Compositions and methods for enhanced protein production in bacillus cells

Abstract

The present disclosure is generally related to recombinant microbial cells expressing heterologous proteins of interest. Certain aspects of the disclosure are therefore related to, inter alia, recombinant Bacillus cells having enhanced protein production capabilities, recombinant polynucleotides encoding heterologous proteins of interest, and related compositions and / or methods thereof. As exemplified herein, the recombinant Bacillus cells of the instant disclosure are particularly suitable for use in the expression, production and secretion of heterologous proteins.

Description

IFF10151-WO-PCTCOMPOSITIONS AND METHODS FOR ENHANCED PROTEIN PRODUCTION IN BACILLUS CELLSFIELD

[0001] The present disclosure is generally related to the fields of microbial cells, molecular biology, fermentation, protein production, and the like. Certain aspects of the disclosure are related to, inter alia, recombinant Bacillus cells having enhanced protein production capabilities.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit to U.S. Provisional Patent Application No. 63 / 721,734, filed November 18, 2024, which is incorporated herein by referenced in its entirety.REFERENCE TO A SEQUENCE LISTING

[0003] The sequence listing text file submitted herewith contains the file “IFF10151-WO-PCT _SequenceListing.xml” created on November 14, 2024, which is 46,080 bytes in size. This sequence listing complies with 37 C.F.R. § 1.52(e) and is incorporated herein by reference in its entirety.BACKGROUND

[0004] Gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and the like are frequently used as microbial factories for the production of industrial relevant proteins, due to their excellent fermentation properties and high yields (e.g., up to 25 grams per liter culture; Van Dijl and Hecker, 2013). For example, Bacillus sp. cells are well known for their production of amylases (Jensen et al., 2000; Raul etal., 2014) and proteases (Brode etal., 1996) necessary for food, textile, laundry, medical instrument cleaning, pharmaceutical industries and the like (Westers et al., 2004). Because these non-pathogenic Gram-positive bacteria produce proteins that completely lack toxic by-products (e.g., lipopolysaccharides; LPS, also known as endotoxins) they have obtained the “Qualified Presumption of Safety” (QPS) status of the European Food Safety Authority, and many of their products gained a “Generally Recognized As Safe” (GRAS) status from the US Food and Drug Administration (Olempska-Beer et al., 2006; Earl et al., 2008; Caspers et al., 2010).

[0005] Thus, the production of proteins (e.g., enzymes, antibodies, receptors, etc.) in Gram-positive bacterial cells is an area of high interest in the biotechnological arts, wherein small improvements in protein yield are quite significant when the protein is produced in large industrial quantities. As described hereinafter, the instant disclosure is related to the highly desirable and unmet needs for obtaining,IFF10151-WO-PCTconstructing, producing and the like, Gram-positive host cells having increased protein production capabilities.SUMMARY

[0006] As set forth herein, certain embodiments of the disclosure are related to recombinant Grampositive bacterial cells (strains) having enhanced protein production phenotypes. Certain embodiments therefore provide, inter alia, methods and compositions for constructing modified Bacillus sp. cells having enhanced protein production phenotypes, methods and compositions for fermenting / cultivating / growing modified Bacillus sp. cells having enhanced protein production phenotypes, modified Bacillus sp. cells having an inactivated htrB gene, modified Bacillus sp. cells having an introduced htrB expression cassette comprising an upstream heterologous promoter operably linked to a downstream htrB gene open reading frame (ORF), modified Bacillus sp. cells expressing / producing / secreting mature proteases of interest, and the like.

[0007] Thus, in certain one or more embodiments, the disclosure provides modified Bacillus sp. cells comprising an inactivated wild-type (endogenous) htrB gene and an introduced (synthetic) htrB expression cassette. In other embodiments, modified Bacillus sp. cells comprise one or more introduced expressions cassettes encoding a protease of interest. In other embodiments, an introduced htrB cassette comprises an upstream heterologous (constitute) promoter. In other embodiments, an introduced htrB cassette comprises a 5 '-untranslated region (5'-UTR) polynucleotide sequence downstream and operably linked to the heterologous promoter. In certain other embodiments, an htrB ORF comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2. In yet other embodiments, an htrB ORF encodes an HtrB protein having at about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 24. In certain other embodiments, an inactivated wild-type (endogenous) htrB gene comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the htrB ORF (SEQ ID NO: 2). In particular embodiments, the wild-type (endogenous) htrB gene present in the cell is inactivated by disrupting the htrB gene, deleting the htrB gene, downregulating the htrB gene and the like. In another embodiment, modified cells comprising the inactivated wild-type htrB gene are at least about 95% to 100% (j.e., completely) deficient in the production of the native HtrB protein. In certain embodiments, a protease of interest is a subtilisin protease, e.g., a native subtilisin and functional subtilisin variants thereof. In certain related embodiments, a mature subtilisin comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 26. In certain preferred embodiments, a modified cell described herein produces at least about 5% or more of the protease of interest as compared (relative) to a control cell fermented under the same conditions.IFF10151-WO-PCT

[0008] Certain other embodiments of the disclosure are related to, infer alia, methods for the enhanced production of proteases of interest in modified Bacillus sp. cells. For instance, certain one or more embodiments are related to methods for constructing Bacillus sp. cells for the enhanced expression / production of proteases of interest. In certain embodiments of the methods, the modified Bacillus sp. cells comprise an inactivated (e.g., deleted, disrupted, downregulated, etc.) wild-type (endogenous) htrB gene and an introduced synthetic htrB expression cassette, wherein the modified cells are fermented under conditions for the production of a protease of interest. In certain related embodiments of the methods, the modified Bacillus sp. cell produces an increased amount of a protease as compared to a control (isogenic) Bacillus sp. cell fermented under the same conditions, wherein the control Bacillus sp. cell expresses the same protease of interest and comprises the same inactivated (e.g., deleted, disrupted, downregulated, etc.) wild-type htrB gene, but does not comprise the introduced synthetic htrB expression cassette. In certain other embodiments of the methods, a protease of interest is a subtilisin protease. In certain other embodiments of the methods, the mature protease is secreted into the fermentation broth. In yet other embodiments, the modified Bacillus sp. cells produce at least about 5% or more of the mature protease as compared to the control cell fermented under the same conditions.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 shows the insertion of three (3) stop codons (“TAATGATAA”) into the htrB gene to inactivate the native htrB coding sequence (CDS).

[0010] Figure 2 shows the general design of three htrB expression constructs. In particular, FIG. 2A shows the design of the fbp promoter construct in which the fbp promoter was used to drive expression of the htrB 5'-UTR and htrB CDS linked to the spoVG terminator, FIG. 2B shows the design of the yqgS promoter construct in which the yqgS promoter was used to drive expression of the htrB 5'-UTR and htrB CDS linked to the spoVG terminator, and FIG. 2C shows the design of the rpoB promoter construct in which the rpoB promoter was used to drive expression of the htrB 5'-UTR and htrB CDS . As shown in FIG. 2, all expression constructs were integrated at the B. subtilis ppsC gene locus (ppsC upstream; ppsC downstream).BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0011] SEQ ID NO: 1 is a DNA sequence comprising a Bacillus subtilis htrB 5' untranslatedregion (5'-UTR).

[0012] SEQ ID NO: 2 is a DNA sequence comprising a B. subtilis htrB gene open reading frame (ORF).

[0013] SEQ ID NO: 3 is a DNA sequence comprising a B. subtilis fbp promoter.

[0014] SEQ ID NO: 4 is a DNA sequence comprising a B. subtilis yqgS promoter.

[0015] SEQ ID NO: 5 is a DNA sequence comprising a B. subtilis rpoB promoter.

[0016] SEQ ID NO: 6 is a DNA sequence comprising a B. subtilis spoVG terminator.IFF10151-WO-PCT

[0017] SEQ ID NO: 7 is a DNA sequence comprising a htrB gene 5' (upstream) flanking region (htrB_5'-FR).

[0018] SEQ ID NO: 8 is a DNA sequence comprising a htrB gene 3' (downstream) flanking region (htrB_3'~ FR).

[0019] SEQ ID NO: 9 is a DNA sequence comprising a ppsC gene 5' (upstream) flanking region (ppsC_5'-FR).

[0020] SEQ ID NO: 10 is a DNA sequence comprising a ppsC gene 3' (downstream) flanking region (ppsC_3'-FR).

[0021] SEQ ID NO: 11 is a DNA sequence comprising a spoIIIAE gene 5' (upstream) flanking region lspoIIIAA_5’-Y ).

[0022] SEQ ID NO: 12 is a DNA sequence comprising a spoIIIAE gene 3' (downstream) flanking region (spoIIIAE_3'-V\C).

[0023] SEQ ID NO: 13 is a DNA sequence comprising an aprE gene 5' (upstream) flanking region («prE_5'-FR).

[0024] SEQ ID NO: 14 is a DNA sequence comprising an aprE gene 3' (downstream) flanking region (aprE 3' -FBI).

[0025] SEQ ID NO: 15 is a DNA sequence comprising a P2 promoter.|0026| SEQ ID NO: 16 is a DNA sequence comprising an aprE 5'-UTR (transcriptional leader).

[0027] SEQ ID NO: 17 is a DNA sequence encoding a B. suhtilis aprE signal sequence (aprE_ss).

[0028] SEQ ID NO: 18 is a DNA sequence comprising an alrA gene.

[0029] SEQ ID NO: 19 is a DNA sequence comprising an alrA gene promoter.

[0030] SEQ ID NO: 20 is a DNA sequence comprising a nprE transcriptional terminator.

[0031] SEQ ID NO: 21 is a synthetic DNA encoding a subtilisin protease pro-region.

[0032] SEQ ID NO: 22 is a synthetic DNA encoding a mature subtilisin (reporter) protease.

[0033] SEQ ID NO: 23 is a synthetic DNA comprising a transcriptional terminator.

[0034] SEQ ID NO: 24 is the amino acid sequence of the HtrB protein encoded by SEQ ID NO: 2.

[0035] SEQ ID NO: 25 is the amino acid sequence of the protease pro region encoded by SEQ ID NO: 21.

[0036] SEQ ID NO: 26 is the amino acid sequence the mature subtilisin reporter protease.DETAILED DESCRIPTION

[0037] As described herein, the instant disclosure addresses numerous ongoing and unmet needs in the art, particularly as related to the industrial scale production recombinant proteins. Certain embodiments of the instant disclosure provide, inter alia, recombinant Bacillus sp. cells capable expressing / producing increased amounts of proteins of interest, recombinant Bacillus sp. cells comprising introduced nucleic acids (e.g., plasmids, vectors, expression cassettes) encoding proteins of interest, recombinant Bacillus sp. cellsIFF10151-WO-PCTcomprising a deleted endogenous (wild-type) htrB gene, recombinant Bacillus sp. cells comprising an introduced htrB expression cassette comprising a heterologous promoter driving the expression of the htrB gene coding sequence, methods and compositions for fermenting / cultivating / growing recombinant Bacillus sp. cells for the expression / production heterologous proteins of interest, and the like.I. DEFINITIONS

[0038] Prior to describing the present strains, compositions and methods in further detail, the following terms and phrases are defined.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present compositions and methods apply. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present compositions and methods, representative illustrative methods and materials are now described. All publications and patents cited herein are incorporated by reference in their entirety.

[0040] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only”, “excluding”, “not including”, “does not comprise”, and the like, in connection with the recitation of claim elements, or use of a “negative” limitation or proviso thereof. For instance, in certain embodiments, a control Bacillus sp. cell “does not” comprise an introduced htrB expression cassette, e.g., relative to a modified cell.

[0041] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present compositions and methods described herein. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0042] 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.

[0043] As used herein, the genus “Bacillus” includes all species within the genus “Bacillus’” as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continuesIFF10151-WO-PCTto undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. ste rothermophilus, which is now named “Geobacillus stearothermophilus” .

[0044] As used herein, the terms “recombinant” or “non-natural” refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has at least one engineered genetic alteration, or has been modified by the introduction of a heterologous nucleic acid molecule, or refer to a cell (e.g., a Gram-positive cell) that has been altered such that the expression of a heterologous nucleic acid molecule or an endogenous nucleic acid molecule or a gene can be controlled. Recombinant also refers to a cell that is derived from a non-natural cell, or is progeny of a non-natural cell having one or more such modifications. Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, or other nucleic acid molecule additions, deletions, substitutions or other functional alteration of a cell’s genetic material. For example, recombinant cells may express genes or other nucleic acid molecules (e.g., polynucleotide expression constructs) that are not found in identical or homologous form within a native (wild-type) cell, or may provide an altered expression pattern of endogenous genes, such as being over-expressed, under-expressed, minimally expressed, or not expressed at all. “Recombination”, “recombining” or generating a “recombined” nucleic acid is generally the assembly of two or more nucleic acid fragments wherein the assembly gives rise to a chimeric DNA sequence that would not otherwise be found in the genome.

[0045] The term “derived” encompasses the terms “originated”, “obtained”, “obtainable”, and “created” and generally indicates that one specified material or composition finds its origin in another specified material or composition, or has features that can be described with reference to the other specified material or composition.

[0046] As used herein, “nucleic acid” refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, as well as to DNA, cDNA, and RNA of genomic or synthetic origin, which may be doublestranded or single-stranded, whether representing the sense or antisense strand. It will be understood that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences may encode a given protein.

[0047] It is understood that the polynucleotides (or nucleic acid molecules) described herein include “genes”, “vectors” and “plasmids”.

[0048] Accordingly, the term “gene”, refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all, or part of a protein coding sequence, and may include regulatory (nontranscribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions (UTRs), including introns, 5 '-untranslated regions (UTRs), and 3'-UTRs, as well as the coding sequence.IFF10151-WO-PCT

[0049] As used herein, an “endogenous gene” refers to a gene in its natural location in the genome of an organism.

[0050] As used herein, a “heterologous” gene, a “non-endogenous” gene, or a “foreign” gene refer to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. The term “foreign” gene(s) comprises native genes inserted into a non-native organism and / or chimeric genes inserted into a native or non-native organism.

[0051] As used herein, the terms “signal sequence” and “signal peptide” refer to a sequence of amino acid residues that may participate in the secretion or direct transport of a mature protein or precursor form of a protein. The signal sequence is typically located N-terminal to the precursor or mature protein sequence. The signal sequence may be endogenous or exogenous. A signal sequence is normally absent from the mature protein. A signal sequence is typically cleaved from the protein by a signal peptidase during translocation.

[0052] As used herein, the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA, derived from a nucleic acid molecule of the disclosure. Expression may also refer to translation of mRNA into a polypeptide. Thus, the term “expression” includes any steps involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, secretion and the like.|0053| As used herein, the term “coding sequence” (abbreviated, “CDS”) 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 (ORF), which usually begins with an ATG start codon. The coding sequence typically includes DNA, cDNA, and recombinant nucleotide sequences.

[0054] As used herein, the terms “promoter”, “promoter element”, “promoter sequence” and the like, refer to a nucleic acid (DNA) sequence capable of controlling the transcription of a gene coding sequence (CDS) into messenger RNA (mRNA) when the promoter region sequence is placed upstream (5') and operably linked to the downstream (3') gene CDS. As generally understood by of skilled in the art, promoters typically provide a site for specific binding by RNA polymerase and the initiation of transcription. In certain aspects, the term “promoter” refers to the minimal portion of the promoter nucleic acid sequence required to initiate transcription (i.e., comprising RNA polymerase binding sites). For example, a promoter generally comprises a “-10” (consensus sequence) element and a “-35” (consensus sequence) element, which are upstream (5') and relative to the +1 transcription start site (TSS) of the gene CDS to be transcribed. The core promoter -10 and -35 elements are generally referred to in the art as the “TATAAT” (Pribnow box) consensus region and the “TTGACA” consensus region, respectively. The spacing of theIFF10151-WO-PCTcore promoter (10 and -35) regions are generally separated (spaced) by about fifteen-twenty (15-20) intervening base pairs (nucleotides).

[0055] Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters can be constitutive promoters, inducible promoters, tunable promoters, hybrid promoters, synthetic promoters, tandem promoters, etc. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0056] As used herein, a “functional promoter sequence controlling the expression of a gene of interest linked to the gene of interest’s protein coding sequence” refers to a promoter sequence which controls the transcription and translation of the coding sequence in a desired Gram-positive host cell. For example, in certain embodiments, the present disclosure provides polynucleotides comprising an upstream (5 ') promoter (or 5' promoter region, or tandem 5' promoters and the like) functional in a Gram-positive cell, wherein the functional promoter region is operably linked to a nucleic acid sequence encoding a protein of interest. |0057| As used herein, the term “precursor protein” refers to an inactive form of a protein. In certain aspects, a full-length protein is synthesized as precursor, in the form of a pro-sequence and mature protein (abbreviated, “pre-protein”). In other aspects, a full-length protein is synthesized as precursor, in the form of a signal peptide sequence, a pro-sequence and mature protein (abbreviated, “pre -pro-protein”). For example, pre-sequences usually act as signal peptides for transport, and pro-sequences are typically essential for the correct folding of the associated (mature) protein.

[0058] As used herein, the term “mature protein” refers to an active form of a protein, in contrast to the inactive precursor (full-length) protein.

[0059] As used herein, the terms “signal sequence”, “secretion signal” and “signal peptide” may be used interchangeably and refer to a sequence of amino acid residues that may participate in the secretion or direct transport of a precursor protein. Tire signal (pre) sequence is typically cleaved from the precursor protein by a signal peptidase during translocation. The signal (pre) sequence is typically located N-terminal to the mature protein sequence, or located N-terminal to the pro-region (pro) sequence when a signal (pre) sequence and a pro-region (pro) sequence are used in operable combination and upstream (5 ') of the mature POI sequence.

[0060] As used herein, the terms “pro sequence”, “pro-sequence” and “pro-region sequence” may be used interchangeably and abbreviated as “PRO” sequence, “Pro” sequence, “pro” sequence and the like. TheIFF10151-WO-PCTterm pro-sequence as used herein has the same meaning as understood in the art. For example, the B. subtilis alkaline serine protease “subtilisin’’ is first produced as a pre-pro-subtilisin, which consists of a signal (pre) sequence for protein secretion followed by a pro-region (pro) sequence followed by the amino acid sequence encoding the mature subtilisin (e.g., pre-pro-subtilisin). Pro-sequences are often essential for the correct folding of the associated (mature) protein, acting as an intra-molecular chaperone (e.g., catalyzing the protein-folding reaction directly). Likewise, pro-sequences may be required for both folding and intracellular transport (or secretion) of the mature protein of interest, suggesting that these two functionalities are intimately related. For instance, in certain embodiments a DNA sequence encoding proregion may be derived from a wild-type or variant B. lentus pro-region (DNA) sequence set forth as SEQ ID NO: 21.

[0061] The term “operably linked” as used herein refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (z.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0062] A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a secretory leader (i.e., a signal sequence), is operably linked to DNA for a polypeptide if it is expressed as a pre-protein 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 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. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0063] As used herein, “suitable regulatory sequences” refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, transcription leader sequences, RNA processing site, effector binding site and stem-loop structures.

[0064] As used herein, a wild-type B. subtilis “htrB 5 '-untranslated region” (abbreviated hereinafter, “htrB 5'-UTR”) comprises a nucleic acid (DNA) sequence set forth as SEQ ID NO: 1, and a wild-type B. subtilis “htrB” gene comprises an open reading frame (htrB ORF) sequence set forth as SEQ ID NO: 2. The htrB ORF encodes a native HtrB protein set forth as SEQ ID NO: 24.IFF10151-WO-PCT

[0065] As used herein, exemplary proteases may be referred to as “reporter proteins”. In certain one or more embodiments, exemplary reporter proteins are expressed / produced by one or more recombinant (modified) cells of the disclosure. In certain embodiments, reporter proteins include, but are not limited to, native and variant Bacillus sp. subtilisins. In certain one or more embodiments, exemplary subtilisin reporters include, but are not limited to, the native B. clausii subtilisin and functional variants thereof, the native B. gibsonii subtilisin and functional variants thereof, the native B. lentus subtilisin and functional variants thereof, the native B. licheniformis subtilisin (AprL) and functional variants thereof, the native B. subtilis subtilisin (AprE) and functional variants thereof, the native B. amyloliquefaciens subtilisin (BPN') and functional variants thereof, and the like. In certain aspects, exemplary B. clausii, B. gibsonii and / or B. lentus subtilisin reporters may be referred to as alkaline proteases. For instance, alkaline subtilisins generally have an isoelectric point (pl) of about 9.5, whereas the B. licheniformis, B. subtilis and B. amyloliquefaciens sublilisins have a pl of about 6.5.

[0066] As used herein, the term “subtilisin” refers to any member of the S8 serine protease family as described in MEROPS — The Peptidase Data base (Rawlings et al., 2006). The term subtilisin includes a wide variety of Bacillus subtilisins which have been identified and sequenced e.g., subtilisin 168, subtilisin BPN', subtilisin Carlsberg, etc., and includes mutant (variant) proteases derived therefrom and the like.

[0067] As used herein, phrases such as “subtilisin reporter”, “reporter protease” and “reporter” may be used interchangeably, and particularly refer to the B. gibsonii subtilisin set forth in SEQ ID NO: 26, and functional variants thereof. One of skill may readily design, construct, screen, and identity functional subtilisin variants using routine methods known in the art. In particular, PCT Publication Nos. W02010 / 056634, WO2011 / 130222, WO2015 / 089447, WO2016 / 202839, WO2017 / 207762 and WO2023 / 114936 (each incorporated herein by reference in its entirety) describe suitable methods and compositions for constructing functional subtilisin variants derived from a native B. clausii subtilisin, functional subtilisin variants derived from a native B. amyloliquefaciens subtilisin, functional subtilisin variants derived from a native B. gibsonii subtilisin, and the like.

[0068] As used herein, a “host cell” refers to a cell that has the capacity to act as a host or expression vehicle for a newly introduced DNA sequence. This, in certain embodiments of the disclosure, the host cells are Gram-positive cells (e.g., Bacillus sp.) and / or Gram-negative cells (e.g., E. coll).

[0069] As used herein, a “modified cell” refers to a recombinant cell that comprises at least one genetic modification which is not present in the reference or control cell from which the modified cell is derived.

[0070] As used herein, when the expression and / or production of a protein of interest (POI) in a recombinant (modified) cell is being compared to the expression and / or production of the same POI in an reference (control) cell, it will be understood that the modified and control cells areIFF10151-WO-PCTgrown / cultivated / fermented under the same conditions (e.g., the same conditions such as media, temperature, pH and the like).

[0071] As used herein, an “increased amount”, when used in phrases such as a “recombinant cell ‘expresses / produces an increased amount’ of a protein of interest relative to a control cell”, particularly refers to an “increased amount” of a protein of interest (POI) expressed / produced in by the recombinant cell, which “increased amount” is always relative to the control cell expressing / producing the same POI, wherein the modified and control cells are grown / cultured / fermented under the same conditions.

[0072] As used herein, “increasing” protein production, or “increased” protein production, is meant an increased amount of protein produced (e.g., a protein of interest). The protein may be produced inside the host cell, or secreted (or transported) into the culture medium. In certain embodiments, the protein of interest is produced (secreted) into the culture medium. Increased protein production may be detected for example, as higher maximal level of protein or enzymatic activity (e.g., such as protease activity), or total extracellular protein produced as compared to the control cell.

[0073] As used herein, the terms “modification” and “genetic modification” are used interchangeably and include: (a) the introduction, substitution, or removal of one or more nucleotides in a gene (or an ORF thereof), or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the gene or ORF thereof, (b) a gene disruption, (c) a gene conversion, (d) a gene deletion, (e) the down-regulation of a gene, (f) specific mutagenesis and / or (g) random mutagenesis of any one or more the genes disclosed herein.

[0074] As used herein, the term “introducing”, as used in phrases such as “introducing into a Gram-positive bacterial cell a ‘gene’, a ‘polynucleotide’, an ‘open reading frame’ (ORF), a ‘gene coding sequence (CDS), a ‘vector’, an ‘expression cassette’”, and the like, includes methods known in the art for introducing polynucleotides (DNA) into a cell, including, but not limited to protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation and the like.

[0075] As used herein, “transformed” or “transformation” mean a cell has been transformed by use of recombinant DNA techniques. Transformation typically occurs by insertion of one or more nucleotide sequences (e.g., a polynucleotide, an ORF or gene) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence that is not naturally occurring in cell that is to be transformed). Transformation therefore generally refers to introducing an exogenous DNA into a host cell so that the DNA is maintained as a chromosomal integrant or a self-replicating extra-chromosomal vector.

[0076] As used herein, “transforming DNA”, “transforming sequence”, and “DNA construct” refer to DNA that is used to introduce sequences into a host cell or organism. Transforming DNA is DNA used to introduce sequences into a host cell or organism. The DNA may be generated in vitro by PCR or any other suitable techniques. In some embodiments, the transforming DNA comprises an incoming sequence, whileIFF10151-WO-PCTin other embodiments it further comprises an incoming sequence flanked by homology boxes. In yet a further embodiment, the transforming DNA comprises other non-homologous sequences, added to the ends (z.e., stuffer sequences or flanks). The ends can be closed such that the transforming DNA forms a closed circle, such as, for example, insertion into a vector.

[0077] As used herein, “disruption of a gene” or a “gene disruption”, are used interchangeably and refer broadly to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., a protein). Thus, as used herein, a gene disruption includes, but is not limited to, frameshift mutations, premature stop codons (z.e., such that a functional protein is not made), substitutions eliminating or reducing activity of the protein internal deletions (such that a functional protein is not made), insertions disrupting the coding sequence, mutations removing the operable link between a native promoter required for transcription and the open reading frame, and the like.

[0078] As used herein “an incoming sequence” refers to a DNA sequence that is introduced into the bacterial cell chromosome. In some embodiments, the incoming sequence is part of a DNA construct. In other embodiments, the incoming sequence encodes one or more proteins of interest. In some embodiments, the incoming sequence comprises a sequence that may or may not already be present in the genome of the cell to be transformed (z.e., it may be either a homologous or heterologous sequence). In some embodiments, the incoming sequence encodes one or more proteins of interest, a gene, and / or a mutated or modified gene. In alternative embodiments, the incoming sequence encodes a functional wildtype gene or operon, a functional mutant gene or operon, or a nonfunctional gene or operon. In some embodiments, the non-functional sequence may be inserted into a gene to disrupt function of the gene. In another embodiment, the incoming sequence includes a selective marker. In a further embodiment the incoming sequence includes two homology boxes.

[0079] As used herein, “homology box” refers to a nucleic acid sequence, which is homologous to a sequence in the bacterial cell chromosome. More specifically, a homology box is an upstream or downstream region having between about 80 and 100% sequence identity, between about 90 and 100% sequence identity, or between about 95 and 100% sequence identity with the immediate flanking coding region of a gene or part of a gene to be deleted, disrupted, inactivated, down-regulated and the like, according to the invention. These sequences direct where in the bacterial cell chromosome a DNA construct is integrated and directs what part of the chromosome is replaced by the incoming sequence. While not meant to limit the present disclosure, a homology box may include about between 1 base pair (bp) to 200 kilobases (kb). Preferably, a homology box includes about between 1 bp and 10.0 kb: between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb, and between 0.25 kb and 2.5 kb. A homology box may also include about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb and 0.1 kb. In someIFF10151-WO-PCTembodiments, the 5' and 3' ends of a selective marker are flanked by a homology box wherein the homology box comprises nucleic acid sequences immediately flanking the coding region of the gene.

[0080] As used herein, a host cell “genome”, a bacterial (host) cell “genome”, or a Bacillus sp. (host) cell “genome” includes chromosomal and extrachromosomal genes.

[0081] As used herein, the terms “plasmid”, “vector” and “cassette” refer to extrachromosomal elements, often carrying genes which are typically not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a singlestranded or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0082] As used herein, the term “plasmid” refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes. In some embodiments, plasmids become incorporated into the genome of the host cell, in some embodiments plasmids exist in a parental cell and are lost in the daughter cell.

[0083] A used herein, a “transformation cassette” refers to a specific vector comprising a gene (or ORF thereof), and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.

[0084] As used herein, the term “vector” refers to any nucleic acid that can be replicated (propagated) in cells and can carry new genes or DNA segments into cells. Thus, the term refers to a nucleic acid construct designed for transfer between different host cells. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), PLACs (plant artificial chromosomes), and the like, that are “episomes” (i.e., replicate autonomously or can integrate into a chromosome of a host organism).

[0085] An “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA in a cell. Many prokaryotic and eukaryotic expression vectors are commercially available and know to one skilled in the art. Selection of appropriate expression vectors is within the knowledge of one skilled in the art.

[0086] As used herein, the terms “expression cassette” and “expression vector” refer to a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell (i.e., these arc vectors or vector elements, as described above). The recombinant expression cassette can be incorporated into a plasmid, chromosome,IFF10151-WO-PCTmitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, DNA constructs also include a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. In certain embodiments, a DNA construct of the disclosure comprises a selective marker and an inactivating chromosomal or gene or DNA segment as defined herein.

[0087] As used herein, a “targeting vector” is a vector that includes polynucleotide sequences that are homologous to a region in the chromosome of a host cell into which the targeting vector is transformed and that can drive homologous recombination at that region. For example, targeting vectors find use in introducing mutations into the chromosome of a host cell through homologous recombination. In some embodiments, the targeting vector comprises other non-homologous sequences, e.g., added to the ends (i.e., stuffer sequences or flanking sequences). The ends can be closed such that the targeting vector forms a closed circle, such as, for example, insertion into a vector. For example, in certain embodiments, a parental B. licheniformis (host) cell is modified (e.g., transformed) by introducing therein one or more “targeting vectors”.

[0088] As used herein, the term “protein of interest” or “POI” refers to a polypeptide of interest that is desired to be expressed in a modified (recombinant) Gram-positive host cell, wherein the POI is preferably expressed at increased levels (i.e., relative to the “unmodified” (parental or control) cell). Thus, as used herein, a POI may be an enzyme, a substrate -binding protein, a surface-active protein, a structural protein, a receptor protein, and the like. In certain embodiments, a modified cell of the disclosure produces an increased amount of a heterologous protein of interest relative to the control cell. In particular embodiments, an increased amount of a protein of interest produced by a modified cell of the disclosure is at least a 0.5% increase, at least a 1.0% increase, at least a 5.0% increase, or a greater than 5.0% increase, relative to the control cell.

[0089] Similarly, as defined herein, a “gene of interest” or “GOI” refers a nucleic acid sequence (e.g., a polynucleotide, a gene or an ORF) which encodes a POI. A “gene of interest” encoding a “protein of interest” may be a naturally occurring gene, a mutated gene or a synthetic gene.

[0090] As used herein, the terms “polypeptide” and “protein” are used interchangeably, and refer to polymers of any length comprising amino acid residues linked by peptide bonds. The conventional one (1) letter or three (3) letter codes for amino acid residues are used herein. The polypeptide may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term polypeptide also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within theIFF10151-WO-PCTdefinition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0091] In certain embodiments, a gene of the instant disclosure encodes a commercially relevant industrial protein of interest, such as an enzyme (e.g., a acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, P-galactosidases, a-glucanases, glucan lysases, endo-P-glucanases, glucoamylases, glucose oxidases, a- glucosidases, P-glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, peptidases, rhamno-galacturonases, ribonucleases, transferases, transport proteins, transglutaminases, xylanases, hexose oxidases, and combinations thereof).

[0092] As used herein, a “variant” polypeptide refers to a polypeptide that is derived from a parent (or reference) polypeptide by the substitution, addition, or deletion of one or more amino acids, typically by recombinant DNA techniques. Variant polypeptides may differ from a parent polypeptide by a small number of amino acid residues and may be defined by their level of primary amino acid sequence homology / identity with a parent (reference) polypeptide.

[0093] In certain one or more embodiments, variant polypeptides have at least about 40% to about 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity with a parent (reference) polypeptide sequence.

[0094] As used herein, a “variant” polynucleotide refers to a polynucleotide having a specified degree of sequence homology / identity with a parent (or reference) polynucleotide, or hybridizes with a parent polynucleotide (or a complement thereof) under stringent hybridization conditions. In certain one or more embodiments, variant polynucleotides of the disclosure comprise at least about 40% to at least about 100% nucleotide sequence identity with a parent (reference) polynucleotide sequence. In certain other embodiments, a variant polynucleotide comprises at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity with a parent (reference) polynucleotide sequence.

[0095] As used herein, a “mutation” refers to any change or alteration in a nucleic acid sequence. Several types of mutations exist, including point mutations, deletion mutations, silent mutations, frame shiftIFF10151-WO-PCTmutations, splicing mutations and the like. Mutations may be performed specifically (e.g., via site directed mutagenesis) or randomly (e.g., via chemical agents, passage through repair minus bacterial strains).

[0096] As used herein, in the context of a polypeptide or a sequence thereof, the term “substitution” means the replacement (i.e., substitution) of one amino acid with another amino acid.

[0097] As used herein, the term “homology” relates to homologous polynucleotides or polypeptides. If two or more polynucleotides or two or more polypeptides are homologous, this means that the homologous polynucleotides or polypeptides have a “degree of identity” of at least 60%, more preferably at least 70%, even more preferably at least 85%, still more preferably at least 90%, more preferably at least 95%, and most preferably at least 98%. Whether two polynucleotide or polypeptide sequences have a sufficiently high degree of identity to be homologous as defined herein, can suitably be investigated by aligning the two sequences using a computer program known in the art, such as “GAP” provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman and Wunsch, (1970). Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

[0098] As used herein, the term “percent (%) identity” refers to the level of nucleic acid or amino acid sequence identity between the nucleic acid sequences that encode a polypeptide or the polypeptide's amino acid sequences, when aligned using a sequence alignment program.

[0099] As used herein, “specific productivity” is total amount of protein produced per cell per time over a given time period.

[0100] As used herein, the terms “purified”, “isolated” or “enriched” are meant that a biomolecule (e.g., a polypeptide or polynucleotide) is altered from its natural state by virtue of separating it from some, or all of, the naturally occurring constituents with which it is associated in nature. Such isolation or purification may be accomplished by art-recognized separation techniques such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulphate precipitation or other protein salt precipitation, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis or separation on a gradient to remove whole cells, cell debris, impurities, extraneous proteins, or enzymes undesired in the final composition. It is further possible to then add constituents to a purified or isolated biomolecule composition which provide additional benefits, for example, activating agents, anti-inhibition agents, desirable ions, compounds to control pH or other enzymes or chemicals.IL RECOMBINANT BACILLUS CELLS FOR THE ENHANCED PRODUCTION OF PROTEINS OF INTERESTIFF10151-WO-PCT

[0101] As generally understood, the htrA and htrB genes of Bacillus encode membrane bound (anchored) proteases (HtrA and HtrB), wherein the HtrA and HtrB protease domains are located on the trans side of the membrane and believed to play a role in protein quality control (Harwood and Kikuchi, 2022). As contemplated and described herein, Applicant designed, constructed and screened recombinant (modified) Bacillus strains (cells) deficient in the production the native HtrB protease. More specifically, as set forth in the Examples below, Applicant constructed three (3) distinct modified Bacillus sp. strains (i.e., AP490, AP491 and AP503) comprising (a) an inactivated wild-type (endogenous) htrB gene, (b) one or more introduced cassettes encoding a protease of interest and (c) an introduced htrB cassette constitutively expressing varying amounts of HtrB. The three strains constructed were subsequently tested via small-scale assays for up to about 46 hours and compared to a control strain, wherein the control strain comprises one or more introduced cassettes encoding the protease of interest, but does not comprise the introduced htrB cassette constitutively expressing varying amounts of HtrB or an inactivated wild-type (endogenous) htrB gene. In particular, as presented in TABLE 1-3 (Example 1), a significant beneficial effect was not detected in two of the three strains constructed (i.e., AP490 and AP491). However, the AP503 strain surprisingly outperformed the control strain when comparing protease production measured by an AAPF activity assay (TABLE 2) and relative productivity (TABLE 3). In particular, the improved AP503 strain demonstrated at least about 5% higher protease product ivity than the control strain.|0102| Based on the foregoing, certain embodiments of the disclosure are directed to provide, inter alia, recombinant Bacillus sp. cells capable expressing / producing increased amounts of proteins of interest, recombinant Bacillus sp. cells comprising introduced nucleic acids encoding proteins of interest, recombinant Bacillus sp. cells comprising a deleted endogenous (wild-type) htrB gene, recombinant Bacillus sp. cells comprising an introduced htrB expression cassette comprising a heterologous promoter driving the expression of the htrB gene coding sequence, methods and coniposilions for fermenting / cultivating / growing recombinant Bacillus sp. cells for the expression / production heterologous proteins of interest, and the like.

[0103] As used herein, phrases such as “inactivated wild-type htrB gene” and / or “inactivated endogenous htrB gene” refer to the wild-type htrB gene present in Bacillus sp. cells, which endogenous htrB gene has been genetically modified herein to render recombinant (modified) cells thereof deficient in expression / production of the functional HtrB protein.

[0104] As used herein, the phrase “deficient in expression / production of a functional HtrB protein” means a modified cell that produces less than no detectable HtrB protein activity, which may be determined using methods described known in the art. . In certain embodiments, modified Bacillus sp. cells are at least about 95% to 100% deficient in production of the HtrB protein as compared to control (isogenic) cells which do not comprise the inactivated htrB gene. In other embodiments, the inactivated htrB gene comprises at leastIFF10151-WO-PCTabout 75%, 80%, 81%, 82%, 83%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the htrB gene ORF of SEQ ID NO: 2.

[0105] In certain embodiments, modified Bacillus sp. cells comprise an introduced htrB expression cassette comprising an upstream (5') heterologous promoter operably linked to a downstream 5'-UTR having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 operably linked to a downstream htrB ORF encoding a HtrB protein. In certain embodiments, the downstream htrB ORF comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2. In certain other embodiments, the introduced htrB expression cassette comprises a transcriptional terminator sequence positioned downstream and operably linked to the htrB ORF. In particular embodiments, the transcriptional terminator sequence comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6.

[0106] Likewise, certain other embodiments provide protease of interest expression cassettes. In certain one or more embodiments, the protease of interest subtilisin. In certain embodiments, the mature subtilisin comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 26. III. MOLECULAR BIOLOGY

[0107] As generally set forth above, certain embodiments of the disclosure are related to nucleic acids, polynucleotides, vectors, expression constructs and the like. More particularly, one or more nucleic acid sequences of the disclosure can be generated by using any suitable synthesis, manipulation, and / or isolation techniques, or combinations thereof.

[0108] For example, one or more polynucleotides described herein may be produced using standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques that are well-known to those skilled in the art. In such techniques, fragments of up to fifty (50) or more nucleotide bases are typically synthesized, then joined (e.g., by enzymatic or chemical ligation methods) to form essentially any desired continuous nucleic acid sequence. The synthesis of the one or more polynucleotide described herein can be also facilitated by any suitable method known in the art, including but not limited to chemical synthesis using the classical phosphoramidite method and automated synthetic methods. One or more polynucleotides described herein can also be produced by using an automatic DNA synthesizer. Customized nucleic acids can be ordered from a variety of commercial sources (e.g., ATUM (DNA 2.0), Newark, CA, USA; Life Tech (GeneArt), Carlsbad, CA, USA; GenScript, Ontario, Canada; Base Clear B. V., Leiden, Netherlands; Integrated DNA Technologies, Skokie, IL, USA; Ginkgo Bioworks (Gen9), Boston, MA, USA; and Twist Bioscience, San Francisco, CA, USA). Other techniques for synthesizing nucleic acids and related principles are described and known in the art.IFF10151-WO-PCT

[0109] Recombinant DNA techniques useful in modification of nucleic acids are well known in the art, such as, for example, restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more polynucleotides described herein may also be obtained by screening cDNA libraries using one or more oligonucleotide probes that can hybridize to or PCR-amplify polynucleotides which encode one or more variants described herein. Procedures for screening and isolating cDNA clones and PCR amplification procedures are well known to those of skill in the art and described in standard references known to those skilled in the art. One or more polynucleotides described herein can be obtained by altering a naturally occurring polynucleotide backbone (e.g., that encodes one or more valiant pro-region sequences described herein) by, for example, a known mutagenesis procedure (e.g., site-directed mutagenesis, site saturation mutagenesis, and in vitro recombination). A variety of methods are known in the art that are suitable for generating modified polynucleotides described herein that encode one or more variants described herein, including, but not limited to, for example, sitesaturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.

[0110] As generally set forth above and further described below in the Examples, certain embodiments of the disclosure are related to recombinant (modified) Gram-positive cells capable of producing increased amounts of heterologous proteins of interest. Certain embodiments are therefore related to methods for constructing such recombinant Gram-positive cells having increased protein production capabilities. In certain embodiments, one or more expression cassettes encoding a protein of intertest are introduced into Gram-positive cells of the disclosure. In exemplary embodiments, the cassettes are integrated into the genome of the cell. Thus, certain embodiments are related to nucleic acid molecules, polynucleotides (e.g., vectors, plasmids, expression cassettes), regulatory elements, and the like, suitable for use in constructing recombinant (modified) Gram-positive host cells.

[0111] Accordingly, as presented in the Examples and generally described herein, recombinant cells of the disclosure may be constructed by one of skill using standard and routine recombinant DNA and molecular cloning techniques well known in the art. Methods for genetic modification include, but are not limited to, (a) the introduction, substitution, or removal of one or more nucleotides in a gene, or the introduction, substitution, or removal of one or more nucleotides in a regulatory element required for the transcription or translation of the gene, (b) a gene disruption, (c) a gene conversion, (d) a gene deletion, (e) a gene downregulation, (f) site specific mutagenesis and / or (g) random mutagenesis.

[0112] In certain embodiments, modified cells of the disclosure may be constructed by reducing or eliminating the expression of a gene, using methods well known in the art, for example, insertions,IFF10151-WO-PCTdisruptions, replacements, or deletions. The portion of the gene to be modified or inactivated may be, for example, the coding region or a regulatory element required for expression of the coding region.

[0113] An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, (z.e., a part which is sufficient for affecting expression of the nucleic acid sequence). Other control sequences for modification include, but are not limited to, a leader sequence, a pro-peptide sequence, a signal sequence, a transcription terminator, a transcriptional activator and the like.

[0114] In certain other embodiments a modified cell is constructed by gene deletion to eliminate or reduce the expression of the gene. Gene deletion techniques enable the partial or complete removal of the gene(s), thereby eliminating their expression, or expressing a non-functional (or reduced activity) protein product. In such methods, the deletion of the gene(s) may be accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3’ regions flanking the gene. The contiguous 5' and 3' regions may be introduced into a cell, for example, on a temperature- sensitive plasmid in association with a second selectable marker at a permissive temperature to allow the plasmid to become established in the cell. The cell is then shifted to a non-permissive temperature to select for cells that have the plasmid integrated into the chromosome at one of the homologous flanking regions. Selection for integration of the plasmid is affected by selection for the second selectable marker. After integration, a recombination event at the second homologous flanking region is stimulated by shifting the cells to the permissive temperature for several generations without selection. The cells are plated to obtain single colonies and the colonies are examined for loss of both selectable markers. Thus, a person of skill in the art may readily identify nucleotide regions in the gene’s coding sequence and / or the gene’s non-coding sequence suitable for complete or partial deletion.

[0115] In other embodiments, a modified cell is constructed by introducing, substituting, or removing one or more nucleotides in the gene or a regulatory element required for the transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a frame-shift of the open reading frame. Such a modification may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Thus, in certain embodiments, a gene of the disclosure is inactivated by complete or partial deletion.

[0116] In another embodiment, a modified cell is constructed by the process of gene conversion. For example, in the gene conversion method, a nucleic acid sequence corresponding to the gene(s) is mutagenized in vitro to produce a defective nucleic acid sequence, which is then transformed into the parental cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. It may be desirable that the defective gene or gene fragment also encodes a marker which may be used for selection of transformants containing the defective gene. ForIFF10151-WO-PCTexample, the defective gene may be introduced on a non-replicating or temperature-sensitive plasmid in association with a selectable marker. Selection for integration of the plasmid is affected by selection for the marker under conditions not permitting plasmid replication. Selection for a second recombination event leading to gene replacement is affected by examination of colonies for loss of the selectable marker and acquisition of the mutated gene. Alternatively, the defective nucleic acid sequence may contain an insertion, substitution, or deletion of one or more nucleotides of the gene, as described below.

[0117] In other embodiments, a modified cell is constructed by established anti-sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the gene. More specifically, expression of the gene by a Gram-positive cell may be reduced (down-regulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary ann-sensc nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated. Such anti-sense methods include, but are not limited to RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, and the like, all of which are well known to the skilled artisan.

[0118] In other embodiments, a modified cell is produced / constructed via CRISPR-Cas9 editing. For example, a gene encoding a protein of interest can be edited or disrupted (or deleted or down-regulated) by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g., Cas9) and Cpfl or a guide DNA (e.g., NgAgo), which recruits the endonuclease to the target sequence on the DNA, wherein the endonuclease can generate a single or double stranded break in the DNA. This targeted DNA break becomes a substrate for DNA repair, and can recombine with a provided editing template to disrupt or delete the gene. For example, the gene encoding the nucleic acid guided endonuclease (for this purpose Cas9 from S. pyogenes) or a codon optimized gene encoding the Cas9 nuclease is operably linked to a promoter active in the Gram-positive cell and a terminator active in Grampositive cells, thereby creating a Gram-positive cell Cas9 expression cassette. l ikewise, one or more target sites unique to the gene of interest are readily identified by a person skilled in the art. For example, to build a DNA construct encoding a gRNA -directed to a target site within the gene of interest, the variable targeting domain (VT) will comprise nucleotides of the target site which are 5' of the (PAM) proto-spacer 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 a VT domain and the DNA encoding the CER domain thereby generate a DNA encoding a gRNA. Thus, a Gram-positive expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in Gram-positive cells and a terminator active in Gram-positive cells.IFF10151-WO-PCT

[0119] In certain embodiments, the DNA break induced by the endonuclease is repaired / replaced with an incoming sequence. For example, to precisely repair the DNA break generated by the Cas9 expression cassette and the gRNA expression cassette described above, a nucleotide editing template is provided, such that the DNA repair machinery of the cell can utilize the editing template. For example, about 500bp 5' of targeted gene can be fused to about 500bp 3' of the targeted gene to generate an editing template, which template is used by the Gram-positive host’s machinery to repair the DNA break generated by the RGEN.

[0120] The Cas9 expression cassette, the gRNA expression cassette and the editing template can be codelivered to filamentous fungal cells using many different methods (e.g., protoplast fusion, electroporation, natural competence, or induced competence). The transformed cells are screened by PCR amplifying the target gene locus, by amplifying the locus with a forward and reverse primer. These primers can amplify the wild-type locus or the modified locus that has been edited by the RGEN. These fragments are then sequenced using a sequencing primer to identify edited colonies.

[0121] In yet other embodiments, a modified cell is constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis and transposition. Modification of the gene may be performed by subjecting the parental cell to mutagenesis and screening for mutant cells in which expression of the gene has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods.

[0122] Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), N-methyl-N’ -nitrosoguanidine (NTG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the parental cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutant cells exhibiting reduced or no expression of the gene.

[0123] PCT Publication No. W02003 / 083125 discloses methods for modifying Gram-positive (Bacillus) cells, such as the creation of Bacillus deletion strains and DNA constructs using PCR fusion to bypass E. coli. PCT Publication No. W02002 / 14490 discloses methods for modifying Bacillus cells including (1) the construction and transformation of an integrative plasmid (pComK), (2) random mutagenesis of coding sequences, signal sequences and pro-peptide sequences, (3) homologous recombination, (4) increasing transformation efficiency by adding non-homologous flanks to the transformation DNA, (5) optimizing double cross-over integrations, (6) site directed mutagenesis and (7) marker-less deletion.IFF10151-WO-PCT

[0124] Those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., Gram-negative cells, Gram-positive cells). Indeed, such methods as transformation including protoplast transformation and congression, transduction, and protoplast fusion are known and suited for use in the present disclosure. Methods of transformation are particularly preferred to introduce a DNA construct of the present disclosure into a host cell.

[0125] In addition to commonly used methods, in some embodiments, host cells are directly transformed (z.e., an intermediate cell is not used to amplify, or otherwise process, the DNA construct prior to introduction into the host cell). Introduction of the DNA construct into the host cell includes those physical and chemical methods known in the ail to introduce DNA into a host cell, without insertion into a plasmid or vector. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, liposomes and the like. In additional embodiments, DNA constructs are co-transformed with a plasmid without being inserted into the plasmid. In further embodiments, a selective marker is deleted or substantially excised from the modified Bacillus strain by methods known in the art. In some embodiments, resolution of the vector from a host chromosome leaves the flanking regions in the chromosome, while removing the indigenous chromosomal region.

[0126] Promoters and promoter sequence regions for use in the expression of genes, coding sequences (CDS), open reading frames (ORFs) and / or variant sequences thereof in Gram-positive cells are generally known on one of skill in the art. Promoter sequences of the disclosure are generally chosen so that they are functional in the Gram-positive cells. 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 a-amylase promoter (amyE) of B. subtilis, the a-amylase promoter (amyE) of B. licheniformis, the a-amylase promoter of B. amyloliquefaciens, the neutral 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 creating promoter libraries with a range of activities (promoter strength) in Bacillus cells is describe in Publication No. W02002 / 14490.IV. PROTEINS OF INTEREST

[0127] A protein of interest (POI) of the instant disclosure can be any endogenous or heterologous protein, and it may be a variant of such a POI. The protein can contain one or more disulfide bridges or is a protein whose functional form is a monomer or a multimcr, i.e., the protein has a quaternary structure and is composed of a plurality of identical (homologous) or non-identical (heterologous) subunits, wherein the POI or a variant POI thereof is preferably one with properties of interest.

[0128] For example, in certain embodiments, a modified Gram-positive cell of the disclosure produces at least about 0.1% more, at least about 0.5% more, at least about 1% more, at least about 5% more, at leastIFF10151-WO-PCTabout 6% more, at least about 7% more, at least about 8% more, at least about 9% more, or at least about 10% or more of a POI, relative to its unmodified (reference or control) cell.

[0129] In certain embodiments, a modified Gram-positive cell of the disclosure exhibits an increased specific productivity (Qp) of a POI relative the control cell. For example, the detection of specific productivity (Qp) is a suitable method for evaluating protein production. The specific productivity (Qp) can be determined using the following equation:“Qp = gP / gDCW*hr”wherein, “gP” is grams of protein produced in the tank; “gDCW” is grams of dry cell weight (DCW) in the tank and “hr” is I'ermenlalion time in hours from the time of inoculation, which includes the time of production as well as growth time.

[0130] Thus, in certain other embodiments, a modified Gram-positive cell of the disclosure comprises a specific productivity (Qp) increase of at least about 0.1 %, at least about 1 %, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% or more, relative to the unmodified (parental) cell.

[0131] In certain embodiments, a POI or a variant POI thereof is selected from the group consisting of acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, |3-galactosidases, a-glucanases, glucan lysases, endo-P-glucanases, glucoamylases, glucose oxidases, a-glucosidases, [3-glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, ligases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, peptidases, rhamno-galacturonases, ribonucleases, transferases, transport proteins, transglutaminases, xylanases, hexose oxidases, and combinations thereof.

[0132] Thus, in certain embodiments, a POI or a variant POI thereof is an enzyme selected from Enzyme Commission (EC) Number EC 1, EC 2, EC 3, EC 4, EC 5, or EC 6.

[0133] There are various assays known to those of ordinary skill in the art for detecting and measuring activity of intracellularly and extracellularly expressed proteins.V. FERMENTING GRAM-POSITIVE CELLS FOR THE PRODUCTION OF PROTEINS

[0134] As generally described above, certain embodiments are related to compositions and methods for constructing and obtaining Gram-positive cells having increased protein production phenotypes. Thus, certain embodiments are related to methods of producing proteins of interest in Gram-positive cells byIFF10151-WO-PCTfermenting the cells in a suitable medium. Fermentation methods well known in the art can be applied to ferment Gram-positive cells of the disclosure.

[0135] In some embodiments, the cells are cultured under batch or continuous fermentation conditions. A classical batch fermentation is a closed system, where the composition of the medium is set at the beginning of the fermentation and is not altered during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism(s). In this method, fermentation is permitted to occur without the addition of any components to the system. Typically, a batch fermentation qualifies as a “batch” with respect to the addition of the carbon source, and attempts are often made to control factors such as pH and oxygen concentration. The metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped. Within typical batch cultures, cells can progress through a static lag phase to a high growth log phase, and finally to a stationary phase, where growth rate is diminished or halted. If untreated, cells in the stationary phase eventually die. In general, cells in log phase are responsible for the bulk of production of product.

[0136] A suitable variation on the standard batch system is the “fed-batch” fermentation system. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression likely inhibits the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors, such as pH, dissolved oxygen and the partial pressure of waste gases, such as CO2. Batch and fed-batch fermentations are common and known in the art.

[0137] Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density, where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, a limiting nutrient, such as the carbon source or nitrogen source, is maintained at a fixed rate and all other parameters are allowed to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the ail of industrial microbiology.

[0138] In certain embodiments, a protein of interest cxprcsscd / produccd by a Gram-positive cell of the disclosure may be recovered from the culture medium by conventional procedures including separating theIFF10151-WO-PCThost cells from the medium by centrifugation or filtration, or if necessary, disrupting the cells and removing the supernatant from the cellular fraction and debris. Typically, after clarification, the proteinaceous components of the supernatant or filtrate are precipitated by means of a salt, e.g., ammonium sulfate. The precipitated proteins are then solubilized and may be purified by a variety of chromatographic procedures, e.g., ion exchange chromatography, gel filtration.

[0139] In some embodiments, the cells are cultured under batch or continuous fermentation conditions. A classical batch fermentation is a closed system, where the composition of the medium is set at the beginning of the fermentation and is not altered during the fermentation. At the beginning of the fermentation, the medium is inoculated with the desired organism(s). In this method, fermentation is permitted to occur without the addition of any components to the system. Typically, a batch fermentation qualifies as a “batch” with respect to the addition of the carbon source, and attempts are often made to control factors such as pH and oxygen concentration. The metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped. Within typical batch cultures, cells can progress through a static lag phase to a high growth log phase, and finally to a stationary phase, where growth rate is diminished or halted. If untreated, cells in the stationary phase eventually die. In general, cells in log phase are responsible for the bulk of production of product.

[0140] A suitable variation on the standard batch system is the “fed-batch” fermentation system. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression likely inhibits the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors, such as pH, dissolved oxygen, and the partial pressure of waste gases, such as CO2. Batch and fed-batch fermentations are common and known in the art.

[0141] Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density, where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, a limiting nutrient, such as the carbon source or nitrogen source, is maintained at a fixed rate and all other parameters are allowed to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors forIFF10151-WO-PCTcontinuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[0142] In certain embodiments, a protein of interest expressed / produced by a Gram-positive cell of the disclosure may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, or if necessary, disrupting the cells and removing the supernatant from the cellular fraction and debris. Typically, after clarification, the proteinaceous components of the supernatant or filtrate are precipitated by means of a salt, e.g.. ammonium sulfate. The precipitated proteins are then solubilized and may be purified by a variety of chromatographic procedures, e.g., ion exchange chromatography, gel filtration.VI. EXEMPLARY EMBODIMENTS

[0143] Non-limiting embodiments of the disclosure include, but are not limited to:

[0144] 1. A method for the enhanced production of a protease in a Bacillus sp. cell comprising: constructing (or obtaining) a Bacillus sp. expressing a protease of interest, modifying the cell by inactivating the wild-type (endogenous) htrB gene present in the cell, introducing a synthetic htrB expression cassette into the cell, and fermenting the cell the modified cell under conditions for the production of the protease of interest.

[0145] 2. The method of embodiment 1, wherein the modified cell produces an increased amount of the protease relative to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell expresses the same protease of interest and has the same inactivated wild-type htrB gene, but does not comprise the introduced synthetic htrB cassette.

[0146] 3. The method of embodiment 1, wherein the introduced htrB cassette comprises an upstream constitutive promoter operably linked to a downstream 5 '-untranslated region (5'-UTR) having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 operably linked to a downstream htrB open reading frame (ORF).

[0147] 4. The method of embodiment 3, wherein the htrB cassette further comprises a transcriptional terminator sequence positioned downstream and operably linked to the ORF.

[0148] 5. The method of embodiment 4, wherein the transcriptional terminator sequence comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6.

[0149] 6. The method of embodiment 3, wherein the htrB ORF comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2.

[0150] 7. The method of embodiment 3, wherein the wherein the htrB ORF encodes an HtrB protein comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 24.IFF10151-WO-PCT

[0151] 8. The method of embodiment 1, wherein the inactivated wild-type (endogenous) htrB gene comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the htrB ORF of SEQ ID NO: 2.

[0152] 9. The method of embodiment 1, wherein the protease of interest is a subtilisin.

[0153] 10. The method of embodiment 9, wherein the subtilisin comprises a mature amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 26.

[0154] 11. The method of embodiment 1 , wherein the protease of interest is secreted into the fermentation broth.

[0155] 12. The method of embodiment 2, wherein the modified cell produces at least about 5% or more of the protease of interest as compared to the control cell.

[0156] 13. The method of embodiment 1, wherein the Bacillus sp. cell is a B. subtilis cell or a B. licheniformis cell.

[0157] 14. A modified Bacillus sp. cell comprising an inactivated wild-type htrB gene and an introduced htrB expression cassette, wherein the htrB cassette comprises an upstream promoter operably linked to a downstream 5'-UTR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 operably linked to a downstream htrB open reading frame (ORF).|0158| 15. The modified cell of embodiment 14, comprising at least one, or at least two introduced expression cassettes encoding a protease of interest.

[0159] 16. The modified cell of embodiment 14, wherein the htrB cassette comprises a transcriptional terminator sequence positioned downstream and operably linked to the htrB ORF.

[0160] 17. The modified cell of embodiment 16, wherein the transcriptional terminator sequence comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6.

[0161] 18. The modified cell of embodiment 14, wherein the htrB ORF comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2.

[0162] 19. The modified cell of embodiment 14, wherein the htrB ORF encodes an HtrB protein having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 24.

[0163] 20. The modified cell of embodiment 14, wherein the inactivated wild-type htrB gene comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the htrB ORF of SEQ ID NO: 2.

[0164] 21. The modified cell of embodiment 15, wherein protease of interest is a subtilisin.

[0165] 22. The modified cell of embodiment 15, wherein the cassette, or cassettes encoding the protease of interest comprise an upstream (heterologous) promoter operably linked to a downstream polynucleotideIFF10151-WO-PCTencoding a signal (pre) sequence operably linked a downstream polynucleotide encoding a pro-region sequence operably linked a downstream polynucleotide encoding a mature protease of interest.

[0166] 23. The modified cell of embodiment 22, wherein the polynucleotide encoding the signal sequence comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 17.

[0167] 24. The modified cell of embodiment 22, wherein the polynucleotide encoding the pro-region sequence comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 21.

[0168] 25. The modified cell of embodiment 22, wherein the polynucleotide encoding a mature protease encodes a mature subtilisin.

[0169] 26. The modified cell of embodiment 25, wherein the polynucleotide comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 22.

[0170] 27. The modified cell of embodiment 25, wherein the subtilisin is a native subtilisin, or a functional subtilisin variant thereof, comprising 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 SEQ ID NO: 26.

[0171] 30. The modified cell of embodiment 15, producing at least about 5% or more of the protease as compared to a control cell fermented under the same conditions, wherein the control cell expresses the same protease of interest and has the same inactivated wild-type htrB gene, but does not comprise the introduced synthetic htrB cassette.EXAMPLES

[0172] It should be understood that the following Examples, while indicating embodiments of the disclosure, are given by way of illustration only. From the above discussion and these Examples, one of skill in the art can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Such modifications are also intended to fall within the scope of the claimed invention. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).EXAMPLE 1EXPRESSION OF PROTEASE

[0173] In the present example, Applicant engineered three distinct strains designed to constitutively express varying amounts of htrB in addition to a protease molecule of interest. More particularly, all three strains comprise two introduced expression cassettes encoding an industrially relevant protease molecule. Likewise, the native htrB gene was inactivated in all three strains by the insertion of three stop codons (i.e., “TAATGATAA”) after the first twelve codons in the coding region of the htrB gene (FIG. 1). AdditionalIFF10151-WO-PCTmutations were made to introduce the htrB 5' untranslated region (5'-UTR; SEQ ID NO: 1) and the htrB coding sequence (CDS; SEQ ID NO: 2) under the control of one of three promoters, including a native B. subtilis fbp promoter sequence (SEQ ID NO: 3), a native B. subtilis yqgS promoter sequence (SEQ ID NO: 4), or a native B. subtilis rpoB promoter sequence (SEQ ID NO: 5) at the ppsC gene locus (FIG. 2).All constructs included the spoVG transcriptional terminator (SEQ ID NO: 6) operably linked to the 3' end of the htrB gene sequence at the ppsC locus. These three different promoter sequences were used to create three unique constructs and three unique strains, AP490, AP491 and AP503 which respectively carry the rpoB construct, / bp construct, and yqgS construct.

[0174] More specifically, the three strains were tested small-scale assay for up to 45.75 hours and compared to the control strain, which is genetically identical (isogenic) except for the modifications to the htrB expression. For instance, as presented below in TABLES 1-3, a significant beneficial effect was not detectable in two of the three strains (z.e., strains AP490, AP491). However, as shown in TABLES 1-3, the AP503 strain significantly outperformed the control strain when comparing protease expression as measured by an AAPF activity assay (TABLE 2), and productivity (TABLE 3), as measured by activity per optical density (OD) at 600 nm wavelength (OD600). More particularly, the improved AP503 strain exhibited between about 8% and 31% higher productivity than the control strain as a result of the inactivation of the native htrB gene and the introduction of the yqgS construct (i.e., comprising an upstream (5') yqgS constitutive promoter sequence operably linked to a downstream htrB 5'-UTR sequence operably linked to a downstream htrB CDS integrated at the ppsC locus.TABLE 1OD600 OF STRAINS AP490, AP490, AP503 RELATIVE TO CONTROL STRAINIFF10151-WO-PCTTABLE 2AAPF ACTIVITY OF STRAINS AP490, AP490, AP503 RELATIVE TO CONTROL STRAINTABLE 3PRODUCTIVITY OF STRAINS AP490, AP490, AP503 RELATIVE TO CONTROL STRAINIFF10151-WO-PCTREFERENCES PCT Publication No. W02002 / 14490PCT Publication No. W02003 / 083125PCT Publication No. WO2010 / 056634PCT Publication No. WO2011 / 130222PCT Publication No. WO2015 / 089447PCT Publication No. WO2016 / 202839PCT Publication No. WO2017 / 207762PCT Publication No. WO2023 / 114936Harwood and Kikuchi, “The ins and outs of Bacillus proteases: activities, functions and commercial significance”, FEMS Microbiology Reviews, Vol. 46, No. 1, 2022.Ausubel et al., “Current Protocols in Molecular Biology”, published by Greene Publishing Assoc, and Wiley -Inter science (1987).Brode et al., “Subtilisin BPN' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity”, Biochemistry, 35(10):3162-3169, 1996.Caspers el al., “Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide”, Appl. Microbiol. Biotechnol., 86(6): 1877-1885, 2010.Earl et al., “Ecology and genomics of Bacillus subtilis”, Trends in Microbiology., 16(6):269-275, 2008.Olempska-Beer et al., “Food-processing enzymes from recombinant microorganisms-a review”’ Regul. Toxicol. Pharmacol., 45(2): 144-158, 2006.Raul et al., “Production and partial purification of alpha amylase from Bacillus subtilis (MTCC 121) using solid state fermentation”, Biochemistry Research International, 2014.Sambrook et al., “Molecular Cloning: A Laboratory Manual” Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), (2001) and (2012).Van Dijl and Hecker, “Bacillus subtilis: from soil bacterium to super- secreting cell factory”, Microbial Cell Factories, 12(3). 2013.

Claims

IFF10151-WO-PCTCLAIMS1. A modified Bacillus sp. cell comprising an inactivated wild-type htrB gene and an introduced htrB expression cassette, wherein the htrB cassette comprises an upstream heterologous promoter operably linked to a downstream 5 '-untranslated region (5'-UTR) operably linked to a downstream htrB open reading frame (ORF).

2. The modified cell of claim 1, comprising at least one, or at least two, introduced expression cassettes encoding a protease of interest.

3. The modified cell of claim 1, wherein the htrB ORF comprises at least about 90% identity to SEQ ID NO: 2.

4. The modified cell of claim 1 , wherein the inactivated wild-type htrB gene comprises at least about 90% identity to SEQ ID NO: 2.

5. The modified cell of claim 2, wherein protease of interest is a subtilisin.

6. The modified cell of claim 2, producing at least about 5% or more of the protease relative to a control cell fermented under the same conditions, wherein the control cell expresses the same protease of interest and has the same inactivated wild-type htrB gene, but does not comprise the introduced htrB cassette.

7. A method for the enhanced production of a protease in a Bacillus sp. cell comprising constructing a Bacillus sp. expressing a protease of interest, modifying the cell by inactivating the wild-type htrB, introducing a htrB expression cassette into the cell, and fermenting the modified cell under conditions for the production of the protease of interest.

8. The method of claim 7, wherein the modified cell produces an increased amount of the protease relative to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell expresses the same protease of interest and has the same inactivated wild-type htrB gene, but does not comprise the introduced htrB cassette.

9. The method of claim 7, wherein the introduced htrB cassette comprises an upstream heterologous promoter operably linked to a downstream 5 '-untranslated region (5'-UTR) operably linked to a downstream htrB open reading frame (ORF).

10. The method of claim 7, wherein the htrB ORF comprises at least about 90% identity to SEQ ID NO: 2.

11. The method of claim 7, wherein the inactivated wild-type htrB gene comprises at least 90% identity to SEQ ID NO: 2.IFF10151-WO-PCT12. The method of claim 7, wherein the protease of interest is a subtilisin.

13. The method of claim 7, wherein the protease of interest is secreted into the fermentation broth.

14. The method of claim 8, wherein the modified cell produces at least about 5 % or more of the protease relative to the control cell.

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