, etc., are used interchangeably and specifically refer to the natural Bacillus amyloliquefaciens subtilisin or a functional variant thereof listed in SEQ ID NO: 22.

[0090] As used herein, phrases such as “subtilisin-3”, “subtilisin-3 reporter”, “reporter-3”, and “reporter-3 protease” are used interchangeably and specifically refer to the natural Bacillus giganteus subtilisin or a functional variant thereof listed in SEQ ID NO: 23.

[0091] In some embodiments, this disclosure relates to one or more variants of subtilisin proteases derived from parental (natural) subtilisin protease sequences, such as natural Bacillus subtilis subtilisin (e.g., 168), natural Bacillus amyloliquefaciens (e.g., BPN'), natural Bacillus licheniformis subtilisin (e.g., Carlsberg), natural Bacillus tarda subtilisin (e.g., 309), Bacillus alcalophilus subtilisin (e.g., PB92), etc. For example, in some embodiments, this disclosure provides recombinant expression cassettes encoding functional subtilisin protease variants derived from natural Bacillus clausti subtilisin (SEQ ID NO: 21), natural Bacillus amyloliquefaciens subtilisin (SEQ ID NO: 22), natural Bacillus giganteus subtilisin (SEQ ID NO: 23), etc. More particularly, those skilled in the art can readily design, construct, screen, and identify functional subtilisin protease variants using conventional methods known in the art. In particular, PCT Publications WO 2010 / 056634, WO 2011 / 130222, WO 2015 / 089447, WO 2016 / 202839, WO 2017 / 207762 and WO 2023 / 114936 (each incorporated herein by reference in its entirety) describe suitable methods and compositions for constructing functional subtilisin variants derived from natural Bacillus clausti subtilisin (SEQ ID NO: 21), functional subtilisin variants derived from natural Bacillus amyloliquefaciens subtilisin (SEQ ID NO: 22), functional subtilisin variants derived from natural Bacillus giganteus subtilisin (SEQ ID NO: 23), etc.

[0092] As used herein, the Bacillus subtilis strain named “CZ437” contains two (2) introduced (integrated) expression cassettes encoding the reporter-1 protease, wherein these cassettes comprise, in a 5' to 3' orientation and in an operably combined manner: an artificial “P2” promoter sequence (SEQ ID NO: 24) linked to a DNA sequence (ss) encoding an aprE signal peptide sequence (SEQ ID NO: 17), which is linked to a DNA sequence encoding a pre-region sequence (SEQ ID NO: 18), which is linked to a DNA sequence encoding a reporter-1 (SEQ ID NO: 21). In some embodiments, strain CZ437 may be referred to as a control (isogenic) cell, particularly when compared with one or more modified strains (e.g., strains BPC0123, BPC0182, BPC0184) overexpressing the SecG, SecE, and SecY transloson proteins.

[0093] As used herein, the Bacillus subtilis strain named “BPC0178” contains three (3) introduced (integrated) expression cassettes encoding reporter-2 protease, wherein these cassettes comprise, in a 5' to 3' orientation and in an operably combined manner: an artificial “P4” promoter sequence (SEQ ID NO: 26) linked to a DNA sequence (ss) encoding a BPN' signal peptide sequence (SEQ ID NO: 19), which is linked to a DNA sequence encoding a BPN' pre-region sequence (SEQ ID NO: 20), which is linked to a DNA sequence encoding a reporter-2 sequence (SEQ ID NO: 22). In some embodiments, strain BPC0178 may be referred to as a control cell, particularly when compared with one or more modified strains overexpressing SecG, SecE, and SecY transloson proteins (e.g., strains BPC0166, BPC0167, BPC0168).

[0094] As used herein, the Bacillus subtilis strain named “BPC0229” contains two (2) introduced (integrated) expression cassettes encoding reporter-3 protease, wherein these cassettes comprise, in a 5' to 3' orientation and in an operably combined manner: an artificial “P2-0078” promoter sequence (SEQ ID NO: 25) linked to a DNA sequence (ss) encoding an aprE signal peptide sequence (SEQ ID NO: 17), which is linked to a DNA sequence encoding a pre-region sequence (SEQ ID NO: 18), which is linked to a DNA sequence encoding reporter-3 (SEQ ID NO: 23). In some embodiments, strain BPC0229 may be referred to as a control cell, particularly when compared with one or more modified strains (e.g., strains AL394, AL395, AL396) overexpressing SecG, SecE, and SecY transloson proteins.

[0095] As used herein, “host cell” refers to a cell that has the ability to serve as a host or expression medium for a newly introduced DNA sequence. Thus, in some embodiments of this disclosure, the host cell is a Gram-positive (e.g., bacillus) and / or a Gram-negative (e.g., Escherichia coli) cell.

[0096] As used herein, “modified cell” means a recombinant cell containing at least one genetic modification that is not present in the parental cell, reference cell, or control cell from which the modified cell is derived.

[0097] As used herein, when comparing the expression and / or production of a target protein (POI) in recombinant (modified) cells with the expression and / or production of the same POI in unmodified (control) cells, it should be understood that modified and unmodified cells were grown / cultured / fermented under the same conditions (e.g., the same conditions such as culture medium, temperature, pH, etc.).

[0098] As used herein, when in phrases such as “recombinant cells express / produce an increased amount of the target protein relative to unmodified (control) cells,” “increased amount” specifically refers to an “increased amount” of the target protein (POI) expressed / produced in recombinant cells, which is always relative to unmodified (control) cells that express / produce the same POI, wherein modified and unmodified cells are grown / cultured / fermented under the same conditions.

[0099] As used herein, “increased” protein production or “enhanced” protein production means an increase in the amount of protein (e.g., the target protein) produced. Proteins can be produced within host cells or secreted (or transported) into a culture medium. In some embodiments, the target protein is produced (secreted) into a culture medium. Increased protein production can be detected, for example, as a higher maximum level of protein or enzyme activity (e.g., like protease activity) compared to the parent host cell, or as the total amount of extracellular protein produced.

[0100] As used herein, the terms “modification” and “genetic modification” are used interchangeably and include: (a) the introduction, substitution, or removal of one or more nucleotides in a gene (or its ORF), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for transcription or translation of a gene or its ORF; (b) gene disruption; (c) gene conversion; (d) gene deletion; (e) gene downregulation; (f) specific mutagenesis of any one or more genes disclosed herein; and / or (g) random mutagenesis of any one or more genes disclosed herein.

[0101] In some respects, genetic modification specifically refers to the introduction, substitution, or removal of one or more nucleotides in a nucleic acid (DNA) sequence encoding the pre-region (amino acid) sequence disclosed herein. For example, in some embodiments, a DNA sequence encoding the natural pre-region amino acid sequence shown in SEQ ID NO: 18 is genetically modified as described herein.

[0102] As used herein, such as in phrases such as “introducing a ‘gene’, ‘polynucleotide’, ‘reading frame (ORF)’, ‘gene coding sequence’, ‘vector’, ‘expression cassette’ into Gram-positive bacterial cells,” the term “introducing” includes methods known in the art for introducing polynucleotides (DNA) into cells, including but not limited to protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation, etc.

[0103] As used herein, “transformed” or “transformation” means the transformation of cells using 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 can be a heterologous nucleotide sequence (i.e., a sequence that is not naturally present in the cell to be transformed). Therefore, transformation generally refers to the introduction of foreign DNA into a host cell, such that the DNA remains an integrative chromosome or a self-replicating extrachromosomal vector.

[0104] As used herein, “transforming DNA,” “transforming sequence,” and “DNA construct” refer to DNA used to introduce a sequence into a host cell or organism. Transforming DNA is DNA used to introduce a sequence into a host cell or organism. The DNA can be generated in vitro by PCR or any other suitable technique. In some embodiments, the transforming DNA comprises an input sequence, while in other embodiments, it further comprises an input sequence flanked by a homology cassette. In yet another embodiment, the transforming DNA comprises additional non-homologous sequences added to the ends (i.e., filler sequences or flanks). The ends can be closed, such that the transforming DNA forms a closed loop, as in an insert vector.

[0105] As used herein, “gene disruption” or “gene damage” is used interchangeably and broadly refers to any genetic modification that substantially prevents the 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, premature stop codons (i.e., preventing the production of functional proteins), substitutions that eliminate or reduce protein activity, internal deletions (preventing the production of functional proteins), insertions that disrupt coding sequences, mutations that remove the operational link between the natural promoter and the read frame required for transcription, etc.

[0106] As used herein, “input sequence” refers to a DNA sequence introduced into the chromosome of a bacterial cell. In some embodiments, the input sequence is part of a DNA construct. In other embodiments, the input sequence encodes one or more target proteins. In some embodiments, the input sequence comprises a sequence that may or may not be present in the genome of the cell to be transformed (i.e., it may be a homologous or heterologous sequence). In some embodiments, the input sequence encodes one or more target proteins, genes, and / or mutated or modified genes. In alternative embodiments, the input sequence encodes a functional wild-type gene or operon, a functional mutant gene or operon, or a non-functional gene or operon. In some embodiments, a non-functional sequence may be inserted into a gene to disrupt its function. In another embodiment, the input sequence includes a selection marker. In yet another embodiment, the input sequence includes two homology boxes.

[0107] As used herein, a "homology cassette" refers to a nucleic acid sequence homologous to a sequence in a bacterial cell chromosome. More specifically, according to the invention, a homology cassette is an upstream or downstream region having approximately 80% to 100%, approximately 90% to 100%, or approximately 95% to 100% sequence identity with a directly flanking coding region of a gene or part of a gene to be deleted, disrupted, inactivated, downregulated, etc. These sequences guide the integration site of the DNA construct in the bacterial cell chromosome and guide which part of the chromosome is replaced by the input sequence. While not intended to limit this disclosure, a homology cassette may comprise between approximately 1 base pair (bp) and 200 kilobases (kb). Preferably, a homology cassette comprises between approximately 1 bp and 10.0 kb; between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb; and between 0.25 kb and 2.5 kb. The homology cassette may also 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 flanking portions of the 5' and 3' ends of the selective marker are homology cassettes, wherein the homology cassette contains a nucleic acid sequence closely adjacent to the coding region of the gene.

[0108] As used in this article, the host cell “genome,” the bacterial (host) cell “genome,” or the Bacillus species (host) cell “genome” includes chromosomal genes and extrachromosomal genes.

[0109] As used herein, the terms “plasmid,” “vector,” and “box” refer to extrachromosomal elements that typically carry genes that are not part of the cell’s central metabolism and are usually in the form of circular double-stranded DNA molecules. Such elements can be linear or circular autonomously replicating sequences, genome-integrated sequences, bacteriophage sequences, or nucleotide sequences derived from any source of single-stranded or double-stranded DNA or RNA, wherein multiple nucleotide sequences have been linked or recombined into a single construct that can introduce a promoter fragment and DNA sequence for a selected gene product, along with an appropriate 3' untranslated sequence, into the cell.

[0110] 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 is incorporated into the genome of a host cell. In some embodiments, the plasmid is present in the parent cell and lost in the daughter cell.

[0111] As used in this article, a “transformation cassette” refers to a specific vector that contains a gene (or its ORF) and, in addition to the exogenous gene, has elements that promote the transformation of a specific host cell.

[0112] As used herein, the term "vector" refers to any nucleic acid that can replicate (spread) within a cell and carry new genes or DNA segments into the cell. Therefore, the term refers to a nucleic acid construct designed for transfer between different host cells. Vectors include viruses, bacteriophages, proviruses, plasmids, phage particles, transposons, and artificial chromosomes such as YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), and PLAC (plant artificial chromosome), which are considered "attachments" (i.e., those that replicate autonomously or can integrate into the chromosome of a host organism).

[0113] "Expression vector" refers to a vector that has the ability to be incorporated into cells and express heterologous DNA within the cells. Many prokaryotic and eukaryotic expression vectors are commercially available and are known to those skilled in the art. The selection of an appropriate expression vector is within the knowledge of those skilled in the art.

[0114] As used herein, the terms "expression cassette" and "expression vector" refer to a nucleic acid construct that is recombinantly or synthetically produced and has a designated set of nucleic acid elements (i.e., vectors or vector elements, as described above) that allow a specific nucleic acid to be transcribed in target cells. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plasso 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 further includes a designated set of nucleic acid elements that allow a specific nucleic acid to be transcribed in target cells. In some embodiments, the DNA constructs of this disclosure include selective markers and inactivated chromosomes, or genes, or DNA segments as defined herein.

[0115] As used herein, a “targeting vector” is a vector comprising a polynucleotide sequence homologous to a region in the chromosome of a host cell transformed therein and capable of driving homologous recombination at that region. For example, a targeting vector can be used to introduce mutations into the chromosome of a host cell via homologous recombination. In some embodiments, the targeting vector includes, for example, additional non-homologous sequences added to the ends (i.e., filler sequences or flanking sequences). The ends can be closed, such that the targeting vector forms a closed loop, as in an insert vector. For example, in some embodiments, the cell is modified (e.g., transformed) by introducing one or more “targeting vectors” into a parent *C. chlamydiae* (host) cell.

[0116] As used herein, the term "target protein" or "POI" refers to a target polypeptide desired to be expressed in modified (recombinant) Gram-positive host cells, wherein the POI is preferably expressed at an increased level (i.e., relative to "unmodified" (parental, control, syngeneic) cells). Thus, as used herein, a POI can be an enzyme, substrate-binding protein, surfactant protein, structural protein, receptor protein, etc. In some embodiments, the modified cells of this disclosure produce an increased amount of the heterologous target protein relative to control cells. In particular embodiments, the increase in the amount of target protein produced by the modified cells of this disclosure is at least 0.5%, at least 1.0%, at least 5.0%, or more than 5.0% increase relative to control cells.

[0117] Similarly, as defined herein, a “target gene” or “GOI” refers to a nucleic acid sequence (e.g., a polynucleotide, gene, or ORF) that encodes a “target protein”. A “target gene” can be a naturally occurring gene, a mutated gene, or a synthetic gene.

[0118] As used herein, the terms “peptide” and “protein” are used interchangeably and refer to a polymer of any length containing amino acid residues linked by peptide bonds. Conventional one (1)-letter or three (3)-letter codes for amino acid residues are used herein. A peptide may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term peptide also covers amino acid polymers that have been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation to a marker component. This definition also includes, for example, peptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art.

[0119] As used herein, a "variant" polypeptide is a polypeptide typically derived from a parent (or reference) polypeptide by means of substitution, addition, or deletion of one or more amino acids using recombinant DNA technology. Variant polypeptides may differ from the parent polypeptide by a small number of amino acid residues and can be defined by the level of homology / identity of their primary amino acid sequence with that of the parent (reference) polypeptide. Preferably, the variant polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity with the parent (reference) polypeptide sequence.

[0120] As used herein, a "variant" polynucleotide refers to a polynucleotide that has a specified degree of sequence homology / identity with a parent polynucleotide, or a polynucleotide that hybridizes with a parent polynucleotide (or its complementary sequence) under strict hybridization conditions. Preferably, the variant polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% nucleotide sequence identity with the parent (reference) polynucleotide sequence.

[0121] As used herein, “mutation” refers to any change or alteration in a nucleic acid sequence. Several types of mutations exist, including point mutations, deletion mutations, silent mutations, frameshift mutations, splicing mutations, etc. Mutations can occur specifically (e.g., via site-directed mutagenesis) or randomly (e.g., via chemical reagents, through repair subtraction of bacterial strain passages).

[0122] As used herein, in the context of a polypeptide or its sequence, the term “substitution” means that one amino acid is replaced by another amino acid (i.e., substitution).

[0123] As used herein, the term “homology” refers to homologous polynucleotides or polypeptides. If two or more polynucleotides or two or more polypeptides are homologous, this means that the homologous polynucleotides or polypeptides have a degree of “identity” of at least 60%, more preferably at least 70%, even more preferably at least 85%, still more preferably at least 90%, more preferably at least 95%, and most preferably at least 98%. Whether two polynucleotide or polypeptide sequences have a sufficiently high degree of homology as defined herein can be suitably investigated by comparing the two sequences using a computer program known in the art, such as “GAP” provided in the GCG package (Program Manual for the Wisconsin Package, 8th Edition, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman and Wunsch, (1970)). DNA sequence comparison was performed using a GAP with the following settings: GAP generates a penalty of 5.0 and GAP extension penalty of 0.3.

[0124] As used herein, the term “percentage of identity (%)” refers to the level of identity between nucleic acid or amino acid sequences encoding a polypeptide when compared using sequence alignment procedures.

[0125] As used in this article, “comparative productivity” refers to the total amount of protein produced per cell per time period.

[0126] As used herein, the terms “purified,” “isolated,” or “enriched” mean that a biomolecule (e.g., a polypeptide or polynucleotide) has been altered from its native state by separating it from some or all of its naturally occurring components associated with it in nature. Such separation or purification can be accomplished using art-recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation or other protein salt precipitation, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove unwanted whole cells, cell debris, impurities, foreign proteins, or enzymes from the final composition. Further additions that provide additional benefits, such as activators, inhibitors, desired ions, pH-controlling compounds, or other enzymes or chemicals, can then be added to the purified or isolated biomolecule composition.

[0127] II. Overexpression of the synthetic SecGEY operon in Gram-positive bacterial cells enhances protein production.

[0128] As briefly described above, the production of many heterologous proteins remains challenging and unpredictable in terms of protein titer and yield. In particular, bottlenecks in the production of secreted proteins can vary depending on the host strain and protein sequence. In the context of Gram-positive bacterial hosts, the initial steps of protein secretion involve the recognition of signal peptides and the targeting and translocation of preproteins. For example, as reviewed by Pohl and Harwood (2010), the secretory pathway in Bacillus includes the Sec-dependent (Sec) and diarginine translocation (Tat) pathways, with the Sec pathway responsible for most protein secretion. As Pohl and Harwood (2010) describe, preproteins target membrane-embedded Sec translocators, which are heterotrimeric protein complexes that form the main channel for translocation of secreted proteins across the cytoplasmic membrane, named SecYEG. Protein secretion via the Sec pathway is typically a complex system involving substrate recognition, intracellular chaperones and guidance to Sec translocases, translocation postfolding events, and more.

[0129] In some respects, Freudl (2018) has outlined the secretion of recombinant proteins into the culture supernatant of bacterial (host) expression systems, with a particular focus on protein efflux systems requiring Sec-specific or Tat-specific signal peptides fused to the N-terminus (N) end of the desired target protein. For example, as concluded in this publication, the most promising way to find the optimal signal peptide for the desired protein is to screen for the maximum possible diversity of signal peptides generated by signal peptide mutations using a large signal peptide library, or alternatively, to optimize the production of a given signal peptide by using site-directed or random mutagenesis strategies. PCT Publication WO 1999 / 04006 describes expression vectors encoding SecG proteins from Gram-positive microorganisms, and related methods for secreting proteins in Gram-positive hosts including vectors encoding SecG proteins. PCT Publication WO 2008 / 141281 describes a modified secretion system for Gram-positive microbial hosts comprising an introduced vector encoding a heterologous truncated SecG protein capable of promoting the secretion of desired proteins, wherein the introduced vector may replace or supplement the endogenous (secG) gene encoding the native SecG protein. US Patent Publication US 2009 / 0029417 describes a recombinant microorganism that produces cellulase and overexpresses the Bacillus subtilis secY gene. PCT Publication WO 2008 / 126929 describes a recombinant microorganism that produces cellulase and overexpresses the Bacillus subtilis secY gene, wherein the microorganism further requires the deletion of one or more sporulation-related genes.

[0130] In other respects, Mulder et al. (2013) described a recombinant Gram-positive bacterial host system for use with IPTG-induced (P... grac The promoter is used to express the α-amylase (amyQ) gene and secrete Bacillus amyloliquefaciens α-amylase. For example, as described in this publication, recombinant host cells include P grac The amyQ gene cassette under promoter control and in xylose-inducible (P) xyl The secYEG gene cassette (for expressing SecG, SecE, and SecY proteins) is controlled by the promoter, where the synthesis of SecYEG and AmyQ is induced by 0.5% xylose and 100 μg IPTG, respectively. Chen et al. (2015) described a study aimed at identifying bottlenecks in the Sec pathway and improving the secretion of heterologous proteins using so-called molecular genetic techniques. For example, Chen et al. (2015) described recombinant cells expressing two α-amylases (AmyL and AmyS), both of which are expressed in strongly constitutive P HpaIIUnder the control of the promoter and followed by its natural signal peptide (SP) amyL and SP amyS Twenty-three (23) major genes or gene operons involved in or closely related to the Sec pathway were overexpressed, leading to the conclusion that the lack of PrsA lipoprotein and DnaK series molecular chaperones is the main rate-limiting factor for heterologous protein secretion.

[0131] Recent studies from various laboratories have described the essential or non-essential contributions of various Sec mechanism components to protein secretion in Bacillus subtilis. In some respects, Neef et al. (2020) compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases to the secretion efficiency of three proteins expressed at high levels: α-amylase AmyE from Bacillus subtilis, AmyL from Bacillus licheniformis, and serine protease BPN' from Bacillus amyloliquefaciens. For example, as described by Neef et al. (2020), syngeneic Bacillus subtilis strains lacking the following genes were constructed: molecular chaperone DnaK, transloase subunits SecDF or SecG or signal peptidases SipS, SipT, SipU, SipV or SipW, and cell envelope-associated proteases SppA, TepA, PrsW, WprA, YqeZ, HtrA or HtrB. As summarized in this publication, the results show that mutations in secDF, secG, or rasP significantly affect the secretion of AmyE, AmyL, and BPN', but the actual effect size depends on the protein studied. Furthermore, as Neef et al. (2020) concluded, the molecular chaperone DnaK is crucial for BPN' secretion, while the secretion of AmyE or AmyL is unaffected by dnaK deletion, and deletions of certain sip genes reveal strong differential effects of specific signal peptidases on the magnitude of secretory stress responses. Therefore, the production of recombinant proteins in Bacillus subtilis cells remains challenging due to bottlenecks in the universal sec pathway and the inherent ability of this bacterium to secrete highly efficient mixtures of proteases (Neef et al., 2021). In particular, Neef et al. (2021) considered and summarized the advantages and disadvantages of Gram-positive bacterial cell factories Bacillus subtilis and Lactococcus lactis, and concluded that Bacillus subtilis and closely related bacilli are best suited for mass production of recombinant proteins. However, they also pointed out that Bacillus subtilis is highly proteolytic, which represents a serious disadvantage because it can lead to product loss and / or accumulation of lysis product derivatives. This can be overcome by the deletion of protease genes, in which the resulting strains are generally more sensitive to autolysis, resulting in an increase in the amount of contaminating cytoplasmic proteins in the fermentation broth.

[0132] Based on the foregoing, certain embodiments of this disclosure relate to unexpected and unforeseen results set forth and illustrated below. More specifically, as described herein, the applicant has conceived, designed, and constructed recombinant Gram-positive (bacterial) cells / strains that overexpress the SecG, SecE, and SecY translocase proteins from an introduced non-natural (synthetic) secGEY operon and express one or more introduced cassettes encoding a Bacillus subtilis protease reporter protein. In particular, as shown in Examples 1-6 of this disclosure, the applicant has unexpectedly observed that overexpression of the complete complementary sequences of the SecG, SecE, and SecY translocase proteins resulted in enhanced production of certain reporter proteins. As further described in Examples 1, 3, and 5, control and modified Bacillus subtilis cells / strains were constructed and summarized in Table 1 below.

[0133] Table 1

[0134] strain name and genetic modification

[0135]

[0136] 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 in the absence of SecGEY overexpression. More specifically, three (3) SecGEY overexpressing strains (BPC0123, BPC0182, BPC0184) and control strain CZ437 were constructed, and reporter-1 yield was evaluated. For example, in the first experiment, control strain CZ437 and SecGEY overexpressing strain BPC0123 were cultured, and protease activity was measured after 24 (24) hours and 40 (40) hours of growth, as shown in Tables 2 and 3 below (Example 2). As presented in Table 3, it was unexpectedly observed that the reporter-1 protein yield was significantly increased (i.e., approximately 60%) when SecGEY protein was overexpressed from the secG promoter in strain BPC0123 compared to control strain CZ437 after 40 hours of culture. Similarly, in the second experiment (Tables 4 and 5), control strain CZ437 and SecGEY overexpressing strains BPC0123, BPC0182, and BPC0184 were cultured, and protease activity was measured after 22 hours of growth (Example 2). In particular, as shown in Table 5, it was unexpectedly observed that, compared to the CZ437 control strain, the reporter-1 protein yield was significantly increased (i.e., approximately 30%–40%) for all three SecGEY overexpressing strains when considering protease yield / OD600. Likewise, as the data presented in Example 2 show, suitable promoter region sequences for expressing the non-natural secGEY operon typically include promoter region sequences that function in host Gram-positive bacterial cells (e.g., secG promoter sequences, spoVG promoter sequences, hbs promoter sequences, etc.).

[0137] 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 (3) SecGEY-overexpressing strains (BPC0166, BPC0167, and BPC0168) and a control strain BPC0178 were constructed, and reporter-2 yields were evaluated. For example, as presented in Tables 6 and 7 (Example 4), the BPC0178 control strain and the SecGEY-overexpressing strains (BPC0166, BPC0167, and BPC0168) were cultured and sampled after 20 and 40 hours of growth to detect reporter-2 activity, where the reporter-2 protein yield was equivalent or lower for all three SecGEY-overexpressing strains.

[0138] 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 (3) SecGEY-overexpressing strains (AL394, AL395, and AL396) and a control strain BPC0229 were constructed, and reporter-3 yields were evaluated. For example, as presented in Tables 8 and 9 (Example 6), the BPC0229 control strain and the SecGEY-overexpressing strains (AL394, AL395, and AL396) were cultured, and samples were taken after 16, 24, and 40 hours of growth to detect reporter-3 activity. More specifically, as shown in Table 9 (Example 6), when protease yield / OD600 was taken into account, reporter-3 protein yields were significantly increased (i.e., approximately 10%–25%) for all three SecGEY-overexpressing strains.

[0139] Therefore, as summarized above and illustrated in the examples below, recombinant Gram-positive bacterial cells (e.g., Bacillus sp. cells) overexpressing SecG, SecE, and SecY proteins via the introduced non-natural secGEY operon are able to express / produce certain reporter proteases (e.g., reporter-1 and reporter-3) at significantly increased levels compared to other reporter proteases (e.g., reporter-2). For example, not wishing to be bound by theories, mechanisms, or modes of operation, the applicant unexpectedly observed in this paper that recombinant Gram-positive bacterial cells overexpressing SecG, SecE, and SecY translocation proteins (i.e., containing the introduced secGEY operon) are able to produce increased amounts of alkaline subtilisin (e.g., Bacillus clausii subtilisin SEQ ID NO: 21 and Bacillus giganteus subtilisin SEQ ID NO: 23) compared to control cells expressing the same alkaline subtilisin, where the control cells do not contain the introduced secGEY operon. In contrast, recombinant cells overexpressing the SecG, SecE, and SecY translocation proteins (i.e., containing the introduced secGEY operon) produced an equivalent amount of Bacillus amyloliquefaciens subtilis protease (BPN'; SEQ ID NO: 22) compared to control cells expressing the same BPN' subtilisin, where the control cells did not contain the introduced secGEY operon.

[0140] Based on the foregoing, some embodiments involve recombinant Gram-positive bacterial cells expressing a non-natural secGEY operon and alkaline subtilisin. Therefore, some embodiments involve recombinant Gram-positive bacterial cells comprising an introduced (synthetic) secGEY operon, wherein these recombinant cells express / produce / secrete alkaline subtilisin having at least about 80% to 100% sequence identity with the natural subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0141] In some embodiments, a natural subtilase (and its functional variants) having at least about 80% identity with SEQ ID NO: 21 or SEQ ID NO: 23 is referred to as a basic subtilase, which is subgroup I-S2, compared to the so-called “true” subtilase (subgroup I-S1). For example, as outlined in Siezen and Leunissen (1997), a subgroup of serine proteases designated as “subtilases” has been proposed, which is defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilase-like proteases. In particular, the Siezen and Leunissen publication presents an overview of the subtilisin class of serine proteases (Table 1, Gram-positive bacteria), which mainly comprises enzymes from the genus Bacillus, with subgroups of true subtilisin proteases (>64% identity), high-alkaline proteases (>55% identity), and intracellular proteases (>37% identity), showing numerous subtle variants of true subtilisin proteases and high-alkaline proteases (Table 2). More specifically, a subgroup of the subtilisin class, named "I-S1" (or "true" subtilisin proteases), includes classic subtilisin proteases such as Bacillus subtilis 168 subtilisin proteases (aprA), Bacillus amyloliquefaciens subtilisin proteases (BPN'), Bacillus licheniformis subtilisin proteases (Carlsberg), etc. The second subgroup of the subtilisin class, named "I-S2" (or alkaline subtilisin), includes, for example, the following subtilisins: *Bacillus alkalophilus* PB92 alkaline subtilisin (PB92), *Bacillus tarda* 309 alkaline subtilisin (309; Savinase™), *Bacillus tarda* 147 alkaline subtilisin (147; Esperase™), etc. For example, such as... Figure 2 As shown, the protein sequence of the mature alkaline subtilis protease from natural Bacillus clausti is... Figure 2A, SEQ ID NO: 21) contains 269 amino acid residues and has an isoelectric point (pI) of approximately 9.30, and is a natural Bacillus girdren alkaline subtilis protease (mature) protein sequence ( Figure 2 C, SEQ ID NO: 23, contains 269 amino acid residues and has a pI of approximately 9.57, while the mature protein sequence of the natural Bacillus amyloliquefaciens "true" subtilisin (C) is similar to that of the Bacillus subtilis protease. Figure 2 B (SEQ ID NO: 22) contains 275 amino acid residues and has a pI of approximately 6.30. More specifically, the isoelectric point is a theoretical value calculated at average resolution using the Expasy (Swiss bioinformatics resource portal) "Calculate pI / Mw tool".

[0142] In some embodiments, one or more suitable pre-region sequences of this disclosure are derived from native (wild-type, Ref.) subtilisin. For example, in some embodiments, suitable pre-region sequences are derived from native pre-region amino acid sequences located upstream (5') of the mature subtilisin ORF. In other embodiments, particularly suitable pre-region sequences are the native Bacillus tarda subtilisin pre-region sequence (and its functional variants) of SEQ ID NO: 18. For example, PCT Publication WO 2008 / 112258 outlines recombinant Gram-positive bacterial cells and methods for producing serine proteases (e.g., modified (variant) pre-region sequences derived from full-length native Bacillus tarda basic serine (Maxacal) protease). Similarly, PCT Publication WO 2010 / 123754 describes compositions and methods for producing serine proteases using one or more modified pre-region sequences derived from full-length native Bacillus tarda serine protease or full-length native Bacillus tarda serine (GG36) protease. PCT Publication No. WO 2011 / 014278 further describes a modified (variant) pre-region sequence suitable for producing serine proteases (such as native (or variant) Bacillus amyloliquefaciens serine (BPN') protease).

[0143] Therefore, in some or more embodiments, this 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 examples below, artificial secGEY operons and / or expression cassettes encoding a subtilisin reporter protein are typically 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-Dalgarno (SD) sequences, signal peptide (secretory) sequences, pre-region sequences, open reading frame (ORF) sequences, terminator sequences, etc.

[0144] In some or more embodiments, an expression cassette encoding an exemplary reporter protein is constructed and introduced into the cells of this disclosure. In some or more embodiments, the exemplary reporter protein of this disclosure is a protease, including but not limited to native Bacillus subtilis protease (and its functional variants), native Bacillus giganteus protease (and its functional variants), native Bacillus tarda protease (and its functional variants), etc. In particular, according to one or more embodiments of this disclosure, the DNA sequence encoding native subtilis protease (and its functional variants) is generally available and suitable for use.

[0145] In some embodiments, the modified cells produce an increased amount of subtilisin (protease) relative to control (or parental) cells, wherein the increase is at least about 0.01%, at least about 0.10%, at least about 0.50%, at least about 1.0%, at least about 2.0%, at least about 3.0%, at least about 4.0%, at least about 5.0%, or more than 5.0%. In some aspects, the increased amount of subtilisin is determined by measuring enzyme activity, measuring protein function, measuring / quantitative productivity (Qp), etc. For example, those skilled in the art can utilize conventional methods and techniques known in the art to detect, determine, and measure protein expression, production, secretion, etc.

[0146] III. Recombinant Polynucleotides and Molecular Biology

[0147] Certain embodiments of this disclosure particularly provide recombinant Gram-positive bacterial cells expressing subtilisin and comprising an introduced (non-natural) secGEY operon overexpressing SecG, SecE, and SecY proteins, and methods for producing subtilisin in Gram-positive bacterial cells comprising an introduced (non-natural) secGEY operon, nucleic acid (DNA) sequences encoding natural SecG, SecE, and SecY proteins (e.g., secG ORF encoding a natural or functional variant of the SecG protein, secE ORF encoding a natural or functional variant of the SecE protein, secY ORF encoding a natural or functional variant of the SecY protein), etc.

[0148] As outlined herein and illustrated in the examples below, recombinant polynucleotides (vectors, expression cassettes, etc.), recombinant (modified) Bacillus strains, etc., can be readily constructed using conventional molecular biology and microbiology techniques and methods known to those skilled in the art. Therefore, this disclosure generally relies on conventional 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, suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., Escherichia coli, Bacillus, etc.) are well known to those skilled in the art.

[0149] Therefore, in some embodiments, the polynucleotides (genes, vectors, plasmids, DNA elements, etc.) of this disclosure may be genetically modified, wherein genetic modification includes, but is not limited to, (a) introducing, substituting, or removing one or more nucleotides in a gene (or its ORF), or introducing, substituting, or removing one or more nucleotides in a regulatory element required for transcription or translation of a gene or its ORF, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation (e.g., interfering RNA), (f) specific mutagenesis of any one or more genes disclosed herein, and / or (g) random mutagenesis. In some embodiments, the modified Bacillus cells of this disclosure are constructed by increasing gene expression and / or decreasing (or eliminating) gene expression using methods well known in the art (e.g., 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 coding region expression. Examples of such regulatory or control sequences may be promoter sequences or functional portions thereof (i.e., portions sufficient to affect the expression of nucleic acid sequences). Other control sequences used for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminator sequences, and transcription activator sequences.

[0150] Gene deletion technology enables the partial or complete removal of one or more genes, thereby eliminating their expression or expressing nonfunctional (or reduced-activity) protein products. In such methods, the deletion of one or more genes can be accomplished by homologous recombination using plasmids constructed to sequentially contain 5′ and 3′ regions flanked by the gene. Sequential 5′ and 3′ regions can be introduced into Bacillus cells, for example, at an allowable temperature on a temperature-sensitive plasmid (such as pE194) associated with a second selectable marker, to allow plasmid establishment in the cells. The cells are then transferred to an unallowable temperature to select cells for integration of the plasmid into one of the chromosomal homologous flanking regions. The selection of plasmid integration is influenced by selecting a second selectable marker. After integration, recombination events at the second homologous flanking region are stimulated by transferring the cells to an allowable temperature for several generations without selection. The cells are plated to obtain single colonies, and the colonies are examined for loss of both selectable markers. Thus, those skilled in the art can readily identify nucleotide regions in the coding and / or non-coding sequences of a gene (suitable for complete or partial deletion). In other embodiments, the modified Bacillus cells of this disclosure are constructed by introducing, substituting, or removing one or more nucleotides into a gene or a regulatory element required for its transcription or translation.

[0151] In some embodiments, modified Bacillus cells are constructed via CRISPR-Cas9 editing. For example, a wild-type gene encoding a natural target protein (or a functional variant of the target protein) can be modified via CRISPR-Cas9 editing using a nucleic acid-guided endonuclease. This endonuclease finds its target DNA by binding to a guide RNA (e.g., Cas9) and either Cpfl or a guide DNA (e.g., NgAgo). This recruits the endonuclease to a target sequence on the DNA, where the endonuclease can produce single-strand or double-strand breaks. The targeted DNA break becomes a substrate for DNA repair and can be recombinated with a provided editing template (e.g., an editing template that replaces the natural gene promoter sequence with a heterologous promoter). For example, a gene encoding a nucleic acid-directed endonuclease (for this purpose, Cas9 from *Streptococcus pyogenes*) or a codon-optimized gene encoding a Cas9 nuclease can be operatively linked to a promoter active in *Bacillus* cells and a terminator active in *Bacillus* cells, thereby producing a *Bacillus* Cas9 expression cassette. Similarly, those skilled in the art can readily identify one or more target sites specific to the target gene. For example, to construct a DNA construct encoding a gRNA targeting a target site within a target gene using *Streptococcus pyogenes* Cas9, a variable targeting (VT) domain would contain nucleotides at the target site located at the 5' of the protospacer adjacent motif (NGG), these nucleotides being fused with DNA encoding the Cas9 endonuclease recognition domain (CER) of *Streptococcus pyogenes* Cas9. Combining the DNA encoding the VT domain and the DNA encoding the CER domain generates DNA encoding the gRNA. Therefore, a Bacillus expression cassette for gRNA is generated by operatively linking DNA encoding gRNA to a promoter and a terminator that are active in Bacillus cells.

[0152] In some embodiments, DNA breaks induced by nucleases are repaired / replaced with an input sequence. For example, to precisely repair DNA breaks generated by the Cas9 expression cassette and gRNA expression cassette described above, a nucleotide editing template is provided, which the cell's DNA repair apparatus can utilize. For example, approximately 500-bp 5' of the target gene can be fused with approximately 500-bp 3' of the target gene to generate an editing template, which is used by the apparatus of the Bacillus host to repair DNA breaks generated by RGEN. Many different methods can be used to co-deliver the Cas9 expression cassette, gRNA expression cassette, and editing template to the cell. Transformed cells are screened by amplifying the target gene loci via PCR using forward and reverse primers. These primers can amplify wild-type loci or modified loci already edited by RGEN. These fragments are then sequenced using sequencing primers to identify the edited colonies.

[0153] In other embodiments, modified Bacillus cells are constructed by random or specific mutagenesis using methods well known in the art (including, but not limited to, chemical mutagenesis and transposition). Gene modification can be performed by mutagenesis of parental cells and screening for mutant cells in which gene expression has been altered. Mutagenesis, which can be specific or random, can be performed, for example, by using suitable physical or chemical mutagens, using suitable oligonucleotides, or subjecting the DNA sequence to PCR-generated mutagenesis. Furthermore, mutagenesis can be performed by any combination of these mutagenesis methods. Examples of physical or chemical mutagens suitable for the purposes of this invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When such reagents are used, mutagenesis is typically carried out by incubating the parental cells to be mutated under suitable conditions in the presence of a selected mutagen, and selecting mutant cells that exhibit reduced or no gene expression.

[0154] International PCT Publication No. WO 2003 / 083125 discloses methods for modifying Bacillus cells, such as using PCR fusion to generate Bacillus deletion strains and DNA constructs to bypass Escherichia coli. PCT Publication No. WO 2002 / 14490 discloses methods for modifying Bacillus cells, including (1) constructing and transforming an integrated plasmid (pComK), (2) randomly mutagenesis of coding, signal, and propeptide sequences, (3) homologous recombination, (4) improving transformation efficiency by adding non-homologous flanking to the transformed DNA, (5) optimizing double cross-integration, (6) site-directed mutagenesis, and (7) marker-free deletion. Suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., Escherichia coli and Bacillus) are well known to those skilled in the art. In fact, methods such as transformation, including protoplast transformation and midplate 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.

[0155] In addition to commonly used methods, in some embodiments, host cells are directly transformed (i.e., the DNA construct is not amplified or otherwise processed before introduction into host cells). Introducing the DNA construct into host cells includes those physical and chemical methods known in the art that introduce DNA into host cells without insertion into plasmids or vectors. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, liposomes, etc. In additional embodiments, the DNA construct is co-transformed with a plasmid without insertion into that plasmid. In further embodiments, selective markers are deleted or substantially excised from the modified Bacillus strain using methods known in the art. In some embodiments, the vector is dissociated from the host chromosome, leaving flanking regions on the chromosome while removing the intrinsic chromosomal regions.

[0156] Promoters and promoter sequence regions used for expressing genes, their open reading frames (ORFs), and / or variant sequences in Bacillus cells are generally known to those skilled in the art. The promoter sequences disclosed herein are typically selected such that they are functional in Bacillus cells, and include, but are not limited to, naturally occurring promoter sequences, synthetic promoter sequences, and / or combinations thereof, which are operable / functional in Bacillus cells. Examples of synthetic (engineered) promoters capable of producing heterologous (exogenous) 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). Other exemplary Bacillus promoter sequences include, but are not limited to, the Bacillus subtilis alkaline protease (aprE) promoter, the Bacillus subtilis α-amylase promoter, the Bacillus amylolyticus α-amylase promoter, the Bacillus subtilis neutral protease (nprE) promoter, mutant aprE promoters (e.g., PCT Publication WO 2001 / 51643), the Bacillus licheniformis tuf promoter, the Bacillus licheniformis citZ promoter, or any other functional promoters from Bacillus species cells. A method for screening and generating promoter libraries with a range of activities (promoter strengths) in Bacillus cells is described in PCT Publication WO 2003 / 089604.

[0157] IV. Fermentation of Gram-positive cells used to produce the target protein.

[0158] In some embodiments, this disclosure provides recombinant microbial cells that produce subtilisin. More specifically, some embodiments are (recombinant) Gram-positive bacterial cells expressing relevant genetic modifications of heterologous polynucleotides encoding subtilisin. Therefore, particular embodiments relate to the growth, culture, fermentation, etc., of microbial cells for producing proteins. Typically, fermentation methods well known in the art are used to ferment microbial cells.

[0159] In some embodiments, these cells are grown under batch 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 remains unchanged during fermentation. At the start of fermentation, the desired organism is inoculated into the culture medium. In this method, fermentation occurs without adding any components to the system. Typically, batch fermentation meets the criteria of being “batch” with respect to the addition of a carbon source and often attempts are made to control factors such as pH and oxygen concentration. The metabolite and biomass composition of a batch system changes continuously until fermentation stops. In batch culture, cells progress through a quiescent phase to a high-growth logarithmic phase, and finally to a stationary phase where the growth rate decreases or stops. Cells in the quiescent phase eventually die if left untreated. Typically, cells in the logarithmic phase are responsible for the large-scale production of the product.

[0160] A suitable variation of the standard batch system is the fed-batch fermentation system. In this variation of the typical batch system, substrate is added incrementally as fermentation progresses. Fed-batch systems are useful when catabolite repression might inhibit cellular metabolism and when a limited amount of substrate is desired in the culture medium. Measuring the actual substrate concentration in a fed-batch system is difficult and therefore estimates are based on changes in measurable factors such as pH, dissolved oxygen, and the partial pressure of exhaust gases (e.g., CO2). Batch and fed-batch fermentation are commonly used and well-known in the art.

[0161] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor while an equal volume of conditioned medium is removed for processing. Continuous fermentation typically maintains the culture at a constant high density, with cells primarily in the logarithmic growth phase. Continuous fermentation allows for the regulation of one or more factors affecting cell growth and / or product concentration. For example, in one embodiment, limiting nutrients (such as carbon or nitrogen sources) are maintained at a fixed rate, and all other parameters can be adjusted. In other systems, many factors affecting growth can change continuously while cell concentration, as measured by the turbidity of the medium, remains constant. Continuous systems strive to maintain steady-state growth conditions. Therefore, cell loss due to medium removal should be balanced against the cell growth rate during fermentation. Methods for regulating nutrients and growth factors used in continuous fermentation processes, as well as techniques for maximizing product formation rates, are well known in the field of industrial microbiology.

[0162] Culture / fermentation is typically carried out in a growth medium containing an aqueous mineral salt medium, organic growth factors, carbon and energy sources, molecular oxygen, and of course, the initial inoculum of the microbial host to be used.

[0163] In addition to carbon and energy sources, oxygen, assimilable nitrogen, and microbial inoculum, it is also necessary to supply appropriate amounts of mineral nutrients in the right proportions to ensure proper microbial growth, maximize the assimilation of carbon and energy sources by cells during microbial transformation, and obtain the maximum cell yield and maximum cell density in the fermentation medium.

[0164] The composition of aqueous mineral culture media can vary widely, depending in part on the microorganisms and substrates used, as is known in the art. In addition to nitrogen, these mineral culture media should also include appropriate amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium in suitable soluble and assimilable ionic and combined forms, and preferably should also contain certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, all as known in the art, also in suitable soluble and assimilable forms.

[0165] Fermentation is an aerobic process in which the required molecular oxygen is supplied by oxygen-containing gases such as air, oxygen-enriched air, or even essentially pure molecular oxygen, as long as the contents of the fermentation vessel are kept at a suitable partial pressure of oxygen that can effectively help the microbial species grow vigorously.

[0166] Fermentation temperature can vary slightly, but for most microbial cells, the temperature will typically be in the range of about 20°C to 40°C.

[0167] Microorganisms also require assimilable nitrogen sources. Assimilable nitrogen sources can be any nitrogen-containing compound or a compound that releases nitrogen in a form suitable for microbial metabolism. While various organic nitrogen sources, such as protein hydrolysates, can be used, 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 ammonia compounds) are commonly available. Ammonia itself is convenient for large-scale operations and can be used in appropriate quantities by bubbling through the aqueous fermentation medium. It can also be used to aid in pH control.

[0168] The pH range of the aqueous microbial ferment (fermentation mixture) should be within the exemplary range of about 2.0 to 8.0. The preference for the microbial pH range depends to some extent on the culture medium used and the specific microorganisms, and thus varies slightly with changes in the culture medium, as can be readily determined by those skilled in the art.

[0169] Preferably, fermentation is carried out in a manner that allows carbon-containing substrates to be controlled as a limiting factor, thereby providing cells with good conversion of carbon-containing substrates and preventing these cells from being contaminated by a basic amount of unconverted substrates. The latter is not a problem for water-soluble substrates, as any remaining trace amounts can be easily washed away. However, this can be problematic with insoluble substrates and requires additional product treatment steps, such as suitable washing steps.

[0170] As mentioned above, the time to reach this level is not critical and can vary depending on the specific microorganism and the fermentation process. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether the desired carbon source level has been reached.

[0171] If desired, some or all of the carbon source and energy material and / or a portion of the assimilable nitrogen source (such as ammonia) may be added to the aqueous mineral medium before feeding it into the fermenter.

[0172] Preferably, each feed stream introduced into the reactor is controlled at a predetermined rate, or in response to needs determined by monitoring factors such as the concentration of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the exhaust gas from the fermenter, cell density measurable by stem cell weight, light transmittance, etc. The feed rates of various materials can be varied to achieve the fastest possible cell growth rate consistent with the efficient utilization of carbon and energy sources, thereby obtaining the highest possible microbial cell yield relative to substrate variations.

[0173] In batch or preferred fed-batch operations, all equipment, reactors or fermentation units, vessels or containers, piping, and associated circulation or cooling equipment are initially sterilized, typically by steaming at approximately 121°C for at least 15 minutes. Then, in the presence of all necessary nutrients, including oxygen and carbonaceous substrate, the sterilized reactor is inoculated with a culture of the selected microorganism. The type of fermenter used is not critical.

[0174] V. Exemplary Model

[0175] Non-limiting examples of the compositions and methods disclosed herein are as follows:

[0176] 1. A recombinant Gram-positive (host) cell comprising an introduced secGEY operon and an expression cassette encoding a heterologous subtilisin.

[0177] 2. The recombinant cell as described in Example 1, wherein 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.

[0178] 3. The recombinant cell as described in Example 1, wherein the introduced secGEY operon comprises a secE ORF sequence having at least about 80% to 100% identity with SEQ ID NO: 31, wherein the secE ORF encodes a functional secE protein.

[0179] 4. The recombinant cell as described in Example 1, wherein the introduced secGEY operon comprises a secY ORF sequence having at least about 80% to 100% identity with SEQ ID NO: 32, wherein the secY ORF encodes a functional secY protein.

[0180] 5. The recombinant cells as described in Example 1, wherein the introduced secGEY operon encodes a functional secG protein having at least about 80% to 100% amino acid identity with SEQ ID NO: 28.

[0181] 6. The recombinant cells as described in Example 1, wherein the introduced secGEY operon encodes a functional secE protein having at least about 80% to 100% amino acid identity with SEQ ID NO: 29.

[0182] 7. The recombinant cells as described in Example 1, wherein the introduced secGEY operon encodes a functional secY protein having at least about 80% to 100% amino acid identity with SEQ ID NO: 30.

[0183] 8. The recombinant cell as described in Example 1, wherein 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 SEQ ID NO: 1, which are operatively linked to a downstream secGEY ORF.

[0184] 9. The recombinant cell as described in Example 1, wherein the introduced secGEY operon comprises a heterologous upstream (5') promoter and a 5'-UTR sequence operatively linked to a downstream secGEY ORF.

[0185] 10. The recombinant cells as described in Example 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.

[0186] 11. The recombinant cells as described in Example 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.

[0187] 12. The recombinant cells as described in Example 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.

[0188] 13. The recombinant cell as described in Example 1, wherein the recombinant cell comprises at least two introduced cassettes encoding the same subtilisin or different heterologous subtilisin, or comprises at least three introduced expression cassettes encoding the same subtilisin or different heterologous subtilisin.

[0189] 14. Recombinant cells as described in Example 1 or Example 13, wherein the one or more cassettes encode alkaline subtilisin.

[0190] 15. The recombinant cell as described in Example 1 or Example 13, wherein the cassette encoding subtilisin contains an upstream promoter region sequence operatively linked to a downstream nucleic acid encoding a protein signal sequence, the downstream nucleic acid encoding the protein signal sequence being operatively linked to a downstream nucleic acid encoding a pre-coding region sequence, and the downstream nucleic acid encoding the pre-coding region sequence being operatively linked to a downstream nucleic acid encoding mature subtilisin.

[0191] 16. The recombinant cells as described in Example 15, wherein the promoter sequence is a strong promoter that functions in Gram-positive cells.

[0192] 17. The recombinant cells as described in Example 15, wherein the signal sequence is a natural subtilisin signal sequence or a functional variant thereof.

[0193] 18. The recombinant cells as described in Example 15, wherein the signal sequence has at least about 95% to 100% amino acid identity with the aprE signal sequence of SEQ ID NO: 17, or at least about 95% to 100% amino acid identity with the BPN' signal sequence of SEQ ID NO: 19.

[0194] 19. The recombinant cells as described in Example 15, wherein the pre-region sequence is a natural subtilisin pre-region sequence or a functional variant thereof.

[0195] 20. The recombinant cells as described in Example 15, wherein the pre-region sequence has at least about 95% to 100% amino acid identity with the pre-region of SEQ ID NO: 18.

[0196] 21. The recombinant cells as described in Example 15, wherein the mature subtilisin has at least about 80% to 100% amino acid identity with the mature subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0197] 22. A polynucleotide construct encoding a synthetic secGEY operon, wherein the polynucleotide comprises at least an upstream promoter sequence operatively linked to a downstream nucleic acid encoding a secG protein having at least about 80% to 100% identity with SEQ ID NO: 28, the downstream nucleic acid encoding the secG protein being operatively linked to a downstream nucleic acid encoding a secE protein having at least about 80% to 100% identity with SEQ ID NO: 29, and the downstream nucleic acid encoding the secE protein being operatively linked to a downstream nucleic acid encoding a secY protein having at least about 80% to 100% identity with SEQ ID NO: 30.

[0198] 23. The polynucleotide as described in Example 22, wherein the nucleic acid encoding the secG protein has at least about 80% to 100% identity with the secG ORF of SEQ ID NO: 2.

[0199] 24. The polynucleotide as described in Example 22, wherein the nucleic acid encoding the secE protein has at least about 80% to 100% identity with the secE ORF of SEQ ID NO: 31.

[0200] 25. The polynucleotide as described in Example 22, wherein the nucleic acid encoding the secY protein has at least about 80% to 100% identity with the secY ORF of SEQ ID NO: 32.

[0201] 26. The polynucleotide as described in Example 24, wherein the nucleic acid encoding the secE protein includes a secE ribosome binding site (RBS) located upstream of the secE ORF and operatively linked to the secE ORF.

[0202] 27. The polynucleotide as described in Example 26, which has at least about 80% to 100% identity with secERBS and secEORF of SEQ ID NO: 3.

[0203] 28. The polynucleotide as described in Example 25, wherein the nucleic acid encoding the secY protein includes a secY ribosome binding site (RBS) located upstream of the secY ORF and operatively linked to the secY ORF.

[0204] 29. The polynucleotide as described in Example 28, which has at least about 80% to 100% identity with secY RBS and secYORF of SEQ ID NO: 4.

[0205] 30. A method for producing heterologous subtilisin in modified Gram-positive bacterial cells, the method 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 producing subtilisin.

[0206] 31. A method for producing heterologous subtilisin in modified Gram-positive bacterial cells, the method 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 producing subtilisin.

[0207] 32. The method as described in Example 30 or Example 31, wherein when fermentation is carried out under conditions suitable for producing subtilisin, subtilisin is secreted into the fermentation broth.

[0208] 33. The method as described in Example 30, wherein when fermented under the same conditions, the modified cells produce an increased amount of subtilisin compared to control cells, wherein the control cells do not contain the introduced synthetic secGEY operon.

[0209] 34. The method as described in Example 31, wherein when fermented under the same conditions, the modified cells produce an increased amount of subtilisin compared to control cells, wherein the control cells contain the same introduced expression cassette encoding the same heterologous subtilisin, and wherein the control cells do not contain the introduced synthetic secGEY operon.

[0210] 35. The method as described in Example 30 or Example 31, wherein 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.

[0211] 36. The method as described in Example 30 or Example 31, wherein 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.

[0212] 37. The method as described in Example 30 or Example 31, wherein 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.

[0213] 38. The method as described in Example 30 or Example 31, wherein the introduced secGEY operon encodes a functional secG protein having at least about 80% to 100% amino acid identity with SEQ ID NO: 28.

[0214] 39. The method as described in Example 30 or Example 31, wherein the introduced secGEY operon encodes a functional secE protein having at least about 80% to 100% amino acid identity with SEQ ID NO: 29.

[0215] 40. The method as described in Example 30 or Example 31, wherein the introduced secGEY operon encodes a functional secY protein having at least about 80% to 100% amino acid identity with SEQ ID NO: 30.

[0216] 41. The method as described in Example 30 or Example 31, wherein 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 SEQ ID NO: 1, which are operatively connected to a downstream secGEY ORF.

[0217] 42. The method as described in Example 30 or Example 31, wherein the introduced secGEY operon comprises a heterogeneous upstream (5') promoter and a 5'-UTR sequence operatively connected to a downstream secGEY ORF.

[0218] 43. The method as described in Example 42, wherein the heterologous promoter and 5'-UTR sequence have at least about 95% to 100% identity with the spoVG promoter and spoVG-5'-UTR of SEQ ID NO: 10.

[0219] 44. The method as described in Example 42, wherein the heterologous promoter and 5'-UTR sequence have at least about 95% to 100% identity with the hbs promoter and spoVG-5'-UTR of SEQ ID NO: 12.

[0220] 45. The method as described in Example 30 or Example 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.

[0221] 46. ​​The method of Example 31, wherein the cell contains at least two introduced cassettes encoding the same subtilisin or different heterologous subtilisin, or contains at least three introduced expression cassettes encoding the same subtilisin or different heterologous subtilisin.

[0222] 47. The method as described in Example 31 or Example 46, wherein the one or more boxes encode alkaline subtilisin.

[0223] 48. The method as described in Example 31 or Example 46, wherein the one or more cassettes encoding subtilisin contain an upstream promoter region sequence operatively linked to a downstream nucleic acid encoding a protein signal sequence, the downstream nucleic acid encoding the protein signal sequence being operatively linked to a downstream nucleic acid encoding a pre-coding region sequence, and the downstream nucleic acid encoding the pre-coding region sequence being operatively linked to a downstream nucleic acid encoding mature subtilisin.

[0224] 49. The method as described in Example 48, wherein the promoter sequence is a strong promoter that functions in Gram-positive cells.

[0225] 50. The method as described in Example 48, wherein the signal sequence is a natural subtilisin signal sequence or a functional variant thereof.

[0226] 51. The method as described in Example 50, wherein the signal sequence has at least about 95% to 100% amino acid identity with the aprE signal sequence of SEQ ID NO: 17, or at least about 95% to 100% amino acid identity with the BPN' signal sequence of SEQ ID NO: 19.

[0227] 52. The method as described in Example 48, wherein the pre-region sequence is a natural subtilisin pre-region sequence or a functional variant thereof.

[0228] 53. The method as described in Example 48, wherein the pre-region sequence contains at least about 90% to 100% amino acid identity with the pre-region of SEQ ID NO: 18.

[0229] 54. The method as described in Example 48, wherein the mature subtilisin has at least about 80% to 100% amino acid identity with the mature subtilisin of SEQ ID NO: 21 or SEQ ID NO: 23.

[0230] 55. The method as described in Example 33 or Example 34, wherein when fermented under the same conditions, the increase in subtilisin is at least about 5% relative to the control cells.

[0231] 56. The method as described in Example 33 or Example 34, wherein the increase in subtilisin is determined by suc-AAPF-pNA assay.

[0232] Example

[0233] Certain aspects of the invention can be further understood from the following examples, which should not be construed as limiting. Modifications to the materials and methods will be apparent 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).

[0234] Example 1

[0235] Construction of Bacillus subtilis cells expressing heterologous proteins and overexpressing SECG, SECE, and SECY

[0236] This example describes the construction of Bacillus subtilis cells overexpressing SecG, SecE, and SecY in a host background expressing a heterologous reporter-1 protein. More specifically, a control Bacillus subtilis strain named CZ437 contains two (2) introduced reporter-1 expression cassettes containing a promoter sequence (P2) located upstream (5') of and operatively linked to the open reading frame (ORF) encoding the reporter-1 protein, wherein the cassette is integrated 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 the Bacillus protease reporter-1 and overexpressing the artificial (non-natural) secGEY operon of this disclosure are described below.

[0237] The first modified Bacillus subtilis strain was constructed that expresses the proteins SecG, SecE, and SecY from its natural locus and as a non-natural operon (PsecG-secGEY), which is initiated by the secG promoter integrated into the pksR locus of Bacillus subtilis. More specifically, the non-natural operon PsecG-secGEY was constructed by overlap extension PCR and contains a Bacillus subtilis secG promoter located upstream (5') of secG ORF (SEQ ID NO: 2) and operably linked to secG ORF (SEQ ID NO: 2) and secG 5'-UTR (SEQ ID NO: 1), which is operably linked to secE ribosome binding sequence (RBS) and ORF (SEQ ID NO: 3), which is operably linked to secY RBS and ORF (SEQ ID NO: 4), which is operably linked to BPN' terminator (SEQ ID NO: 5) from Bacillus amyloliquefaciens. An integration cassette containing a downstream (3') homologous region of Bacillus subtilis pksR (SEQ ID NO: 6) was constructed by overlap extension PCR. This downstream (3') homologous region of Bacillus subtilis pksR was operatively linked to the non-natural operon PsecG-secGEY (SEQ ID NO: 7). The non-natural operon PsecG-secGEY was operatively linked to the Bacillus subtilis alarA gene (SEQ ID NO: 8). The Bacillus subtilis alarA gene was operatively linked to the upstream (5') homologous region of Bacillus subtilis pksR (SEQ ID NO: 9). The integration cassette was then transformed into a Bacillus subtilis strain with alarA deletion containing a copy of the =reporter-1 expression cassette (skf::P2-reporter-1) integrated at the skf locus. The resulting strain was transformed with the second copy of the reporter-1 cassette integrated at the aprE locus (aprE::P2-reporter-1) to produce Bacillus subtilis strain BPC0123.

[0238] A second modified strain was constructed that expresses SecG, SecE, and SecY from its natural locus as a non-natural operon (PspoVG-secGEY), which is initiated by the spoVG promoter (PspoVG) integrated into the pksR locus. More specifically, the non-natural operon PspoVG-secGEY was constructed by overlap extension PCR and contains a Bacillus subtilis spoVG promoter located upstream (5') of secGORF (SEQ ID NO: 2) and operably linked to secG ORF (SEQ ID NO: 2) and spoVG 5'-UTR (SEQ ID NO: 10), which is operably linked to secE RBS and ORF (SEQ ID NO: 3), which is operably linked to secY RBS and ORF (SEQ ID NO: 4), which is operably linked to BPN' terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a downstream (3') homologous region of Bacillus subtilis pksR (SEQ ID NO: 6) was constructed by overlap extension PCR. This downstream (3') homologous region of Bacillus subtilis pksR was operatively linked to the non-natural operon PspoVG-secGEY (SEQ ID NO: 11). The non-natural operon PspoVG-secGEY was operatively linked to the Bacillus subtilis alarA gene (SEQ ID NO: 8). The Bacillus subtilis alarA gene was operatively linked to the upstream (5') homologous region of Bacillus subtilis pksR (SEQ ID NO: 9). The integration cassette was then transformed into a Bacillus subtilis strain with alarA deletion containing a 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 Bacillus subtilis protease variant-1 expression cassette integrated at the aprE locus (aprE::P2-reporter-1) to produce Bacillus subtilis strain BPC0182.

[0239] A third modified strain was constructed that expresses SecG, SecE, and SecY from its natural locus as a non-natural operon (Phbs-secGEY), which is initiated by the hbs promoter (Phbs) integrated into the pksR locus. More specifically, the non-natural operon Phbs-secGEY was constructed by overlap extension PCR and contains the Bacillus subtilis hbs promoter located upstream (5') of secG ORF (SEQ ID NO: 2) and operably linked to secG ORF (SEQ ID NO: 2) and Bacillus subtilis spoVG 5'-UTR (SEQ ID NO: 12), which is operably linked to secE RBS and ORF (SEQ ID NO: 3), which is operably linked to secY RBS and ORF (SEQ ID NO: 4), which is operably linked to BPN' terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing a downstream (3') homologous region of Bacillus subtilis pksR (SEQ ID NO: 6) was constructed by overlap extension PCR. This downstream (3') homologous region of Bacillus subtilis pksR was operatively linked to the non-natural operon Phbs-secGEY (SEQ ID NO: 13). The non-natural operon Phbs-secGEY was operatively linked to the Bacillus subtilis alrA gene (SEQ ID NO: 8). The Bacillus subtilis alrA gene was operatively linked to the upstream (5') homologous region of Bacillus subtilis pksR (SEQ ID NO: 9). The integration cassette was then transformed into a Bacillus subtilis strain with alrA deletion containing a 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 Bacillus subtilis protease variant-1 expression cassette integrated at the aprE locus (aprE::P2-reporter-1) to produce Bacillus subtilis strain BPC0184.

[0240] Example 2

[0241] Reporter-1 protease expression in Bacillus subtilis cells overexpressing SECG, SECE, and SECY

[0242] In this example, the expression of heterologous reporter-1 protein in the presence of SecGEY overexpression will be compared with the expression of the same reporter-1 protein in the absence of SecGEY overexpression. More specifically, the reporter-1 yield of three (3) SecGEY-overexpressing strains (BPC0123, BPC0182, BPC0184) and the control strain CZ437 was evaluated under small-scale conditions as follows. Precultures in tryptone soybean broth (1.7% tryptone, 0.3% soybean peptone, 0.25% glucose, 0.5% sodium chloride, 0.25% dipotassium hydrogen phosphate) were inoculated with single colonies and grown at 37°C and 250 RPM. The preculture was inoculated with 20 ml of 0.5X MPS2 medium at a 1:1000 dilution, supplemented with 80 mMMOPS adjusted to pH 7.3, and the culture was grown in flasks at 37°C and 250 RPM. MPS2 medium consists of the following: a medium based on 10% v / v 10x MOPS (8.4% w / v MOPS, 2.9% w / v sodium chloride, 1.5% w / v potassium hydroxide, 0.05% w / v potassium sulfate, 0.05% w / v magnesium chloride, 0.7% w / v tris(hydroxymethyl)glycine, 10% v / v micronutrients), 10% w / v Maltrin M150, 6% w / v soybean peptone, 0.78% w / v dipotassium phosphate, 0.36% w / v urea, 0.2% w / v dipotassium hydrogen phosphate, and 0.06% w / v trisodium citrate dihydrate, with pH adjusted with potassium hydroxide. The micronutrients were prepared in 100X stock solution in one (1) liter, comprising: 1.47 g sodium citrate 2H2O, 1.47 g CaCl2 2H2O, 400 mg FeSO4 7H2O, 100 mg MnSO4 H2O, 100 mg ZnSO4 H2O, 50 mg CuCl2 2H2O, 100 mg CoCl2 6H2O, and 100 mg Na2MoO4 2H2O.

[0243] Essentially as described in WO 2020112609 (incorporated hereby by reference), the amount of reporter-1 protease in culture supernatant was determined using the suc-AAPF-pNA assay. The substrate was N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (suc-AAPF-pNA). After the protease hydrolyzed the peptide substrate, 4-nitroaniline was cleaved to produce 4-nitroaniline as a yellow chromophore. Therefore, absorbance was measured at 405 nm, and the slope of the absorbance change (mOD / min) was directly correlated with the amount of protease in the analyzed 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 solution diluted in Tris buffer (100 mg / ml AAPF stock solution dissolved in DMSO, diluted 100X 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.

[0244] In the first experiment, control strain CZ437 and SecGEY overexpressing strain BPC0123 were cultured, and protease activity was measured after 24 (24) hours and 40 (40) hours of growth. For example, reporter-1 protein production of strain BPC0123 relative to control strain CZ437 (and associated coefficients of variation (CV; n ≥ 4)) is presented in Table 2 below. Similarly, Table 3 below shows the relative reporter-1 production of strains CZ437 and BPC0123 normalized to the amount of cell clusters in the culture, as measured by OD600. Thus, as generally shown in Tables 2 and 3, reporter-1 protein production increases with overexpression of SecGEY from the secG promoter.

[0245] Table 2

[0246] Small-scale production of proteases

[0247]

[0248] Table 3

[0249] Small-scale production of proteases relative to culture OD600 normalization

[0250]

[0251] In the second experiment, control strain CZ437 and SecGEY overexpressing strains BPC0123, BPC0182, and BPC0184 were cultured and sampled for protease activity after 22 hours of growth. More specifically, the reporter-1 yield and associated coefficient of variation (CV; n = 6) of the SecGEY overexpressing strains BPC0123, BPC0182, and BPC0184 relative to the control strain CZ437 are presented in Table 4 below. Similarly, the relative reporter-1 yield normalized relative to cell mass in culture, as measured by OD600, is presented in Table 5 below. As shown in Tables 4 and 5, when protease yield / OD600 is taken into account, the reporter-1 protein yield is increased for all three SecGEY overexpressing strains (see Table 5).

[0252] Table 4

[0253] Small-scale production of proteases

[0254]

[0255] Table 5

[0256] Small-scale production of proteases relative to culture OD600 normalization

[0257]

[0258] Example 3

[0259] Construction of Bacillus subtilis cells expressing heterologous proteins and overexpressing SECG, SECE, and SECY

[0260] This example 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 (3) introduced reporter-2 expression cassettes containing a promoter sequence (P4) located upstream (5') of and operatively linked to the open reading frame (ORF) encoding the Bacillus protease reporter-2 protein, wherein the three cassettes are integrated 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 are described below.

[0261] The first modified Bacillus subtilis strain was constructed that expresses SecG, SecE, and SecY proteins from its natural locus as a non-natural operon (PsecG-secGEY), which is initiated by the secG promoter integrated into the Bacillus subtilis aprE locus. More specifically, the non-natural operon PsecG-secGEY was constructed by overlap extension PCR and contains a Bacillus subtilis secG promoter located upstream (5') of secG ORF (SEQ ID NO: 2) and secG 5'-UTR (SEQ ID NO: 1), which is operatively linked to secE RBS and ORF (SEQ ID NO: 3), which is operatively linked to secY RBS and ORF (SEQ ID NO: 4), which is operatively linked to BPN' terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette was constructed by overlap extension PCR containing: an upstream (5') homologous region of Bacillus subtilis aprE (SEQ ID NO: 14), which is operatively linked to the non-natural operon PsecG-secGEY (SEQ ID NO: 7), which is operatively linked to the promoter and kanamycin resistance gene (SEQ ID NO: 15), which is operatively linked to the downstream (3') homologous region of Bacillus subtilis aprE (SEQ ID NO: 7). 16), and the integration cassette was transformed into a Bacillus subtilis strain containing three copies of the reporter-2 expression cassette integrated at the skf locus (skf::P4-reporter-2), the nprE locus (nprE::P4-reporter-2), and the ppsC locus (ppsC::P4-reporter-2) to produce Bacillus subtilis strain BPC0166.

[0262] A second modified strain was constructed that expresses SecG, SecE, and SecY from its natural locus as a non-natural operon (PspoVG-secGEY), which is initiated by the spoVG promoter integrated into the aprE locus. More specifically, the non-natural operon PspoVG-secGEY was constructed by overlap extension PCR and contains a Bacillus subtilis spoVG promoter located upstream (5') of secG ORF (SEQ ID NO: 2) and operably linked to secG ORF (SEQ ID NO: 2), which is operably linked to secE RBS and ORF (SEQ ID NO: 3), which is operably linked to secY RBS and ORF (SEQ ID NO: 4), which is operably linked to BPN' terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette was constructed by overlap extension PCR containing: an upstream (5') homologous region of Bacillus subtilis aprE (SEQ ID NO: 14), which is operatively linked to the non-natural operon PspoVG-secGEY (SEQ ID NO: 11), which is operatively linked to the promoter and kanamycin resistance gene (SEQ ID NO: 15), which is operatively linked to the downstream (3') homologous region of Bacillus subtilis aprE (HR; SEQ ID NO: 11). 16), and the integration cassette was transformed into a Bacillus subtilis strain containing three copies of the reporter-2 expression cassette integrated at the skf locus (skf::P4-reporter-2), the nprE locus (nprE::P4-reporter-2), and the ppsC locus (ppsC::P4-reporter-2) to produce Bacillus subtilis strain BPC0167.

[0263] A third modified strain was constructed that expresses SecG, SecE, and SecY from its natural locus as a non-natural operon (Phbs-secGEY), which is initiated by the hbs promoter integrated into the aprE locus. More specifically, the non-natural operon Phbs-secGEY was constructed by overlap extension PCR and contains a Bacillus subtilis hbs promoter located upstream (5') of secG ORF (SEQ ID NO: 2) and operably linked to secG ORF (SEQ ID NO: 2) and a Bacillus subtilis spoVG 5'-UTR (SEQ ID NO: 12), which is operably linked to secE RBS and ORF (SEQ ID NO: 3), which is operably linked to secYRBS and ORF (SEQ ID NO: 4), which is operably linked to a BPN' terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette was constructed using overlap extension PCR. This cassette contained: an upstream (5') homologous region of *Bacillus subtilis* aprE (SEQ ID NO: 14), which was operatively linked to the non-natural operon Phbs-secGEY (SEQ ID NO: 13), which was operatively linked to a promoter and a kanamycin resistance gene (SEQ ID NO: 15), which was operatively linked to a downstream (3') homologous region of *Bacillus subtilis* aprE (SEQ ID NO: 16). 16), and the integration cassette was transformed into a Bacillus subtilis strain containing three copies of the reporter-2 expression cassette integrated at the skf locus (skf::P4-reporter-2), the nprE locus (nprE::P4-reporter-2), and the ppsC locus (ppsC::P4-reporter-2) to produce Bacillus subtilis strain BPC0168.

[0264] Example 4

[0265] Reporter-2 protease expression in Bacillus subtilis cells overexpressing SECG, SECE, and SECY

[0266] In this example, the expression of heterologous reporter-2 protein in the presence of SecGEY overexpression will be compared with the expression of the same reporter-2 protein in the absence of SecGEY overexpression. More specifically, the reporter-2 yield of three (3) SecGEY-overexpressing strains (BPC0166, BPC0167, BPC0168) and the control strain BPC0178 was evaluated under small-scale conditions as follows. Precultures in tryptone soybean broth (1.7% tryptone, 0.3% soybean peptone, 0.25% glucose, 0.5% sodium chloride, 0.25% dipotassium hydrogen phosphate) were inoculated with single colonies and grown at 37°C and 250 RPM. The preculture was inoculated with 20 ml of 0.5X MPS2 medium at a 1:1000 dilution, supplemented with 80 mMMOPS adjusted to pH 7.3, and the culture was grown in flasks at 37°C and 250 RPM. MPS2 medium consists of the following: a medium based on 10% v / v 10x MOPS (8.4% w / v MOPS, 2.9% w / v sodium chloride, 1.5% w / v potassium hydroxide, 0.05% w / v potassium sulfate, 0.05% w / v magnesium chloride, 0.7% w / v tris(hydroxymethyl)glycine, 10% v / v micronutrients), 10% w / v Maltrin M150, 6% w / v soybean peptone, 0.78% w / v dipotassium phosphate, 0.36% w / v urea, 0.2% w / v dipotassium hydrogen phosphate, and 0.06% w / v trisodium citrate dihydrate, with pH adjusted with potassium hydroxide. The micronutrients were prepared in 100X stock solution in one (1) liter, comprising: 1.47 g sodium citrate 2H2O, 1.47 g CaCl2 2H2O, 400 mg FeSO4 7H2O, 100 mg MnSO4 H2O, 100 mg ZnSO4 H2O, 50 mg CuCl2 2H2O, 100 mg CoCl2 6H2O, and 100 mg Na2MoO4 2H2O.

[0267] Essentially as described in WO 2020112609, the amount of reporter-2 protease in culture supernatant was determined using the suc-AAPF-pNA assay. The substrate was N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (suc-AAPF-pNA). After the protease hydrolyzed the peptide substrate, 4-nitroaniline was cleaved to produce 4-nitroaniline as a yellow chromophore. Therefore, absorbance was measured at 405 nm, and the slope of the absorbance change (mOD / min) was directly correlated with the amount of protease in the analyzed 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 solution diluted in Tris buffer (100 mg / ml AAPF stock solution dissolved in DMSO, diluted 100X 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.

[0268] Control strain BPC0178 and SecGEY overexpressing strains BPC0166, BPC0167, and BPC0168 were cultured and sampled at 20 (20) h and 40 (40) h to detect protease activity. More specifically, reporter-2 yields and associated coefficients of variation (CV; n = 3) of SecGEY overexpressing strains BPC0166, BPC0167, and BPC0168 relative to control strain BPC0178 are presented in Table 6 below, and relative reporter-2 yields normalized relative to cell cluster size in culture as measured by OD600 are presented in Table 7 below. As shown in Tables 6 and 7, reporter-2 protein yields were equivalent or lower for all three SecGEY overexpressing strains.

[0269] Table 6

[0270] Small-scale production of proteases

[0271]

[0272] Table 7

[0273] Small-scale production of proteases relative to culture OD600 normalization

[0274]

[0275] Example 5

[0276] Construction of Bacillus subtilis cells expressing heterologous proteins and overexpressing SECG, SECE, and SECY

[0277] This example 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 (2) introduced reporter-3 expression cassettes containing a promoter sequence (P2-00788) located upstream (5') of and operatively linked to the open reading frame (ORF) encoding the Bacillus subtilis protease variant-3 protein, wherein the cassette is integrated into the Bacillus subtilis skf locus (skf::P2-00788-reporter-3) and the Bacillus subtilis pksR locus (pksR::P2-00788-reporter-3). The design and construction of a modified Bacillus subtilis strain expressing reporter-3 proteins and overexpressing the artificial (non-natural) secGEY operon of this disclosure are described below.

[0278] The first modified Bacillus subtilis strain was constructed that expresses SecG, SecE, and SecY proteins from its natural locus as a non-natural operon (PsecG-secGEY), which is initiated by the secG promoter integrated into the Bacillus subtilis aprE locus. More specifically, the non-natural operon PsecG-secGEY was constructed by overlap extension PCR and contains a Bacillus subtilis secG promoter located upstream (5') of secG ORF (SEQ ID NO: 2) and secG 5'-UTR (SEQ ID NO: 1), which is operatively linked to secE RBS and ORF (SEQ ID NO: 3), which is operatively linked to secY RBS and ORF (SEQ ID NO: 4), which is operatively linked to BPN' terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette was constructed using overlap extension PCR. This cassette contained: an upstream (5') homologous region of *Bacillus subtilis* aprE (SEQ ID NO: 14), which was operatively linked to the non-natural operon PsecG-secGEY (SEQ ID NO: 7), which was operatively linked to a promoter and a kanamycin resistance gene (SEQ ID NO: 15), which was operatively linked to a downstream (3') homologous region of *Bacillus subtilis* aprE (SEQ ID NO: 15). 16), and the integration cassette was transformed into a Bacillus subtilis strain containing two copies of the reporter-3 expression cassette integrated at the skf locus (skf::P2-00788-reporter-3) and the pksR locus (pksR::P2-00788-reporter-3) to produce Bacillus subtilis strain AL394.

[0279] A second modified strain was constructed that expresses SecG, SecE, and SecY from its natural locus as a non-natural operon (PspoVG-secGEY), which is initiated by the spoVG promoter integrated into the aprE locus. More specifically, the non-natural operon PspoVG-secGEY was constructed by overlap extension PCR and contains a Bacillus subtilis spoVG promoter located upstream (5') of secG ORF (SEQ ID NO: 2) and operably linked to secG ORF (SEQ ID NO: 2), which is operably linked to secE RBS and ORF (SEQ ID NO: 3), which is operably linked to secY RBS and ORF (SEQ ID NO: 4), which is operably linked to BPN' terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette containing the upstream (5') homologous region of *Bacillus subtilis* aprE (SEQ ID NO: 14) was constructed by overlap extension PCR. This upstream (5') homologous region of *Bacillus subtilis* aprE was operatively linked to the non-natural operon PspoVG-secGEY (SEQ ID NO: 11). The non-natural operon PspoVG-secGEY was operatively linked to the promoter and kanamycin resistance gene (SEQ ID NO: 15). This promoter and kanamycin resistance gene were operatively linked to the downstream (3') homologous region of *Bacillus subtilis* aprE (SEQ ID NO: 15). 16), and the integration cassette was transformed into a Bacillus subtilis strain containing two copies of the reporter-3 expression cassette integrated at the skf locus (skf::P2-00788-reporter-3) and the pksR locus (pksR::P2-00788-reporter-3) to produce Bacillus subtilis strain AL395.

[0280] A third modified strain was constructed that expresses SecG, SecE, and SecY from its natural locus as a non-natural operon (Phbs-secGEY), which is initiated by the hbs promoter integrated into the aprE locus. More specifically, the non-natural operon Phbs-secGEY was constructed by overlap extension PCR and contains a Bacillus subtilis hbs promoter located upstream (5') of secG ORF (SEQ ID NO: 2) and operably linked to secG ORF (SEQ ID NO: 2) and a Bacillus subtilis spoVG 5'-UTR (SEQ ID NO: 12), which is operably linked to secE RBS and ORF (SEQ ID NO: 3), which is operably linked to secYRBS and ORF (SEQ ID NO: 4), which is operably linked to a BPN' terminator from Bacillus amyloliquefaciens (SEQ ID NO: 5). An integration cassette was constructed using overlap extension PCR. This cassette contained: an upstream (5') homologous region of *Bacillus subtilis* aprE (SEQ ID NO: 14), which was operatively linked to the non-natural operon Phbs-secGEY (SEQ ID NO: 13), which was operatively linked to a promoter and a kanamycin resistance gene (SEQ ID NO: 15), which was operatively linked to a downstream (3') homologous region of *Bacillus subtilis* aprE (SEQ ID NO: 16). 16), and the integration cassette was transformed into a Bacillus subtilis strain containing two copies of the reporter-3 expression cassette integrated at the skf locus (skf::P2-00788-reporter-3) and the pksR locus (pksR::P2-00788-reporter-3) to produce Bacillus subtilis strain AL396.

[0281] Example 6

[0282] Reporter-3 protease expression in Bacillus subtilis cells overexpressing SECG, SECE, and SECY

[0283] In this example, the expression of heterologous reporter-3 protein in the presence of SecGEY overexpression will be compared with the expression of the same reporter-3 protein in the absence of SecGEY overexpression. More specifically, the reporter-3 yield of three (3) SecGEY-overexpressing strains (AL394, AL395, AL396) and the control strain BPC0229 was evaluated under small-scale conditions as follows. Precultures in tryptone soybean broth (1.7% tryptone, 0.3% soybean peptone, 0.25% glucose, 0.5% sodium chloride, 0.25% dipotassium hydrogen phosphate) were inoculated with single colonies and grown at 37°C and 250 RPM. The preculture was inoculated with 20 ml of 0.5X MPS2 medium at a 1:1000 dilution, supplemented with 80 mM MOPS and 5 mM CaCl2 adjusted to pH 7.3, and the culture was grown in flasks at 37°C and 250 RPM. MPS2 medium consists of the following: a medium based on 10% v / v 10x MOPS (8.4% w / v MOPS, 2.9% w / v sodium chloride, 1.5% w / v potassium hydroxide, 0.05% w / v potassium sulfate, 0.05% w / v magnesium chloride, 0.7% w / v tris(hydroxymethyl)glycine (Tricine), 10% v / v micronutrients), 10% w / v Maltrin M150, 6% w / v soybean peptone, 0.78% w / v dipotassium phosphate, 0.36% w / v urea, 0.2% w / v dipotassium hydrogen phosphate, and 0.06% w / v trisodium citrate dihydrate, with pH adjusted with potassium hydroxide. The micronutrients were prepared in 100X stock solution in one (1) liter, comprising: 1.47 g sodium citrate 2H2O, 1.47 g CaCl2 2H2O, 400 mg FeSO4 7H2O, 100 mg MnSO4 H2O, 100 mg ZnSO4 H2O, 50 mg CuCl2 2H2O, 100 mg CoCl2 6H2O, and 100 mg Na2MoO4 2H2O.

[0284] Essentially as described in WO 2020112609, the amount of reporter-3 protease in culture supernatant was determined using the suc-AAPF-pNA assay. The substrate was N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (suc-AAPF-pNA). After the protease hydrolyzed the peptide substrate, 4-nitroaniline was cleaved to produce 4-nitroaniline as a yellow chromophore. Therefore, absorbance was measured at 405 nm, and the slope of the absorbance change (mOD / min) was directly correlated with the amount of protease in the analyzed 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 solution diluted in Tris buffer (100 mg / ml AAPF stock solution dissolved in DMSO, diluted 100X 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.

[0285] Control strain BPC0229 and SecGEY overexpressing strains AL394, AL395, and AL396 were cultured and sampled after sixteen (16), twenty-four (24), and forty (40) hours of growth to detect reporter-3 protease activity. More specifically, reporter-3 yields and associated coefficients of variation (CV; n = 3) of SecGEY overexpressing strains AL394, AL395, and AL396 relative to control strain BPC0229 are presented in Table 7 below, and relative reporter-3 protease yields normalized relative to cell mass in culture as measured by OD600 are presented in Table 8 below. As shown in Tables 7 and 8, reporter-3 protein yields were increased for all three SecGEY overexpressing strains when protease yield / OD600 was taken into account.

[0286] Table 8

[0287] Small-scale production of proteases

[0288]

[0289] Table 9

[0290] Small-scale production of proteases relative to culture OD600 normalization

[0291] References

[0292] PCT Publication No. WO 1999 / 04006

[0293] PCT Publication No. WO 2008 / 126929

[0294] PCT Publication No. WO 2010 / 056634

[0295] PCT Publication No. WO 2011 / 130222

[0296] PCT Publication No. WO 2015 / 089447

[0297] PCT Publication Number WO 2016 / 202839

[0298] PCT Publication Number WO 2017 / 207762

[0299] PCT Publication Number WO 2020 / 112609

[0300] PCT Publication Number WO 2023 / 114936

[0301] PCT Publication No. WO 2008 / 141281

[0302] US Patent Publication No. US 2009 / 0029417

[0303] 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.

[0304] Freudl, “Signal peptides for recombinant protein secretion inbacterial expression systems”, Microbial Cell Factories, 17:52 2018.

[0305] Mulder et al., “Construction of an artificial secYEG operon allowing high-level secretion of α-amylase”, Protein Expression and Purification, 89, pp. 92-96, 2013.

[0306] Nef 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.

[0307] Neef et al., “Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential”, Essays in Biochemistry, 65: 187-195, 2021.

[0308] Pohl and Harwood, “Heterologous Protein Secretion by Bacillus Species: From the Cradle to the Grave”, Advances in Applied Microbiology, Vol. 73, Chapter I, 2010.

[0309] Rawlings et al., MEROPS: the peptidase database, Nucleic Acids Research, 34 Database Issue, D270-272, 2006.

[0310] Siezen and Leunissen, “Subtilases: The superfamily of subtilisin-like serine proteases”, Protein Science, 6, pp. 501-523, 1997.

Claims

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

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

2.

3. The recombinant cell of 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 of 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 of 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 operably linked to a downstream secGEY read frame, or wherein the introduced secGEY operon comprises a heterologous upstream (5') promoter and a 5'-UTR sequence operably linked to the downstream secGEY read frame (ORF).

6. The recombinant cell of 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. The recombinant cell of claim 1, wherein the recombinant cell comprises at least two introduced cassettes encoding the same or different subtilisin, or comprises at least three introduced expression cassettes encoding the same or different subtilisin.

8. The recombinant cell of claim 1 or claim 7, wherein one or more cassettes encode alkaline subtilisin.

9. The recombinant cell of claim 1 or claim 7, wherein the subtilisin has at least 80% amino acid identity with 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 operatively linked to a downstream nucleic acid encoding a secG protein having at least 80% identity with SEQ ID NO: 28, the downstream nucleic acid encoding the secG protein being operatively linked to a downstream nucleic acid encoding a secE protein having at least 80% identity with SEQ ID NO: 29, and the downstream nucleic acid encoding the secE protein being operatively linked to a downstream (3') nucleic acid encoding a secY protein having at least 80% identity with SEQ ID NO:

30.

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

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

13. A method for producing heterologous subtilisin in modified Gram-positive bacterial cells, the method comprising: (a) Obtaining Gram-positive bacterial cells that produce heterologous subtilisin, and introducing a synthetic secGEY operon into the cells, and (b) Ferment the modified cells under conditions suitable for producing the subtilisin.

14. A method for producing heterologous subtilisin in modified Gram-positive bacterial cells, the method comprising: (a) Obtain Gram-positive bacterial cells and introduce (i) an expression cassette encoding the heterologous subtilisin and (ii) a synthesized secGEY operon into the cells, and (b) Ferment the modified cells under conditions suitable for producing the subtilisin.

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

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

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

2.

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

31.

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

32.

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

21. The method of claim 13 or claim 14, wherein the introduced secGEY operon comprises a heterogeneous upstream (5') promoter and a 5'-UTR sequence operatively connected to the downstream secGEY ORF.

22. The method of claim 13 or claim 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 of claim 14, wherein the cell comprises at least two introduced cassettes encoding the same or different subtilisin, or at least three introduced expression cassettes encoding the same or different subtilisin.

24. The method of claim 14 or claim 23, wherein the one or more boxes encode alkaline subtilisin.

25. The method of claim 14 or claim 23, wherein the one or more cassettes encoding the subtilisin contain an upstream promoter region sequence operatively linked to a downstream nucleic acid encoding a protein signal sequence, the downstream nucleic acid encoding the protein signal sequence being operatively linked to a downstream nucleic acid encoding a pre-coding region sequence, and the downstream nucleic acid encoding the pre-coding region sequence being operatively linked to a downstream nucleic acid encoding mature subtilisin.

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

23.

27. The method of claim 15, wherein when fermented under the same conditions, the increase in the amount of the subtilisin protease increases by at least about 5% relative to the control cells.

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