Intracellular co-expression of protease inhibitor in bacillus improves cell viability and enhances protease production

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, novel protein signal sequences, recombinant polynucleotides encoding heterologous proteins of interest, and related compositions and / or methods thereof. Thus, 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

IFF10126-WO-PCTINTRACELLULAR CO-EXPRESSION OF PROTEASE INHIBITOR IN BACILLUS IMPROVES CELL VIABILITY AND ENHANCES PROTEASE PRODUCTIONFIELD

[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 recombinant Bacillus cells having enhanced protease production capabilities.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims benefit to U.S. Provisional Patent Application No. 63 / 735,112, filed December 17, 2024, which is incorporated herein by referenced in its entiretyREFERENCE TO A SEQUENCE LISTING

[0003] The sequence listing text file submitted herewith contains the file “IFF10126-WO- PCT _SequenceListing.xml” created on December 16, 2024, which is 68 kilobytes (KB) 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, B. subtilis is well known for its production of a-amylases (Jensen et al., 2000; Raul et al., 2014) and proteases (Brode et al., 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). Thus, the production of proteins (e.g., enzymes, antibodies, receptors, etc.) in Bacillus sp. cells is of particular interest in the biotechnological arts. Likewise, the optimization of Bacillus host cells for the production and secretion of one or more protein(s) of interest is of high relevance, particularly in the industrial biotechnology setting, wherein small improvements in protein yield are quite significant when the protein is produced in large industrial quantities. As described hereinafter, the instant disclosure is related to the highly desirable and unmet needs for obtaining, constructing, producing and the like, genetically modified (recombinant) Bacillus sp. cells having increased protein production capabilities.IFF10126-WO-PCTSUMMARY

[0005] As generally set forth and described herein, certain embodiments of the disclosure are related compositions and methods for enhanced production of proteins of interest in Gram-positive bacterial cells. More particularly, certain one or more embodiments of the disclosure are related to, inter alia, methods and compositions for enhanced production of proteases in Bacillus sp. cells. Thus, certain embodiments provide methods for enhanced production of proteases in a Bacillus sp. cells, such methods generally comprising obtaining a Bacillus sp. cell producing a protease of interest, introducing into the cell an expression cassette encoding a protease inhibitor (polypeptide), and fermenting the modified cell under conditions for production of the protease, wherein the protease is secreted into the fermentation broth, and the protease inhibitor is retained intracellularly. In certain embodiments of the methods, the modified cell produces at least 5% or more of the protease as compared to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell expresses and secretes the same protease of intertest, but does not comprise the introduced cassette encoding the protease inhibitor.

[0006] In certain other embodiments, the disclosure provides methods for enhanced production of a protease in a Bacillus sp. cell comprising introducing into a Bacillus sp. cell an expression cassette encoding a mature protease and an expression cassette encoding a protease inhibitor polypeptide, and fermenting the modified cell under conditions for production of the protease, wherein the protease is secreted into the fermentation broth, and the protease inhibitor is retained intracellularly. In certain embodiments of the methods, the modified cell produces at least 5% or more of the protease as compared to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell comprises the same introduced cassette encoding the protease, but does not comprise the introduced cassette encoding the protease inhibitor. In other embodiments of the methods, the cell comprises at least two, three, or four introduced cassettes encoding the mature protease. In yet other embodiments of the methods, the cassette encoding the protease inhibitor (polypeptide) comprises an upstream heterologous promoter region operably linked to a polynucleotide encoding a chymotrypsin inhibitor (CI-2) polypeptide having at least 90% identity to SEQ ID NO: 6. In other embodiments of the methods, the modified cell has at least a 5% increase in biomass after 48 hours of fermentation relative to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell comprises the same introduced cassette encoding the protease, but does not comprise the introduced cassette encoding the protease inhibitor. In other embodiments of the methods, the cassettes encoding the mature protease comprises upstream and downstream flanking sequences homologous to gene loci of interest (GLOI), wherein the cassettes are integrated into the GLOI. In certain other embodiments of the methods, the mature protease comprises at least 80% identity to any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44.IFF10126-WO-PCT

[0007] Certain other embodiments of the disclosure provide, inter alia, modified Gram-positive bacterial cells co-expressing a heterologous protease and a heterologous protease inhibitor (polypeptide). Thus, certain one or more embodiments are related to modified (recombinant) Bacillus sp. cells co-expressing a heterologous protease and protease inhibitor (polypeptide), wherein the mature protease is secreted extracellularly, and the protease inhibitor is retained intracellularly, when the modified cells are fermented under conditions for the production of the protease. In other certain embodiments, the modified cell produces at least 5% or more of the protease relative to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell expresses the same protease, but does not co-express the heterologous protease inhibitor. In other one or more embodiments, the modified cell comprises at least one, two, three, or four introduced expression cassettes encoding the heterologous protease. In related embodiments, the cassettes encoding the heterologous protease are integrated into the genome of the cell. In certain other embodiments, the modified cell comprises an introduced expression cassette encoding the protease inhibitor polypeptide, wherein the cassette comprises an upstream heterologous promoter region operably linked to a polynucleotide encoding a chymotrypsin inhibitor (CI-2) polypeptide having at least about 75% to 100% identity to SEQ ID NO: 6. In certain other embodiments, the expression cassette encoding a CI-2 polypeptide comprises an upstream heterologous promoter region operably linked to a polynucleotide encoding a CI-2 polypeptide having at least about 75% to 100% identity to SEQ ID NO: 45. In yet other embodiments, the cassettes encoding the mature protease comprise a polypeptide sequence having at least 80% identity to any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44.

[0008] Other useful embodiments of the disclosure are described hereinafter, and further described in the Examples.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 shows an SDS PAGE gel of the secreted protease inhibitors. As presented in FIG. 1, the secreted protease inhibitors were analyzed by 4-12 % SDS-PAGE gels. The description of each lane is listed in the table on the right. All protease inhibitors could be successfully expressed in B. subtilis. Purified Eglin C was used as a standard to determine the concentration of all inhibitors using gel densitometry with Imagequant software

[0010] Figure 2 presents a schematic diagram of an exemplary subtilisin-chymotrypsin inhibitor (CI-2) expression cassette. As shown in FIG. 2, the cassette was constructed for intracellular overexpression of the CI-2 polypeptide, wherein the cassette comprises an upstream (5') nucleic acid (DNA) comprising a heterologous promoter sequence (FIG. 2; veg promoter, bglC promoter) operably linked to downstream DNA comprising a Shine-Dalgarno (SD) sequence (FIG. 2; grey shaded “SD”) operably linked to aIFF10126-WO-PCT downstream open reading frame (ORF) sequence (FIG. 2; black shaded “CI-2”) encoding the CI-2 polypeptide.

[0011] Figure 3 shows a Sequence Logo created by the alignment of one-hundred (100) chymotrypsin inhibitor 2 (CI-2) proteins (FIG. 3A) and a consensus CI-2 sequence derived therefrom (FIG. 3B). As presented in the consensus sequence (FIG. 3B) amino acid residues shown as “X” have no clear consensus amino acid across all sequences being compared at that specific residue location.

[0012] Figure 4 presents graphs showing the dynamic expression of veg (FIG. 4; top panel) and bglC (FIG. 4; bottom panel) in the B. subtills strains expressing proteases (i.e., BPN'-Vl, BPN'-V2, BG46-V1 and GG36-V1 proteases) throughout the fermentation. Fermentation samples from each protease production strain were collected every two hours from 8 hours to 36 hours intervals for RNA-seq analysis.BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0013] SEQ ID NO: 1 is the amino acid sequence of a Streptomyces nigrescens metalloprotease inhibitor (SMPI).|0014| SEQ ID NO: 2 is the amino acid sequence of a Streptomyces caespitosus neutral protease inhibitor (ScNPI).

[0015] SEQ ID NO: 3 is the amino acid sequence of a Hordeum vulgare wheat Subtilisin / Chymotrypsin inhibitor (WSCI).

[0016] SEQ ID NO: 4 is the amino acid sequence of a Aequorivila sublilhincola Azu-Kazal (type serine protease inhibitor).

[0017] SEQ ID NO: 5 is the amino acid sequence of a Streptomyces albogriseolus streptomyces subtilisin inhibitor (SSI).

[0018] SEQ ID NO: 6 is the amino acid sequence of a Hordeum vulgare chymotrypsin inhibitor 2 (CI-2).

[0019] SEQ ID NO: 7 is the amino acid sequence of a Hordeum vulgare bailey amylase subtilisin inhibitor(BASI).

[0020] SEQ ID NO: 8 is a DNA sequence comprising a Bacillus subtilis veg promoter.

[0021] SEQ ID NO: 9 is a DNA sequence comprising a Bacillus subtilis bglc promoter.

[0022] SEQ ID NO: 10 is a DNA sequence encoding a Bacillus amyloliquefaciens mature BPN' (subtilisin) variant named “BPN'-Vl” protease.

[0023] SEQ ID NO: 11 is a DNA sequence comprising a Bacillus subtilis P2 promoter.

[0024] SEQ ID NO: 12 is a DNA sequence comprising a Bacillus subtilis aprE 5'-UTR.

[0025] SEQ ID NO: 13 is a DNA sequence encoding a Bacillus subtilis AprE signal (peptide) sequence.

[0026] SEQ ID NO: 14 is a DNA sequence encoding a variant Bacillus amyloliquefaciens BPN' (subtilisin) pro-region.IFF10126-WO-PCT

[0027] SEQ ID NO: 15 is a DNA sequence comprising a Bacillus amyloliquefaciens transcriptional terminator.

[0028] SEQ ID NO: 16 is a DNA sequence comprising a Bacillus subtilis 5' skfA flanking region.

[0029] SEQ ID NO: 17 is a DNA sequence comprising a Bacillus subtilis 3' skfH' flanking region.

[0030] SEQ ID NO: 18 is a DNA sequence comprising a Bacillus subtilis 5' sunT flanking region.

[0031] SEQ ID NO: 19 is a DNA sequence comprising a Bacillus subtilis 3' sunT flanking region.

[0032] SEQ ID NO: 20 is a DNA sequence comprising a Bacillus subtilis 5' aprE flanking region.

[0033] SEQ ID NO: 21 is a DNA sequence comprising a Bacillus subtilis alanine racemase gene.

[0034] SEQ ID NO: 22 is a DNA sequence comprising a Bacillus subtilis 3' aprE flanking region.

[0035] SEQ ID NO: 23 is a DNA sequence comprising a Bacillus subtilis spoVG Shine-Dalgarno region.

[0036] SEQ ID NO: 24 is a DNA sequence comprising a Bacillus subtilis 5' pksL flanking region.

[0037] SEQ ID NO: 25 is a DNA sequence comprising a Bacillus subtilis 3' pksL flanking region.

[0038] SEQ ID NO: 26 is a DNA sequence encoding a Bacillus amyloliquefaciens mature BPN' (subtilisin) variant named “BPN'-V2” protease.

[0039] SEQ ID NO: 27 is a DNA sequence comprising a Bacillus subtilis synthetic PspoVG-Prml-Pscr promoter.

[0040] SEQ ID NO: 28 is a DNA sequence encoding a Bacillus amyloliquefaciens BPN' signal (peptide) sequence.

[0041] SEQ ID NO: 29 is a DNA sequence comprising a Bacillus subtilis spoVG transcriptional terminator.

[0042] SEQ ID NO: 30 is a DNA sequence comprising a Bacillus subtilis 5' ppsC flanking region.

[0043] SEQ ID NO: 31 is a DNA sequence comprising a Bacillus subtilis 3' ppsC flanking region.

[0044] SEQ ID NO: 32 is a DNA sequence encoding a variant Bacillus clausii GG36 (subtilisin) pro-region.

[0045] SEQ ID NO: 33 is a DNA sequence encoding a Bacillus gibsonii mature BG46 (subtilisin) variant named “BG46-V1” protease.

[0046] SEQ ID NO: 34 is a DNA sequence comprising a Bacillus subtilis 5' spoJIJAE flanking sequence.

[0047] SEQ ID NO: 35 is a DNA sequence comprising a Bacillus subtilis 3' spoIIIAE flanking sequence.

[0048] SEQ ID NO: 36 is a DNA sequence encoding a Bacillus gibsonii mature BG46 (subtilisin) variant named “BG46-V2” protease.

[0049] SEQ ID NO: 37 is a DNA sequence encoding a Paenibacillus hunanensis metalloprotease named “PHD22”.

[0050] SEQ ID NO: 38 is the amino acid sequence of a Bacillus amyloliquefaciens mature BPN' (subtilisin) variant named “BPN'-Vl” protease.

[0051] SEQ ID NO: 39 is the amino acid sequence of a Bacillus amyloliquefaciens mature BPN' (subtilisin) variant named “BPN'-V2” protease.IFF10126-WO-PCT

[0052] SEQ ID NO: 40 is the amino acid sequence of a Bacillus gibsonii mature BG46 (subtilisin) variant named “BG46-V1” protease.

[0053] SEQ ID NO: 41 is the amino acid sequence of a Bacillus gibsonii mature BG46 (subtilisin) variant named “BG46-V2” protease.

[0054] SEQ ID NO: 42 is a DNA sequence encoding a variant Bacillus clausii GG36 (sublilisin) proregion.

[0055] SEQ ID NO: 43 is a DNA sequence encoding a Bacillus clausii mature GG36 (subtilisin) variant named “GG36-V1” protease.

[0056] SEQ ID NO: 44 is the amino acid sequence of a Bacillus clausii mature GG36 (subtilisin) variant named “GG36-V1” protease.

[0057] SEQ ID NO: 45 is a CI-2 amino acid consensus sequence.DETAILED DESCRIPTION

[0058] As described herein, certain embodiments of the disclosure are related to recombinant (genetically modified) Gram-positive bacterial cells for use in the commercial scale production of proteins (polypeptides). Certain one or more embodiments are therefore related to Gram-positive bacterial cells expressing heterologous proteins of interest, Gram-positive bacterial cells secreting heterologous proteins of interest into the fermentation media (broth), polynucleotide (DNA) sequences (e.g., expression cassettes) encoding heterologous proteases, DNA sequences (e.g., expression cassettes) encoding heterologous protease inhibitor polypeptides, Gram-positive bacterial cells comprising one or more introduced expression cassettes encoding one or more heterologous proteases and a heterologous protease inhibitor polypeptide, and the like.I. DEFINITIONS

[0059] Prior to describing the present strains, compositions and methods in further detail, the following terms and phrases are defined. Terms not defined should be accorded their ordinary meaning as used and known to one skilled in the art.

[0060] 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.

[0061] All publications and patents cited herein arc incorporated by reference in their entirety.

[0062] 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”,IFF10126-WO-PCT“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 (isogenic) Bacillus sp. cell “does not” comprise an introduced cassette encoding a protease inhibitor (i.e., relative to a modified cell).

[0063] 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.

[0064] 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.

[0065] 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. steam thermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named “Geobacillus stearothermophilus” .

[0066] 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 nucleicIFF10126-WO-PCT acid fragments wherein the assembly gives rise to a chimeric DNA sequence that would not otherwise be found in the genome.

[0067] 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.

[0068] As used herein, a “Streptomyces nigrescens metalloprotease inhibitor” may be abbreviated as “SMPI”, wherein the SMPI polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0069] As used herein, a “Streptomyces caespitosus neutral protease inhibitor” may be abbreviated as “ScNPI”, wherein the ScNPI polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0070] As used herein, a “Hordeum vulgare wheat subtilisin / chymotrypsin inhibitor” may be abbreviated as “WSCI”, wherein the WSCI polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0071] As used herein, a “Aequorivita sublithincola Azu-Kazal inhibitor” or “Kazul-type serine protease inhibitor” may be abbreviated as “Azu-Kazal”, wherein the Azu-Kazal polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0072] As used herein, a “Streptomyces albogriseolus streptomyces subtilisin inhibitor” may be abbreviated as “SSI”, wherein the SSI polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.100731 As used herein, a “Hordeum vulgare chymotrypsin inhibitor 2” may be abbreviated as “CI-2”, wherein the CI-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0074] As used herein, a “Hordeum vulgare barley amylase subtilisin inhibitor” may be abbreviated as “BASI”, wherein the BASI polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0075] As used herein, ceriain variants of the native Bacillus amyloliquefaciens BPN' (subtilisin) protease are named “BPN'-Vl” protease and “BPN'-V2” protease. Nucleic acid (DNA) sequences encoding the mature BPN'-Vl and BPN'-V2” proteases are set forth in SEQ ID NO: 10 and SEQ ID NO: 26, respectively. In particular, PCT Publication Nos. WO2011 / 072099 and WO2024 / 163584 (each incorporated herein by reference in its entirety) generally describes methods and compositions for the design and construction of native and variant BPN' (subtilisin) proteases. Although the BPN'-Vl and BPN'-V2 proteases are exemplified herewith, one skilled in the art may readily construct and express other suitable proteases in the recombinant cells of the disclosure.

[0076] As used herein, a variant of the native Bacillus gibsonii subtilisin protease is named “BG46-V1”, wherein the variant BG46-V1 protease is encoded by the nucleic acid (DNA) sequence set forth in SEQ ID NO: 33. In particular, PCT Publication No. WO2023 / 192953 (incorporated herein by reference in its entirety) generally describes methods and compositions for the design and construction of native and variant B. gibsoniiIFF10126-WO-PCT(subtilisin) proteases. Although the BG46-V1 protease is exemplified herewith, one skilled in the art may readily construct and express other suitable proteases in the recombinant cells of the disclosure.

[0077] As used herein, a variant of the native Bacillus clausii subtilisin protease is named “GG36-V1”, wherein the variant GG36-V 1 protease is encoded by the nucleic acid (DNA) sequence set forth in SEQ ID NO: 43. In particular, PCT Publication No. W02010 / 056634 (incorporated herein by reference in its entirety) generally describes methods and compositions for the design and construction of native and variant B. clausii (subtilisin) proteases. Although the GG36-V1 protease is exemplified herewith, one skilled in the art may readily construct and express other suitable proteases in the recombinant cells of the disclosure

[0078] As used herein, a Paenibacillus hunanensis metalloprotease named “PHD22” is encoded by the nucleic acid (DNA) sequence set forth in SEQ ID NO: 37.

[0079] 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.

[0080] It is understood that the polynucleotides (or nucleic acid molecules) described herein include “genes”, “vectors” and “plasmids”.|0081 | 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.

[0082] As used herein, an “endogenous gene” refers to a gene in its natural location in the genome of an organism.

[0083] 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.

[0084] 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.IFF10126-WO-PCT

[0085] 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.

[0086] 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.

[0087] 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 the core promoter (10 and -35) regions are generally separated (spaced) by about fifteen-twenty (15-20) intervening base pairs (nucleotides).

[0088] 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.

[0089] 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 theIFF10126-WO-PCT 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.

[0090] 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.

[0091] As used herein, the term “mature protein” refers to an active form of a protein, in contrast to the inactive precursor (full-length) protein.

[0092] 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. The signal (pre) sequence is typically cleaved from the precursor protein by a signal peptidase during translocation. The signal (pre) sequence is typically located N-terminal to the mature protein sequence, or located N-terminal to the pro-region (pro) sequence when a signal (pre) sequence and a pro-region (pro) sequence are used in operable combination and upstream (5') of the mature POI sequence.

[0093] 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. The term pro-sequence as used herein has the same meaning as understood in the art. For example, the B. subtilis alkaline serine protease “subtilisin’’ is first produced as a pre-pro-subtilisin, which consists of a signal (pre) sequence for protein secretion followed by a 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.

[0094] 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 (i.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.IFF10126-WO-PCT

[0095] 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 (z.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 tire 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.

[0096] 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.

[0097] As used herein, exemplary proteases may be referred to as “reporter proteases”. In certain one or more embodiments, exemplary proteases are expressed / produced by one or more recombinant (modified) cells of the disclosure. In certain embodiments, reporter proteases include, but are not limited to, native and functional variant Bacillus sp. subtilisin proteases. 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 subtilisins have a pl of about 6.5.

[0098] 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. One of skill may readily design, construct, screen, and identity functional subtilisin variants using routine methods known in the art. In particular, PCT Publication Nos. WO2010 / 056634, W02011 / 130222, WO2015 / 089447, WO2016 / 202839, WO2017 / 207762 and WO2023 / 114936 (each incorporated herein byIFF10126-WO-PCT 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.

[0099] 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. coli).

[0100] 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.

[0101] As used herein, when the expression and / or production of a protein of interest (POI, e. g. , a protease) 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 are grown / cultivated / fermented under the same conditions (e.g., the same conditions such as media, temperature, pH and the like).

[0102] 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 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.

[0103] 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.

[0104] 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-regularion of a gene, (f) specific mutagenesis and / or (g) random mutagenesis of any one or more the genes disclosed herein.

[0105] 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 introducingIFF10126-WO-PCT 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.

[0106] 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.

[0107] As used herein, “transforming DNA”, “transforming sequence”, and “DNA construct” refer to DNA that is used to introduce sequences into a host cell or organism. Transforming DNA is DNA used to introduce sequences into a host cell or organism. The DNA may be generated in vitro by PCR or any other suitable techniques. In some embodiments, the transforming DNA comprises an incoming sequence, while in other embodiments it further comprises an incoming sequence flanked by homology boxes. In yet a further embodiment, the transforming DNA comprises other non-homologous sequences, added to the ends (i.e., stuffer sequences or flanks). The ends can be closed such that the transforming DNA forms a closed circle, such as, for example, insertion into a vector.

[0108] As used herein “an incoming sequence” refers to a DNA sequence that is introduced into the bacterial cell chromosome. In some embodiments, the incoming sequence is part of a DNA construct. In other embodiments, the incoming sequence encodes one or more proteins of interest. In some embodiments, the incoming sequence comprises a sequence that may or may not already be present in the genome of the cell to be transformed (i.e., it may be either a homologous or heterologous sequence). In some embodiments, the incoming sequence encodes one or more proteins of interest, a gene, and / or 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.

[0109] 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 notIFF10126-WO-PCT meant to limit the present disclosure, a homology box may include about between 1 base pair (bp) to 200 kilobases (kb). Preferably, a homology box includes about between 1 bp and 10.0 kb; between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb, and between 0.25 kb and 2.5 kb. A homology box may also include about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb and 0.1 kb. In some embodiments, the 5' and 3' ends of a selective marker are flanked by a homology box wherein the homology box comprises nucleic acid sequences immediately flanking the coding region of the gene.

[0110] As used herein, a host cell “genome”, a bacterial (host) cell “genome”, or a Bacillus sp. (host) cell “genome” includes chromosomal and extrachromosomal genes.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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” (z.e., replicate autonomously or can integrate into a chromosome of a host organism).

[0115] 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.IFF10126-WO-PCT

[0116] As used herein, the terms “expression cassette” and “expression vector” refer to a nucleic acid construct generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell (i.e., these are vectors or vector elements, as described above). The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, DNA constructs also include a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. In certain embodiments, a DNA construct of the disclosure comprises a selective marker and an inactivating chromosomal or gene or DNA segment as defined herein.

[0117] 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., staffer 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”.

[0118] 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.

[0119] 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.

[0120] 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 orIFF10126-WO-PCT branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term polypeptide also encompasses an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0121] 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).|0122| As used herein, a “mutation” refers to any change or alteration in a nucleic acid sequence. Several types of mutations exist, including point mutations, deletion mutations, silent mutations, frame shift mutations, splicing mutations and the like. Mutations may be performed specifically (e.g., via site directed mutagenesis) or randomly (e.g., via chemical agents, passage through repair minus bacterial strains).

[0123] 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.

[0124] 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 about 40%, 50%, 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.IFF10126-WO-PCT

[0125] As set forth above, the term “identical” in the context of two polynucleotide or polypeptide sequences refers to the nucleotides or amino acids in the two sequences that are the same when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms described below and known in the art. The phrase “percent (%) identity” (abbreviated, “PID”) refers to polynucleotide (nucleic acid) or polypeptide (amino acid) sequence identity. Percent identity may be determined using standard techniques known in the art. In certain embodiments, the percent amino acid identity shared by sequences of interest can be determined by aligning the sequences to directly compare the sequence information, e.g., by using an alignment program / algorithm such as BLAST, MUSCLE, or CLUSTAL. For example, the BLAST algorithm has been described in Altschul et al. (1990) and Karlin et al. (1993). In particular, a percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “reference” sequence including any gaps created by the program for optimal / maximum alignment. BLASI’ algorithms refer to the “reference” sequence as the “query” sequence.

[0126] The BLAST program uses several search parameters, most of which are set to the default values. The NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity but is not recommended for query sequences of less than 20 residues (Altschul et al., 1997 and Schaffer et al., 2001). Exemplary default BLAST parameters for a nucleic acid sequence searches include: Neighboring words threshold=ll; E-value cutoff=10; Scoring Matrix=NUC.3.1 (match=L mismatch=-3);Gap Opening=5; and Gap Extension=2. Exemplary default BLAST parameters for amino acid sequence searches include: Word size = 3; E-value cutoff=10; Scoring Matrix=BLOSUM62; Gap Opening=ll; and Gap extension=l. Using this information, protein sequences can be grouped and / or a phylogenetic tree built therefrom. Amino acid sequences can be entered in a program such as the Vector NTI Advance suite and a Guide Tree can be created using the Neighbor Joining (NJ) method (Saitou and Nei, 1987). The tree construction can be calculated using Kimura’s correction for sequence distance and ignoring positions with gaps. A program such as AlignX can display the calculated distance values in parenthesis following the molecule name displayed on the phylogenetic tree.

[0127] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (Thompson et al., 1994). Default parameters for the CLUSTAL W algorithm include: Gap opening penalty=10.0; Gap extension penalty=0.05; Protein weight matrix=BLOSUM series; DNA weight matrix=IUB; Delay divergent sequences %=40; Gap separation distance=8; DNA transitions weight=0.50; List hydrophilic residues=GPSNDQEKR; Use negative matrix=OFF; Toggle Residue specific penalties=ON; Toggle hydrophilic penalties=ON; and Toggle end gap separation penalty=OFF. In CLUSTAL algorithms, deletions occurring at cither terminus arc included. For example, a variant with a five amino acid deletion at either terminus (or within the polypeptide) of a polypeptide of 500 amino acids would have a percentIFF10126-WO-PCT sequence identity of 99% (495 / 500 identical residues x 100) relative to the “reference” polypeptide. Such a variant would be encompassed by a variant having “at least 99% sequence identity” to the polypeptide.

[0128] As used herein, a “variant” polynucleotide (protein) refers to a polynucleotide having a specified degree of sequence homology / identity with a parent (reference or control) polynucleotide, or hybridizes with a parent (reference or control) polynucleotide (or a complement thereof) under stringent hybridization conditions. In certain embodiments, a variant polynucleotide comprises at least about 40% to 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% to about 100% nucleotide sequence identity with a reference polynucleotide sequence.

[0129] As used herein, a “variant” polypeptide (protein) refers to a polypeptide that is derived from a parent (or reference or control) 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. 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.

[0130] Certain embodiments provide polynucleotides (e.g., gene expression constructs, expression cassettes) encoding proteins of interest, polynucleotides encoding protease inhibitor proteins (polypeptides)), and the like. In certain embodiments, a protease inhibitor protein sequence comprises an amino acid sequence derived from a reference chymotrypsin inhibitor 2 (CI-2) protein sequence set forth as SEQ ID NO: 6. In certain embodiments, amino acid modifications of the CI-2 sequence described herein are numbered by reference to amino acid positions 1-65 of SEQ ID NO: 6. In other embodiments, a CI-2 protein sequence comprises an amino acid sequence derived from a reference CI-2 consensus sequence set forth as SEQ ID NO: 45. In certain embodiments, amino acid modifications of the of the CI-2 sequence described herein are numbered by reference to amino acid positions 1-65 of SEQ ID NO: 45 (e.g., FIG. 3 ft ). For example, the amino acid sequence of a CI-2 sequence variant described herein can be aligned with the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 45 using an alignment algorithm, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with an amino acid residue in SEQ ID NO: 6 or SEQ ID NO: 45 is conveniently numbered by reference to the numerical position of that corresponding amino acid residue.

[0131] As used herein, “specific productivity” is total amount of protein produced per cell per time over a given time period.IFF10126-WO-PCT

[0132] 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.

[0133] As used herein, the terms “broth”, “cultivation broth” and “fermentation broth” may be used interchangeably, and particularly refer to a whole fermentation broth.

[0134] The term “whole fermentation broth” as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and / or purification. For example, whole fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of proteins by host cells) and secretion into cell culture medium. Typically, the whole fermentation broth is unfractionated and comprises spent cell culture medium, extracellular polypeptides, and microbial cells.

[0135] “Cell debris” refers to cell walls and other insoluble cellular components that are released after disruption of the cell membrane, e.g., after lysis of microbial cells.II. CO-EXPRESSION OF INTRACELLULAR PROTEASE INHIBITORS ANDEXTRACELLAR PROTEASES

[0136] As generally understood, Bacillus subtilis stands out as an atractive host for protease expression / production due to its unique features. However, intracellular accumulation of proteases can lead to adverse effects, including cell death and degradation of translation complexes if the B. subtilis cells faced secretion bottlenecks when expressing extracellular proteases. As describe herein, to mitigate these challenges, Applicant explored intracellular co-expression of protease inhibitors alongside protease molecules of interest. More particularly, as presented below in the Examples and briefly described herein, Applicant has surprisingly observed that co-expressing certain protease inhibitors (e.g., chymotrypsin inhibitor II) can significantly improve both cell viability and protease production. For instance, rather than relying solely on pro-peptide co-expression or optimizing secretion machinery, Applicant leveragedIFF10126-WO-PCT different classes of known protease inhibitors (e.g., Azu-Kazal, BASI, WSCI, CI-2, ScNPI, SSI, and SMPI SEQ ID NO: 1) as promising candidates.

[0137] More particularly, as presented in Example 1, Applicant evaluated the expression of various protease inhibitor polypeptides in B. subtilis, and tested / screened the binding affinity of the protease inhibitors with subtilisin proteases and metalloproteases. For instance, expression constructs (cassettes) encoding mature protease inhibitor sequences (e.g., SEQ ID NOs: 2-7) were synthesized, cloned into B. subtilis expression vector pJM103, and introduced into a B. subtilis protease deleted strain. In particular, as described in this example, activity assays of subtilisin variants BG46-V2 and BPN'-V2 were performed using a range of concentrations of subtilisin protease substrate (suc-AAPF-pNA) in the presence of a range of concentrations of protease inhibitor supernatants. Likewise, activity assays of metalloprotease PHD22 were performed with metalloendopeptidase substrate (Abz-AGLA-Nba) in the presence of various concentrations of protease inhibitor supernatants. As presented in TABLE 1 (Example 1), the inhibition constants (Ki) of the protease inhibitors in the presence of different proteases (BPN'-V2, BG46-V2 and PHD22). More particularly, as shown in TABLE 1, with the exception of the BASI and SMPI inhibitors, all other protease inhibitors demonstrated tight binding inhibition with subtilisin protease (BPN'-V2 and BG46-V1).

[0138] As further described in Example 2, Applicant screened / tested protease inhibitor polypeplides as a potential candidates for intracellular expression (e.g., to improve cell viability, maintain the biomass during fermentation, etc.). In particular, a modified B. subtilis strain encoding copies of a mature subtilisin protease (BPN'-Vl; SEQ ID NO: 10) and a protease inhibitor polypeptide (CI-2) was constructed and screened for cell viability. For instance, as described in this example, the protease inhibitor polypeptide was intracellularly expressed in B. subtilis under the control of suitable promoters such as a veg promoter (Pveg; SEQ ID NO: 8), a bglC promoter (BglC; SEQ ID NO: 9), and the like. More particularly, as shown in TABLE 2 (Example 2), co-expression of a protease inhibitor polypeptide intracellularly with the subtilisin protease (BPN'-Vl) results in a much higher biomass relative to the control strain expressing the subtilisin protease (BPN'-Vl) without CI-2 expression. In addition, as shown below in TABLE 3 (Example 2), co-expression of CL2 with subtilisin protease results in more viable cells relative to the strain expressing the subtilisin protease without CI-2 expression.

[0139] Example 3 of the disclosure describes the construction of modified B. subtilis strains co-expressing other subtilisin proteases (BPN'-V2 and BG46-V1) and the CI-2 protease inhibitor polypeptide (retained intracellularly). For instance, as described in this example, Applicant surprisingly observed that coexpression of CI-2 polypeptide inhibitor intracellularly with an optimal strength promoter particularly enhances (increases) production of the subtilisin proteases of interest. More particularly, as shown in TABLE 4 (Example 3), modified Bacillus strains expressing the protease inhibitor polypeptide (CI-2)IFF10126-WO-PCT intracellularly demonstrate significantly enhanced protease production phenotype (e.g., about 10% to 30% higher) relative to the control Bacillus strain without CI-2 co-expression.

[0140] Thus, as briefly described above, certain embodiments are related to compositions and methods for enhanced protein production in Gram-positive bacterial (host) cells. More particularly, certain one or more embodiments of the disclosure provide, inter alia, Gram-positive bacterial cells (strains) expressing heterologous proteins of interest, Gram-positive bacterial cells secreting heterologous proteins of interest into the fermentation media (broth), polynucleotide (DNA) sequences (e.g., expression constructs / cassettes) encoding heterologous proteases, DNA sequences (e.g., expression constructs / cassettes) encoding heterologous protease inhibitor polypeptides, Gram-positive cells comprising one or more introduced expression cassettes encoding one or more heterologous proteases and a heterologous protease inhibitor polypeptide, and the like.

[0141] For instance, certain embodiments are related to polynucleotide constructs (e.g., expressions cassettes) encoding a protease of interest. In certain embodiments, a protease of interest is a native subtilisin or a functional subtilisin variant thereof. In particular embodiments, a polynucleotide construct comprises an expression cassette encoding a protease of interest, wherein the cassette comprises upstream (5') and downstream (3') flanking sequences homologous to a gene locus of interest (GLOI), e.g., a GLOI as described in the Examples section below. In certain other embodiments, one or more expression cassettes encoding a protease of interest comprise an upstream (heterologous) promoter operably linked to a polynucleotide (DNA) comprising a 5 '-untranslated region (5'-UTR) operably linked to a polynucleotide encoding a signal (secretion) sequence operably linked to a polynucleotide encoding a subtilisin pro-region sequence operably linked to a polynucleotide encoding the mature subtilisin protease. In related embodiments, the cassettes comprise a polynucleotide comprising a transcriptional terminator sequence downstream and operably linked to the polynucleotide ending the mature subtilisin. In certain embodiments, a polynucleotide encoding a mature subtilisin comprises at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a polynucleotide of any one of SEQ ID NOs: 10, 26, 33, 36 and 43. For instance, in certain embodiments mature subtilisin variants having at least about 75% identity to SEQ ID NOs: 38, 39, 40, 41 and 44 are screened, evaluated and identified as generally described below in Examples 1-3. In certain one or more embodiments, functional / active mature subtilisin variants encoded by a polypeptide having at least about 75% identity to SEQ ID NOs: 38, 39, 40, 41 and 44 are identified via subtilisin (protease) activity assays described herein.

[0142] As set forth above, certain other embodiments are directed to expressions cassettes encoding protease inhibitor polypeptides. In related embodiments, expressions cassettes encoding protease inhibitor polypeptides comprise an upstream (heterologous) promoter region operably linked to the polynucleotideIFF10126-WO-PCT encoding the protease inhibitor. More particularly, expression cassettes encoding one or more protease inhibitor polypeptides are constructed / designed for intracellular retention of the expressed protease inhibitor polypeptides. In certain embodiments, a polynucleotide (DNA) encoding a protease inhibitor polypeptide comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the polynucleotide of SEQ ID NO: 6. For instance, in certain embodiments protease inhibitor polypeptide variants having at least 90% identity to SEQ ID NO: 6 are screened, evaluated and identified as generally described below in Examples 13. Thus, in certain one or more embodiments, functional / active protease inhibitor variants of SEQ NO: 6 are identified by testing their binding affinity with sublilisin proteases described herein. In particular embodiments, the primary amino acid sequence of the H. vulgare chymotrypsin inhibitor 2 (CI-2) polypeptide set forth in SEQ ID NO: 6 may be analyzed in silico such that tertiary structure of the native CI-2 polypeptide may be visualized.[0143 J The crystal structure of the complex of subtilisin BPN' with chymotrypsin inhibitor 2 (Cl-2) has been deposited in the protein data bank (PDB “ 1LW6”). The nali ve CI-2 inhibitor protein comprises eighty- three (83) amino acid residues and was originally identified in Hiproly barley. However, as described by Radisky and Koshland (2002), truncation of twenty (20) amino acid residues from the N-terminus of the CI-2 protein (63 AA residues) was shown to retain the complete structure and function relative to the full- length (native) CI-2 protein. The structure of the CI-2 inhibitor is therefore crucial for its function as a serine protease inhibitor. In particular, key regions of the Cl-2 protein (i.e., based on PDB 1LW6) include, alpha-helices (residues 10-20 and 30-40), beta-sheets (residues 1-10, 40-50 and 60-65), the reactive site loop (approximately residues 50-60). The reactive site loop is the primary interaction site with the protease, generally acting as a pseudo-substrate, fitting into the active site of the protease and blocking its activity. For instance, the flexibility and specific amino acid composition of this loop are essential for effective inhibition, and as such, changes here can drastically affect the inhibitor’s ability to bind to the protease. The alpha-helices and beta-sheets usually maintain the hydrophobic core and hydrogen bonding to preserve integrity. As further set forth in FIG. 3, Applicant generated a sequence logo by aligning one-hundred ( 100) chymotrypsin protease inhibitor sequences (FIG. 3A). In particular, the CI-2 consensus sequence (SEQ ID NO: 45) has been annotated in FIG. 3B to show conserved amino acid residues in bold font followed by their position in subscript.

[0144] Promoters and promoter sequence regions for use in the expression of genes, open reading frames (ORFs) thereof and / or variant sequences thereof in Bacillus cells are generally known on one of skill in the art. Promoter sequences of the disclosure are generally chosen so that they are functional in the Bacillus cells, and include, but are not limited to, naturally occurring promoter sequences, synthetic promoter sequences, and / or promoter sequence combinations thereof and the like, which promoter (sequences) are operable / functional in Bacillus cells. Certain exemplary promoter sequences include, but are not limitedIFF10126-WO-PCT to, the B. subtilis alkaline protease (aprE) promoter, the a-amylase promoter of B. subtilis, the a-amylase promoter of B. amyloliquefaciens, the neutral protease (nprE) promoter from B. subtilis, a mutant aprE promoter (e.g., PCT Publication No. W02001 / 51643), a B licheniformis tuf promoter, a B licheniformis citZ promoter, or any other functional promoter from Bacillus sp. cells. Methods for screening and creating promoter libraries with a range of activities (promoter strength) in Bacillus cells is described in PCT Publication No. W02003 / 089604. For instance, in certain embodiments, promoters for the intracellular expression of protease inhibitor polypeptides are exemplified herein with veg and bglC promoters, which are not meant to be limiting, as one skilled in the art may readily select other promoters suitable for expressing protease inhibitor polypeptides intracellularly at a desired level. Theoretically, the accumulation of protease molecules may occur throughout fermentation. Thus, depending on the amount of accumulated protease in the cells, the veg promoter (e.g., maximal expression around 2000-4000 TPM) or the bglC promoter (e.g., maximal expression around 100-300 TPM) is selected to ensure that the intracellular expression of the protease inhibitor CI-2 gradually increases until the end of fermentation. In particular, the dynamic expression of veg and bglC in the B. subtills strain expressing BPN'-Vl, BPN'-V2, BG46-V1 and GG36-V 1 proteases throughout the fermentation is presented in FIG. 4, wherein fermentation samples from each protease production strain were collected every two hours from 8 hour to 36 hour intervals for RNA-seq analysis.III. RECOMBINANT NUCLEIC ACIDS AND MOLECULAR BIOLOGY

[0145] Certain embodiments of the disclosure are related to nucleic acids, polynucleotides, vectors, expression constructs, and the like. More particularly, as described herein and the Examples section below, 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. 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 phosphoramiditc 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, SanIFF10126-WO-PCTFrancisco, C A, USA). Other techniques for synthesizing nucleic acids and related principles are described and known in the art.

[0146] 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 variant 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.|0147| Thus, in certain embodiments, a polynucleotide (genes, vectors, plasmids, DNA elements, etc.) of the disclosure may be genetically modified, wherein genetic modifications include, but are not limited to, the introduction, substitution, or removal of one or more nucleotides in a gene (or an ORF / CDS 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, a gene disruption, a gene conversion, a gene deletion, the down-regulation of a gene (e.g., interfering RNA), specific mutagenesis, random mutagenesis and the like of any one or more the genes disclosed herein. In certain embodiments, a modified Bacillus cell of the disclosure is constructed by increasing the expression of a gene and / or by reducing (or eliminating) the expression of a gene, using methods well known in the art, for example, insertions, disruptions, replacements, or deletions. The portion of the gene to be modified or inactivated may be, for example, the coding region (CDS, ORF) or a regulatory (DNA) element required for expression of the coding region. An example of such a regulatory or control sequences may be a promoter sequence or a functional part thereof, (i.e., a part which is sufficient for affecting expression of the nucleic acid sequence). Other control sequences for modification include, but are not limited to, a leader sequence, a pro-peptideIFF10126-WO-PCT sequence, a signal sequence, a transcription terminator sequence, a transcriptional activator sequence and the like.

[0148] Gene deletion techniques enable the partial or complete removal of gene(s), thereby eliminating their expression, or expressing a non-functional (or reduced activity) protein product. In such methods, the deletion of the gene(s) may be accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3' regions flanking the gene. The contiguous 5' and 3' regions may be introduced into a Bacillus cell, for example, on a temperature-sensitive plasmid, such as pE194, in association with a second selectable marker at a permissive temperature to allow the plasmid to become established in the cell. The cell is then shifted to a non-permissive temperature to select for cells that have the plasmid integrated into the chromosome at one of the homologous flanking regions. Selection for integration of the plasmid is affected by selection for the second selectable marker. After integration, a recombination event at the second homologous flanking region is stimulated by shifting the cells to the permissive temperature for several generations without selection. The cells are plated to obtain single colonies and the colonies are examined for loss of both selectable markers. Thus, a person of skill in the art may readily identify nucleotide regions in the gene’s coding sequence and / or the gene’s non-coding sequence suitable for complete or partial deletion. In other embodiments, a modified Bacillus cell of the disclosure is constructed by introducing, substituting, or removing one or more nucleotides in the gene or a regulatory element required for the transcription or translation thereof.

[0149] In certain embodiments, a modified Bacillus cell is constructed via CRISPR-Cas9 editing. For example, a wild-type gene encoding a native protein of interest (or functional variant protein of interest thereof) may be modified via CRISPR-Cas9 editing, by means of nucleic acid guided endonucleases, that find their target DNA by binding either a guide RNA (e.g., Cas9) and Cpfl or a guide DNA (e.g., NgAgo), which recruits the endonuclease to the target sequence on the DNA, wherein the endonuclease can generate a single or double stranded break in the DNA. This targeted DNA break becomes a substrate for DNA repair, and can recombine with a provided editing template (e.g., an editing template to replace the native gene promoter sequence with a heterologous promoter). For example, the gene encoding the nucleic acid guided endonuclease (for this purpose Cas9 from 5. pyogenes) or a codon optimized gene encoding the Cas9 nuclease is operably linked to a promoter active in the Bacillus cell and a terminator active in Bacillus cell, thereby creating a Bacillus Cas9 expression cassette. Likewise, one or more target sites unique to the gene of interest are readily identified by a person skilled in the art. For example, to build a DNA construct encoding a gRNA-directed to a target site within the gene of interest using Streptococcus pyogenes Cas9, the variable targeting domain (VT) will comprise nucleotides of the target site which are 5’ of the (PAM) proto-spacer adjacent motif (NGG), which nucleotides arc fused to DNA encoding the Cas9 endonuclease recognition domain for S. pyogenes Cas9 (CER). The combination of the DNA encoding a VT domain andIFF10126-WO-PCT the DNA encoding the CER domain thereby generate a DNA encoding a gRNA. Thus, a Bacillus expression cassette for the gRNA is created by operably linking the DNA encoding the gRNA to a promoter active in Bacillus cells and a terminator active in Bacillus cells.

[0150] In certain embodiments, the DNA break induced by the endonuclease is repaired / replaced with an incoming sequence. For example, to precisely repair the DNA break generated by the Cas9 expression cassette and the gRNA expression cassette described above, a nucleotide editing template is provided, such that the DNA repair machinery of the cell can utilize the editing template. For example, about 500-bp 5' of targeted gene can be fused to about 500-bp 3' of the targeted gene to generate an editing template, which template is used by the Bacillus host’s machinery to repair the DNA break generated by the RGEN. The Cas9 expression cassette, the gRNA expression cassette and the editing template can be co-delivered to the cells using many different methods. The transformed cells are screened by PCR amplifying the target gene locus, by amplifying the locus with a forward and reverse primer. These primers can amplify the wild-type locus or the modified locus that has been edited by the RGEN. These fragments are then sequenced using a sequencing primer to identify edited colonies.

[0151] In other embodiments, a modified Bacillus cell is constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis and transposition. Modification of the gene may be performed by subjecting the parental cell to mutagenesis and screening for mutant cells in which expression of the gene has been altered. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods. Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'- nitrosoguanidine (NTG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the parental cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutant cells exhibiting reduced or no expression of the gene.

[0152] International PCT Publication No. W02003 / 083125 discloses methods for modifying Bacillus cells, such as the creation of Bacillus deletion strains and DNA constructs using PCR fusion to bypass E. coli. PCT Publication No. 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) optimizingIFF10126-WO-PCT double cross-over integrations, (6) site directed mutagenesis and (7) marker-less deletion. Those of skill in the art are well aware of suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli and Bacillus). Indeed, such methods as transformation including protoplast transformation and congression, transduction, and protoplast fusion are known and suited for use in the present disclosure. Methods of transformation are particularly preferred to introduce a DNA construct of the present disclosure into a host cell.

[0153] 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.

[0154] 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 least about 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 protease relative to its unmodified (reference or control) cell.

[0155] In certain embodiments, a modified Gram-positive cell exhibits an increased specific productivity (Qp) of a protease 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 fermentation time in hours from the time of inoculation, which includes the time of production as well as growth time.

[0156] In certain other embodiments, a modified Gram-positive cell 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 (reference or control) cell.IFF10126-WO-PCT

[0157] As described above, in certain one or more embodiments, the protease of interest is a subtilisin. There are various assays known to those of ordinary skill in the art for detecting and measuring activity of intracellularly and extracellularly expressed proteins (e.g., see Examples 1-3).V. FERMENTING GRAM-POSITIVE CELLS FOR THE PRODUCTION OF PROTEINS

[0158] 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 by fermenting the cells in a suitable medium. Fermentation methods well known in the art can be applied to ferment Gram-positive cells of the disclosure.

[0159] 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 qualities 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.

[0160] 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.

[0161] Continuous fermentation is an open system where a defined fermentation medium is added continuously to a biorcactor, 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 continuouslyIFF10126-WO-PCT while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the art of industrial microbiology.

[0162] 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.VII. EXEMPLARY EMBODIMENTS|0163| Non-limiting embodiments of the disclosure include, but are not limited to:

[0164] 1. A method for the enhanced production of a protease in a Bacillus sp. cell comprising obtaining a Bacillus sp. cell producing a protease of interest, introducing into the cell an expression construct (cassette) encoding a protease inhibitor polypeptide, and fermenting the modified cell under conditions for production of the protease, wherein the protease is secreted into the fermentation broth, and the protease inhibitor is retained intracellularly.

[0165] 2. The method of embodiment 1 , wherein the modified cell produces at least 5 % or more of the protease as compared to a control (isogenic) Bacillus sp. cell fermented under the same conditions, wherein the control cell expresses and secretes the same protease of intertest, but does not comprise an introduced expression construct (cassette) encoding the protease inhibitor.

[0166] 3. A method for the enhanced production of a protease in a Bacillus sp. cell comprising introducing into a Bacillus sp. cell an expression construct (cassette) encoding a mature protease and an expression construct (cassette) encoding a protease inhibitor polypeptide, and fermenting the modified cell under conditions for production of the protease, wherein the protease is secreted into the fermentation broth, and the protease inhibitor is retained intracellularly.

[0167] 4. The method of embodiment 4, wherein the modified cell produces at least 5% or more of the protease as compared to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell comprises the same introduced expression construct (cassette) encoding the protease, but does not comprise an introduced expression construct (cassette) encoding the protease inhibitor.

[0168] 5. The method of embodiment 3, wherein the expression construct (cassette) encoding the mature protease comprises an upstream heterologous promoter region operably linked to a polynucleotideIFF10126-WO-PCT comprising a 5'-UTR operably linked to a polynucleotide encoding a signal (secretion) sequence operably linked to a polynucleotide encoding a protease pro-region operably linked to a polynucleotide encoding a mature protease.

[0169] 6. The method of embodiment 3, wherein the cell comprises at least two, three, or four introduced expression constructs (cassettes) encoding the mature protease.

[0170] 7. The method of embodiment 5, or embodiment 6, wherein the cassette, or cassettes, further comprise a polynucleotide comprising a transcriptional terminator downstream and operably linked to the polynucleotide encoding the mature protease.

[0171] 8. The method of embodiment 1, wherein the expression construct (cassette) encoding the protease inhibitor p< >1 ypeplide comprises an upslream heterologous promoter region operably linked to a polynucleotide encoding a chymotrypsin inhibitor (CI-2) polypeptide having at least 75% identity to SEQ ID NO: 6 or SEQ ID NO: 45.

[0172] 9. The method of embodiment 3, wherein the modified cell has at least a 5% increase in biomass after 48 hours of fermentation relative to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell comprises the same introduced expression construct (cassette) encoding the protease, but does not comprise an introduced expression construct (cassette) encoding the protease inhibitor.101731 10. The method of embodiment 3, wherein the expression construct (cassette) encoding the mature protease comprises upstream and downstream flanking sequences homologous to a gene locus of interest (GLOI), wherein the cassette is integrated into the GLOI.

[0174] 11. The method of embodiment 6, wherein the at least two, three, or four expression constructs (cassettes) encoding the mature protease each comprise different upstream and downstream flanking sequences homologous to at least two, three, or four gene loci of interest (GLOI), wherein the constructs (cassettes) are integrated into the at least two, three, or four GLOI.

[0175] 12. The method of embodiment 3, wherein the polynucleotide encoding the mature protease comprises at least 75% identity to a polynucleotide selected from SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 36 and SEQ ID NO: 43.

[0176] 13. The method of embodiment 1 or 3, wherein the protease comprises at least 75% identity to any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44.

[0177] 14. The method of embodiment 5, wherein the polynucleotide comprising the 5'-UTR has at least 90% identity to the B. subtilis aprE 5'-UTR of SEQ ID NO: 11.

[0178] 15. The method of embodiment 5, wherein the polynucleotide encoding the signal sequence comprises at least 90% identity to the B. subtilis aprE signal sequence of SEQ ID NO: 11 or at least 90% identity to the B. amyloliquefaciens BPN' signal sequence SEQ ID NO: 28.IFF10126-WO-PCT

[0179] 16. The method of embodiment 5, wherein the polynucleotide encoding the protease pro-region comprises at least 90% to the B. amyloliquefaciens BPN' pro-region of SEQ ID NO: I4or at least 90% identity to the B. gibsonii pro-region of SEQ ID NO: 32 .

[0180] 17. The method of embodiment 1 or embodiment 3, wherein the cell comprises one or more genetic modifications rendering the cell deficient in the production of one or more endogenous proteins.

[0181] 18. A modified Gram-positive bacterial cell co-expressing a heterologous protease and a heterologous protease inhibitor polypeptide.

[0182] 19. The modified cell of embodiment 18, wherein the mature protease is secreted extracellularly, and the protease inhibitor is retained intracellularly, when fermented under conditions for the production of the protease.

[0183] 20. The modified cell of embodiment 18, wherein the cell produces at least 5% or more of the protease relative to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell expresses the same protease, but does not co-express the heterologous protease inhibitor.

[0184] 21. The modified cell of embodiment 18, comprising at least one, two, three, or four introduced expression constructs (cassettes) encoding the heterologous protease.

[0185] 22. The modified cell of embodiment 21, wherein the constructs (cassettes) encoding the heterologous protease are integrated into the genome of the cell.|0186| 23. The modified cell of embodiment 18, comprising an introduced expression construct (cassette) encoding the protease inhibitor polypeptide.

[0187] 24. The modified cell of embodiment 23, wherein the construct (cassette) encoding the protease inhibitor comprises an upstream heterologous promoter region operably linked to a polynucleotide encoding a chymotrypsin inhibitor (CI-2) polypeptide comprising at least 75% identity to SEQ ID NO: 6 or SEQ ID NO: 45.

[0188] 25. The modified cell of embodiment 21, wherein the constructs (cassettes) encoding the mature protease comprise a polypeptide sequence having at least 75% identity to any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44.

[0189] 26. A method for the enhanced viability of Bacillus sp. cells producing a protein of interest (POI), the method comprising: introducing into a Bacillus sp. cell an expression cassette encoding a POI and an expression cassette encoding a protease inhibitor polypeptide comprising at least 75% identity to SEQ ID NO: 6 or SEQ ID NO: 45, and fermenting the modified cells under conditions for production of the POI, wherein the ratio of live to dead cells at 44 hours fermentation is increased relative to a control (isogenic) Bacillus sp. cell fermented under the same conditions, wherein the controlIFF10126-WO-PCT cells express the same protease, but do not co-express the protease inhibitor polypeptide comprising at least 75% identity to SEQ ID NO: 6 or SEQ ID NO: 45.EXAMPLES

[0190] Certain aspects of the present disclosure may be further understood in light of the following examples, which should not be construed as limiting. Modifications to materials and methods will be apparent to those skilled in the art. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art (Ausubel et al., 1987; Sambrook et al., 1989).EXAMPLE 1EXPRESSION OF PROTEASE INHIBITORS IN BACILLUS CELLS AND THEIR BINDING AFFINITY TO DIFFERENT CLASSES OF PROTEASES

[0191] A. Overview

[0192] As briefly set forth in the preceding sections, the intracellular accumulation of proteases can lead to adverse effects, such as cell death, degradation of translation complexes and the like. For instance, to mitigate such adverse effects, the instant example investigates the intracellular expression (i.e., coexpression) of protease inhibitors and proteases in a recombinant host Bacillus sp. host strains. More particularly, Applicant has evaluated the expression of several protease inhibitors in B. subfilis and further tested their binding affinity with subtilisin variants and metalloproteases.

[0193] B. Bacillus Strain Construction

[0194] The DNA sequences encoding mature protease inhibitor sequences including Azu-Kazal (SEQ ID NO: 4), BASI (SEQ ID NO: 7), WCSI (SEQ ID NO: 3), CI-2 (SEQ ID NO: 6) , ScNPI (SEQ ID NO: 2), SSI (SEQ ID NO: 5) and SMPI (SEQ ID NO: 1) were synthesized by GeneArt (ThermoFisher Scientific) and individually cloned into a B. subtilis expression vector p.IM103 (Vogtentanz, 2007). In particular, the pJM103 plasmid contains an upstream aprE promoter an AprE signal sequence used to target extracellular protein secretion in B. subtilis. as well as a downstream LAT transcriptional terminator and chloramphenicol antibiotic resistance cassette.

[0195] The pJM103 derived plasmid was then introduced into B. subtilis protease deleted cells (i.e., degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Ampr, AispA, Abpr) and plated on Luria Agar media supplemented with 5 ppm chloramphenicol. Colonies were picked and subjected to fermentation in a 250 mL shake flask using a soytone medium (which is a MOPS-based defined medium, supplemented with additional 5 M CaCE). More particularly, the whole broth cultures were spun down and the supernatant from each modified cell was collected, wherein protein concentrations were determined for supernatants of each culture using MES running buffer (BioRad). Purified Eglin C was used as a standard to determine the concentration of all protease inhibitor proteins using gel densitometry with Imagequant software.IFF10126-WO-PCT

[0196] C. Activity Assays

[0197] The activity assays of subtilisin variants BG46-V2 and BPN'-V2 were performed in 0.1 M Tris (pH 8.6), 0.005% Tween-80 using a range of concentrations of subtilisin / proteases substrate suc-AAPF-pNA (Millipore Sigma) in the presence of a range of concentrations of inhibitor supernatants with 333 nM purified proteases subtilisin BPN'-V2 and BG46-V2. AAPF, 100 mg / niL stock in dimethyl sulfoxide (DMSO), was diluted to various concentrations in 0. 1 M Tris pH 8.6, 0.005% Tween containing a range of inhibitor concentrations. Five (5) pL protease solution was added to 95 pL AAPF containing working solution in 96-well plates (Corning 9017). The samples were then mixed for five (5) seconds and monitored in kinetic read mode at an absorbance wavelength of 405 nm.

[0198] The activity assays of metalloprotease PHD22 were performed in 0.1 M HEPES (pH 7.3), 2.6 mM CaCF with metalloendopeptidase substrate Abz-AGLA-Nba (Bachem) in the presence of various concentrations of inhibitors. To prepare the 2.4 mM Abz-AGLA-Nba working solution, one (1) mL of Abz-AGLA-Nba stock solution 48 mM Abz-AGLA-Nba in dimethylformamide (DMF) was added to 19 mL of HEPES buffer and mixed thoroughly for at least ten (10) seconds. The assay was performed in disposable black polystyrene flat-bottom 96-well microtiter plates suitable for fluorescence reading (Corning 3915). One hundred ninety-five (195) gL of 2.4 mM Abz-AGLA-Nba working solution containing a range of protease inhibitor concentrations were added to each well of the 96-well microtiter assay plates, followed by the addition of five (5) gL of diluted protease samples. The solutions were mixed for five (5) seconds, and the fluorescence changes were measured in kinetic mode at 25 °C (excitation wavelength 350 nm and emission wavelength 415 nm) using a microplate spectrofluorometer (SpectraMAX Gemini EM, Molecular Devices).

[0199] For instance, TABLE 1 presented below shows the inhibition constants (Ki) of the protease inhibitors in the presence of different proteases (i.e., subtilisins BPN'-V2, BG46-V2 and metalloprotease PHD22). In particular, Kaleidagraph software was used to fit the Michaelis-Menten equation to the data to determine the KMapp at each inhibitor concentration for B ASI. Thus, the slope of the linear fit of KMapp versus protease inhibitor concentration provided the slope (KM / K), wherein Ki was solved assuming that inhibition was competitive.

[0200] As shown in TABLE 1, with the exception of the BASI inhibitor and SMPI, all other protease inhibitors including Azu-Kazal, WSCI, CI2, ScNPI and SSI demonstrated tight binding inhibition with subtilisin variants BPN'-V2 and BG46-V2, and therefore, the inhibition constants (K) were less than the enzyme concentration used in the assay (333nM). Notably, only SMPI demonstrated tight binding inhibition for the metalloprotease PHD22. Likewise, as shown in FIG. 1, the secreted protease inhibitors were analyzed by SDS-PAGE, demonstrating that the inhibitors could be successfully expressed in Bacillus sp. cells.IFF10126-WO-PCTTABLE 1INHIBITION CONSTANTS FOR PROTEASES WITH INHIBITORSEXAMPLE 2INTRACELLULAR EXPRESSION OF CI-2 INHIBITOR IMPROVES BACILLUS CELL GROWTH AND VIABILITY

[0201] A. Overview

[0202] As set forth in the preceding example, Applicant cultured modified B. subtilis strains expressing / secreting a protease inhibitor polypepride (FIG. 1; Azu-Kazal, BASI, WSCI, CI-2, ScNPI, SSI and SMPI), and screened the culture supernatants thereof in protease inhibition assays (TABLE 1) using three candidate subtilisin proteases ( BPN'-V2 and BG46-V2) and metalloprotease(PIID22). In the present example, Applicant has found that accumulating active subtilisin variant intracellularly may cause cell death, degradation of translation complexes, etc., and further impacts protein translation and cell growth. As described herein, the CI-2 protease inhibitor polypeptide was further analyzed as a potential candidate for intracellular expression to improve cell viability and maintain the biomass during fermentation.

[0203] B. Protease cassettes

[0204] In particular, the mature BPN'-Vl subtilisin was expressed in B. subtilis as follows. DNA fragments were amplified and assembled by standard fusion PCR methods. These assembled fragments then served as linear DNA expression cassettes for integration into B. subtilis strains. For instance, the construction of protease expression cassettes was earned out as described below, wherein the expression cassettes comprise in the 5' to 3' direction, a polynucleotide (DNA) sequence comprising a B. subtilis P2 promoter (SEQ ID NO: 11) operably linked to a DNA sequence comprising an aprE 5'-UTR (SEQ ID NO: 12) operably linked to a DNA sequence (SEQ ID NO: 13) encoding an AprE signal sequence operably linked to a DNA sequence encoding a variant BPN' (subtilisin) pro-region sequence (SEQ ID NO: 14)IFF10126-WO-PCT operably linked to a DNA sequence encoding a variant BPN' mature (subtilisin) protease (SEQ ID NO: 10) operably linked to DNA comprising a BPN' (subtilisin) transcriptional terminator (SEQ ID NO: 15).

[0205] In particular, a first DNA fragment comprising an upstream (5') skjA flanking sequence (SEQ ID NO: 16) was operably linked to the protease expression cassette followed by a downstream (3') skfH flaking sequence (SEQ ID NO: 17). A second DNA fragment, comprising an upstream (5') sunT flanking sequence (SEQ ID NO: 18) was operably linked to the protease expression cassette followed by a downstream (3') sunT flaking sequence (SEQ ID NO: 19). A third DNA fragment comprising an upstream (5') aprE flanking sequence (SEQ ID NO: 20) was operably linked to the protease expression cassette followed by a B. subtilis alanine racemase gene (SEQ ID NO: 21) and downstream (3') aprE flaking sequence (SEQ ID NO: 22).

[0206] C. CI-2 Inhibitor cassete

[0207] As shown in FIG. 2, the CI-2 protease inhibitor expression cassette comprises an upstream (5') veg promoter (Pveg; SEQ ID NO: 8) operably linked to DNA comprising a Shine-Dalgarno (SD) sequence (SEQ ID NO: 23) operably linked to DNA encoding the CI-2 protease inhibitor (SEQ ID NO: 6) operably linked to DNA comprising a BPN' transcriptional terminator (SEQ ID NO: 15). In particular, the CI-2 fragment comprises 5' pksL flanking sequence (SEQ ID NO: 24) was operably linked to the CI-2 expression cassette followed by a 3' pksL flanking sequence (SEQ ID NO: 25).

[0208] Thus, a modified B. subtilis strain comprising the introduced BPN'-Vl protease cassettes and introduced CI-2 protease inhibitor cassette was constructed by integrating all three protease fragments and CI-2 fragment described above into the genome of the host strain. Subsequently, the strains with, or without the CI-2 inhibitor cassette were fermented using standard fermentation conditions in a 24-well deep well plate with 0.5X MPS2 media (pH 7.3).

[0209] D. Cell Viability Assays

[0210] To screen for cell viability, Applicant performed cell viability assays wherein the whole cell broths were stained with propidium iodide and SYTO™ 9 following manufacturer’s instructions (Thermo Fisher). For instance, the fermented cells with a compromised membrane that are dead or dying will stain red with propidium iodide, whereas viable cells with an intact membrane will stain green with SYTO™ 9 propidium iodide. Live / dead cell ratio was analyzed by flow cytometry. The protease inhibitor CI-2 was intracellularly expressed in . subtilis using either the veg promoter (Pveg; SEQ ID NO: 8) or the bglC promoter (PbglC; SEQ ID NO: 9), as these promoters play a crucial role in regulating optimal gene expression, ensuring that CI-2 inhibitor production occurs effectively within the B. subtilis protease production host.

[0211] For example, as shown below in TABLE 2, co-expression of CL2 intracellularly with BPN'-Vl delivers a much higher biomass at 48 hours of fermentation compared to the strain without CI-2 coexpression.IFF10126-WO-PCTTABLE 2 RELATIVE IMPROVEMENT IN THE GROWTH OF BACILLUS STRAIN COEXPRESSING A PROTEASE AND CL2 INHIBITOR

[0212] In addition, as shown below in TABLE 3, co-expression of CI-2 with subtilisin BPN'-Vl results in more viable cells and less dead cells compared to the strain without CI-2 co-expression at 44 hours of fermentation.TABLE 3CELL VIABILITY ASSAY FOR BACILLUS STRAINS EXPRESSING A PROTEASE WITH OR WITHOUT CL2 INHIBITOR INTRACELLULAR EXPRESSIONEXAMPLE 3INTRACELLULAR EXPRESSION OF CI-2 INHIBITOR IMPROVES PROTEASE PRODUCTION IN BACILLUS CELLS

[0213] A. Overview

[0214] As set forth above in Example 2, Applicant constructed recombinant B. subtilis strains coexpressing an exemplary subtilisin protease (BPN'-V 1 ) and a protease inhibitor (CI-2) polypeptide, wherein the CL2 polypeptide was expressed and retained within the B. subtilis cell (intracellularly). In the present example, Applicant has constructed recombinant B. subtilis strains co-expressing other subtilisin proteases (e.g., BPN'-V2 and BG46-V1) and the CI-2 protease inhibitor polypeptide, wherein the co-expressed CI-2 polypeptide is retained intracellularly. More particularly, as set forth below, Applicant has discovered that co-expression of CI-2 polypeptide inhibitor intracellularly (e.g. with an optimal strength promoter) particularly supports multicopy of gene of interest expression, and enhances production of the subtilisin proteases.

[0215] B. Protease CassettesIFF10126-WO-PCT

[0216] The general construction of expression cassettes encoding the BPN'-V2, BG46-V1 and GG36-V1 variant proteases are described hereinafter. For instance, in the case of the mature BPN'-V2 protease, a first DNA fragment comprising an upstream (5') s A flanking sequence (SEQ ID NO: 16) of B. subtilis was operably linked to a nucleic acid (expression cassette) comprising an upstream B. subtilis PspoVG- Prrnl-Pscr promoter (SEQ ID NO: 27) operably linked to a DNA sequence comprising an aprE 5'-UTR (SEQ ID NO: 12) operably linked to a DNA sequence (SEQ ID NO: 28) encoding a B. amyloliquefaciens (BPN') signal sequence operably linked to a DNA sequence (SEQ ID NO: 14) encoding a B. amyloliquefaciens (BPN') pro-peptide sequence operably linked to a DNA sequence (SEQ ID NO: 26) encoding the mature BPN'-V2 protease operably linked to a spoVG transcriptional terminator (SEQ ID NO:29) operably linked to a downstream (3') skfH flanking region (SEQ ID NO: 17).

[0217] A second DNA fragment containing an upstream (5') ppsC flanking sequence (SEQ ID NO: 30) of B. subtilis was operably linked to a nucleic acid (expression cassette) comprising an upstream B. subtilis PspoVG-Prrnl-Pscr promoter (SEQ ID NO: 27) operably linked to a DNA sequence comprising an aprE 5'-UTR (SEQ ID NO: 12) operably linked to a DNA sequence (SEQ ID NO: 28) encoding a B. amyloliquefaciens (BPN') signal sequence operably linked to a DNA sequence (SEQ ID NO: 14 ) encoding a B. amyloliquefaciens pro-peptide sequence operably linked to a DNA sequence (SEQ ID NO: 26) encoding the mature BPN'-V2 protease operably linked to a downstream DNA sequence comprising a spoVG transcriptional terminator (SEQ ID NO: 29) which was operably linked to a downstream (3') ppsC flanking region (SEQ ID NO: 31).

[0218] A third DNA fragment containing an upstream (5') sunT flanking sequence (SEQ ID NO: 18) of B. subtilis was operably linked to an expression cassette comprising an upstream (5') B. subtilis PspoVG- Prrnl-Pscr promoter (SEQ ID NO: 27) operably liked to a DNA sequence comprising an aprE 5'-UTR (SEQ ID NO: 12) operably linked to a DNA sequence (SEQ ID NO: 28) encoding the BPN' signal sequence operably linked to a DNA sequence (SEQ ID NO: 14) encoding a B. amyloliquefaciens pro-peptide sequence operably linked to a DNA sequence (SEQ ID NO: 26) encoding a mature BPN'-V2 protease operably linked to a DNA sequence comprising spoVG transcriptional terminator (SEQ ID NO: 29) operably linked to a B. subtilis alanine racemase gene (SEQ ID NO: 21) which was operably linked to a downstream (3') sunT flanking region (SEQ ID NO: 19).

[0219] A fourth DNA fragment containing an upstream (5') yhfN flanking sequence (SEQ ID NO: 20) of B. subtilis was operably linked to an expression cassette comprising an upstream (5’) B. subtilis PspoVG- Prrnl-Pscr promoter (SEQ ID NO: 20) operably linked to a DNA sequence (SEQ ID NO: 12) comprising an aprE 5'-UTR operably linked to a DNA sequence (SEQ ID NO: 28) encoding the B. subtilis BPN' signal sequence operably linked to a DNA sequence (SEQ ID NO: 14) encoding pro-peptide sequence operably linked to a DNA sequence (SEQ ID NO: 26) encoding a mature BPN'-V2 protease operably linked to aIFF10126-WO-PCT downstream DNA sequence comprising a spoVG transcriptional terminator (SEQ ID NO: 29) which was operably linked to a downstream (3') aprE flanking region (SEQ ID NO: 22).

[0220] For example, a modified B. subtilis BPN'-V2 production strain comprising protease expression cassettes was constructed by integrating all four (4) protease fragments into the genome.

[0221] Similarly, in the case of the mature BG46-V1 protease, a first DNA fragment containing an upstream (5') skfA flanking sequence (SEQ ID NO: 16) of B. subtilis was operably linked to an expression cassette comprising an upstream (5') B. subtilis P2 promoter (SEQ ID NO: 11) operably linked to a DNA sequence comprising an aprE 5'-UTR (SEQ ID NO: 12) operably linked to a DNA sequence (SEQ ID NO: 13) encoding an AprE signal sequence operably linked to a DNA sequence (SEQ ID NO: 32) encoding a B. gibsonii pro-peptide sequence operably linked to a DNA sequence (SEQ ID NO: 33) encoding the mature BG46-V 1 protease operably linked to a DNA sequence comprising a BPN’ transcriptional terminator (SEQ ID NO: 15) which was operably linked to a downstream (3') skfH flanking region (SEQ ID NO: 17).

[0222] A second DNA fragment containing an upstream(5') spoIIIAE flanking sequence (SEQ ID NO: 34) of B. subtilis was operably linked to an expression cassette comprising an upstream B. subtilis P2 promoter (SEQ ID NO: 11) operably linked to a DNA sequence (SEQ ID NO: 12) comprising an aprE 5'-UTR operably linked to an DNA sequence (SEQ ID NO: 13) encoding an AprE signal sequence operably linked to an DNA sequence (SEQ ID NO: 32 ) encoding B. gibsonii pro-peptide sequence operably linked to a DNA sequence (SEQ ID NO: 33) encoding the mature BG46-V1 protease operably linked to a DNA sequence (SEQ ID NO: 15) comprising a BPN’ transcriptional terminator operably linked to a B. subtilis alanine racemase gene (SEQ ID NO: 21) which was operably linked to a downstream (3') spoIIIAE flanking region (SEQ ID NO: 35).

[0223] A third DNA fragment containing an upstream (5') yhfN flanking sequence (SEQ ID NO: 20) of B. subtilis was operably linked to an expression cassette comprising an upstream (5') B. subtilis P2 promoter (SEQ ID NO: 11) operably linked to a DNA sequence (SEQ ID NO: 12) comprising an aprE 5' untranslated region (5’ UTR) operably linked to a DNA sequence (SEQ ID NO: 13) encoding an AprE signal sequence operably linked to a DNA sequence (SEQ ID NO: 32) encoding B. gibsonii pro-peptide sequence operably linked to a DNA sequence (SEQ ID NO: 33) encoding the mature BG46-V1 protease operably linked to a DNA sequence (SEQ ID NO: 15) comprising a BPN' terminator which was operably linked to a downstream (3') aprE flanking region (SEQ ID NO: 22).

[0224] For example, a modified B. subtilis BG46-V1 production strain comprising protease expression cassettes was constructed by integrating all three (3) protease fragments into the genome.

[0225] Similarly, in the case of the mature GG36-V1 protease, a first DNA fragment containing an upstream (5') skjA flanking sequence (SEQ ID NO: 16) of B. subtilis was operably linked to an expression cassette comprising an upstream (5') B. subtilis P2 promoter (SEQ ID NO: 11) operably linked to a DNAIFF10126-WO-PCT sequence comprising an aprE 5'-UTR (SEQ ID NO: 12) operably linked to a DNA sequence (SEQ ID NO: 13) encoding an AprE signal sequence operably linked to a DNA sequence (SEQ ID NO: 42) encoding a B. clausii pro-peptide sequence (AGKAEE-GG36 Pro_E30G) operably linked to a DNA sequence (SEQ ID NO: 43) encoding the mature GG36-V 1 protease operably linked to a DNA sequence comprising a BPN’ transcriptional terminator (SEQ ID NO: 15) which was operably linked to a downstream (3') skfH flanking region (SEQ ID NO: 17).

[0226] A second DNA fragment containing an upstream(5') sunT flanking sequence (SEQ ID NO: 18) of B. subtilis was operably linked to an expression cassette comprising an upstream B. subtilis P2 promoter (SEQ ID NO: 11) operably linked to a DNA sequence (SEQ ID NO: 12) comprising an aprE 5'-UTR operably linked to an DNA sequence (SEQ ID NO: 13) encoding an AprE signal sequence operably linked to an DNA sequence (SEQ ID NO: 42 ) encoding B. clausii pro-peptide sequence (AGKEE-GG36 Pro_E30G) operably linked to a DNA sequence (SEQ ID NO: 3) encoding the mature GG36-V1 protease operably linked to a DNA sequence (SEQ ID NO: 15) comprising a BPN’ transcriptional terminator which was operably linked to a downstream (3') sunT flanking region (SEQ ID NO: 19).

[0227] A third DNA fragment containing an upstream (5') yhfN flanking sequence (SEQ ID NO: 20) of B. subtilis was operably linked to an expression cassette comprising an upstream (5') B. subtilis P2 promoter (SEQ ID NO: 11) operably linked to a DNA sequence (SEQ ID NO: 12) comprising an aprE 5' untranslated region (5’ UTR) operably linked to a DNA sequence (SEQ ID NO: 13) encoding an AprE signal sequence operably linked to a DNA sequence (SEQ ID NO: 42) encoding B. clausii pro-peptide sequence (AGKEE- GG36 Pro_E30G) operably linked to a DNA sequence (SEQ ID NO: 43) encoding the mature GG36-V1 protease operably linked to a DNA sequence (SEQ ID NO: 15) comprising a BPN' terminator operably linked to a B. subtilis alanine racemase gene (SEQ ID NO: 21) which was operably linked to a downstream (3') aprE flanking region (SEQ ID NO: 22).

[0228] For example, a modified B. subtilis GG36-V1 production strain comprising protease expression cassettes was constructed by integrating all three (3) protease fragments into the genome.

[0229] Thus, as further described below, the BPN'-V2, BG46-V1 and GG36-V1 production strains were further modified by the introduction of an expression cassette encoding the CI-2 protease inhibitor polypeptide.

[0230] C. Inhibitor Cassete

[0231] As described above, the B. subtilis BPN'-V2, BG46-V1 and GG36-V1 protease production strains were further modified by introducing CI-2 cassettes therein.

[0232] More particularly, in the case of BPN'-V2 protease strain, the CI-2 protease inhibitor cassette (FIG. 2) comprises an upstream (5') bglC promoter (PbglC SEQ ID NO: 9) operably linked to a Shinc-Dalgarno (SD) sequence (SEQ ID NO: 23) operably linked to a DNA (ORF) sequence encoding the CI-2 inhibitorIFF10126-WO-PCT(SEQ ID NO: 6) operably linked to a BPN' transcriptional terminator (SEQ ID NO: 15). The CI-2 fragment comprising 5' pksL flanking sequence (SEQ ID NO: 24) was operably linked to CI-2 expression cassette followed by 3' pksL flanking sequence (SEQ ID NO: 25). In the case of the BG46-V1 and GG36-V1 protease strain, the CI-2 protease inhibitor cassette (FIG. 2) was integrated into the genome, wherein the cassette is driven by the veg promoter (Pveg; SEQ ID NO:8)

[0233] D. Fermentation and Protease Production

[0234] The protease production strains with, or without CI-2 expression, were fermented about 60 hours using standard fermentation condition in a 24-well deep well plate with 0.5X MPS2 media (pH7.3), wherein the expression of protease was determined by standard AAPF assay. More particularly, as shown below in TABLE 4, the modified strains with intracellular CI-2 expression demonstrate significantly improved / enhanced protease production (i.e., at least about 10% to 30% higher) compared to the strain without CI-2 co-expression.TABLE 4 RELATIVE IMPROVEMENT IN PROTEASE PRODUCTION OF BACILLUS STRAINS CO-EXPRESSING CI-2 INHIBITOR INTRACELLULARLYIFF10126-WO-PCTREFERENCESPCT Publication No. W02002 / 14490PCT Publication No. W02003 / 083125PCT Publication No. W02008 / 112459PCT Publication No. W02010 / 056634PCT Publication No. WO2011 / 072099PCT Publication No. WO2011 / 130222PCT Publication No. WO2015 / 089447PCT Publication No. WO2016 / 202839PCT Publication No. WO2017 / 207762PCT Publication No. WO2020 / 242858PCT Publication No. WO2023 / 023642PCT Publication No. WO2023 / 114936PCT Publication No. WO2023 / 114939PCT Publication No. WO2024 / 163584Armenteros et al., “SignalP 5.0 improves signal peptide predictions using deep neural networks”, Nature Biotechnology, 37: 420-423, 2019.Ausubel et al., “Current Protocols in Molecular Biology”, published by Greene Publishing Assoc, and Wiley-Interscience (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 et al., “Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus sublilis by saturation mutagenesis of the N-domain of the AmyE signal peptide”, Appl. Microbiol. Biotechnol., 86(6):1877-1885, 2010.Chen et al., “Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression”, Microbial Cell Factories, 14(1 ):92, 2015.IFF10126-WO-PCTEarl 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’” Regal. Toxicol. Pharmacol., 45(2): 144-158, 2006.Radisky and Koshland, “A clogged gutter mechanism for protease inhibitors”. Proc Natl Acad Sci USA, 99(16): 10316-10321, 2002.Raul et al., “Production and parrial 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).Tjalsma et al., “Signal Peptide-Dependent Protein Transport in Bacillus subtilis: a Genome-Based Survey of the Sccrctomc”, Microbiology and Molecular Biology Reviews, 64: 515-547, 2000.Van Dijl and Hecker, “Bacillus subtilis: from soil bacterium to super-secreting cell factory”, Microbial Cell Factories, 12(3). 2013.Vogtentanz, Protein Expr. Purif., 55:40-52, 2007.

Claims

IFF10126-WO-PCTCLAIMS1. A modified Bacillus sp. cell co-expressing a heterologous subtilisin protease and a heterologous protease inhibitor polypeptide comprising at least 75% identity to SEQ ID NO: 6 or SEQ ID NO: 45.

2. The modified cell of claim 1, wherein the mature protease is secreted extracellularly and the protease inhibitor polypeptide is retained intracellularly, when fermented under conditions for the production of the protease.

3. The modified cell of claim 1 , wherein the cell produces at least 5% or more of the protease relative to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell expresses the same protease, but does not co-express the protease inhibitor.

4. The modified cell of claim 1, comprising at least one, two, three, or four introduced expression cassettes encoding the protease.

5. The modified cell of claim 4, wherein the cassettes are integrated into the genome of the cell .

6. The modified cell of claim 4, wherein the cassettes comprise a polynucleotide encoding a mature protease, wherein the polynucleotide comprises at least 75% identity to any one of SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 36 and SEQ ID NO: 43.

7. The modified cell of claim 1, comprising an introduced expression cassette encoding the protease inhibitor polypeptide, wherein the cassette comprises an upstream heterologous promoter region operably linked to a downstream polynucleotide encoding a heterologous protease inhibitor polypeptide comprising at least 75% identity to SEQ ID NO: 6 or SEQ ID NO: 45.

8. The modified cell of claim 1, wherein the cell comprises one or more genetic modifications rendering the cell deficient in the production of one or more endogenous proteins.

9. The modified cell of claim 1, wherein the protease comprises at least 75% identity to any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44.

10. A method for the enhanced production of a protease in a Bacillus sp. cell comprising: introducing into a Bacillus sp. cell an expression cassette encoding a mature subtilisin protease and an expression cassette encoding a protease inhibitor polypeptide comprising at least 75% identity to SEQ ID NO: 6 or SEQ ID NO: 45, and fermenting the modified cell under conditions for production of the protease, wherein the mature protease is secreted into the fermentation broth and the protease inhibitor is retained intracellularly.IFF10126-WO-PCT11. The method of claim 10, wherein the modified cell produces at least 5% or more of the protease as compared to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell comprises the same introduced cassette encoding the protease, but does not comprise an introduced cassette encoding the protease inhibitor.

12. The method of claim 10, wherein the modified cell comprises at least two, three, or four introduced cassettes encoding the mature protease.

13. The method of claim 10, wherein the modified cell has at least a 5% increase in biomass after 48 hours of fermentation relative to a control Bacillus sp. cell fermented under the same conditions, wherein the control cell comprises the same introduced cassette encoding the protease, does not comprise an introduced cassette encoding the protease inhibitor.

14. The method of claim 10, wherein the cassette encoding the mature protease comprises upstream and downstream flanking sequences homologous to a gene locus of interest (GLOI), wherein the cassette is integrated into the GLOT.

15. The method of claim 10 or claim 12, wherein the cassette, or cassettes encoding the mature subtilisin comprise a polynucleotide sequence having at least 75% identity to a polynucleotide selected from any one of SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 36 and SEQ ID NO: 43.

16. The method of claim 10 or claim 12, wherein the mature subtilisin comprises at least 75% identity to any one of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 44.

17. The method of claim 10, wherein the cell comprises one or more genetic modifications rendering the cell deficient in the production of one or more endogenous proteins.

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