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

Abstract

The present disclosure relates broadly to Gram-positive bacterial strains that comprise an enhanced protein production phenotype. Accordingly, certain aspects relate to compositions and methods for constructing recombinant (engineered) Gram-positive bacterial strains for enhanced production of a protein of interest.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 299,159, filed January 13, 2022, which is incorporated by reference in its entirety.

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

[0003] Sequence Listing Reference The contents of the electronic submission of the sequence listing text file entitled "NB41845-WO-PCT_SequenceListing.xml" was created on January 6, 2023, is 35KB in size, and is incorporated by reference herein in its entirety. [Background technology]

[0004] Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens are frequently used as microbial factories to produce industrially relevant proteins due to their excellent fermentation properties and high yields (e.g., up to 25 grams per liter of culture; Van Dijl and Hecker, 2013). For example, host cells of Bacillus sp. are well known for their production of enzymes (e.g., amylases, cellulases, mannanases, pectate lysases, proteases, pullulanases, etc.) required for food, textile, laundry applications, medical equipment cleaning, pharmaceutical industries, etc. These non-pathogenic Gram-positive bacteria produce proteins that are completely free of harmful by-products (e.g., lipopolysaccharides (LPS), also known as endotoxins), and thus have been awarded a “Qualified Presumption of Safety” (QPS) rating by the European Food Safety Authority (EFSA), and many of their products have earned a “Generally Recognized As Safe” (GRAS) rating from the US Food and Drug Administration (Olempska-Beer et al., 2006; Earl et al., 2008; Caspers et al., 2010).

[0005] Therefore, the production of proteins (e.g., enzymes, antibodies, receptors, etc.) via microbial host cells is of particular interest in the biotechnology field. Similarly, the optimization of Bacillus host cells to produce and secrete one or more proteins of interest is particularly relevant in the context of industrial biotechnology, where small improvements in protein yields are of crucial importance when the protein is to be industrially mass-produced. For example, the expression of many heterologous proteins can still be difficult and unpredictable in terms of yield, etc. As described herein below, the present disclosure relates to a highly desirable and unmet need to obtain and construct Gram-positive cells (e.g., protein-producing hosts) with enhanced protein production capabilities. Summary of the Invention [Means for solving the problem]

[0006] As outlined herein, applicants have surprisingly observed that overexpression of the yvyD gene is particularly relevant for enhanced production of a protein of interest in a gram-positive bacterial cell. Accordingly, certain aspects of the present disclosure relate to compositions and methods for producing a protein of interest. More specifically, certain embodiments of the present disclosure provide, inter alia, recombinant (modified) gram-positive bacterial cells (strains) overexpressing the yvyD gene, recombinant gram-positive bacterial cells expressing / producing one or more proteins of interest, recombinant gram-positive bacterial cells expressing a protein of interest and overexpressing the yvyD gene, polynucleotide constructs suitable for introduction into bacterial strains (e.g., plasmids, vectors, expression cassettes, etc.), etc. Accordingly, certain aspects relate to recombinant gram-positive bacterial cells overexpressing the yvyD gene, where the recombinant cells produce increased amounts of a protein of interest, exhibit enhanced specific productivity (Qp) of the protein produced when cultured under suitable conditions, exhibit enhanced carbon utilization of the protein produced when cultured under suitable conditions, etc. [Brief description of the drawings]

[0007] [Figure 1] Schematic maps of integration cassettes are shown, with FIG. 1A showing the integration cassette "yvzG-yvyD intergenic::lox-SpecR-lox-PspoVG-yvyD" (SEQ ID NO: 19) and FIG. 1B showing the integration cassette "yvzG-yvyD intergenic::lox-SpecR-lox-Phbs-yvyD" (SEQ ID NO: 20). For example, as shown in FIG. 1A, the integration cassette "yvzG-yvyD intergenic::lox-SpecR-lox-PspoVG-yvyD" (SEQ ID NO: 19) contains an upstream (5') "fliS" sequence, followed by a "fliT" sequence, followed by a "yvzG" gene sequence, which are upstream of a lox-SpecR-lox sequence, followed by a "PspoVG-yvyD" sequence, followed by a downstream (3') "secA" sequence. As shown, the integration cassette "yvzG-yvyD intergenic::lox-SpecR-lox-Phbs-yvyD" (SEQ ID NO:20) of Figure 1B contains an upstream (5') "fliS" sequence, followed by a "fliT" sequence, followed by a "yvzG" gene sequence, which is upstream of a lox-SpecR-lox sequence, followed by a "Phbs-spoVGSD-yvyD" sequence, followed by a downstream (3') "secA" sequence.

[0008] [Diagram 2] Protease activity (arbitrary units) of sample aliquots taken at the indicated time points is shown (Example 3). Protease productivity (Protease-1) of the control strain (2x Protease-1) and the engineered strain (2x Protease-1+PspoVG-yvyD) fermented under the same conditions was compared (Figure 2).

[0009] [Diagram 3] Protease activity (arbitrary units) of sample aliquots taken at the indicated time points is shown (Example 3). Protease productivity (protease-2) of the control strain (2x protease-2) and the engineered strain (2x protease-2+PspoVG-yvyD) fermented under the same conditions was compared (Figure 3).

[0010] [Figure 4]Protease activity (arbitrary units) of sample aliquots taken at the indicated time points is shown (Example 3). Protease productivity (protease-2) of the control strain (2x protease-2) and the engineered strain (2x protease-2+Phbs-yvyD) fermented under the same conditions was compared (Figure 4).

[0011] [Diagram 5] Protease activity (arbitrary units) of sample aliquots taken at the indicated time points is shown (Example 3). Protease productivity (protease-3) of the control strain (2x protease-3) and the engineered strain (2x protease-3+Phbs-yvyD) fermented under the same conditions was compared (Figure 5).

[0012] [Figure 6] 6 shows the amino acid sequence of the native B. subtilis YvyD protein (SEQ ID NO: 26). As shown in FIG. 6, the YvyD protein (SEQ ID NO: 26) contains an N-terminal conserved RaiA superfamily domain (SEQ ID NO: 27) with amino acid residues shown underlined, and a C-terminal conserved ribosomal S30AE_C superfamily domain (SEQ ID NO: 28) with amino acid residues shown in bold.

[0013] [Figure 7] 7A-7C show the nucleic acid sequence of the Phbs promoter region set forth in SEQ ID NO:29. Specifically, the Phbs promoter region (SEQ ID NO:29; FIG. 1A) comprises an upstream (5') hbs promoter sequence (SEQ ID NO:22; FIG. 7B) operably linked to a downstream (3') spoVG Shine-Dalgarno (SD) sequence (SEQ ID NO:25; FIG. 7C). As shown in FIG. 1A, the nucleotides of the hbs promoter sequence (Phbs; SEQ ID NO:22) are underlined and the nucleotides of the Shine-Dalgarno sequence (SD; SEQ ID NO:25) are bolded. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] A brief description of biological sequences SEQ ID NO:1 is the synthetic DNA sequence of a primer designated "343".

[0015] SEQ ID NO:2 is the synthetic DNA sequence of a primer designated "402".

[0016] SEQ ID NO:3 is the synthetic DNA sequence of a primer designated "400".

[0017] SEQ ID NO:4 is the synthetic DNA sequence of a primer designated "370".

[0018] SEQ ID NO:5 is the synthetic DNA sequence of a primer designated "539".

[0019] SEQ ID NO:6 is the synthetic DNA sequence of a primer designated "246".

[0020] SEQ ID NO:7 is the synthetic DNA sequence of a primer designated "540".

[0021] SEQ ID NO:8 is the synthetic DNA sequence of a primer designated "754".

[0022] SEQ ID NO: 9 is a primer for a synthetic DNA sequence (36 bp) of the spoVG promoter region.

[0023] SEQ ID NO:10 is the synthetic DNA sequence of a primer designated "675".

[0024] SEQ ID NO:11 is the synthetic DNA sequence of a primer designated "307".

[0025] SEQ ID NO:12 is the synthetic DNA sequence of a primer designated "674".

[0026] SEQ ID NO:13 is the synthetic DNA sequence of a primer designated "345".

[0027] SEQ ID NO:14 is the synthetic DNA sequence of a primer designated "348".

[0028] SEQ ID NO:15 is the synthetic DNA sequence of a primer designated "346".

[0029] SEQ ID NO:16 is the synthetic DNA sequence of a primer designated "300".

[0030] SEQ ID NO:17 is the synthetic DNA sequence of a primer designated "573".

[0031] SEQ ID NO:18 is the open reading frame (DNA) sequence of the yvyD gene CDS.

[0032] SEQ ID NO:19 is a polynucleotide (integration) cassette designated "yvzG-yvyD intergenic::lox-SpecR-lox-PspoVG-yvyD."

[0033] SEQ ID NO:20 is a polynucleotide (integration) cassette designated "yvzG-yvyD intergenic::lox-SpecR-lox-Phbs-yvyD."

[0034] SEQ ID NO:21 is the spoVG promoter region (PspoVG) comprising the upstream (5') spoVG promoter operably linked to the downstream (3') spoVG Shine-Dalgarno (SD) sequence.

[0035] SEQ ID NO:22 is the hbs promoter (Phbs) sequence.

[0036] SEQ ID NO:23 is the yvyD gene (DNA) sequence of B. subtilis, including the yvyD promoter region and the yvyD gene coding sequence (CDS, i.e., SEQ ID NO:18).

[0037] SEQ ID NO:24 is the DNA sequence of the upstream (5') yvyD promoter region of the yvyD gene set forth in SEQ ID NO:23.

[0038] SEQ ID NO:25 is a DNA sequence containing the native spoVG Shine-Dalgarno (SD) sequence.

[0039] SEQ ID NO:26 is the amino acid sequence of the native YvyD protein of B. subtilis.

[0040] SEQ ID NO:27 is the amino acid sequence of the N-terminal conserved RaiA superfamily domain of the native YvyD protein (SEQ ID NO:26).

[0041] SEQ ID NO: 28 is the amino acid sequence of the C-terminal conserved ribosomal S30AE_C superfamily domain of the native YvyD protein (SEQ ID NO: 26).

[0042] SEQ ID NO:29 is the hbs promoter region sequence, which comprises the hbs promoter (Phbs) sequence (SEQ ID NO:22) operably linked to the SD sequence (SEQ ID NO:25).

[0043] Detailed Description As described herein, certain embodiments of the present disclosure relate to compositions and methods for enhancing protein production in Gram-positive bacterial (host) cells / strains. More specifically, as described herein below and in more detail in the Examples below, the recombinant (genetically modified) Gram-positive bacterial cells of the present disclosure are particularly useful for enhancing the production of a protein of interest. Certain embodiments of the present disclosure relate, inter alia, to recombinant polynucleotides that increase yvyD gene expression in Gram-positive bacterial cells, recombinant Gram-positive cells that overexpress a yvyD gene coding sequence (CDS, e.g., yvyD ORF; SEQ ID NO: 18), recombinant Gram-positive cells that overexpress the yvyD gene CDS and express one or more (multiple) copies of a gene encoding a protein of interest, and the like.

[0044] I. Definition In view of the disclosed recombinant (modified) cells and methods thereof described herein, the following terms and phrases are defined. Terms not defined herein should be given the meaning commonly used in the art.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the compositions and methods of the present invention belong. Although any methods and materials similar or equivalent to those described herein can also be used to carry out or test the compositions and methods of the present invention, exemplary methods and materials are described below. All publications and patents cited herein are incorporated herein by reference in their entirety.

[0046] It is further noted that the claims may be written to exclude optional elements. Thus, this statement is intended to serve as a prelude to the use of exclusive terminology such as "solely," "only," "excluding," "not including," or the use of "negative" limitations or qualifications thereof in connection with the recitation of claim elements. For example, in certain embodiments, a "control cell / strain" of Gram-positive bacteria produces a protein of interest but "does not contain" an overexpressed yvyD gene.

[0047] As will be apparent to one of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated or combined with the features of any of the other embodiments without departing from the scope or spirit of the compositions and methods described herein. Any described method may be carried out in the order of events recited or in any other order which is logically possible.

[0048] As used herein, the phrases "gram-positive bacteria", "gram-positive cells", "gram-positive bacterial strains", and / or "gram-positive bacterial cells" have the same meaning as used in the art. For example, gram-positive bacterial cells include all strains of Actinobacteria and Firmicutes. In certain embodiments, such gram-positive bacteria are of the classes Bacilli, Clostridia, and Mollicutes. As used herein, the term "recombinant" or "non-naturally occurring" refers to an organism, microorganism, cell, nucleic acid molecule, or vector that has at least one engineered genetic change or has been modified by the introduction of a heterologous nucleic acid molecule, or a cell (e.g., a microbial cell) that has been altered such that expression of a heterologous or endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from or is the descendant of a non-naturally occurring cell that has one or more such modifications. Genetic changes include, for example, modifications that introduce expressible nucleic acid molecules that code for proteins, additions, deletions, substitutions of nucleic acid molecules, or other functional changes to the genetic material of a cell. For example, recombinant cells may express genes or other nucleic acid molecules that are not found in the same or homologous form in the native (wild-type) cell (e.g., fusion or chimeric proteins), or may provide an altered expression pattern of an endogenous gene that is over-expressed, under-expressed, minimally expressed, or not expressed at all. "Recombinant," "recombining," or producing a "recombinant" nucleic acid is generally the assembly of two or more nucleic acid fragments, which assembly gives rise to a chimeric gene.

[0049] As used herein, the term "yvyD" or "yvyD gene" refers to a gene (or yvyD gene homolog) encoding the "YvyD" protein (or a YvyD protein homolog). As outlined in Section II below, the YvyD protein is believed to function as a "general stress factor" or "ribosomal hibernation promoting factor." The term yvyD gene includes synonyms such as the "hpf" gene and / or the "yviI" gene.

[0050] As used herein, the phrase "overexpression of yvyD" or "increased yvyD expression" refers to increased expression of the yvyD gene coding sequence (CDS). In certain embodiments, the yvyD gene CDS comprises at least 80% sequence identity to the yvyD open reading frame (ORF; SEQ ID NO: 18). For example, in certain embodiments, increased expression of yvyD can be achieved by replacing the native upstream (5') "yvyD promoter region" with a suitable heterologous (alternative) promoter region, and expression of the downstream yvyD gene CDS is controlled by the heterologous (alternative) promoter. In certain embodiments, the yvyD gene CDS comprises at least about 50%-100% identity to the yvyD ORF of SEQ ID NO: 18. In certain embodiments, the yvyD gene CDS comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the yvyD ORF of SEQ ID NO:18.

[0051] As used herein, a native "yvyD promoter region" (abbreviated "PyvyD") refers to a "yvyD gene promoter" that comprises at least about 60%-100% sequence identity to the native B. subtilis yvyD gene promoter region sequence of SEQ ID NO: 24. In certain embodiments, the yvyD gene promoter region (PyvyD) comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to PyvyD of SEQ ID NO: 24.

[0052] As used herein, the term "hbs promoter sequence" (abbreviated "Phbs") refers to a nucleic acid comprising at least about 60%-100% identity to SEQ ID NO: 22. In certain embodiments, the hbs promoter (Phbs) sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 22.

[0053] As used herein, the term "spoVG Shine-Dalgarno (SD) sequence" refers to a nucleic acid comprising at least about 60%-100% identity to SEQ ID NO: 25. In certain embodiments, the spoVG SD sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:25.

[0054] As used herein, the term "hbs promoter region sequence" (or "Phbs region sequence" for short) refers to a nucleic acid comprising at least about 60%-100% identity to SEQ ID NO:29. For example, as shown in FIG. 7A, the Phbs promoter region (SEQ ID NO:29) sequence comprises a Phbs promoter (SEQ ID NO:22) disposed upstream of and in operable combination with an SD sequence (SEQ ID NO:25). Thus, in certain embodiments, the Phbs promoter region comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:29.

[0055] As used herein, the term "spoVG promoter region" (abbreviated "PspoVG") refers to a nucleic acid comprising at least about 60%-100% identity to SEQ ID NO: 21. In certain embodiments, the spoVG promoter region (PspoVG) comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the PspoVG of SEQ ID NO:21.

[0056] As used herein, exemplary "yvyD gene overexpression (integration) cassettes" are shown generally in FIG. 1, where the integration cassettes designated "yvzG yvyD intergenic::lox-SpecR-lox-PspoVG-yvyD" (FIG. 1A; SEQ ID NO: 19) and "yvzG yvyD intergenic::lox-SpecR-lox-Phbs-yvyD" (FIG. 1B; SEQ ID NO: 20) are shown.

[0057] As used herein, the term "promoter swap," when used in phrases such as "increased expression of the yvyD gene by "promoter swap" integration," refers to replacing (substituting) a yvyD gene promoter region sequence upstream (5') of the native yvyD gene coding sequence (CDS) with a "heterologous (alternative) promoter" sequence, where the promoter-swapped yvyD gene CDS is expressed (overexpressed) under the control of the heterologous (alternative) promoter.

[0058] As used herein, genes encoding exemplary reporter proteases may be listed as a "protease-1" gene, a "protease-2" gene, a "protease-3" gene, etc., with the encoded proteases listed as "protease-1," "protease-2," "protease-3," etc., respectively.

[0059] As used herein, phrases such as "two copies of protease-1" and "encoding two copies of protease-1" may be abbreviated as "2x protease-1" and "2x Protease-1," respectively; "two copies of protease-2" and "encoding two copies of protease-2" may be abbreviated as "2x protease-2" and "2x Protease-2," respectively; "two copies of protease-3" and "encoding two copies of protease-3" may be abbreviated as "2x protease-1" and "2x Protease-1," respectively, etc.

[0060] As used herein, the reporter protease of the name "Protease-1" refers to a variant Bacillus lentus subtilisin (protease) described in PCT Publication No. WO 2012 / 151534, which is incorporated by reference in its entirety.

[0061] As used herein, the reporter protease of the name "Protease-2" refers to the variant Bacillus gibsonii protease described in PCT Publication No. WO 2020 / 243738, which is incorporated by reference in its entirety.

[0062] As used herein, the reporter protease of the name "Protease-3" refers to the variant Bacillus amyloliquefaciens BPN' protease described in PCT Publication No. WO 2011 / 72099A, which is incorporated by reference in its entirety.

[0063] Thus, in certain aspects, the Gram-positive cells of the present disclosure comprise an endogenous yvyD gene encoding a native YvyD protein, wherein the yvyD gene comprises between about 50% and 100% identity to the yvyD gene of SEQ ID NO: 23. In other embodiments, the yvyD gene comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23.

[0064] SEQ ID NO: 26 is the amino acid sequence of the native YvyD protein of B. subtilis, SEQ ID NO: 27 is the amino acid sequence of the N-terminal conserved RaiA superfamily domain of the native YvyD protein (SEQ ID NO: 26), and SEQ ID NO: 28 is the amino acid sequence of the C-terminal conserved ribosomal S30AE_C superfamily domain of the native YvyD protein (SEQ ID NO: 26).

[0065] As used herein, "host cell" refers to a cell that has the ability to act as a host or expression vehicle for a newly introduced DNA sequence. Thus, in certain embodiments of the present disclosure, the host cell is a Gram-positive (e.g., Bacilli) and / or Gram-negative (e.g., E. coli) cell.

[0066] As used herein, the phrases "modified Gram-positive cell" and / or "Gram-positive (daughter) cell" refer to a recombinant Gram-positive cell that contains at least one genetic modification that is not present in the parent (control) Gram-positive cell from which the modified Gram-positive cell is derived. For example, when comparing expression / production of a protein of interest (POI) in a Gram-positive "control" cell with expression / production of the same POI in an "modified" (recombinant) cell, it will be understood that the "control" and "modified" cells are grown / cultured / fermented under identical conditions (e.g., identical conditions of medium, temperature, pH, etc.).

[0067] As used herein, "increasing" protein production or "increased" protein production refers to an increased amount of a protein (e.g., a protein of interest) produced. The protein may be produced inside the host cell or secreted (or transported) into the culture medium. In certain embodiments, the protein of interest is produced (secreted) into the culture medium. Increased protein production may be detected, for example, as a higher maximum level of protein or enzyme activity (e.g., protease activity, amylase activity, pullulanase activity, cellulase activity, etc.) or as total extracellular protein produced, as compared to the parent cell.

[0068] 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 the transcription or translation of a gene or its ORF; (b) gene disruption; (c) gene conversion; (d) gene deletion; (e) gene downregulation; (f) directed mutagenesis; and / or (g) random mutagenesis of any one or more genes disclosed herein.

[0069] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecule of the present disclosure. Expression can also refer to the translation of mRNA into a polypeptide. Thus, the term "expression" includes any process involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0070] As used herein, "nucleic acid" refers to nucleotide or polynucleotide sequences, and fragments or portions thereof, and DNA, cDNA and RNA of genomic or synthetic origin, which may be double-stranded or single-stranded, whether representing the sense or antisense strand. It will be understood that, as a result of the degeneracy of the genetic code, many nucleotide sequences can code for a given protein.

[0071] The polynucleotides (or nucleic acid molecules) described herein are understood to include "genes," "vectors," and "plasmids."

[0072] Thus, the term "gene" refers to a polynucleotide that codes for a particular sequence of amino acids, including all or part of a coding sequence for a protein, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, that determine the conditions under which the gene is expressed. The transcribed region of a gene may include introns, untranslated regions (UTRs), including 5'-untranslated regions (UTRs) and 3'-UTRs, and the coding sequence.

[0073] As used herein, the term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of the coding sequence are generally determined by an open reading frame (hereinafter "ORF"), which usually begins with the ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0074] The term "promoter" as used herein refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' (downstream) of the promoter sequence. Promoters may be derived entirely from a native gene, or may be composed of different elements derived from different naturally occurring promoters, or may even comprise synthetic nucleic acid segments. Those skilled in the art understand that different promoters may direct the expression of genes in different cell types, or at different developmental stages, or in response to different environmental or physiological conditions. The promoters that most often cause expression of genes in most cell types are generally referred to as "constitutive promoters". Furthermore, it is recognized that DNA fragments of different lengths may have the same promoter activity, since in most cases the exact boundaries of regulatory sequences are not completely clear.

[0075] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence (e.g., an ORF) if it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation.

[0076] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader (i.e., signal peptide) is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0077] As used herein, "a functional promoter sequence (or open reading frame thereof) that controls expression of a gene of interest linked to a protein coding sequence of the gene of interest" refers to a promoter sequence that controls the transcription and translation of a coding sequence in Bacillus. For example, in certain embodiments, the present disclosure is directed to a polynucleotide that includes a 5' promoter (or a 5' promoter region or tandem 5' promoters, etc.), where the promoter region is operably linked to a nucleic acid sequence (e.g., an ORF) that encodes a protein.

[0078] As used herein, "suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing or stability or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0079] As used herein, the term "introducing," when used in phrases such as introducing a gene, polynucleotide, vector, cassette, etc. into a Gram-positive bacterial cell, includes methods known in the art for introducing a polynucleotide into a cell, including, but not limited to, protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation, and the like.

[0080] As used herein, "transformed" or "transformation" refers to a cell that has been transformed by the use of recombinant DNA technology. Transformation generally occurs by inserting one or more nucleotide sequences (e.g., polynucleotides, ORFs, or genes) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence that does not naturally occur in the cell being transformed). Thus, transformation generally refers to the introduction of exogenous DNA into a host cell such that the DNA is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector.

[0081] As used herein, "transforming DNA," "transforming sequence," and "DNA construct" refer to DNA used to introduce a sequence into a host cell or organism. Transforming DNA is DNA used to introduce a sequence into a host cell or organism. This DNA can be generated in vitro by PCR or any other suitable technique. In some embodiments, the transforming DNA includes the incoming sequence, while in other embodiments, the transforming DNA further includes the incoming sequence flanked by homology boxes. In yet other embodiments, the transforming DNA includes other non-homologous sequences (i.e., stuffer sequences or flanking sequences) added to the ends. The ends can be closed such that the transforming DNA forms a closed circle, such as, for example, by insertion into a vector.

[0082] As used herein, "gene disruption" or "gene disruption" are used interchangeably and refer broadly to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., protein).Thus, as used herein, gene disruption includes, but is not limited to, frameshift mutations, premature stop codons (i.e., so that no functional protein is produced), substitutions that eliminate or reduce the activity of protein internal deletions (so that no functional protein is produced), insertions that disrupt coding sequences, mutations that remove the operable link between the native promoter and the open reading frame required for transcription, and the like.

[0083] As used herein, "incoming sequence" refers to a DNA sequence that is introduced into the chromosome of a Gram-positive bacterial cell. In some embodiments, the incoming sequence is part of a DNA construct. In other embodiments, the incoming sequence encodes one or more proteins of interest. In some embodiments, the incoming sequence comprises a sequence that may or may not already be present in the genome of the cell to be transformed (i.e., it may be a homologous or heterologous sequence). In some embodiments, the incoming sequence encodes one or more proteins of interest, genes and / or mutant or modified genes. In alternative embodiments, the incoming sequence encodes a functional wild-type gene or operon, a functional mutant gene or operon, or a non-functional gene or operon. In some embodiments, a non-functional sequence may be inserted into a gene to disrupt the function of the gene. In another embodiment, the incoming sequence comprises a selection marker. In yet another embodiment, the incoming sequence comprises two homology boxes.

[0084] As used herein, a "homology box" refers to a nucleic acid sequence that is homologous to a sequence in a Gram-positive bacterial cell chromosome. More specifically, a homology box is an upstream or downstream region that has about 80-100% sequence identity, about 90-100% sequence identity, or about 95-100% sequence identity with the immediately adjacent coding region of a gene or part of a gene to be deleted, disrupted, inactivated, downregulated, etc., according to the present invention. These sequences direct where in the Gram-positive bacterial cell chromosome the DNA construct is integrated and direct what part of the chromosome the incoming sequence replaces. Without intending to limit the disclosure, a homology box can include from about 1 base pair (bp) to 200 kilobases (kb). Preferably, the homology box includes from about 1 bp to 10.0 kb; 1 bp to 5.0 kb; 1 bp to 2.5 kb; 1 bp to 1.0 kb, and 0.25 kb to 2.5 kb. A homology box can also comprise about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb, and 0.1 kb. In some embodiments, the 5' and 3' ends of the selectable marker are flanked by homology boxes, where the homology box comprises nucleic acid sequences that are immediately adjacent to the coding region of a gene.

[0085] As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence capable of being expressed in a host cell, where expression of the selectable marker confers to cells containing the expressed gene the ability to grow in the presence of a corresponding selective agent or in the absence of an essential nutrient.

[0086] As used herein, the terms "selectable marker" and "selection marker" refer to a nucleic acid (e.g., a gene) that can be expressed in a host cell to facilitate the selection of those hosts that contain a vector. Examples of such selectable markers include, but are not limited to, antimicrobial agents. Thus, the term "selectable marker" refers to a gene that provides an indication that a host cell has taken up an incoming DNA of interest or that some other reaction has occurred. Generally, a selectable marker is a gene that confers an antimicrobial resistance or a metabolic advantage to a host cell that allows cells containing foreign DNA to be distinguished from cells that have not received the foreign sequence during transformation.

[0087] An "present selectable marker" is a marker that is located on the chromosome of the microorganism being transformed. The present selectable marker encodes a different gene than the selectable marker on the transforming DNA construct. Selectable markers are well known to those of skill in the art. As noted above, markers include antimicrobial resistance markers (e.g., amplicons ... R , phleo R , spec R , kan R ,ery R , tet R , cmp R and neo R In some embodiments, the present invention provides a chloramphenicol resistance gene (e.g., a gene present on pC194). This resistance gene is particularly useful in embodiments involving chromosomal integration cassettes and chromosomal amplification of the integration plasmid. Other markers useful according to the present invention include, but are not limited to, auxotrophic markers such as serine, lysine, tryptophan, and detectable markers such as β-galactosidase.

[0088] As defined herein, a host cell "genome" and / or a Gram-positive bacterial cell "genome" includes chromosomal genes and extrachromosomal genes.

[0089] As used herein, the terms "plasmid," "vector," and "cassette" refer to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell and are usually in the form of circular double-stranded DNA molecules. Such elements may be linear or circular, single-stranded or double-stranded, autonomously replicating sequences of DNA or RNA, genome-integrating sequences, phages, or nucleotide sequences from any source in which multiple nucleotide sequences have been joined or recombined into a unique structure that can introduce into a cell a promoter fragment and a DNA sequence for a selected gene product along with appropriate 3' untranslated sequences.

[0090] As used herein, the term "plasmid" refers to a circular, double-stranded (ds) DNA construct that is used as a cloning vector and forms an extrachromosomal, self-replicating genetic element in many bacteria and some eukaryotes. In some embodiments, the plasmid becomes integrated into the genome of the host cell, and in some embodiments, the plasmid is present in the parent cell and lost in the daughter cells.

[0091] As used herein, a "transformation cassette" refers to a specific vector that contains a gene (or its ORF) and has elements in addition to a foreign gene that facilitate transformation of a particular host cell.

[0092] As used herein, the term "vector" refers to any nucleic acid that can replicate (multiply) in a cell and can carry new genes or DNA segments into the cell. Thus, the term refers to a nucleic acid construct designed for transport between various host cells. Vectors include viruses, bacteriophages, proviruses, plasmids, phagemids, transposons, which are "episomal" (i.e., they can replicate autonomously or be integrated into the chromosomes of the host organism), as well as artificial chromosomes, such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes).

[0093] "Expression vector" refers to a vector capable of incorporating and expressing heterologous DNA in a cell. Many prokaryotic and eukaryotic expression vectors are commercially available and known to those skilled in the art. The selection of an appropriate expression vector is within the knowledge of one of ordinary skill in the art.

[0094] As used herein, the terms "expression cassette" and "expression vector" refer to a nucleic acid construct (i.e., they are vectors or vector elements as described above) that is recombinantly or synthetically produced with a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, a chromosome, mitochondrial DNA, plastid DNA, a virus, or a nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, the DNA construct also includes a set of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. In certain embodiments, the DNA construct of the present disclosure includes a selectable marker and an inactivated chromosomal segment or gene segment or DNA segment as defined herein.

[0095] As used herein, a "targeting vector" is a vector that contains a polynucleotide sequence that is homologous to a region in a host cell chromosome into which the targeting vector is transformed and can drive homologous recombination at that region. For example, a targeting vector is used to introduce a mutation into a host cell chromosome by homologous recombination. In some embodiments, the targeting vector contains other non-homologous sequences (i.e., stuffer sequences or flanking sequences), for example, added to the ends. The ends can be closed such that the targeting vector forms a closed circle, for example, by insertion into a vector. For example, in certain embodiments, parent Gram-positive (host) cells are modified (e.g., transformed) by introducing one or more "targeting vectors" into them.

[0096] As used herein, the term "protein of interest" or "POI" refers to a polypeptide of interest that is desired to be expressed in a Gram-positive bacterial cell. Thus, as used herein, a POI can be an enzyme, a substrate binding protein, a surfactant protein, a structural protein, a receptor protein, a protein biologic, and the like. In certain embodiments, the engineered Gram-positive cells of the present disclosure produce an increased amount of a heterologous or endogenous POI compared to the parent. In certain embodiments, the increased amount of POI produced by the engineered cells is at least a 0.5% increase, at least a 1.0% increase, at least a 5.0% increase, or more than a 5.0% increase compared to the parent.

[0097] Similarly, as defined herein, a "gene of interest" or "GOI" refers to a nucleic acid sequence (e.g., polynucleotide, gene, ORF) that encodes a POI. A "GOI" that encodes a "POI" may be a naturally occurring gene, a mutated gene, or a synthetic gene.

[0098] As used herein, the terms "polypeptide" and "protein" are used interchangeably and refer to polymers of any length that contain amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein. Polypeptides may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term polypeptide also encompasses amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety. Also included within the scope of this definition are, for example, polypeptides that contain one or more analogs of an amino acid, including, for example, unnatural amino acids, and other modifications known in the art.

[0099] In certain embodiments, the genes of the present disclosure are those encoding enzymes (e.g., acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lysase, lysase), endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, laccase, lipase, lyase, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methyl esterase, pectin degrading enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phytase, polygalacturonase, protease, peptidase, rhamno-galacturonase, ribonuclease, transferase, transport protein, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0100] As used herein, a "variant" polypeptide refers to a polypeptide derived from a parent (or reference) polypeptide by one or more amino acid substitutions, additions, or deletions, typically by recombinant DNA techniques. A variant polypeptide may differ from the parent polypeptide by a small number of amino acid residues and may be defined by the level of primary amino acid sequence homology / identity with the parent (reference) polypeptide.

[0101] Preferably, a variant polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with a parent (reference) polypeptide sequence. As used herein, a "variant" polynucleotide refers to a polynucleotide that encodes a variant polypeptide, which has a particular degree of sequence homology / identity with a parent polynucleotide or hybridizes to a parent polynucleotide (or its complement) under stringent hybridization conditions. Preferably, the variant nucleotides have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% nucleotide sequence identity with the parent (reference) polynucleotide sequence.

[0102] As used herein, the term "mutation" refers to any change or alteration in a nucleic acid sequence. There are several types of mutations, including point mutations, deletion mutations, silent mutations, frameshift mutations, splicing mutations, etc. Mutations can be made specifically (e.g., by site-directed mutagenesis) or randomly (e.g., by chemical agents, repair minus passaging through bacterial strains).

[0103] As used herein, in reference to a polypeptide or sequence thereof, the term "substitution" refers to the replacement (ie, substitution) of one amino acid with another.

[0104] As defined herein, "endogenous gene" refers to a gene that is present in its natural location in the genome of an organism.

[0105] As defined herein, a "heterologous" gene, "non-endogenous" gene or "foreign" gene refers to a gene (or ORF) that is not normally found in the host organism, but which has been introduced into the host organism by gene transfer. As used herein, the term "foreign" gene includes a native gene (or ORF) inserted into a non-native organism and / or a chimeric gene inserted into a native or non-native organism.

[0106] As defined herein, a "heterologous control sequence" refers to a gene expression control sequence (e.g., a promoter or enhancer) that does not function in nature to regulate (control) the expression of a gene of interest. Generally, heterologous nucleic acid sequences are not endogenous (natural) to the cell or part of the genome in which they are present, but have been added to the cell by infection, transfection, transformation, microinjection, electroporation, etc. A "heterologous" nucleic acid construct may contain a control sequence / DNA coding (ORF) sequence combination that is the same or different from the control sequence / DNA coding sequence combination found in the native host cell.

[0107] As used herein, the terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that may be involved in the secretion or direct transport of a mature protein or a precursor form of a protein. A signal sequence is generally located at the N-terminus of a precursor or mature protein sequence. A signal sequence may be endogenous or exogenous. A signal sequence is not usually present in a mature protein. A signal sequence is generally cleaved from a protein by a signal peptidase after the protein has been transported.

[0108] The term "derived from" includes the terms "originating from," "obtained from," "available from," and "made from," and generally indicates that one particular material or composition has characteristics that can be found its origin in another material or composition, or that can be described with reference to that other particular material or composition.

[0109] As used herein, the term "homology" refers to homologous polynucleotides or homologous polypeptides. When two or more polynucleotides or two or more polypeptides are homologous, this means that the homologous polynucleotides or polypeptides have a "degree of identity" of at least 60%, more preferably at least 70%, even more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, and most preferably at least 98%. Whether two polynucleotide or polypeptide sequences have a sufficiently high degree of identity to be homologous as defined herein can be conveniently determined by aligning the two sequences using a computer program known in the art, for example, "GAP" provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman and Wunsch, (1970). For DNA sequence comparison, GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0110] As used herein, the term "percent identity" refers to the level of nucleic acid or amino acid sequence identity between nucleic acid sequences encoding a polypeptide or between the amino acid sequences of a polypeptide when aligned using a sequence alignment program.

[0111] As used herein, the term "specific productivity" refers to the total amount of protein produced per cell per unit time over a given period of time.

[0112] As defined herein, the terms "purified," "isolated," or "enriched" refer to a biomolecule (e.g., a polypeptide or polynucleotide) that has been altered from its native state by separation from some or all of the naturally occurring components with which it is naturally associated. Such isolation or purification can be accomplished by separation techniques known in the art, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation or other protein salting out, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove unwanted whole cells, cell debris, impurities, extraneous proteins, or enzymes in the final composition. Purified or isolated biomolecule compositions can then be supplemented with components that confer additional benefits, such as activators, anti-inhibitors, desirable ions, pH adjusting compounds, or other enzymes or chemicals.

[0113] As used herein, "flanking sequence" refers to any sequence that is either upstream or downstream of the sequence under consideration (e.g., in gene ABC, gene B is flanked by gene sequences A and C). In certain embodiments, the incoming sequence is flanked on each side by a homology box. In another embodiment, the incoming sequence and the homology box comprise a unit that is flanked on each side by a stuffer sequence. In some embodiments, the flanking sequences are present on only one side (3' or 5'), but in preferred embodiments, they are present on each side of the flanked sequence. The sequence of each homology box is homologous to a sequence in the Gram-positive bacterial cell chromosome. These sequences direct where in the chromosome the new construct will be integrated and which part of the chromosome will be replaced by the incoming sequence. In other embodiments, the 5' and 3' ends of the selection marker are flanked by polynucleotide sequences that comprise a portion of the inactivated chromosomal segment. In some embodiments, the flanking sequences are present on only one side (3' or 5'), but in other embodiments, they are present on each side of the flanked sequence.

[0114] II. Overexpression of yvyD in Gram-positive bacterial cells enhances protein production As briefly mentioned above, the yvyD gene of B. subtilis encodes a protein that is thought to function as a "general stress factor" or "ribosomal hibernation-promoting factor". For example, σ , which shows an atypical induction profile, as reviewed in Drzewiecki et al. (1998), B Analysis of two-dimensional protein gels of Hst23-dependent general stress proteins identified the protein YvyD (formerly known as "Hst23") as the product of the yvyD gene in B. subtilis. As explained in this publication, the atypical σ B In addition to the stress- and starvation-inducible pattern of dependence, yvyD also H It was also induced in response to amino acid starvation via a β-dependent promoter activation. In a study to elucidate the biological functions of the B. subtilis small (p)ppGpp synthases YjbM and YwaC, Tagami et al. (2012) described the structure of a B. subtilis mutant (e.g. triple mutant; ΔrelA ΔyjbM ΔywaC) in which the YvyD protein was suggested to be essential for 70S ribosome dimerization. More recently, the cryo-EM structure of the B. subtilis 100S (Bs100S) particle revealed a binding site for the B. subtilis hibernation-promoting factor (BsHPF), named YvyD (Beckert et al., 2017). JP 2009-225711 A describes deleting the yvyD gene of B. subtilis, and states that the yvyD gene is not directly involved in the production of the protein of interest, and that the YvyD protein is not required for the growth of the microorganism in normal industrial production media. Therefore, it is hypothesized that in wild-type B. subtilis cells, YvyD binds to inactive ribosomes during stress conditions, normally protecting the ribosomes.

[0115] As outlined herein and described in the Examples below, Applicant has surprisingly observed that overexpression of the yvyD gene is particularly relevant for enhancing the production of a protein of interest in a Gram-positive bacterial cell. More specifically, as described in Example 1, Applicant has designed and constructed a yvyD (gene) overexpression (integration) cassette for introduction into an exemplary Gram-positive (Bacillus) host cell (see, e.g., FIG. 1). For example, a fragment of the integration cassette was designed to integrate at the yvzG-yvyD intergene region to replace (substitute) the native yvyD gene promoter with a heterologous promoter (alternative). As shown in FIG. 1, the yvzG-yvyD integration cassette contains the promoter region of spoVG (PspoVG; FIG. 1A, "PspoVG-yvyD") or hbs (Phbs; FIG. 1B, "Phbs-yvyD"). Specifically, as shown in Figures 7A to 7C, the Phbs promoter region sequence (SEQ ID NO: 29) comprises an upstream hbs promoter (Phbs) sequence (SEQ ID NO: 22) operably linked to a downstream spoVG SD sequence (SEQ ID NO: 25).

[0116] Example 2 further describes the construction of Gram-positive bacterial cells overexpressing yvyD. More specifically, the promoter swap integration cassette described in Example 1 was introduced into a recombinant bacillus cell containing two copies of a gene encoding an exemplary reporter protein (e.g., two copies of a gene encoding protease-1, protease-2, or protease-3). As shown in Example 3, Applicant evaluated the PspoVG-yvyD cassette for increasing yvyD expression via the production of reporter protease-1 in the bacillus strain described in Example 2. More specifically, sample aliquots were taken from the 2xprotease-1 (control) and 2xprotease-1 (yvyD overexpression; PspoVG-yvyD) strains at 12, 20, 36, 45, 61, 68, 73, and 84 h time points, and protease activity assays were performed to determine the effect of yvyD overexpression driven by the spoVG promoter (PspoVG) on the production of protease-1. The results of the protease assay (Figure 2) show that protease-1 production tends to increase at the end of fermentation, and production is significantly enhanced from 68 h to approximately 73 h due to overexpression of yvyD.

[0117] Similarly, Example 3 evaluated the PspoVG-yvyD cassette for increasing yvyD expression via the production of reporter protease-2 in the B. subtilis strain described in Example 2. More specifically, sample aliquots were taken from the 2× protease-2 (control) and 2× protease-2 (yvyD overexpression; PspoVG-yvyD) strains at 16, 22, 39, 46, 64, and 89 hours, and protease activity assays were performed to determine the effect of yvyD overexpression from the spoVG promoter (PspoVG) on the production of protease-2. The results of the protease assay ( FIG. 3 ) show that the increase in protease-2 production started from about 39 hours and tended to continue until the end of fermentation, with the production of protease-2 being significantly enhanced due to the overexpression of yvyD at 39 and 46 hours.

[0118] As further described in Example 3, Applicants evaluated the Phbs-yvyD cassette for increasing yvyD expression via the production of reporter protease-2 in the B. subtilis strain described in Example 2. More specifically, sample aliquots were taken from the 2× protease-2 (control) and 2× protease-2 (yvyD overexpression; Phbs-yvyD) strains at 11, 23, 37, 50, and 65 hours and protease activity assays were performed to determine the effect of yvyD overexpression from the Phbs promoter (Phbs) on the production of protease-2. The results of the protease assay ( FIG. 4 ) show that the increase in protease-2 production started from about 37 hours and tended to continue until the end of the fermentation, with the production of protease-2 being significantly enhanced due to the overexpression of yvyD at 37 hours.

[0119] As further shown in Example 3, Applicants evaluated the Phbs-yvyD cassette for increasing yvyD expression via the production of reporter protease-3 in the B. subtilis strain described in Example 2. More specifically, sample aliquots were taken from the 2× protease-3 (control) and 2× protease-3 (yvyD overexpression; Phbs-yvyD) strains at time points of expression driven by the Phbs promoter (Phbs) for the production of protease-3 at 14, 22, 37, 46, and 65. The results of the protease assay ( FIG. 5 ) show that the increase in protease-3 production started from about 22 hours and tended to continue until the end of the fermentation, with the production of protease-3 being significantly enhanced due to the overexpression of yvyD at 22 and 37 hours.

[0120] Thus, as described herein, certain embodiments of the present disclosure relate to the surprising and unexpected observation that overexpression of the yvyD gene CDS enhances production of a protein of interest in a Gram-positive bacterial cell. Thus, certain aspects of the present disclosure relate to recombinant Gram-positive bacterial cells / strains expressing the yvyD gene CDS from a promoter that produces a higher steady-state level of mRNA than the native yvyD promoter. For example, as reviewed in Zhu and Stuelke (2017), the steady-state mRNA level of spoVG expressed from its native promoter is higher than the steady-state yvyD mRNA level.

[0121] Specifically, the results of Zhu and Stulke's (2017) study carried out under four different growth conditions are reproduced in Table 1 below, where the conditions exponential growth + glucose (labeled "LBGexp"), fermentation (labeled "Ferm"), time to glucose exhaustion (labeled "T"), and exponential growth (labeled "LBexp") are shown in the first column (Table 1), and the steady-state mRNA levels of yvyD, spoVG, and hbs under the indicated conditions are shown in the second through fourth columns (Table 1), respectively. More specifically, in more than 80% of the transcriptome data points from 53 experimental conditions, the steady-state levels of spoVG mRNA are higher than the yvyD (hpf) mRNA levels (Zhu and Stulke, 2017). Importantly, the growth conditions most suitable for industrial fermentation of Gram-positive strains show higher steady-state mRNA levels of spoVG than yvyD (Table 1). [Table 1]

[0122] As shown in Table 1, a similar trend was observed for hbs steady-state mRNA levels relative to yvyD (hpf), with over 85% of hbs mRNA transcriptome data points from 53 experimental conditions being higher than the steady-state mRNA levels of yvyD (Zhu and Stulke, 2017). Thus, under LBGexp, Ferm, time to glucose exhaustion (T), and LBexp growth conditions, the steady-state levels of hbs mRNA are higher than those of yvyD, as summarized by the data reproduced in Table 1.

[0123] More specifically, as described in the Examples below, Applicants have experimentally demonstrated that overexpression of yvyD in recombinant Gram-positive bacterial cells increases the amount of three different reporter proteins produced (see, e.g., Figures 2-5). For example, the impact of increasing steady-state yvyD mRNA on protein production was demonstrated for two different yvyD overexpression cassettes (PspoVG-yvyD; Phbs-yvyD) that use promoter swap mutations to increase the relative amount of steady-state mRNA (Table 1). Similarly, as described and discussed herein, Applicants expect that other means of increasing steady-state mRNA levels of yvyD will have similar beneficial effects on the production of a protein of interest.

[0124] For example, other possible means of increasing yvyD mRNA levels include, but are not limited to, yvyD overexpression cassettes using promoters from other genes to increase yvyD steady-state mRNA levels over native yvyD steady-state mRNA levels, yvyD overexpression cassettes using non-Bacillus subtilis heterologous promoters to increase yvyD steady-state mRNA levels over native yvyD steady-state mRNA levels, plasmid-based expression cassettes of yvyD from its native promoter (PyvyD-yvyD), integration of multiple copies of PyvyD-yvyD into the genome, moving the yvyD locus to a genomic region that increases yvyD expression, host modifications (e.g., mRNA degradation pathways) that increase yvyD mRNA steady-state levels, mutations in transcribed yvyD that affect mRNA stability, etc. Additionally, mutations in transcribed yvyD that affect mRNA translation (e.g., more efficient ribosome binding sites) are expected to increase YvyD levels in cells and increase production of the protein of interest.

[0125] Thus, without wishing to be bound by any theory, mechanism, or mode of action, the applicants herein contemplate that increased YvyD protein levels enhance the production of a target protein of interest. More specifically, as shown in the Examples, recombinant Gram-positive cells expressing increased levels of YvyD produce increased amounts of reporter protein (compared to control cells), which demonstrates increased specific productivity (Qp) and increased carbon utilization of the reporter protein produced when cultured under suitable conditions. In certain one or more aspects or embodiments, the enhanced production of the protein of interest described in the Examples (Figures 2-5) can be explained by at least two possible mechanisms resulting from increased yvyD expression. In the first possible mechanism, YvyD promotes the stability of ribosome-associated proteins via the YvyD ribosome dimerization function (Feaga et al., 2020). In a second possible mechanism, the pool of free ribosomes is reduced by (YvyD) ribosome dimerization (e.g., via higher than normal (native) YvyD levels that promote translation of highly expressed gene of interest (GOI) mRNAs and / or GOI mRNAs with efficient ribosome binding sites).

[0126] In certain other aspects or embodiments, the Gram-positive bacterial yvyD gene comprises sequence homology to the B. subtilis yvyD gene of SEQ ID NO: 23. In certain other embodiments, the overexpressed yvyD gene comprises sequence homology to the B. subtilis yvyD gene of SEQ ID NO: 23 (e.g., comprises at least about 50% sequence identity to SEQ ID NO: 23) and encodes a functional YvyD protein. In other embodiments, the overexpressed yvyD gene encodes a YvyD protein that comprises sequence homology to the native B. subtilis YvyD protein of SEQ ID NO: 26. In certain embodiments, the overexpressed yvyD gene encodes a functional YvyD protein that comprises at least about 50% sequence identity to SEQ ID NO: 26.

[0127] In a related aspect, the Gram-positive bacterial yvyD gene (or yvyD gene homolog) encodes a functional "general stress factor protein" (or "ribosomal hibernation-promoting factor") that contains sequence homology to the YvyD protein (or its YvyD homolog) of SEQ ID NO: 26. For example, as outlined above, mechanisms for ribosome maintenance in dormant bacteria have been characterized in certain bacteria (Franklin et al., 2020), which include "ribosomal accessory proteins" such as ribosomal regulatory factors (RMFs), hibernation-promoting factors (HPFs), and HPF paralogs (YfiA), and structures of ribosomes containing HPFs and / or RMFs in their active sites have been elucidated for several different bacterial species.

[0128] In certain embodiments, Gram-positive bacterial yvyD genes (homologs) can be identified by sequence alignment. For example, the sequence of the native B. subtilis YvyD protein (amino acids) is shown in FIG. 6 (SEQ ID NO: 26), where the full-length protein sequence includes the conserved N-terminal domain (underlined residues; RaiA superfamily domain) and the conserved C-terminal domain (bold residues; ribosomal S30AE_C superfamily domain). Specifically, the conserved N-terminal domain RaiA superfamily domain present in the B. subtilis YvyD protein (SEQ ID NO: 26) is set forth in SEQ ID NO: 27, and the conserved C-terminal domain ribosomal S30AE_C superfamily domain present in the B. subtilis YvyD protein (SEQ ID NO: 26) is set forth in SEQ ID NO: 28.

[0129] For example, the "ribosome-associated inhibitor A" (RaiA) protein is a known stress response protein that binds to the ribosomal subunit interface and halts translation by preventing aminoacyl-tRNA binding to the ribosomal A site, and the RaiA fold is structurally similar to the double-stranded RNA-binding domain (dsRBD). Similarly, the ribosomal S30AE_C superfamily domain often occurs at the C-terminus of ribosomal stress response proteins (e.g., Sigma 54 regulatory / S30EA ribosomal protein).

[0130] Thus, in certain aspects, the Gram-positive bacterial yvyD gene encodes a YvyD protein that comprises at least about 50%-100% identity to the B. subtilis N-terminal RaiA superfamily domain of SEQ ID NO: 27. In other embodiments, the Gram-positive bacterial yvyD gene encodes a YvyD protein that comprises at least about 50%-100% identity to the B. subtilis C-terminal ribosomal S30AE_C superfamily domain of SEQ ID NO: 28. In certain other embodiments, the Gram-positive bacterial yvyD gene encodes a YvyD protein that comprises at least about 50%-100% identity to SEQ ID NO: 27 and at least about 50%-100% identity to SEQ ID NO: 28.

[0131] III. Microbial host cells As outlined above, certain embodiments relate to recombinant microbial (host) cells expressing genes encoding proteins of interest, etc. In certain aspects, Gram-positive bacterial cells (strains) include those from the classes Bacilli, Clostridia, and Mollicutes (e.g., Lactobacillales, including Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Oscillospiraceae, Streptococcaceae), and those from the classes Lactobacillaceae, ... In certain embodiments, the gram-positive bacterial cell (strain) may include the genus Streptomyces.

[0132] The species of the Bacillaceae family include the genera Alkalibacillus, Amphibacillus, Anoxybacillus, Bacillus, Caldalkalibacillus, Cerasilbacillus, Exiguobacterium, Filobacillus, Geobacillus, Gracilibacillus, Halobacillus, Halolactibacillus, Jeotgalibacillus, Lentibacillus, Examples of the genera include Maribacillus, Oceanobacillus, Ornithinibacillus, Paraliobacillus, Paucisalibacillus, Pontibacillus, Pontibacillus, Saccharococcus, Salibacillus, Salinibacillus, Tenuibacillus, Thalassobacillus, Ureibacillus, and Virgibacillus.

[0133] As used herein, the term "Bacillus" includes all species within the genus "Bacillus" known to those skilled in the art, such as, for example, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophii, B. arginini ... Examples of species that may be classified as Bacillus include, but are not limited to, B. lus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomic reorganization. Thus, the genus is intended to include organisms such as species that have been reclassified, for example, but not limited to, "B. stearothermophilus," which is now referred to as "Geobacillus stearothermophilus."

[0134] In certain embodiments, the cells of the Bacillus sp. include B. acidiceler, B. acidicola, B. acidocaldarius, B. acidoterrestris, B. aeolius, B. aerophilus, B. agaradhaerens, B. agri, B. aidingensis, B. akibai ... B.akibai, B.alcalophilus, B.algicola, B.alginolyticus, B.alkalidiazo-trophicus, B.alkalinitrilicus, B.alkalitelluris, B.altitudinis, B.alveayuensis ), B.alvei, B.amylolyticus, B.aneurinilyticus, B.aneurinolyticus, B.anthracia, B.aquimaris, B.arenosi, B.arseniciselenatis, B.arsenicoselenatis, B. B.arsenicus, B.arvi, B.asahii, B.atrophaeus, B.aurantiacus, B.axarquiensis, B.azotofixans, B.azotoformans, B.badius, B.barbaricus, B.bataviensis, B.bataviensis, B. beijingensis, B. benzoevorans, B. bogoriensis, B. boroniphilus, B. borstelenis, B. butanolivorans, B. carboniphilus, B. cecembensis, B. cellulosilyticus ticus, B. centrosporus, B. chagannorensis, B. chitinolyticus, B. chondroitinus, B. choshinensis, B. cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B. coagulans B. coahuilensis, B. cohnii, B. curdianolyticus, B. cycloheptanicus, B. decisifrondis, B. decolorationis, B. dipsosauri, B. drentensis, B. edaphicus, B. ehimensis ), B.endophyticus, B.farraginis, B.fastidiosus, B.firmus, B.plexus, B.foraminis, B.fordii, B.formosus, B.fortis, B.fumarioli, B.funiculus, B.fusiformisfusiformis, B.galactophilus, B.galactosidilyticus, B.gelatini, B.gibsonii, B.ginsengi, B.globisporus, B.globisporus subsp.globisporus, B.globisporus subsp.marinus subsp.marinus, B.glucanolyticus, B.gordonae, B.halmapalus, B.haloalkaliphilus, B.halodenitrificans, B.halodurans, B.halophilus, B.hemicellulosilyticus, B.herbersteinensis, B.horikoshii, B.horti, B.hemi, B.hwajinpoensis, B.idriens is), B. indicus, B. infantis, B. infernus, B. insolitus, B. isabeliae, B. jeotgali, B. kaustophilus, B. kobensis, B. koreensis, B. kribbensis, B. krulwichiae, B. laevolacticus, B. larvae, B. laterosporus, B. lautus, B. lehensis, B. lentimorbuslentimorbus, B. lentus, B. litoralis, B. luciferensis, B. macauensis, B. macerans, B. macquariensis, B. maciae, B. malacitensis, B. mannanilyticus, B. marinus, B. marisif B. marisflavi, B. marismortui, B. massiliensis, B. methanolicus, B. migulanus, B. mojavensis, B. mucilaginosus, B. muralis, B. murimartini, B. mycoides, B. naganoens is), B.nealsonii, B.neidei, B.niabensis, B.niacini, B.novalis, B.odysseyi, B.okhensis, B.okuhidensis, B.oleronius, B.oshimensis, B.pabuli, B.pallidus, B. .pallidus (illeg.), B. panaciterrae, B. pantothenticus, B. parabrevis, B. pasteurii, B. patagoniensis, B. peoriae, B. plakortidis, B. pocheonensis, B. polygoni, B. polymyxa, B.polymyxa, B. popilliae, B. pseudocaliphilus, B. pseudofirmus, B. pseudomycoides, B. psychrodurans, B. psychrophilus, B. psychrosaccarolyticus, B. psychrotolerans ans), B. pulvifaciens, B. pycnus, B. qingdaonensis, B. reuszeri, B. runs, B. safensis, B. salarius, B. salexigens, B. saliphilus, B. schlegelii, B. selenatars enatis, B. selenitrireducens, B. seohaeanensis, B. shackletonii, B. silvestris, B. simplex, B. siralis, B. smithii, B. soli, B. sonorensis, B. sphaericus, B. sporothermodurans B. sporothermodurans, B. stearothermophilus, B. stratosphericus, B. subterraneus, B. subtilis subsp. spizizenii, B. subtilis subsp. subtilis, B. taeanensis, B. tequilensis, B. thermanthalcuticus, B.thermantarcticus, B. thermoaerophilus, B. thermoamylovorans, B. thermoantarcticus, B. thermocatenulatus. nulatus, B. thermocloacae, B. thermodenitrificans, B. thermoglucosidasius, B. thermoleovorans, B. thermoruber, B. thermosphaericus, B. thiaminolyticus, B. thioparans, B. thuringiensis Examples of suitable microbial species include, but are not limited to, B. thuringiensis, B. tusciae, B. validus, B. vallismortis, B. vedderi, B. velezensis, B. vietnamensis, B. vireti, B. vulcani, B. wakoensis, and B. weihenstephanensis.

[0135] IV. Recombinant Polynucleotides and Molecular Biology Suitable nucleic acid (DNA) control sequences, regulatory sequences, etc. for constructing a yvyD overexpression polynucleotide cassette include promoter sequences and functional portions thereof (i.e., portions sufficient to affect expression of a nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, etc. In certain embodiments, promoter region sequences are generally selected to be functional in Gram-positive bacterial cells and to overexpress the yvyD gene CDS relative to expression of the yvyD gene CDS from its wild-type yvyD promoter region (SEQ ID NO:24).

[0136] For example, promoters useful for driving gene expression in Bacillus cells include, but are not limited to, the B. subtilis alkaline protease (aprE) promoter, the B. subtilis α-amylase promoter (amyE), the B. licheniformis α-amylase promoter (amyL), the B. amyloliquefaciens α-amylase promoter, the native protease (nprE) promoter from B. subtilis, a mutant aprE promoter, or any other promoter from B. licheniformis or other related Bacilli. Methods for screening and generating promoter libraries with different activities (promoter strengths) in Bacillus cells are described in WO 2002 / 14490.

[0137] An example of such a regulatory or control sequence may be a promoter sequence or a functional portion thereof (i.e., a portion sufficient to affect the expression of a nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, and the like.

[0138] Bacillus host cells. Certain embodiments therefore relate to a polynucleotide (e.g., expression cassette) that includes an upstream (5') promoter (pro) sequence operably linked to a downstream nucleic acid sequence (ss) encoding a modified (protein) signal sequence operably linked to a downstream (3') nucleic acid sequence (poi) encoding a protein of interest.

[0139] Certain embodiments of the present disclosure are directed to isolated nucleic acids (polynucleotides). Thus, certain aspects relate to plasmids, vectors, expression cassettes, etc., that contain polynucleotide sequences encoding the proteins of the present disclosure. Similarly, other embodiments are directed to recombinant microbial cells (strains) that express one or more heterologous proteins. More specifically, in certain embodiments, genes, polynucleotides, open reading frames, etc. of the present disclosure are genetically modified. In certain aspects, genetic modifications include, but are not limited to, (a) the introduction, substitution, or removal of one or more nucleotides in a gene, gene coding sequence (CDS), open reading frame (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 gene CDS), (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation (e.g., interfering RNA), and / or (g) random mutagenesis of any one or more genes or polynucleotides disclosed herein.

[0140] Those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli, Bacillus sp., etc.), filamentous fungal cells (e.g., Aspergillus sp., Trichoderma sp., etc.), yeast cells (e.g., Saccharomyces spp.), and the like (i.e., microbial cells).

[0141] As generally specified above, certain embodiments of the present disclosure are directed to expressing, producing, and / or secreting one or more proteins of interest that are heterologous to a microbial host cell. Thus, the present disclosure generally relies on routine techniques in the field of recombinant genetics. Basic texts that disclose the general methods of use in the present disclosure include Sambrook et al., (1989; 2011; 2012); Kriegler (1990) and Ausubel et al., (1987; 1994).

[0142] In certain embodiments, the present disclosure relates to a recombinant (modified) nucleic acid comprising a genetic CDS encoding a YvyD protein. For example, in certain aspects, the recombinant nucleic acid is a polynucleotide expression cassette suitable for expressing a YvyD protein.

[0143] In certain other embodiments, the recombinant nucleic acid (polynucleotide) comprises one or more selectable markers. Selectable markers for use in gram-negative bacteria, gram-positive bacteria, filamentous fungi, and yeast are commonly known in the art. Thus, in certain embodiments, the polynucleotide construct encoding the YvyD protein and / or the polynucleotide construct encoding the protein of interest (POI) comprises a nucleic acid sequence operably linked thereto that encodes a selectable marker.

[0144] In other embodiments, the nucleic acid comprising the gene or gene CDS encoding the YvyD protein further comprises an operably linked regulatory or control sequence. An example of a regulatory or control sequence can be a promoter sequence or a functional portion thereof (i.e., a portion sufficient to affect expression of the nucleic acid sequence). Other control sequences include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, and the like. Thus, in certain embodiments, the recombinant (modified) polynucleotide comprises an upstream (5') promoter (pro) sequence that drives expression of the YvyD protein or gene coding sequence (CDS) encoding the POI of the present disclosure. More specifically, in certain embodiments, the promoter is a constitutive or inducible promoter that is active (functional) in the microbial host cell. For example, one of skill in the art can use any suitable promoter capable of driving expression of the gene of interest in the microbial expression host cell. Thus, in certain aspects, a recombinant nucleic acid of the present disclosure includes a promoter (pro) sequence that is 5' (upstream) and operably linked to a nucleic acid sequence encoding a YvyD protein (gene CDS) (e.g., 5'-[pro]-[gene CDS]-3').

[0145] In certain other aspects, a recombinant nucleic acid (e.g., an expression cassette) comprises an upstream (5') promoter (pro) sequence operably linked to a downstream (3') nucleic acid sequence (gene CDS) encoding a YvyD protein (or encoding a POI), and further comprises a terminator (term) sequence operably linked downstream thereto. For example, in certain aspects, a recombinant nucleic acid of the present disclosure comprises a promoter (pro) sequence (e.g., 5'-[pro]-[gene CDS]-[term]-3') operably linked to a nucleic acid sequence (gene CDS) encoding a YvyD protein (or POI), which is 5' (upstream) and operably linked to a downstream terminator (term) sequence.

[0146] Suitable promoters for driving the expression of a gene of interest in the microbial host cell of the present disclosure are generally known in the art. For example, exemplary Bacillus sp. promoters include, but are not limited to, tac promoter sequence, β-lactamase promoter sequence, aprE promoter sequence, groES promoter sequence, ftsH promoter sequence, tufA promoter sequence, secDF promoter sequence, minC promoter sequence, spoVG promoter sequence, veg promoter sequence, hbs promoter sequence, amylase promoter sequence, P43 promoter sequence, etc., and exemplary filamentous fungal promoters include, but are not limited to, Trichoderma sp. promoters (e.g., cellobiohydrolase promoter, endoglucanase promoter, β-glucosidase promoter, xylanase promoter, glucoamylase promoter), Aspergillus sp. promoters (e.g., trpCase promoter, glucoamylase promoter), etc. However, it is not intended that the present disclosure be limited to any particular promoter, as any suitable promoter known to one of skill in the art finds use in the present invention.

[0147] Thus, certain other embodiments relate to culturing (fermenting) a microbial host cell expressing a POI, where the expressed POI is secreted into the culture (fermentation) broth. For example, in certain other embodiments, the recombinant nucleic acid comprises an upstream (5') heterologous promoter (pro) sequence operably linked to a downstream (3') nucleic acid sequence (ss) encoding a protein signal sequence operably linked to a downstream (3') nucleic acid sequence encoding a protein of interest (GOI) (e.g., 5'-[pro]-[ss]-[GOI]-3').

[0148] Any suitable (protein) signal sequence (signal peptide) that is functional in the microbial cell of choice can be used for secretion (transport) of the mature protein of interest. The signal sequence is generally located at the N-terminus of the precursor or mature protein sequence. For example, signal sequences suitable for use include, but are not limited to, signal sequences derived from secreted proteases, peptidases, amylases, glucoamylases, cellulases, lipases, esterases, arabinases, glucanases, chitosanases, lyases, xylanases, nucleases, phosphatases, transport and binding proteins, and the like. In certain embodiments, the signal sequence is selected from the aprE signal sequence, the nprE signal sequence, the vpr signal sequence, the bglC signal sequence, the bglS signal sequence, the sacB signal sequence, the amylase signal sequence, a heterologous signal sequence, and / or a synthetic signal sequence.

[0149] Therefore, in certain embodiments, standard methods for the transformation of microbial cells (known to those skilled in the art) are used to transform the microbial host cells of the present disclosure. Thus, the method of introducing DNA constructs or vectors into host cells includes transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection-mediated and DEAE-dextrin-mediated transfection), incubation with calcium phosphate DNA precipitates, high-velocity gun using DNA-coated microprojectiles, gene gun or biolistic transformation, protoplast fusion, and other methods. General transformation techniques are known in the art.

[0150] In certain embodiments, heterologous genes, polynucleotides, or ORFs are cloned into intermediate vectors before being transformed into microbial (host) cells for replication and / or expression. These intermediate vectors can be, for example, prokaryotic vectors, such as plasmids or shuttle vectors. Thus, expression vectors / constructs usually contain a transcription unit or expression cassette that includes all the additional elements required for the expression of the heterologous sequence. For example, a typical expression cassette contains a 5' promoter operably linked to the heterologous nucleic acid sequence encoding the protein of interest, and may further include sequence signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

[0151] The particular expression vector used to transport genetic information into cells is not particularly important. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include bacteriophages λ and M13, as well as plasmids such as pBR322-based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags, such as c-myc, can also be added to recombinant proteins to provide convenient isolation methods. Elements that can be included in expression vectors can also include replicons, genes encoding antibiotic resistance to allow for selection of bacteria carrying recombinant plasmids, or unique restriction sites in non-essential regions of the plasmid to allow for insertion of heterologous sequences.

[0152] The transformation method of the present invention may result in stable integration of all or part of the transformation vector into the genome of the microbial cell. However, transformation resulting in the maintenance of an autonomously replicating extrachromosomal transformation vector is also contemplated. Any known procedure for introducing foreign nucleotide sequences into a host cell may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors, and any other method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, for example, Sambrook et al., supra). Agrobacterium-mediated transfection methods are also useful. It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the heterologous gene.

[0153] After the expression vector is introduced into the cells, the transfected cells are cultured under conditions that favor the expression of the gene of interest. Large batches of transformed cells can be cultured as described herein. Finally, the broth and / or product are harvested from the culture using standard techniques. Thus, the disclosure herein provides for the expression and secretion of a desired protein.

[0154] The microbial cells of the present disclosure can include genetic modifications of one or more endogenous genes and / or one or more introduced (heterologous) genes as described herein. For example, the microbial cells can be constructed to reduce or eliminate expression of an endogenous gene (e.g., a gene encoding a protease) using methods well known in the art, such as, for example, insertion, disruption, substitution, or deletion. The portion of the gene to be modified or inactivated can be, for example, a coding region or a regulatory element required to express the coding region.

[0155] In certain embodiments, the modified cells of the present disclosure are constructed by introducing, substituting, or removing one or more nucleotides in a gene or regulatory element required for its transcription or translation. For example, nucleotides may be inserted or removed to introduce a stop codon, remove a start codon, or cause a frame shift in an open reading frame. Such modifications may be achieved by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art.

[0156] In another embodiment, the modified cell is constructed by a gene conversion process. For example, in gene conversion methods, a nucleic acid sequence corresponding to a gene is mutated in vitro to generate a defective nucleic acid sequence, which is then transformed into a parent cell to generate the defective gene. The defective nucleic acid sequence replaces the endogenous gene by homologous recombination. It may be desirable for the defective gene or gene fragment to also encode a marker that can be used to select transformants containing the defective gene. For example, the defective gene can be introduced on a non-replicating or temperature-sensitive plasmid associated with a selectable marker. Selection for integrating the plasmid is performed by selecting for that marker under conditions that do not allow plasmid replication. Selection of the second recombination event resulting in gene replacement is performed by examining colonies for the loss of the selectable marker and the acquisition of a mutated gene. Alternatively, the defective nucleic acid sequence can contain an insertion, substitution, or deletion of one or more nucleotides of the gene, as described below.

[0157] In other embodiments, modified cells are constructed by established antisense technology using a nucleotide sequence that is complementary to the nucleic acid sequence of a gene. More specifically, the expression of a gene by a cell can be reduced (downregulated) or eliminated by introducing a nucleotide sequence that is complementary to the nucleic acid sequence of this gene, which can be transcribed in the cell and hybridize to the mRNA produced in the cell. Thus, under conditions in which the complementary antisense nucleotide sequence can hybridize to the mRNA, the amount of protein translated is reduced or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, etc., all of which are well known to those skilled in the art.

[0158] In other embodiments, modified cells are produced / constructed by CRISPR-Cas9 editing. For example, genes of interest may be destroyed (or deleted or downregulated) by a guide RNA (e.g., Cas9) that recruits endonucleases to target sequences on the DNA and nucleic acid-guided endonucleases that find their target DNA by binding to Cpf1 or guide DNA (e.g., NgAgo), where the endonucleases can generate single- or double-stranded breaks in the DNA. This targeted DNA break can become a substrate for DNA repair and recombine with the editing template provided to disrupt or delete the gene. For example, a gene encoding a nucleic acid-guided endonuclease (for this purpose, Cas9 from S. pyogenes) or a codon-optimized gene encoding a Cas9 nuclease is operably linked to a promoter active in the microbial cell and a terminator active in the microbial cell, thereby generating a microbial cell Cas9 expression cassette. Similarly, one or more target sites unique to a gene of interest are easily identified by those skilled in the art. For example, to construct a DNA construct encoding a gRNA directed to a target site within a gene of interest, a variable targeting domain (VT) would include the nucleotides of the target site that are 5' to the (PAM) protospacer adjacent motif (TGG), which nucleotides are fused to DNA encoding the Cas9 endonuclease recognition domain for S. pyogenes Cas9 (CER). The combination of the DNA encoding the VT domain and the DNA encoding the CER domain thereby generates a DNA encoding the gRNA. Thus, a microbial cell expression cassette for a gRNA is generated by operably linking the DNA encoding the gRNA to a promoter active in the microbial cell and a terminator active in the microbial cell. The Cas9 expression cassette, the gRNA expression cassette and the editing template can be delivered simultaneously to a cell using a number of different methods (e.g., protoplast fusion, electroporation, innate competence or induced competence).Transformed cells are screened by PCR amplification of the target locus by amplifying the locus with forward and reverse primers. These primers can amplify the wild-type locus or the modified locus edited by RGEN.

[0159] In yet other embodiments, modified cells are constructed by random or directed mutagenesis using methods well known in the art, including but not limited to chemical mutagenesis and transposition. Genetic modification can be performed by subjecting parent cells to mutagenesis and screening for mutant cells in which expression of the gene is reduced or eliminated. Mutagenesis can be directed or random, and can be performed, for example, by using suitable physical or chemical mutagenesis agents, by using suitable oligonucleotides, or by subjecting the DNA sequence to PCR-generated mutagenesis. Furthermore, mutagenesis can be performed by using any combination of these mutagenesis methods. Examples of physical or chemical mutagenesis agents suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When such agents are used, mutagenesis is generally carried out by incubating the parent cells to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting mutant cells that show reduced or no expression of the gene.

[0160] V. Fermentation of Gram-Positive Cells to Produce a Protein of Interest In certain embodiments, the present disclosure provides recombinant microbial cells capable of producing a protein of interest. More specifically, certain embodiments are related genetically modified microbial cells expressing a heterologous polynucleotide encoding a protein of interest, genetically microbial cells co-expressing a heterologous protein of interest and a YvyD protein, etc. Thus, certain embodiments relate to culturing (fermenting) microbial cells to produce a protein of interest.

[0161] Generally, fermentation methods well known in the art are used to ferment microbial cells. In some embodiments, the cells are grown under batch or continuous fermentation conditions. Classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and is not changed during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism. In this method, fermentation can be carried out without adding any components to the system. Generally, batch fermentation is considered to be "batch" with respect to the addition of the carbon source, and factors such as pH and oxygen concentration are often controlled. The metabolite and biomass composition of a batch system changes constantly until the point at which the fermentation is stopped. Within a batch culture, cells progress through a static lag phase to a high growth log phase and eventually to a stationary phase where the growth rate slows or stops. If not treated, the cells in the stationary phase will eventually die. Generally, the cells in the log phase are responsible for the majority of the production of the product.

[0162] A suitable variation of the standard batch system is the "fed-batch fermentation" system. In this variation of the general batch system, substrate is added gradually as the fermentation proceeds. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and when a limited amount of substrate is desired in the medium. Actual substrate concentration is difficult to measure in a fed-batch system and is therefore estimated based on changes in measurable factors such as pH, dissolved oxygen and partial pressure of exhaust gases such as CO2. Batch and fed-batch fermentation are common and well known in the art.

[0163] Continuous fermentation is an open system in which a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant high density, with the cells primarily in logarithmic growth phase. Continuous fermentation allows for the adjustment of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, the limiting nutrient, such as the carbon or nitrogen source, is maintained at a constant ratio, while all other parameters can be adjusted. In other systems, a number of factors that affect growth can be continuously varied, while the cell concentration, measured by the turbidity of the medium, is kept constant. Continuous systems attempt to maintain steady-state growth conditions. Thus, cell loss due to medium removal must be balanced against the cell growth rate during fermentation. Methods for adjusting nutrients and growth factors in continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[0164] Cultivation / fermentation is generally carried out in a growth medium comprising an aqueous inorganic salts medium, organic growth factors, carbon and energy source materials, molecular oxygen, and, of course, a starting inoculum of the microbial host being used.

[0165] To ensure proper microbial growth, maximize the uptake of carbon and energy sources by the cells in the microbial conversion process, and achieve maximum cell yield at maximum cell density in the fermentation medium, suitable amounts of inorganic nutrients must be supplied in appropriate proportions in addition to the carbon and energy sources, oxygen, assimilable nitrogen, and microbial inoculum.

[0166] The composition of the aqueous mineral medium can vary widely depending in part on the microorganism and substrate used, as is known in the art. In addition to nitrogen, the mineral medium will contain suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur, and sodium in suitable soluble, absorbable, ionic complex forms, and preferably also several trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, also in suitable soluble, absorbable forms, all of which are known in the art.

[0167] Fermentation reactions are aerobic processes in which the necessary molecular oxygen is supplied by a molecular oxygen-containing gas, such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermenter at a suitable oxygen partial pressure effective to support vigorous growth of the microbial species.

[0168] Fermentation temperatures can vary somewhat, but for most microbial cells the temperature will generally be in the range of about 20°C to 40°C.

[0169] Microorganisms also require an assimilable nitrogen source. The assimilable nitrogen source can be any nitrogen-containing compound or any compound that can release nitrogen in a form suitable for metabolic utilization by the microorganism. Although various organic nitrogen source compounds such as protein hydrolysates can be used, typically inexpensive nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself is convenient for large-scale operations and can be used by bubbling it through the aqueous fermentation (fermentation medium) in a suitable amount. At the same time, such ammonia can also be used to aid in pH control.

[0170] The pH range of the aqueous microbial fermentation (fermentation mixture) should be in the exemplary range of about 2.0 to 8.0 The preferred pH range of a microorganism will depend to some extent on the medium and the particular microorganism used, and will therefore vary somewhat with changes in medium, as can be readily determined by one of skill in the art.

[0171] It is preferred to carry out the fermentation in such a way that it is possible to control the carbon-containing substrate as the limiting factor, thereby providing good conversion of the carbon-containing substrate to the cells and avoiding contamination of the cells with significant amounts of unconverted substrate. The latter is not a problem with water-soluble substrates, since traces of residues can be easily washed away. However, it can be a problem with water-insoluble substrates, necessitating additional product processing steps, such as suitable washing steps.

[0172] As noted above, the time to reach this level is not critical and may vary depending on the particular microorganism and fermentation process being carried out, however, methods for determining the carbon source concentration in a fermentation medium and whether a desired carbon source level has been achieved are well known in the art.

[0173] If necessary, some or all of the carbon and energy source materials and / or a portion of the assimilable nitrogen source, such as ammonia, can be added to the aqueous mineral medium before it is fed to the fermenter.

[0174] Each of the streams introduced into the reactor is preferably controlled at a predetermined rate or according to needs that can be determined by monitoring the concentrations of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the fermenter exhaust gas, cell density as measured by dry cell weight, light transmittance, etc. The feed rates of the various materials can be varied in order to obtain the fastest possible cell growth rate and the highest possible yield of microbial cells relative to the substrate feed, consistent with efficient utilization of the carbon and energy sources.

[0175] In a batch or, preferably, fed-batch operation, all equipment, reactors, or fermentation means, tanks or vessels, piping, associated circulation or cooling systems, etc. are first sterilized, usually using steam, for example, at about 121° C. for at least about 15 minutes. The sterilized reactor is then inoculated with a culture of the selected microorganism in the presence of all necessary nutrients, including oxygen, and a carbon-containing substrate. The type of fermenter used is not critical.

[0176] VI. Protein of interest The protein of interest (POI) of the present disclosure may be any endogenous or heterologous protein, or may be a variant of such a POI. The protein may contain one or more disulfide bridges, or may be a protein whose functional form is monomeric or multimeric. That is, the protein has a quaternary structure and is composed of multiple identical (homologous) or non-identical (heterologous) subunits, where the POI or the variant POI is preferably a protein with a property of interest. Thus, in certain embodiments, the modified cell of the present disclosure expresses an endogenous POI, a heterologous POI, or a combination of one or more of such POIs.

[0177] In certain embodiments, the modified cells can produce an increased amount of POI (e.g., a protein having DNase activity) compared to the parental (control) cells, the increased amount of POI being at least about 0.01%, at least about 0.10%, at least about 0.50%, at least about 1.0%, at least about 2.0%, at least about 3.0%, at least about 4.0%, at least about 5.0%, or more than 5.0%. In certain embodiments, the increased amount of POI is determined by testing the enzyme activity and / or by testing / quantifying its specific productivity (Qp). Similarly, the skilled artisan can utilize other routine methods and techniques known in the art to detect, test, measure, etc., the expression, production, or secretion of one or more proteins of interest.

[0178] In certain embodiments, the POI or variant POI thereof is selected from the group consisting of acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, aryl esterase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lysase, and the like. lysases), endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, laccase, ligase, lipase, lyase, lysozyme, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methyl esterase, pectin degrading enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phosphodiesterase, phytase, polyesterase, polygalacturonase, protease, peptidase, rhamno-galacturonase, ribonuclease, transferase, transport protein, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0179] Thus, in certain embodiments, the POI or variant POI thereof is an enzyme selected from Enzyme Code (EC) EC1, EC2, EC3, EC4, EC5 or EC6.

[0180] For example, in certain embodiments, the POI is an enzyme selected from oxidoreductases (EC1), transferases (EC2), hydrolases (EC3), lyases (EC4), and isomerases (EC5).

[0181] Thus, in certain embodiments, Gram-positive host cells that produce industrial proteases provide particularly useful expression hosts. Similarly, in certain other embodiments, Gram-positive host cells that produce industrial amylases provide particularly useful expression hosts. For example, proteases typically secreted by Bacillus sp. are of two general types: neutral (or "metalloproteases") and alkaline (or "serine") proteases. For example, Bacillus subtilisin proteins (enzymes) are exemplary serine proteases for use in the present disclosure. A wide variety of Bacillus subtilisins have been identified and sequenced, such as subtilisin 168, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 147, and subtilisin 309. In some embodiments, the engineered Gram-positive cells produce mutant (i.e., variant) proteases. Thus, in certain embodiments, the engineered (recombinant) Gram-positive cell comprises an expression construct encoding a native and / or variant protease.

[0182] In certain other embodiments, the engineered Gram-positive cell comprises an expression construct encoding an amylase. A wide variety of amylase enzymes and variants thereof are known to those of skill in the art. Thus, in certain embodiments, the engineered (recombinant) Gram-positive cell comprises an expression construct encoding a native and / or variant protease.

[0183] In other embodiments, the POI or variant POI expressed and produced in the modified cells of the present disclosure are peptides, peptide hormones, growth factors, clotting factors, chemokines, cytokines, lymphokines, antibodies, receptors, adhesion molecules, microbial antigens (e.g., HBV surface antigen, HPV E7, etc.), and variants thereof, fragments thereof, etc. Other types of proteins of interest (or variants thereof) can be proteins that can provide nutritional value to food or crops. Non-limiting examples include plant proteins that can inhibit the formation of anti-nutritional factors and plant proteins that have a more desirable amino acid composition (e.g., higher lysine content than non-transgenic plants).

[0184] There are various assays known to those skilled in the art for detecting and measuring the activity of intracellularly and extracellularly expressed proteins. In particular, for proteases, there are assays based on the release of acid-soluble peptides from casein or hemoglobin, measured as absorbance at 280 nm or colorimetrically using the Folin method. Other typical assays include the succinyl-Ala-Ala-Pro-Phe-para-nitroanilide assay (SAAPFpNA) and the 2,4,6-trinitrobenzenesulfonic acid sodium salt assay (TNBS assay).

[0185] WO 2014 / 164777 discloses a Ceralpha α-amylase activity assay useful for the amylase activity described herein.

[0186] Means for determining the secretion levels of a protein of interest in a host cell and for detecting the expressed protein include the use of immunoassays using either polyclonal or monoclonal antibodies specific for the protein, examples of which include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), and fluorescence activated cell sorting (FACS).

[0187] VII. Exemplary Embodiments Non-limiting embodiments of the compositions and methods disclosed herein are as follows.

[0188] 1. Recombinant Gram-positive cells overexpressing the yvyD gene.

[0189] 2. A recombinant Gram-positive cell overexpressing the yvyD gene and expressing a gene encoding a protein of interest (POI).

[0190] 3. A recombinant cell according to embodiment 2, which expresses multiple copies of the gene encoding the POI.

[0191] 4. A recombinant cell according to any one of embodiments 1 to 3, wherein the overexpressed yvyD gene comprises at least 50% identity to the yvyD gene of SEQ ID NO: 23.

[0192] 5. A recombinant cell according to any one of embodiments 1 to 3, wherein the overexpressed yvyD gene comprises at least 50% identity to the yvyD gene coding sequence (CDS) of SEQ ID NO: 18.

[0193] 6. A recombinant cell according to any one of embodiments 1 to 3, wherein the yvyD gene encodes a protein comprising at least 50% identity to the YvyD protein of SEQ ID NO: 26.

[0194] 7. A recombinant cell according to any one of embodiments 1 to 3, wherein the yvyD gene encodes a protein comprising at least 50% identity to the RaiA superfamily domain of SEQ ID NO: 27.

[0195] 8. A recombinant cell according to any one of embodiments 1 to 3, wherein the yvyD gene encodes a protein comprising at least 50% identity to the ribosomal S30AE_C superfamily domain of SEQ ID NO: 28.

[0196] 9. The recombinant cell of embodiment 2, wherein the recombinant cell produces an increased amount of the POI compared to a control cell, and when the recombinant cell and the control cell are grown under the same conditions, the control cell expresses the same POI but does not overexpress the yvyD gene.

[0197] 10. The recombinant cell of embodiment 9, wherein the control cell expresses the endogenous yvyD gene CDS under the control of its native yvyD gene promoter.

[0198] 11. A recombinant cell described in any one of embodiments 1 to 3, wherein overexpressing the yvyD gene comprises replacing the native yvyD gene promoter region with a heterologous promoter region operably linked to the downstream (3') yvyD gene CDS, wherein the heterologous promoter region increases expression of the yvyD gene CDS compared to the native yvyD gene promoter.

[0199] 12. The recombinant cell of embodiment 11, wherein the heterologous promoter region is selected from the spoVG gene promoter (PspoVG) region comprising at least 90% identity to SEQ ID NO: 21, and the hbs gene promoter (Phbs) region comprising at least 90% identity to SEQ ID NO: 29.

[0200] 13. A recombinant cell according to embodiment 2 or embodiment 3, wherein the POI is an enzyme.

[0201] 14. The enzyme is acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, aryl esterase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lysase lysases), endo-β-glucanase, glucoamylase, glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase, hydrolase, invertase, isomerase, laccase, ligase, lipase, lyase, lysozyme, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectin depolymerase, pectin 14. The recombinant cell of embodiment 13, wherein the enzyme is selected from the group consisting of cholinesterase, choline methylesterase, pectin degrading enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phosphodiesterase, phytase, polyesterase, polygalacturonase, protease, peptidase, rhamno-galacturonase, ribonuclease, transferase, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0202] 15. The recombinant cell of embodiment 13, wherein the POI is a protease.

[0203] 16. The recombinant cell of embodiment 15, wherein the protease is subtilisin.

[0204] 17. The recombinant cell of embodiment 16, wherein the subtilisin is selected from natural or variant Bacillus lentus subtilisin, natural or variant Bacillus gibsonii subtilisin, and natural or variant Bacillus amyloliquefaciens subtilisin.

[0205] 18. A yvyD expression cassette comprising an upstream (5') yvyD flanking region (FR) nucleic acid sequence operably linked to a downstream heterologous promoter (het-pro) sequence operably linked to a downstream yvyD gene coding sequence (CDS) operably linked to a downstream (3') yvyD flanking region (FR) nucleic acid sequence, generally as described in Formula I. Formula I:5'-[yvyD FR]-[het-pro]-[yvyD CDS]-[yvyD FR]-3'

[0206] 19. A method for producing increased amounts of a protein of interest (POI) in a Gram-positive bacterial cell, comprising obtaining a parent cell containing a yvyD gene having at least 50% identity to the yvyD gene of SEQ ID NO: 23, and genetically modifying the cell to overexpress the yvyD gene.

[0207] 20. The method of embodiment 19, wherein the parent cell or the modified (recombinant) cell comprises an introduced expression cassette encoding the POI.

[0208] 21. The method of embodiment 19, wherein the modified cells overexpressing the yvyD gene produce an increased amount of POI compared to the parent cells when cultured under the same conditions.

[0209] 22. The method of embodiment 19, wherein the yvyD gene of the parent cell comprises at least 50% identity to the yvyD gene coding sequence (CDS) of SEQ ID NO: 18.

[0210] 23. The method of embodiment 19, wherein the yvyD gene of the parent cell encodes a YvyD protein comprising at least 50% identity to the YvyD protein of SEQ ID NO: 26.

[0211] 24. The method of embodiment 23, wherein the YvyD protein comprises at least 50% identity to the RaiA superfamily domain of SEQ ID NO: 27.

[0212] 25. The method of embodiment 23, wherein the YvyD protein comprises at least 50% identity to the ribosomal S30AE_C superfamily domain of SEQ ID NO: 28.

[0213] 26. The method of embodiment 19, wherein the modified cell overexpressing the yvyD gene comprises a heterologous promoter region operably linked to a downstream (3') yvyD gene CDS, and the heterologous promoter region increases expression of the yvyD gene CDS compared to the native yvyD gene promoter.

[0214] 27. The method of embodiment 19, wherein the modified cell overexpressing the yvyD gene comprises an introduced polynucleotide construct comprising an upstream (5') heterologous promoter region sequence operably linked to a downstream (3') yvyD gene CDS comprising at least 50% identity to SEQ ID NO: 18, wherein the heterologous promoter region increases expression of the native yvyD gene CDS compared to the native yvyD gene promoter.

[0215] 28. The method of embodiment 26 or embodiment 27, wherein the heterologous promoter region is selected from the spoVG gene promoter (PspoVG) region comprising at least 90% identity to SEQ ID NO: 21, and the hbs gene promoter (Phbs) region comprising at least 90% identity to SEQ ID NO: 29.

[0216] 29. The method of embodiment 19, wherein the POI is an enzyme.

[0217] 30. The enzyme is acetyl esterase, aminopeptidase, amylase, arabinase, arabinofuranosidase, aryl esterase, carbonic anhydrase, carboxypeptidase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lysase 30. The method of embodiment 29, wherein the enzyme is selected from the group consisting of: beta-endo-beta-glucanase, glucoamylase, glucose oxidase, alpha-glucosidase, beta-glucosidase, glucuronidase, glycosyl hydrolase, hemicellulase, hexose oxidase hydrolase, invertase, isomerase, laccase, ligase, lipase, lyase, lysozyme, mannosidase, oxidase, oxidoreductase, pectate lyase, pectin acetyl esterase, pectin depolymerase, pectin methyl esterase, pectin degrading enzyme, perhydrolase, polyol oxidase, peroxidase, phenol oxidase, phosphodiesterase, phytase, polyesterase, polygalacturonase, protease, peptidase, rhamno-galacturonase, ribonuclease, transferase, transglutaminase, xylanase, hexose oxidase, and combinations thereof.

[0218] 31. The method of embodiment 29, wherein the POI is a protease.

[0219] 32. The method of embodiment 31, wherein the protease is subtilisin.

[0220] 33. The method of embodiment 19, wherein the gram-positive bacterial cell is a Bacillus sp. cell.

[0221] 34. The method of embodiment 33, wherein the Bacillus sp. cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.

[0222] 35. The recombinant Gram-positive bacterial cell of embodiment 1 or embodiment 2, wherein the Gram-positive bacterial cell is a Bacillus sp. cell.

[0223] 36. The recombinant Bacillus sp. cell of embodiment 35, selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. EXAMPLES

[0224] Certain aspects of the present invention can be further understood in view of the following examples, which should not be construed as limiting. Modifications of materials and methods will be apparent to those skilled in the art. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989). As described herein, all expression cassettes were transformed into host strains using the methods described in WO2019 / 040412 (incorporated herein by reference in its entirety).

[0225] Example 1 Construction of a yvyD overexpression integration cassette This example describes the construction of a yvyD (gene) overexpression (integration) cassette (see, e.g., FIG. 1). More specifically, the yvyD overexpression cassette described herein was generated by NEBuilder (New England Biolabs) via assembly of PCR amplified DNA fragments. For example, an integration cassette fragment was designed to integrate at the yvzG-yvyD intergenic region (hereinafter, the "yvzG-yvyD region") to replace (substitute) the native yvyD promoter with a heterologous promoter, where sequences flanking the yvzG-yvyD region were amplified from Bacillus subtilis (e.g., B. subtilis strain 168, ATCC 23857) genomic DNA. The upstream (5') yvzG-yvyD flanking region was amplified with oligonucleotide primers 343 (sequence number 1) and 402 (sequence number 2), and the downstream (3') yvzG-yvyD flanking region was amplified with oligonucleotide primers 400 (sequence number 3) and 370 (sequence number 4), as described in Table 2 below. [Table 2]

[0226] A DNA fragment containing a spectinomycin antibiotic resistance marker (SpecR) flanked by loxP sites was amplified using oligonucleotide primers 539 (Table 2; SEQ ID NO:5) and 246 (Table 2; SEQ ID NO:6). The spoVG promoter (PspoVG) region was amplified using oligonucleotide primers 540 (Table 2; SEQ ID NO:7) and 754 (Table 2; SEQ ID NO:8). Thirty-six base pairs (bp) of the spoVG promoter region adjacent to the spoVG open reading frame (ORF) encompassing the Shine-Dalgarno (SD) sequence (see Figure 1B) were included adjacent to the promoter region of Phbs-yvyD (see Table 3; primer SEQ ID NO:9).

[0227] The hbs promoter region was amplified using the 675 (SEQ ID NO: 10) and 307 (SEQ ID NO: 11) oligonucleotide primer pair as shown in Table 2. The yvyD ORF (SEQ ID NO: 18) was amplified from B. subtilis genomic DNA using oligonucleotide primers 400 (Table 2; SEQ ID NO: 3) and 370 (Table 2; SEQ ID NO: 4) for the PspoVG-yvyD assembly and oligonucleotide primers 674 (Table 3; SEQ ID NO: 12) and 370 (Table 2; SEQ ID NO: 4) for the Phbs-yvyD assembly. NEBuilder assembly was performed as per the manufacturer's instructions using overlapping DNA fragments to generate the complete yvzG-yvyD intergenic::lox-SpecR-lox-PspoVG-yvyD (Figure 1A) and yvzG-yvyD intergenic::lox-SpecR-lox-Phbs-yvyD (Figure 1B) integration cassettes. The complete nucleotide sequence of the assembled integration cassette of PspoVG-yvyD is shown in SEQ ID NO:19, and the complete nucleotide sequence of the assembled integration cassette of Phbs-yvyD is shown in SEQ ID NO:20. [Table 3]

[0228] Example 2 Construction and generation of B. subtilis strains with increased YVYD expression This example describes the construction of B. subtilis cells (strains) that increase yvyD expression. More specifically, recombinant B. subtilis cells were constructed by introducing a cassette that increased expression of the endogenous (native) B. subtilis yvyD gene (SEQ ID NO: 23) by promoter swap integration at the yvzG-yvyD intergenic region, which contains two copies of genes encoding three different exemplary proteases (2x Protease-1; 2x Protease-2; 2x Protease-3), as described in Example 1. Isogenic cells carrying the native yvyD promoter and encoding three different exemplary proteases (control cells; 2x protease-1; 2x protease-2; 2x protease-3) were constructed for comparison purposes. For example, approximately 1-2 μg of the yvzG yvyD intergenic::lox-SpecR-lox-PspoVG-yvyD integration cassette (SEQ ID NO: 19) and the yvzG yvyD intergenic::lox-SpecR-lox-Phbs-yvyD integration cassette (SEQ ID NO: 20) were individually transformed into a comK competent B. subtilis parent strain.

[0229] More specifically, transformed cells were plated on LB (1% tryptone, 0.5% yeast extract, 1.0% sodium chloride, 1.5% agar) and 100 μg / ml spectinomycin, and spectinomycin-resistant colonies were purified by restreaking on LB with 100 mg / L spectinomycin. Integration of each cassette between the yvzG-yvyD genes was confirmed by PCR amplification using Q5 High Fidelity PCR polymerase (NEB) and harvested genomic DNA as template with oligonucleotide primers 345 (SEQ ID NO: 12) and 348 (SEQ ID NO: 13) listed in Table 4 below, which bound outside the integration event. Similarly, the correct sequence of each integration cassette was confirmed by Sanger sequencing using oligonucleotides 345 (Table 4; SEQ ID NO:12), 346 (Table 4; SEQ ID NO:14), 300 (Table 4; SEQ ID NO:15), 573 (Table 4; SEQ ID NO:16), 674 (Table 3; SEQ ID NO:11), and 348 (Table 4; SEQ ID NO:13). [Table 4]

[0230] Furthermore, the spectinomycin antibiotic resistance marker (lox-SpecR-lox) was removed by transformation with a plasmid expressing Cre recombinase. After plasmid loss, spectinomycin-sensitive colonies were identified and the integration cassette was amplified with oligonucleotide primers 346 (Table 4; SEQ ID NO: 14) and 573 (Table 4; SEQ ID NO: 16). Correct recombination of the lox sites was confirmed in each of the yvyD overexpression strains by sequence analysis using oligonucleotide 346 (Table 4: SEQ ID NO: 14). Two cassettes expressing protease-1 (2x protease-1) and two cassettes expressing protease-2 (2x protease-2) were introduced individually into the PspoVG-yvyD overexpression strain. Similarly, two cassettes expressing protease-2 (2x protease-2) and two cassettes expressing protease-3 (2x protease-3) were individually introduced into the Phbs-yvyD overexpressing strain, and simultaneously, two cassettes expressing protease-1, protease-2, and protease-3 were individually introduced into the parent strain expressing yvyD from its native (yvyD) promoter.

[0231] Example 3 Overexpression of yvyD increases protein production in B. subtilis strains expressing two copies of a protein of interest In this example, Applicants evaluated overexpression of yvyD for the production of reporter proteases in two copy protease producing B. subtilis strains described in Example 2 (i.e., 2x Protease-1 and 2x Protease-1 PspoVG-yvyD; 2x Protease-2 and 2x Protease-2 PspoVG-yvyD; 2x Protease-2 and 2x Protease-2 Phbs-yvyD; 2x Protease-3 and 2x Protease-3 Phbs-yvyD). The protease activity assays described herein were performed as described in EP 0283075, which is incorporated herein by reference.

[0232] For example, aliquots were taken from the 2x protease-1 control strain and the 2x protease-1 PspoVG-yvyD strain at 12, 20, 36, 45, 61, 68, 73, and 84 hours. Protease activity assays were performed to determine the effect of increased expression of yvyD from the spoVG promoter (PspoVG) on the production of protease-1. The results of the protease assay (Figure 2) show that there is a trend towards increased protease production at the end of fermentation, and that the production of protease is significantly enhanced at 68 and 73 hours due to increased expression of yvyD.

[0233] Additionally, aliquots were taken from the 2x protease-2 control strain and the 2x protease-2+PspoVG-yvyD strain at 16, 22, 39, 46, 64, and 89 hours. Protease activity assays were performed to determine the effect of overexpression of yvyD from the spoVG promoter (PspoVG) on the production of protease-2. The results of the protease assay (Figure 3) show that the increase in protease production started from about 39 hours and tended to continue until the end of the fermentation, with the production of protease being significantly enhanced due to overexpression of yvyD at 39 and 46 hours.

[0234] Similarly, aliquots were taken from the 2xProtease-2 control strain and the 2xA Protease-2Phbs-yvyD strain at 11, 23, 37, 50, and 65 hours. Protease activity assays were performed to determine the effect of overexpression of yvyD from the hbs promoter (Phbs) on the production of Protease-2. The results of the protease assay (Figure 4) show that the increase in protease production started from about 37 hours and tended to continue until the end of the fermentation, with the production of protease being significantly enhanced due to the overexpression of yvyD at 37 hours.

[0235] Additionally, aliquots were taken from the 2xprotease-3 control and 2xprotease-3Phbs-yvyD strains at 14, 22, 37, 46, 65, and 90 hours. Protease activity assays were performed to determine the effect of overexpression of yvyD from the hbs promoter (Phbs) on the production of protease-3. The results of the protease assay (Figure 5) show that the increase in protease production started from about 22 hours and tended to continue until the end of the fermentation, with the production of protease being significantly enhanced due to overexpression of yvyD at 22 and 37 hours.

[0236] References European Patent Publication No. 0283075 International Publication No. 2014 / 164777 Brochure Beckert et al., “Structure of the Bacillus subtilis hibernating 100S ribosome reveals the basis for 70S dimerization”, The EMBO Journal, 36, pages 2061-2071, 2017. Drzewiecki et al., “The yvyD Gene of Bacillus subtilis Is Under Dual Control of σ B and σ H ”,J.Bacteriology,Vol.180,No.24,pages 6674-6680,1998. Feaga et al., “Ribosome dimerization protects the small subunit” Journal of Bacteriology, Vol. 202, No. 10, pages 9-20, 2020. Franklin et al.,“Functional Characterization of the Pseudomonas aeruginosa Ribosome Hibernation-Promoting Factor”,Journal of Bacteriology,Volume 202,Issue 19,2020. Tagami et al.,“Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome”,Microbiology Open,1(2),pages115-134,2012. Zhu and Stuelke,“SubtiWiki in 2018: from genes and proteins to functional network annotation of the model organism Bacillus subtilis”,Nucleic Acid Research,Volume 46, Issue D1,Pages D743-D748,2017.

Claims

1. Recombinant Gram-positive cells overexpressing the yvyD gene.

2. A recombinant Gram-positive cell overexpressing the yvyD gene and expressing a gene encoding a protein of interest (POI).

3. The recombinant cell of claim 2 , which expresses multiple copies of the gene encoding the POI.

4. The recombinant cell of claim 1, wherein the overexpressed yvyD gene comprises at least 50% identity to the yvyD gene of SEQ ID NO:

23.

5. The recombinant cell of claim 1, wherein the overexpressed yvyD gene comprises at least 50% identity to the yvyD gene coding sequence (CDS) of SEQ ID NO:

18.

6. The recombinant cell of claim 1, wherein the overexpressed yvyD gene encodes a protein comprising at least 50% identity to the YvyD protein of SEQ ID NO:

26.

7. The recombinant cell of claim 2, wherein the recombinant cell produces an increased amount of the POI compared to a control cell expressing the same POI, and the control cell does not overexpress the yvyD gene.

8. The recombinant cell of claim 1, wherein overexpressing the yvyD gene comprises replacing the native yvyD gene promoter region with a heterologous promoter region operably linked to the downstream (3') native yvyD gene CDS, wherein the heterologous promoter region increases expression of the native yvyD gene CDS compared to the native yvyD gene promoter.

9. The recombinant cell of claim 2, wherein overexpressing the yvyD gene comprises replacing the native yvyD gene promoter region with a heterologous promoter region operably linked to the downstream (3') native yvyD gene CDS, wherein the heterologous promoter region increases expression of the native yvyD gene CDS compared to the native yvyD gene promoter.

10. The recombinant cell of claim 2 , wherein the POI is an enzyme.

11. 2. The recombinant cell of claim 1, wherein the Gram-positive bacterial cell is a Bacillus sp. cell.

12. The recombinant cell of claim 2 , wherein the Gram-positive bacterial cell is a Bacillus sp. cell.

13. A method for producing increased amounts of a protein of interest (POI) in a Gram-positive bacterial cell, comprising obtaining a parent cell containing a yvyD gene having at least 50% identity to the yvyD gene of SEQ ID NO:23, and genetically modifying the cell to overexpress the yvyD gene.

14. The method of claim 13, wherein the parent cell or modified (recombinant) cell comprises an introduced expression cassette encoding the POI.

15. The method of claim 13, wherein the modified cells that overexpress the yvyD gene produce an increased amount of the POI compared to the parent cells when cultured under the same conditions.

16. The method of claim 13, wherein the modified cell overexpressing the yvyD gene comprises a heterologous promoter region operably linked to a downstream (3') yvyD gene CDS, the heterologous promoter region increasing expression of the yvyD gene CDS compared to a native yvyD gene promoter.

17. The method of claim 13, wherein the POI is an enzyme.

18. The method of claim 13, wherein the Gram-positive bacterial cell is a Bacillus sp. cell.

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