Improved bacillus cells with inactivated metalloproteases

Abstract

The present invention provides an improved bacillus cell with reduced metalloproteinase gene expression. Inactivation of the metalloproteinase gene reduces proteolytic degradation of the protein of interest produced by the Bacillus cells and thus increases the yield of the protein of interest in such production methods. The invention also relates to a method of inactivating a metalloprotease and to the use of this inactivation method in the production of a protein of interest.

Description

Technical Field

[0001] This invention relates to a modified Bacillus cell for producing proteins of interest. Specifically, this invention relates to a Bacillus cell having reduced expression of a gene encoding a metalloproteinase containing amino acids having at least 55% identity with SEQ ID NO: 08. Background Technology

[0002] Bacillus microorganisms are widely used as an industrial mainstay for the production of valuable compounds such as chemicals, polymers, and proteins, particularly proteins such as detergent and / or cleaning active enzymes or enzymes for feed and food applications. The biotechnological production of these enzymes is carried out via fermentation of these Bacillus strains and subsequent product purification. Bacillus strains are capable of secreting large quantities of protein into the fermentation broth. This allows for a simpler product purification process for the protein of interest compared to intracellular production, explaining the success of Bacillus in industrial applications. Therefore, continuous optimization of Bacillus cells to increase the production of these proteins is highly relevant. Especially in large-scale industrial production environments, even small improvements can have a significant impact on production costs.

[0003] Bacillus cells naturally secrete a variety of extracellular proteins and enzymes, meaning that the protein or enzyme of interest is not the only protein in the supernatant. Most often, these cellular proteins are ignored due to their low abundance compared to the protein of interest. However, because they (especially cellular proteases) are capable of degrading other proteins, this may not be desirable in applications where the protein of interest is unstable in terms of proteolysis or where the protein product must lack any proteolytic activity. The latter includes, for example, dairy applications where milk-derived casein should not be degraded by trace amounts of proteases, or detergent formulations where other co-formulated enzymes will be degraded.

[0004] Kawamura et al. (Karamura and Doi, Journal of Bacteriology, October 1984, Vol. 160, No. 1, pp. 442-444) have recognized the prevention of foreign protein degradation by the deletion of cellular proteases. Here, in the parent host *Bacillus subtilis*, two major host proteases (NprE and AprA (also known as AprE)) have been inactivated, resulting in *Bacillus subtilis* strain DB104. This mutant strain lacking extracellular neutral and extracellular alkaline proteases has been discussed here for the production of foreign proteins and peptides. Wu et al. (Wu et al., Journal of Bacteriology, August 1991, Vol. 173, No. 16, pp. 4952-4958) have deleted other host proteases. Here, a strain already lacking four host proteases (Bacillus subtilis DB428 with inactive genes encoding NprE, AprE, Epr, and Bpf (also known as Bpr)) was used as a template to construct Bacillus subtilis WB600 with inactive genes encoding NprE, AprE, Epr, and Bpr, and further lacking genes encoding Mpr and NprB. Wu et al. also described using this strain to improve the production of heterologous protein β-lactamases. Here, proteolytically sensitive β-lactamases showed significantly higher yields due to reduced loss from degradation after extended culture times. WB600 was optimized by the same group, where the deletion of genes encoding two additional proteases (WprA and Vpr) allowed for the successful secretion of single-chain antibody fragments (Wu et al., Applied and Environmental Microbiology, July 2002, Vol. 68, No. 7, pp. 3261-3269). This strain is known as Bacillus subtilis WB800, which possesses the following inactivated protease genes: nprE, aprE, epr, bpr, mpr, nprB, vpr, and wprA. Similarly, WO09022162 further developed a multi-protease knockout strain, which additionally lacks the genes encoding the quality control proteases htrA and hrtB, thereby increasing the yield and purity of secreted recombinant proteins.

[0005] The concept of deleting host proteases to improve the overexpression of proteins of interest has been described not only for Bacillus subtilis but also for other Bacillus species. Schallmey et al. (Schallmey et al., Canadian Journal of Microbiology, 2004, Vol. 50; pp. 1-17; doi: 10.1139 / W03-07) generally described how the production and secretion of high-yield recombinant proteins in Bacillus hosts are hindered by the degradation of the products by host proteases.

[0006] Vehmannaperä et al. (Vehmannaperä et al.; Journal of Biotechnology, 1991, Vol. 19, pp. 221-240) described improved amylase and dextranase secretion, for example, in Bacillus amyloliquefaciens lacking functional nprE and aprE genes, even in complex media requiring basic protein hydrolytic activity for degradation. In the case of culture techniques using chemically determined media, even more advanced effects can be hypothesized here.

[0007] WO2014206829 describes a Bacillus licheniformis strain that lacks several genes encoding proteases (aprL, mprL, bprAB, epr, vpr, wprA, and additionally ispA) that can be used to produce heterologous enzymes. The latter gene is noteworthy because it encodes the intracellular serine protease IspA. This highlights the need to consider not only the reduction of proteolytic activity in the supernatant by extracellular proteases but also that of intracellular proteases. Similarly, EP1829959 describes the inactivation of the intracellular protease AprX, alone or in combination with the aforementioned proteases, for increasing the yield of recombinant proteins.

[0008] However, proteins of interest that are sensitive to proteolysis may still be degraded by host proteases unknown to those skilled in the art.

[0009] Identification of host proteases presents a significant challenge, as more than 100 genes with proteolytic functions have been described, for example, in Bacillus licheniformis DSM 13. A search for proteases and peptidases of Bacillus licheniformis via the UniProt database (www.uniprot.org; UniProtConsortium. UniProt: Universal Protein Knowledge Base of 2021. Nucleic Acids Res. Jan 8, 2021; 49(D1):D480-D489) yielded 177 entries (search date: May 02, 2023). This database contains entries for "proteases" or "peptidases" and "Bacillus licheniformis" (BioID 279010, with Bacillus licheniformis strains ATCC 14580 / DSM 13 / JCM 2505 / CCUG 7422 / NBRC 12200 / NCIMB9375 / NCTC 10341 / NRRL NRS-1264 / Gibson 4).

[0010] As disclosed herein, selecting genes for inactivation remains very challenging because it is uncertain whether the deletion of specific genes encoding proteins with proteolytic activity is effective in reducing the degradation of proteins of interest.

[0011] In this paper, the inventors surprisingly discovered that inactivation of the gene encoding the putative metalloproteinase in Bacillus cells led to an increase in the yield of the protein of interest produced using the Bacillus cells. Summary of the Invention

[0012] Therefore, the present invention relates to a Bacillus cell having reduced expression of a gene encoding a metalloproteinase selected from the group consisting of: I. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and II. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07.

[0013] Furthermore, the present invention relates to a method for producing a protein of interest, preferably an enzyme, the method comprising the following steps: I. Providing Bacillus cells with reduced expression of genes encoding metalloproteinases selected from the group consisting of: a) Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b) A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. II. An expression cassette encoding the protein of interest, preferably the enzyme, that is sensitive to the metalloproteinase is introduced into the Bacillus cell. III. The Bacillus cells are cultured under conditions that allow for the expression of the protein of interest, thereby forming a fermentation broth containing the protein of interest, and IV. Optionally, the protein of interest may be isolated from the fermentation broth of step III.

[0014] In another embodiment, the present invention relates to a method for inactivating metalloproteinases, wherein the method comprises the following steps: I. Provide metalloproteinases selected from the following groups: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07, and II. Contact the metalloproteinase with a chelating agent, preferably EDTA, MGDA, or EDDS, for a sufficient amount and time to inactivate the metalloproteinase.

[0015] Therefore, in an alternative embodiment, the present invention relates to a method for producing a protein of interest, preferably an enzyme, the method comprising the following steps: I. Provide Bacillus cells expressing a metalloproteinase selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. II. An expression cassette encoding the protein of interest, preferably the enzyme, that is sensitive to the metalloproteinase is introduced into the Bacillus cell. III. The Bacillus cells are cultured under conditions that allow for the expression of both the protein of interest and the metalloproteinase, thereby forming a fermentation broth containing both the protein of interest and the metalloproteinase. IV. Optionally, the protein of interest is isolated from the culture medium to form a solution containing the protein of interest and the metalloproteinase, and V. Contact the fermentation broth from step III and / or the solution from step IV with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a duration that effectively inactivates the metalloproteinase.

[0016] Therefore, the present invention also relates to the use of a chelating agent, preferably EDTA, MGDA, or EDDS, for improving the yield and / or stability of the protein of interest, preferably an enzyme, in a method for producing the protein of interest via Bacillus cells, wherein the Bacillus cells express a metalloproteinase selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. The protein of interest is sensitive to this metalloproteinase. Detailed Implementation

[0017] The invention will be more readily understood by referring to the following definitions, detailed description of embodiments of the invention, and examples included herein. Although the invention will be described with reference to specific embodiments, this description should not be construed as limiting.

[0018] definition Unless otherwise indicated, the terminology used herein should be understood in accordance with the usual usage of those skilled in the art.

[0019] Before describing exemplary embodiments of the invention in detail, definitions important for understanding the invention are provided. Unless otherwise stated or apparent from the nature of the definitions, these definitions apply to all compounds, methods, and uses described herein.

[0020] It should be understood that the word "a (or an)" as used in the specification and claims may mean one or more, depending on the context in which it is used. Thus, for example, reference to "a cell" may mean that at least one cell can be used.

[0021] Furthermore, it should be understood that, as used herein, the term "at least one" means one or more items mentioned after the term can be used according to the invention. For example, if the term indicates that at least one feed solution should be used, this can be understood as one feed solution or more than one feed solution, i.e., two, three, four, five, or any other number of feed solutions. Depending on the item referred to by the term, those skilled in the art will understand the upper limit (if any) that the term may refer to.

[0022] As used herein, the term “about” means with respect to any number referenced after the term, in which there exists an interval of accuracy in which the technical effect can be achieved. Thus, as referred to herein, “about” preferably means a precise numerical value or a range around said precise numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0023] The term “comprising” as used herein should not be construed as restrictive. Rather, it indicates that more than the actual item referred to may be present; for example, if the term refers to a method that includes certain steps, then the presence of additional steps should not be excluded. However, the term “comprising” also covers implementations in which only the item referred to is present, i.e., in the sense of “consisting of”.

[0024] The terms “encoding” and “coding” are used interchangeably in this document. Generally, the term refers to the property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to act as a template for the synthesis of other macromolecules (such as a defined amino acid sequence). Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes a protein.

[0025] The difference between a "variant" protein and a "parent" protein lies in the alteration of certain amino acids, preferably the substitution of amino acids at one or more amino acid positions.

[0026] When describing the polypeptides and variants of the present invention, the abbreviations for single amino acids are used in accordance with the recognized IUPAC single-letter or three-letter amino acid abbreviations.

[0027] As used in this article, "amino acid alteration" refers to the substitution, deletion, or insertion of amino acids.

[0028] A substitution is described by providing the original amino acid, followed by its position number within the amino acid sequence, and then the amino acid that replaced the original amino acid. For example, replacing histidine at position 120 with alanine is termed "His120Ala" or "H120A". A substitution can also be described by simply naming the resulting amino acid without specifying the initial amino acid at that position, such as "X120A", "120A", "Xaa120Ala", or "120Ala". The position of the substitution can also be described by providing only the position number within the amino acid sequence.

[0029] The term "deletion" is described by providing the original amino acid, followed by its position number within the amino acid sequence, and then an asterisk (*). Thus, a glycine deletion at position 150 is named "Gly150*" or "G150*". Alternatively, a deletion is indicated by, for example, "deletion of G184".

[0030] An "insertion" is described by providing the original amino acid, followed by its position number within the amino acid sequence, and then the original amino acid and the additional amino acid. For example, an insertion of lysine immediately adjacent to glycine at position 180 is named "Gly180GlyLys" or "G180GK". When more than one amino acid residue is inserted, such as, for example, inserting Lys and Ala after Gly180, this can be represented as "Gly180GlyLysAla" or "G195GKA".

[0031] When substitution and insertion occur at the same position, this can be represented as "S99A+S99SD" or simply "S99AD". Sequences containing multiple changes are separated by "+", for example, "Arg170Tyr+Gly195Glu", "R170Y+G195E", or "X170Y+X195E" indicate that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively. Alternatively, multiple changes can be separated by spaces or commas, for example, "R170Y G195E" or "R170Y, G195E". When different substitution changes can be introduced at one position, the different changes are separated by commas, for example, "Arg170Tyr, Glu" and "R170T, E" indicate that arginine at position 170 is replaced by tyrosine or glutamic acid, respectively. Substitution at a specific location can also be expressed as “X120A,G,H”, “120A,G,H”, “X120A / G / H”, or “120A / G / H”. Alternatively, different changes or optional substitutions can be indicated in parentheses, such as “Arg170[Tyr,Gly]” or “Arg170{Tyr,Gly}”, or simply “R170[Y,G]” or “R170{Y,G}”.

[0032] Variants of the parent protein can be defined by their sequence identity when compared to the parent protein. Sequence identity is typically provided as "sequence identity %" or "identity %". To determine the percentage of identity between two amino acid sequences in the first step, pairwise sequence alignments are generated between the two sequences, where the two sequences are aligned across their full length (i.e., pairwise global alignments). Such alignments are generated by a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1970) 48, pp. 443-453), preferably using the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with the program's default parameters (vacancy open = 10.0, vacancy extension = 0.5, and matrix = EBLOSUM62). For the purposes of this invention, the preferred alignment is the one from which the highest sequence identity can be determined.

[0033] After comparing the two sequences, in the second step, an identity value should be determined based on the comparison. Therefore, according to the present invention, the following percentage of identity is applicable for calculation: Identity % = (Identical residues / Length of the alignment region showing the corresponding sequence of the invention over its full length) * 100. Therefore, sequence identity related to the comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region showing the corresponding sequence of the invention over its full length. Multiplying this value by 100 yields "Identity %".

[0034] To calculate the percentage of identity between two DNA sequences, the same applies to calculating the percentage of identity between two amino acid sequences with certain specifications. For protein-coding DNA sequences, pairwise alignment should be performed along the full length of the coding region from the start codon to the stop codon (excluding introns). For non-protein-coding DNA sequences, pairwise alignment should be performed along the full length of the sequence of this invention, thus comparing the full sequence of this invention with another sequence or a region outside of another sequence. Furthermore, the preferred alignment procedure for nucleic acid sequences implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443-453) is “NEEDLE” (European Molecular Biology Open Software Suite (EMBOSS)) with the program's default parameters (vacancy open = 10.0, vacancy extension = 0.5, and matrix = EDNAFULL).

[0035] In this document, the substitution of an amino acid by a similar amino acid exchange can be termed "conservative substitution." Similar amino acids according to the present invention are defined as follows: Amino acid A is similar to amino acid S Amino acid D is similar to amino acid E; N Amino acid E is similar to amino acids D; K; Q Amino acid F is similar to amino acid W; Y Amino acid H is similar to amino acid N; Y Amino acid I is similar to amino acids L; M; V Amino acid K is similar to amino acids E; Q; R Amino acid L is similar to amino acids I; M; V Amino acid M is similar to amino acids I; L; V Amino acid N is similar to amino acids D; H; S Amino acid Q is similar to amino acids E; K; R Amino acid R is similar to amino acid K; Q Amino acid S is similar to amino acids A, N, and T. Amino acid T is similar to amino acid S Amino acid V is similar to amino acids I; L; M Amino acid W is similar to amino acid F; Y Amino acid Y is similar to amino acids F, H, and W.

[0036] In this paper, "protein homologs" are defined as proteins that share sequence and functional similarity with another protein due to a common ancestor. The corresponding meaning applies to "gene homologs".

[0037] The term “natural” (or natural, wild-type, or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or polypeptide that is found in nature (i.e., without any human intervention).

[0038] The term "isolated" molecule, such as a polypeptide or polynucleotide, is defined in this document as a molecule that has been isolated from its natural environment.

[0039] The term "heteropeptide" (or exogenous or foreign peptide) is defined herein as a peptide that is not naturally expressed by cells. The term "heteronucleotide" (or exogenous or foreign polynucleotide) is defined herein as a polynucleotide that is not naturally present in cells.

[0040] For the purposes of this invention, “recombination” (or non-natural or unnaturally) in relation to cells or organisms means that the cells or organisms contain polynucleotides introduced using genetic technology or polynucleotides that have been removed from the cells or organisms using genetic technology, or a combination of both. Recombination of polynucleotides or peptides means that the polynucleotides or peptides have been rearranged or rearranged in their genetic environment using recombinant DNA technology. Therefore, recombinant polynucleotides or peptides include peptides or polynucleotides that are native to the cell, whose expression is quantitatively altered or targeted to a genomic location different from that of the native cell due to manipulation of the cell’s DNA by recombinant DNA technology, such as a stronger promoter. Recombinant polynucleotides or peptides can also be heterologous, meaning they can be foreign sequences, but they can also originate from the same organism in which they were introduced.

[0041] Regarding the relationship between two or more polynucleotides or two or more polypeptides, the term "recombination" is used to characterize two or more polynucleotides or two or more polypeptides that do not naturally exist in a specific combination with each other.

[0042] "Modified recombinant" polynucleotides or peptides refer to recombinant polynucleotides or recombinant peptides that have been modified by introducing changes, such as deletions, substitutions, and / or insertions, to alter the natural peptide or natural polynucleotide through the use of recombinant DNA technology.

[0043] "Synthetic" compounds are obtained through in vitro chemical or enzymatic synthesis.

[0044] As used herein, a “gene construct” or “expression cassette” is a nucleic acid molecule consisting of at least one sequence of interest to be expressed, which is operatively linked to one or more control sequences (at least to a promoter) as described herein.

[0045] As used herein, the term "vector" encompasses any kind of construct suitable for carrying a polynucleotide sequence for transfer into a cell or for stable or transient expression within a given cell. This covers any kind of cloning vector, such as, but not limited to, plasmids, phage particles, viral vectors (e.g., bacteriophages), bacterial phages, baculoviruses, cosmids, fosmids, artificial chromosomes, and any other vectors specific to a particular host of interest. The foreign polynucleotide sequence typically contains a sequence encoding a protein of interest, which may be referred to herein as the "gene of interest."

[0046] As used herein, the terms “introduction of polynucleotides” or “conversion of polynucleotides” encompass the transfer of polynucleotides from outside the cell into the cell, regardless of the method used for the transfer. That is, as used herein, the term “conversion of polynucleotides” is independent of the vector, shuttle system, or cell, and it not only refers to polynucleotide transfer methods known in the art (see, for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but it also encompasses any other kind of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0047] The term "expression" or "gene expression" refers to the transcription of one or more specific genes or specific nucleic acid constructs. Specifically, it refers to the transcription of one or more genes or gene constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, which is subsequently translated into or not translated into a protein. This process includes the transcription of DNA and the processing of the resulting mRNA product. The term "protein expression" specifically refers to the transcription of one or more genes or gene constructs into mRNA, which is subsequently translated into a protein.

[0048] Cells expressing recombinant polynucleotides or peptides may exhibit “increased” or “decreased” expression when compared to their corresponding parental cells. As used herein, the terms “increased expression,” “enhanced expression,” or “overexpression” mean any form of expression other than the expression level in parental cells (which may also be absent or unmeasurable expression). “Increased expression,” “enhanced expression,” or “overexpression” as used herein means increased gene expression relative to a control organism, and / or, in the case of a peptide mentioned, increased peptide level and / or increased peptide activity. An increase in expression compared to a control organism may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or 100% or even more.

[0049] The gene-related term "reduced" expression means a gene expression level lower than that in parental cells, such that the gene with reduced expression does not produce active proteins or produces only a small amount of active proteins. The expression level can be reduced by, for example, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or 100%, preferably 100%, compared to parental cell expression. Preferably, the cell does not produce active proteins. Cells with reduced expression levels such that they do not produce active proteins are referred to as "knockout" cells.

[0050] The terms “reduced protein expression” or “reduced protein expression” should mean a small amount of protein produced by the cell, for example, through reduced transcription or reduced translation. The term “non-natural reduction in protein expression” should mean a small amount of protein produced by the cell through any kind of genetic modification, particularly recombinant DNA technology, compared to unmodified cells, i.e., the corresponding parental cells.

[0051] "Parental cell" is a cell that differs from the cell of this invention only in that its gene expression is not reduced; that is, the corresponding host cell possessing the functional gene. Reduced gene expression is preferably achieved through gene deletion. Unless otherwise specified, gene "deletion" encompasses the partial or full-length deletion of a gene, which causes said gene to not express an active protein. Gene deletion can be indicated by a "D" preceding the gene name.

[0052] The protein-related term “inactivation” (also referred to herein as “functional inactivation”) means that the function of a protein has been reduced compared to the function of a protein that has not been inactivated, such that the protein is inactive or its activity is reduced, preferably wherein the protein is inactivated.

[0053] Proteins or genes can be inactivated, or their expression levels can be reduced compared to uninactivated genes or proteins, by any kind of artificial intervention applied to the gene or protein, i.e., non-natural inactivation, particularly by modification using recombinant DNA technology. Gene deletion (complete or partial) should be understood as a non-natural deletion achieved by any kind of artificial intervention applied to the cell, particularly by recombinant DNA technology, resulting in the complete or partial removal of the gene compared to unmodified cells.

[0054] The term "purification" refers to the process in which at least one component, such as the protein of interest, is separated from at least another component, such as particulate matter of the fermentation broth, and transferred to different compartments or phases, wherein the different compartments or phases do not necessarily need to be separated by a physical barrier. Accordingly, examples of such different compartments are two compartments separated by a filter membrane or filter cloth, namely filtrate and osmate; examples of such different phases are pellets and supernatant or filter cake and filtrate. The solution resulting from the purification of the enzyme of interest from the fermentation broth is referred to herein as a "purified enzyme solution".

[0055] The term "metalloproteinase" refers to an enzyme with proteolytic activity that contains one or more metal ions bound to the enzyme, wherein the association of the metal ion with the enzyme is essential for holoenzyme activity. Specifically, the metalloproteinases described herein belong to EC 3.4.24, which characterizes zinc-dependent metalloproteinases belonging to the M12 family of metallopeptidases.

[0056] A chelating agent is a compound that can coordinate with a metal ion at multiple sites to form a stable complex (called a chelate) with the metal ion.

[0057] The term "sensitive to metalloproteinases" for proteins refers to the property of a protein to be degraded by metalloproteinases via proteolytic degradation in the presence of the metalloproteinases and under conditions where the metalloproteinases exhibit proteolytic activity; that is, the protein is readily degraded by metalloproteinases. In this context, "degradation" should refer to partial degradation, i.e., truncation with one or more remaining larger protein fragments, and complete degradation, i.e., digestion into only small peptides and / or single amino acids.

[0058] The term “carbohydrate-binding domain” (CBD) or “carbohydrate-binding module” (CBM) is understood in this paper as a protein domain with carbohydrate-binding activity present in carbohydrate-active enzymes, such as glycoside hydrolases, particularly mannanases.

[0059] Bacillus cells with inactivated metalloproteinases In one embodiment, the present invention relates to a Bacillus cell having reduced expression of a gene encoding a metalloproteinase selected from the group consisting of: I. With SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 have at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity metalloproteinases, and II. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with SEQ ID NO: 07.

[0060] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloproteinase, the metalloproteinase having an amino acid sequence that is at least 55% identical to SEQ ID NO: 08.

[0061] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloproteinase having an amino acid sequence that is at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identical to that of SEQ ID NO: 08.

[0062] Preferably, the Bacillus cell has reduced gene expression encoding a metalloproteinase, the metalloproteinase having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identical to that of SEQ ID NO: 08. More preferably, the Bacillus cell has reduced gene expression encoding a metalloproteinase, the metalloproteinase having an amino acid sequence that is 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%, at least 99%, or 100% identical to that of SEQ ID NO: 08. Most preferably, the Bacillus cell has reduced gene expression encoding a metalloproteinase, the metalloproteinase having an amino acid sequence that is 100% identical to that of SEQ ID NO: 08. It is well understood by those skilled in the art that Bacillus strains use alternative start codons, wherein atg encodes methionine, gtg encodes valine, and ttg encodes leucine. These sequence variants are within the scope of this invention.

[0063] Preferably, the Bacillus cell has reduced gene expression, the gene having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with the gene encoding the metalloproteinase SEQ ID NO: 07. More preferably, the Bacillus cell has reduced gene expression, the gene having 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%, at least 99%, or 100% identity with the gene encoding the metalloproteinase SEQ ID NO: 07. Most preferably, the Bacillus cell has reduced gene expression, the gene having 100% identity with the gene encoding the metalloproteinase SEQ ID NO: 07. It is well understood by those skilled in the art that Bacillus strains use alternative start codons, wherein atg encodes methionine, gtg encodes valine, and ttg encodes leucine. These sequence variants are within the scope of this invention.

[0064] The Bacillus cells according to the present invention are recombinant Bacillus cells.

[0065] Preferably, the reduced expression of the gene encoding the metalloproteinase is achieved through gene deletion (partial or full-length, preferably full-length deletion). Preferably, the Bacillus cell is a metalloproteinase knockout cell, wherein the expression of the gene encoding the metalloproteinase is reduced to prevent the production of active metalloproteinases.

[0066] In one embodiment, the present invention relates to a Bacillus cell comprising a gene knockout encoding a metalloproteinase selected from the group consisting of: I. With SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 have at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity metalloproteinases, and II. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with SEQ ID NO: 07.

[0067] Preferably, the present invention relates to a Bacillus cell containing a gene knockout encoding a metalloproteinase, the metalloproteinase having at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with SEQ ID NO: 08.

[0068] The Bacillus cell may be selected from the group consisting of: *Bacillus hygroscopicus*, *Bacillus amyloliquefaciens*, *Bacillus atrophicus*, *Bacillus spp.*, *Bacillus hygroscopicus*, *Bacillus licheniformis*, *Bacillus nakamura*, *Bacillus basilica*, *Bacillus paralicheniformis*, *Bacillus pumilus*, *Bacillus safortiformis*, *Bacillus sonora*, *Bacillus stratosphericus*, *Bacillus belleus*, and *Cladosporium*, preferably from the group consisting of: *Bacillus licheniformis*, *Bacillus pumilus*, *Bacillus amyloliquefaciens*, and *Bacillus belleus*, more preferably *Bacillus licheniformis* and *Bacillus pumilus*. Most preferably, the Bacillus cell is a *Bacillus licheniformis* cell. Preferably, the Bacillus licheniformis cells belong to Bacillus licheniformis strains ATCC 14580, ATCC 31972, ATCC 53757, ATCC 53926, ATCC 55768, DSM13, DSM 394, DSM 641, DSM 1913, DSM 11259 or DSM 26543.

[0069] Preferably, the Bacillus cell is a Bacillus licheniformis cell with reduced expression of a gene encoding a metalloproteinase, the metalloproteinase having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with SEQ ID NO: 08. Most preferably, the Bacillus cell is a Bacillus licheniformis cell with reduced expression of a gene encoding a metalloproteinase, the metalloproteinase having an amino acid sequence having 100% identity with SEQ ID NO: 08.

[0070] Preferably, the Bacillus cell is a Bacillus licheniformis cell with reduced gene expression, the gene having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with the gene encoding the metalloproteinase SEQ ID NO: 07. Most preferably, the Bacillus cell is a Bacillus licheniformis cell with reduced gene expression, the gene having 100% identity with the gene encoding the metalloproteinase SEQ ID NO: 07.

[0071] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloproteinase, wherein the metalloproteinase is a homolog of the metalloproteinase having the amino acid sequence shown in SEQ ID NO: 08. Preferably, the homolog of the metalloproteinase having the amino acid sequence shown in SEQ ID NO: 08 comprises an amino acid sequence having at least 55% identity with SEQ ID NO: 08. The sequence identity of the homolog of the metalloproteinase described in SEQ ID NO: 08 is shown in the table below.

[0072] Therefore, in one embodiment, the Bacillus cell has reduced expression of a gene encoding a metalloproteinase having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with the following: SEQ ID NO: 08, wherein the Bacillus cell is Bacillus licheniformis; SEQ ID NO: 18, wherein the Bacillus cell is Bacillus highlandii; SEQ ID NO: 19, wherein the Bacillus cell is Bacillus amyloliquefaciens; SEQ ID NO: 20, wherein the Bacillus cell is Bacillus atrophicus; SEQ ID NO: 21, wherein the Bacillus cell is Bacillus spp.; SEQ ID NO: 22, wherein the Bacillus cell is Bacillus spp.; SEQ ID NO: 08, wherein the Bacillus cell is Bacillus spp.; SEQ ID NO: 09, wherein the Bacillus cell is Bacillus spp.; SEQ ID NO: 0 ... SEQ ID NO: 23, wherein the Bacillus cell is *Bacillus haematobium*; SEQ ID NO: 24, wherein the Bacillus cell is *Bacillus nakamura*; SEQ ID NO: 25, wherein the Bacillus cell is *Bacillus basilacea*; SEQ ID NO: 26, wherein the Bacillus cell is *Bacillus paralicheniformis*; SEQ ID NO: 27, wherein the Bacillus cell is *Bacillus pumilus*; SEQ ID NO: 28, wherein the Bacillus cell is *Bacillus safortiformis*; SEQ ID NO: 29, wherein the Bacillus cell is *Bacillus sianna*; SEQ ID NO: 30, wherein the Bacillus cell is *Bacillus sonora*; SEQ ID NO: 31, wherein the Bacillus cell is *Bacillus stratus*; SEQ ID NO: 32, wherein the Bacillus cell is *Bacillus belesii*; or SEQ ID NO: 33, wherein the Bacillus cell is *Craniobacillus* 7505.

[0073] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloproteinase having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identical to the following: SEQ ID NO: 08, wherein the Bacillus cell is Bacillus licheniformis; SEQ ID NO: 19, wherein the Bacillus cell is Bacillus amyloliquefaciens; SEQ ID NO: 27, wherein the Bacillus cell is Bacillus pumilus; or SEQ ID NO: 32, wherein the Bacillus cell is Bacillus belesii.

[0074] In one embodiment, the Bacillus cell has reduced expression of a gene encoding a metalloproteinase having an amino acid sequence that is at least 70% identical to SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus highlandii*; at least 69% identical to SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus amyloliquefaciens*; at least 72% identical to SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus atrophicatum*; at least 69% identical to SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus spp.*; at least 86% identical to SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus spp.*; at least 91% identical to SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus helixeris*; at least 68% identical to SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus nakamura*; and at least 68% identical to SEQ ID NO: 08. SEQ ID NO: 08 has at least 55% identity, wherein the Bacillus cell is *Bacillus paspadias*; has at least 86% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus paralichrysogenus*; has at least 68% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus pumilus*; has at least 69% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus safortiformis*; has at least 69% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus sicca*; has at least 80% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus sonora*; has at least 70% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus stratus*; has at least 67% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus belesi*; or has at least 67% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus belesi*; or has at least 68% identity with SEQ ID NO: 08, wherein the Bacillus cell is *Bacillus belesi*; or has at least 65 ... 08 has at least 56% identity, wherein the Bacillus cell is Bacillus 7505.

[0075] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloproteinase having an amino acid sequence that is at least 78% identical to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus amyloliquefaciens; at least 76% identical to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus pumilus; or at least 76% identical to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus belesii.

[0076] In one embodiment, the Bacillus cell contains additional genetic modifications. Preferably, the Bacillus cell further has reduced expression levels of at least one other gene, preferably reduced expression of at least one other endogenous gene. Preferably, the Bacillus cell further has reduced expression of at least one other gene, preferably an endogenous gene (such as deletion), the at least one other gene being selected from the group consisting of: genes encoding proteases different from the metalloproteinase, genes encoding sporulation factors, genes encoding secretory enzymes different from the protease, genes encoding proteins involved in extracellular matrix formation, or genes encoding autolysins, preferably genes encoding proteases different from the metalloproteinase. Preferably, the Bacillus cell further has reduced gene expression of at least one other gene, the at least one other gene being selected from the group consisting of: genes encoding proteases different from the metalloproteinase, genes encoding sporulation factors, genes encoding secretory enzymes different from the protease, and genes encoding proteins involved in extracellular matrix formation. Preferably, the Bacillus cell further has reduced gene expression of a gene encoding a protease different from the metalloproteinase, the protease being inactivated in Bacillus cells as described herein. Most preferably, the Bacillus cell also has reduced gene expression of genes encoding proteases different from the metalloproteinase, genes encoding sporulation factors, genes encoding secretory enzymes different from the proteases, and genes encoding proteins involved in the formation of the extracellular matrix.

[0077] The expression level of genes encoding proteases different from the metalloproteinase can be further reduced (e.g., deleted). Preferably, these genes are selected from the group consisting of protease-encoding genes aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, more preferably aprE, mpr, bpr, vpr, and ispA. Therefore, preferably, the Bacillus cell further has reduced gene expression of at least one gene selected from the group consisting of aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA. In one embodiment, the Bacillus cell contains a functional wprA and / or epr gene. Therefore, most preferably, the Bacillus cell has reduced gene expression of all protease-encoding genes aprE, mpr, bpr, vpr, and ispA. Preferably, the Bacillus cell contains a functional wprA and / or epr gene.

[0078] In one embodiment, the Bacillus cell has reduced gene expression (e.g., deletion) of genes encoding proteins involved in extracellular matrix formation. Preferably, it is envisioned that modified cells as described herein do not produce poly-γ-glutamic acid (PGA) or produce small amounts of PGA. Therefore, preferably, the Bacillus cell has reduced gene expression of at least one gene involved in PGA production. Preferably, at least one gene involving poly-γ-glutamic acid is at least one gene selected from ywsC (pgsB), ywtA (pgsC), ywtB (pgsA), and ywtC (pgsE). Therefore, preferably, the Bacillus cell further has reduced gene expression of at least one gene selected from the group consisting of ywsC (pgsB), ywtA (pgsC), ywtB (pgsA), and ywtC (pgsE). Preferably, all of the aforementioned genes, namely ywsC (pgsB), ywtA (pgsC), ywtB (pgsA), and ywtC (pgsE), have been inactivated (e.g., deleted). When this document refers to the inactivation of pga genes, any one or more or all of the genes listed above may be inactivated (e.g., deleted).

[0079] Preferably, the Bacillus cell further has reduced gene expression (e.g., deletion) of at least one other gene encoding a sporulation factor. Therefore, in one embodiment, it is envisioned that the modified cell cannot form spores. This can be achieved by inactivating (e.g., deleting) at least one gene involved in sporulation. Genes involved in sporulation are well known in the art (e.g., EP1391502) and include, but are not limited to, sigE, sigF, spoIIGA, spoIIE, sigG, spoIVCB, and yqfD. Therefore, preferably, the Bacillus cell further has reduced gene expression of at least one gene selected from the group consisting of sigE, sigF, spoIIGA, spoIIE, sigG, spoIVCB, and yqfD. In a preferred embodiment, the sigF gene is deleted.

[0080] Preferably, the Bacillus cell further has reduced gene expression (e.g., deletion) of at least one other gene encoding a secretory enzyme different from the protease. In a preferred embodiment, at least one gene encoding a secretory enzyme different from the protease is inactivated, the protease being selected from the group consisting of amylase, cellulase, xylanase, phosphatase, and pullulanase. Specifically, it is envisioned that the modified cell has reduced glycosidase activity. This can be achieved by inactivating (e.g., deleting) at least one gene encoding a glycosidase, including but not limited to α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2 and dextran-1,4-α-maltose hydrolase (EC 3.2.1.133), cellulase (EC 3.2.1.4), endo-1,3-β-xylanase (EC 3.2.1.32), endo-1,4-β-xylanase (EC 3.2.1.8), lactase (EC 3.2.1.108), galactosidase (EC 3.2.1.23 and EC 3.2.1.24), and mannanase (EC 3.2.1.24 and EC 3.2.1.25). In a preferred embodiment, at least one gene encoding an endogenous α-amylase polypeptide is inactivated. Therefore, preferably, the Bacillus cell further has reduced gene expression (such as deletion) of the amyB gene (also known as the amyL gene).

[0081] Preferably, the Bacillus cell further has reduced gene expression (e.g., deletion) of at least one gene encoding an autolysin. Therefore, in one embodiment, it is envisioned that the modified cell cannot lyse or has a reduced lysis rate. This can be achieved by inactivating (e.g., deleting) at least one gene involved in cell lysis. Genes involved in cell lysis include, but are not limited to, genes selected from the group consisting of lytC, lytD, lytE, lytF, cwlD, cwlE, and cwlJ. Therefore, preferably, the Bacillus cell further has reduced gene expression of at least one gene selected from the group consisting of lytC, lytD, lytE, lytF, cwlD, cwlE, and cwlJ.

[0082] Preferably, the Bacillus cells described herein also have reduced expression of at least one other gene selected from the group consisting of sigF, pga, and amyB, preferably all of the sigF, pga, and amyB genes (such as deletions).

[0083] Most preferably, the Bacillus cells described herein further have reduced gene expression (such as deletion) of at least one other gene selected from the group consisting of sigF, pga, amyB, aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, or even more preferably selected from sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, and most preferably have reduced expression of all sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA genes. Preferably, the Bacillus cells contain functional wprA and / or epr genes.

[0084] Even more preferably, the Bacillus cell is a Bacillus licheniformis cell with reduced gene expression of a gene encoding a metalloproteinase having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with SEQ ID NO: 08. The Bacillus licheniformis cell also has reduced gene expression (such as deletion) of at least one other gene selected from the group consisting of sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, most preferably with reduced gene expression of all sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA genes, preferably including a functional wprA and / or epr gene. Most preferably, the Bacillus cell is a Bacillus licheniformis cell with reduced gene expression of a gene encoding a metalloproteinase having an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with SEQ ID NO: 08. The Bacillus licheniformis cell also has reduced gene expression (such as deletion) of at least one other gene selected from the group consisting of sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, most preferably with reduced gene expression of all sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA genes, and includes a functional wprA and / or epr gene, preferably a functional wprA and epr gene.

[0085] The present invention also relates to a method for producing Bacillus cells as described herein. In one embodiment, the present invention relates to a method for producing Bacillus cells, wherein the method includes the following steps: I. Provide Bacillus cells containing a gene encoding a metalloproteinase selected from the group consisting of: a) With SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 have at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity metalloproteinases, and b) A metalloproteinase encoded by a polynucleotide having at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, or 100% identity with SEQ ID NO: 07, and II. Inactivate the metalloproteinase gene.

[0086] Methods for inactivating genes are well known to those skilled in the art. Gene inactivation can be achieved by partially or completely deleting the coding region or a complete gene including the coding region and regulatory sequences, modifying or deleting the promoter region, introducing point mutations that produce inactivating metalloproteinases (e.g., via CRISPR / Cas methods), or gene silencing.

[0087] Gene inactivation can also be achieved through homologous recombination, where the introduced DNA molecule includes sequences homologous to the 5' and 3' flanking sequences of the target sequence on the chromosome of the host cell to be inactivated (e.g., Bacillus). Subsequently, during homologous recombination, the sequence between the flanking sequences is replaced by the homologous sequence of the introduced DNA molecule, i.e., the sequence is deleted from the chromosome. Similarly, "gene integration," i.e., the integration of a DNA sequence, such as a gene expression cassette with or without optional markers, into the chromosome of a bacterial host cell via homologous recombination. Thus, the DNA sequence to be integrated is flanked by DNA sequences homologous to the 5' and 3' flanking sequences on the chromosome. According to the invention, gene integration can also combine gene integration and gene deletion in one step, i.e., the DNA sequence on the chromosome is replaced by the introduced DNA sequence for gene integration.

[0088] Homologous recombination can be achieved by two different methods known in the art, for example based on the well-known temperature-sensitive plasmid pE194: by two rounds of consecutive homologous recombination with circular plasmid DNA (Campbell recombination) (Nahrstedt et al., Strain development in Bacillus licheniformis: construction of biologically contained mutants deficient in sporulation and DNA repair. J Biotechnol. 2005 Sep 29; 119(3):245-54).

[0089] Alternatively, non-replicating "suicide" plasmids can be used, thereby forcing integration through selection on selectable markers. Only cells that integrate the plasmid into the genome via homologous recombination can grow under selective conditions. Removal / excision of the plasmid from the chromosome is achieved through a second homologous recombination, which is accomplished by activating anti-selection markers present on the plasmid.

[0090] The second method of homologous recombination refers to the simultaneous occurrence of two homologous recombination events, also known as "double crossover" or "double homologous recombination." The introduced DNA sequence is linear and can be obtained through PCR, linearization of plasmid DNA, or preparation of chromosomal DNA, which inevitably results in fragmented linear DNA. WO 0308125 uses a linear DNA construct (linearized plasmid or PCR fragment) that includes optional markers flanking 5' and 3' homologous regions for genome integration via double crossover of homologous recombination. It is well known that, adjacent to the optional markers, when said homologous regions are flanked, additional DNA (such as gene expression cassettes) is integrated into the chromosome of the bacterial host cell.

[0091] Homologous recombination requires that the DNA sequence homologous to the 5' and 3' flanking sequences of the target sequence on the host cell chromosome be of sufficient size and therefore contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly identical to the corresponding target sequence to increase the likelihood of homologous recombination (Dubnau, 1993, Genetic exchange and homologous recombination. In Bacillus subtilis and Other Gram-positive Bacteria, pp. 555-584. Edited by AI Sonenshein, JA Hoch & R. Losick, Washington DC, American Society for Microbiology; Michel and Ehrlich, 1984, The EMBO Journal, Vol. 3, pp. 2879-2884).

[0092] Gene inactivation via deletion / insertion / substitution can also be achieved through CRISPR / Cas9 genome editing technology, in which the cutting properties of CRISPR can be used to disrupt genes in the genome of almost any organism with unprecedented ease (Mali P et al. (2013) Science. 339(6121): 819-823; Cong L et al. (2013) Science 339(6121)). It has recently become clear that providing templates for repair (e.g., homologous regions) allows for genome editing at almost any site with almost any desired sequence, thus transforming CRISPR into a powerful gene editing tool (WO 2014 / 150624, WO 2014 / 204728).

[0093] CRISPR-based genome editing systems for use in Gram-positive organisms have been well described, such as single-plasmid systems based on Bacillus species, which include a Cas9 endonuclease, gRNA (e.g., sgRNA or crRNA / tracrRNA), and a repair homologous sequence (donor DNA) on a single E. coli-Bacillus shuttle vector (Altenbuchner, (2016): Applied and environmental microbiology 82 (17), 5421-5427; Zhou et al. (2019): International journal of biological macromolecules 122, 329–337), or dual-plasmid systems or systems with a Cas9 endonuclease integrated into the Bacillus genome, as described, for example, in WO2020 / 206202 and WO 2020 / 206197.

[0094] As an alternative to “directed” methods of inactivation, it should be understood within the scope of this invention that whole-cell mutagenesis by applying mutagenic conditions (such as exposing cells to UV radiation) or chemical mutagenic chemicals (such as NTG (N-methyl-N'-nitro-N-nitrosoguanidine) or EMS (ethyl methanesulfonate) in combination with screening and / or selection of desired properties (e.g., reduced lipase / esterase activity) is a well-known method for achieving functional inactivation.

[0095] Furthermore, genes may be inactivated by gene silencing. Gene silencing can be achieved by introducing an antisense expression construct into the bacterial host cell, which produces antisense RNA complementary to the gene's mRNA, thereby inhibiting the expression of the gene. Alternatively, the expression of the gene can be inhibited by blocking transcription initiation or transcriptional elongation via a CRISPR repression mechanism (WO 18 / 009520).

[0096] Proteins of interest Preferably, Bacillus cells having reduced expression of genes encoding metalloproteinases as described herein express the protein of interest, preferably the protein of interest sensitive to that metalloproteinase. Preferably, the protein of interest is secreted by the Bacillus cell, preferably secreted into the extracellular space. Preferably, the protein of interest is heterologous to the Bacillus cell.

[0097] Preferably, the protein of interest is an enzyme. Therefore, particularly preferably, the protein of interest is sensitive to the metalloproteinase secreted by the Bacillus cell, preferably secreted into the extracellular space, and is a heterologous enzyme to the Bacillus cell.

[0098] Preferably, the protein of interest, preferably the enzyme, comprises or consists of two or more domains (preferably at least two domains connected via a flexible linker) linked by a flexible linker, and preferably, the flexible linker is sensitive to the metalloproteinase.

[0099] Preferably, the enzyme is selected from the group consisting of: mannanase, amylase, pullulanase, protease, lipase, keratinase, acyltransferase, cellulase, endoglucanase, glucosidase, glucosaminidase, cellobiase, lactase, xylanase, xyloglucantransferase, xylosidase, xanthan gum lyase, DNase, dispersin, phytase, phosphatase, xylose isomerase, glucose isomerase, acetolactate decarboxylase, pectinase, pectic acid lyase, pectin methyl esterase, polygalacturonidase, lyase, pectic acid lyase, arabinosease, arabinofuranase, galactanase, laccase, peroxidase, oxidoreductase, and asparaginase.

[0100] More preferably, the protein of interest is a mannanase, preferably a mannanase heterologous to the Bacillus cell. Most preferably, the mannanase, preferably a mannanase heterologous to the Bacillus cell, is secreted by the cell.

[0101] The mannanase according to the invention has mannan-degrading activity and belongs to enzyme class EC 3.2.1.78. In one embodiment, the mannan-degrading activity involves the degradation of at least one galactomannan. Preferably, the at least one galactomannan is characterized by a mannose:galactose ratio of about 1:1, about 2:1, about 3:1, about 4:1, and / or 5:1.

[0102] Mannan degradation activity or mannanase activity can be tested according to standard test procedures known in the art. For example, the mannanase to be tested can be applied to 4 mm diameter wells punched in an agar plate containing 0.2% AZCL galactomannan (carob), the substrate for the determination of endo-1,4-β-D-mannanase, which can be obtained as I-AZGMA from Megazyme (Megazyme's website: http: / / www.megazyme.com / Purchase / index.html). Mannan degradation activity can also be tested in liquid assays using carob galactomannan stained with Remazol brilliant blue, as described in McCleary, BV (1978). Carbohydrate Research, 67(1), 213-221. Another method for testing mannan degradation activity uses the detection of reducing sugars when incubated with substrates such as guar gum or locust bean gum—see Miller, GL Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars. Analytical Chemistry 1959; 31: 426–428 for reference.

[0103] Preferably, the mannanase comprises or consists of two or more domains (preferably at least two domains connected via a flexible linker) linked by a flexible linker, and preferably, the flexible linker is sensitive to the metalloproteinase. The mannanase preferably comprises or consists of a catalytic domain and a carbohydrate-binding domain. Preferably, the catalytic domain and the carbohydrate domain of the mannanase are connected via a flexible linker, and preferably, the flexible linker is sensitive to the proteolytic cleavage of the metalloproteinase.

[0104] The mannanase can be selected from mannanases derived from Bacillus organisms, such as those described in JP-0304706, JP-63056289, JP-63036774, JP-08051975, WO97 / 11164, WO91 / 18974, WO 97 / 11164, and WO2014 / 100018. Suitable mannanases are also described in WO99 / 064619. The mannanase can be selected from mannanases derived from Trichoderma organisms, such as those disclosed in WO93 / 24622. The mannanase can be selected from commercially available mannanases, such as those from Mannaway. ® (Novozymes A / S) or Preferenz ®(M100)(DuPont).

[0105] Preferably, the mannanase is a variant of the parental mannanase (i.e., a mannanase variant). The parental mannanase can be any mannanase. Preferably, the parental mannanase is SEQ ID NO: 12. In a particular embodiment, the mannanase is a mannanase variant comprising one or more amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 12 and with SEQ ID NO: 12. The amino acid sequences numbered 59, 66, 89, 234, 259, 282, 318, 319 and 322 having at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99%, but less than 100% identity.

[0106] Preferably, the mannanase is a variant of the parental mannanase, preferably as shown in SEQ ID NO: 12, wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 12 and having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identity with SEQ ID NO: 12, preferably with SEQ ID NO: 12 The amino acid sequences numbered 59, 66, 89, 234, 259, 282, 318, 319 and 322 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% identity.

[0107] Preferably, the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions selected from the group consisting of: X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G, which are preferably identical to SEQ ID NO: 12 with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%. More preferably, the mannanase variant comprises all amino acid substitutions selected from the group consisting of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to SEQ ID NO: 12. Most preferably, the mannanase variant comprises or consists of the following: having amino acid substitutions X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to SEQ ID NO: 12 and optionally having 1-10, preferably 1-5, conservative substitutions according to SEQ ID NO: 12.

[0108] In one embodiment, the protein of interest is a lipase. “Lipase,” “lipolytic enzyme,” and “lipoesterase” all refer to enzymes of EC class 3.1.1 (“carboxylate hydrolases”). Lipase means an active protein possessing lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), keratinase activity (EC 3.1.1.74; enzymes with keratinase activity may be referred to herein as keratins), sterol esterase activity (EC 3.1.1.13), and / or wax-ester hydrolase activity (EC 3.1.1.50). Lipases include those of bacterial or fungal origin. In one aspect of the invention, a suitable lipase (component (b)) is selected from lipases derived from *Pyrophyllaria* (synonymous thermophilic molds), such as those from *Pyrophyllaria lataniae* (*Pyrophyllaria lataniae*), as described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500. In one embodiment, the lipase is selected from fungal triacylglycerol lipases (EC class 3.1.1.3). The cottony thermophilic mold lipase variant may be selected from variants with lipolytic activity, which, when compared with the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438, has 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%, at least 99%, or 100% identity. Preferably, when compared with amino acids 1-269 of SEQ ID NO: 2 of US5869438, the variant contains one or more, preferably all of the following amino acid substitutions: T231R, N233R, Q4V, V60S, A150G, L227G, P256K. The lipase of *Thermophilus sparsely distributed* may also be selected from variants of the following: containing at least one, preferably more than one, and more preferably all of the following substitutions N11K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q within the polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO2015 / 010009, and having at least 95%, at least 96%, or at least 97% similarity when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO2015 / 010009. In one embodiment, at least one lipase is selected from commercially available lipases, including but not limited to those marketed under the trade name Lipolase. ™ Lipex ™ Lipolex ™ and Lipoclean ™Products sold by Novozymes A / S, Lumafast (originally from Genencor), Preferenz L (DuPont), and Lipomax (Gist-Brocades / now DSM).

[0109] In one embodiment, the protein of interest is a cellulase. A cellulase is an enzyme capable of hydrolyzing cellulose. Cellulases may be selected from cellobiase (1,4-PD-glucan-cellobiase, EC 3.2.1.91), endo-ss-1,4-glucanase (EC 3.2.1.4), and ss-glucosidase (EC 3.2.1.21). Endo-glucanases of EC class 3.2.1.4 may be named endo-glucanase, endo-1,4-ss-D-glucan-4-glucanase, endo-1,4-β-glucanase, carboxymethyl cellulase, and β-1,4-glucanase. The cellulase may be a specific *Pseudomonas* DSM 1800 cellulase complex possessing endo-glucanase, cellobiase, and β-glucosidase activities. The cellulase may be a specific humic mold DSM 1800 endoglucanase (EC 3.2.1.4), which preferably has a polypeptide sequence according to positions 21-435 of SEQ ID NO: 2 disclosed in WO 2018 / 224544 or a variant having at least 80% identity with it. In one embodiment, at least one cellulase is selected from commercially available cellulases, including but not limited to those marketed under the trade name Renozyme. ® Celluzyme ® Celluclean ® Endolase ® and Carezyme ® (Novozymes A / S), Clazinase ™ and Puradax HA ™ (Genencor Int.Inc.) and KAC-500(B) ™ Products sold by (Kao Corporation).

[0110] In one embodiment, the protein of interest is a protease different from the metalloproteinases described herein. Preferably, the protease is a subtilisin or a variant thereof as described in any of WO 89 / 06276 and EP 0283075, WO 89 / 06279, WO 89 / 09830, WO 89 / 09819, WO 91 / 06637 and WO 91 / 02792. Suitable examples of protease variants include proteases derived from SEQ ID NO: 22 as described in EP 1921147 that have amino acid substitutions at one or more of the following positions: 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274 (according to BPN numbers). Preferably, the Bacillus subtilis protease variant is as described in EP 1921147 SEQ ID NO: 22 has at least 80% identity and is characterized by an amino acid substitution at position 101, either alone or in combination with one or more substitutions at positions 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and / or 274 (according to BPN number), such as R101E or R101D, and has proteolytic activity.The Bacillus subtilis protease variant may have an amino acid sequence that is at least 80% identical to SEQ ID NO: 22 as described in EP1921147, and is further characterized by comprising R101E and one or more substitutions selected from the group consisting of: S156D, L262E, Q137H, S3T, R45E, D, Q, P55N, T58W, Y, L, Q59D, M, N, T, G61D, R, S87E, G97S, A98D, E, R, S106A, W, N117E, H120V, D, K, N, S125M, P129D, E136Q, S144W, S161T, S163A, G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D.

[0111] In one embodiment, the protein of interest is an amylase. The amylase may be of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, the amylase is selected from the group including α-amylase (EC 3.2.1.1). The amylase may be derived from Bacillus licheniformis having SEQ ID NO: 2 as described in WO 95 / 10603 and variants thereof having at least 95% nucleotide content. Suitable variants are described in WO 95 / 10603, which contain one or more substitutions at the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444, which have starch-degrading activity. Variants are described in SEQ ID NO:4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424, and WO 99 / 019467. The amylase may further be derived from *Bacillus stearothermophilus* having SEQ ID NO: 6 as disclosed in WO 02 / 10355 or optionally from an amylase having a C-terminal truncation in the wild-type sequence. Suitable variants of SEQ ID NO: 6 include those containing deletions at positions 179 and / or 181 and / or 182 and / or substitutions at position 193. The amylase may further be derived from *Bacillus* genus 707 and its variants having SEQ ID NO: 6 as disclosed in WO99 / 19467, preferably those variants having substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. The amylase may have SEQ ID NO:12 as described in WO 2006 / 002643, or an amylase variant thereof comprising substitutions for Y295F and M202LITV within said SEQ ID NO:12. The amylase may have SEQ ID NO:2 as described in WO 2013 / 001087, or an amylase variant comprising deletions at positions 181+182, 182+183, or 183+184 within said SEQ ID NO:2, optionally comprising one or more modifications at any position corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476, and G477 within said SEQ ID NO:2.The heterozygous amylase can be purported to be from WO 2021 / 032881, comprising an A and B domain derived from α-amylase (from Bacillus spp. A7-7 (DSM 12368)) and a C domain derived from α-amylase (from Bacillus cereus); preferably, the A and B domains have at least 75% identity with the amino acid sequence of SEQ ID NO: 42, and the C domain has at least 75% identity with the amino acid sequence of SEQ ID NO: 44, both sequences being disclosed in WO 2021 / 032881; more preferably, the heterozygous amylase has at least 80% identity with SEQ ID NO: 54 disclosed in WO 2021 / 032881. In one embodiment, at least one amylase is selected from commercially available amylases, including but not limited to those marketed under the trade name Duramyl. ™ Terminyl ™ Fun-gamyl ™ Stainzyme ™ Stainzyme Plus ™ Natalase ™ Liquozyme X and BAN ™ Amplify ™ Amplify Prime ™ (From Novozymes A / S) and Rapidase ™ Purastar ™ Powerase™, Effectiveenz ™ (M100, from DuPont), Preferenz ™ (S1000, S110, and F1000; from DuPont), PrimaGreen ™ (ALL; DuPont), Optisize ™ Products sold by (DuPont).

[0112] Methods for producing proteins of interest In another embodiment, the present invention relates to a method for producing a protein of interest, preferably an enzyme as described herein, the method comprising the following steps: I. Providing Bacillus cells with reduced expression of genes encoding metalloproteinases selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07, and II. An expression cassette encoding the protein of interest, preferably the enzyme, that is sensitive to the metalloproteinase is introduced into the Bacillus cell. III. The Bacillus cells are cultured under conditions that allow for the expression of the protein of interest, thereby forming a fermentation broth containing the protein of interest, and IV. Optionally, the protein of interest may be isolated from the fermentation broth of step III.

[0113] Preferably, the protein of interest is mannanase as described herein.

[0114] In a preferred embodiment, the present invention relates to a method for producing a protein of interest, preferably an enzyme as described herein, the method comprising the following steps: II. Providing Bacillus cells with reduced expression of a gene encoding a metalloproteinase, the metalloproteinase having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08. II. An expression cassette encoding the protein of interest, preferably the enzyme, that is sensitive to the metalloproteinase is introduced into the Bacillus cell. III. The Bacillus cells are cultured under conditions that allow for the expression of the protein of interest, thereby forming a fermentation broth containing the protein of interest, and IV. Optionally, the protein of interest may be isolated from the fermentation broth of step III.

[0115] Preferably, the protein of interest is mannanase as described herein. Preferably, the Bacillus cell is a Bacillus licheniformis cell.

[0116] Typically, this expression cassette comprises three elements: a promoter sequence (including the 5' UTR), an open reading frame, and a 3' untranslated region. Additional regulatory elements may include transcriptional enhancers and translational enhancers. The expression cassette may be part of a vector or may be integrated into the host cell's genome and replicate along with it. Expression vectors may be low-copy-number or high-copy-number vectors. Vectors used herein provide segments for transcription and translation of foreign polynucleotides upon transformation into host cells. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication required for maintenance and / or replication in a specific cell type, one or more optional markers, polyadenylation signals, and suitable sites for inserting foreign coding sequences (such as multiple cloning sites). Non-limiting examples of suitable origins of replication include f1-ori and colE1.

[0117] Vectors can replicate without integrating into the host cell's genome, such as as plasmids in bacterial host cells, or they can integrate part or all of their DNA into the host cell's genome, thereby inducing DNA replication and expression. Polynucleotides encoding the protein of interest can be introduced into vectors using standard recombinant DNA techniques. Once introduced into the vector, the polynucleotides containing the coding sequence are suitable for introduction (transformation, transduction, transfection, etc.) into host cells. Cloning vectors suitable for expressing polynucleotide sequences in host cells can be selected.

[0118] Polynucleotides encoding proteins of interest as described herein can be introduced transiently or stably into host cells and can remain non-integrated, for example, as plasmids or granules. Typically, stable transformation is attributed to the integration of a nucleic acid containing a foreign coding sequence into the chromosome. Transient transformation is typically attributed to the failure to integrate a nucleic acid containing a foreign nucleic acid sequence into the chromosome.

[0119] Methods for introducing nucleic acids into host cells are well known to those skilled in the art. Introducing nucleic acids into host cells can be achieved, for example, but not limited to, by protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, for example, Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278).

[0120] After introducing an expression cassette encoding the protein of interest into Bacillus cells, the Bacillus cells are cultured under conditions that allow for the expression of the protein of interest, thereby forming a fermentation broth containing the protein of interest. As used herein, the term "culture" means keeping the modified cells included in the culture viable and / or proliferating for at least a predetermined time. This term encompasses both the exponential cell growth phase at the onset of growth after inoculation and the stationary growth phase. The culture conditions should allow for the expression of the protein of interest, i.e., the production. Those skilled in the art will readily select such conditions. Exemplary conditions for culturing modified cells are described in WO2020169564 A1 or in Example 3 of the Examples section provided herein. In one embodiment of the method of the invention, the step of culturing the Bacillus cells is performed as a fed-batch culture.

[0121] The method of the present invention (as applied) allows for increased production of the protein of interest. Preferably, production is increased compared to expression in unmodified control cells, preferably Bacillus cells, preferably Bacillus licheniformis control cells, which do not have inactivated metalloproteinases as described herein. In a preferred embodiment, the production of the protein of interest is increased by at least 20%, such as at least 50%, particularly at least 100% or at least 200%, compared to expression in control cells. For example, the production of the protein of interest can be increased by 20% to 300%, such as 100% to 300%, compared to control cells. This expression can be measured by determining the amount of the protein of interest in cells and / or culture medium. Furthermore, production can be assessed by measuring enzyme activity. For example, an enzyme assay can be used to determine the activity of the protein of interest.

[0122] The protein of interest described herein may be secreted from the microbial cells (into the liquid fraction of the fermentation broth) or may not be secreted from the microbial cells (and thus contained within the cells of the fermentation broth). Depending on this, the protein of interest may be recovered from the liquid fraction of the fermentation broth or from the cell lysate. Preferably, the protein of interest is secreted from the cells into the fermentation broth, preferably by means of a secretion signal peptide added to the end of the amino acid sequence of the protein of interest.

[0123] The protein of interest can be separated from the fermentation broth using methods known in the art. For example, the protein of interest can be separated from the fermentation broth using conventional procedures, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. If the product of interest precipitates or crystallizes in the fermentation broth or is at least partially bound to particulate matter in the fermentation broth, additional processing steps may be required to release the protein of interest from the biomass and / or dissolve the crystals and precipitates of the protein of interest. WO0043502A1, WO2008110498 A1, and WO2017097869A1 describe methods for recovering proteins of interest that have precipitated and / or crystallized during fermentation from the fermentation broth. In cases where it is desired that the protein be contained within cells in the fermentation broth, it may be necessary to release the product of interest from the cells. Release from cells can be achieved, for example, but not limited to, by cell lysis using techniques well known to those skilled in the art, such as lysozyme treatment, sonication, French press, or combinations thereof.

[0124] The isolated protein of interest can then be further purified using a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatographic focusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing (IEF), differential solubility methods (e.g., ammonium sulfate precipitation), or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, eds., VCH Publishers, New York, 1989). The purified protein of interest can then be concentrated using procedures known in the art, including but not limited to ultrafiltration and evaporation.

[0125] Methods to inactivate metalloproteinases In an alternative embodiment of reducing gene expression encoding a metalloproteinase in Bacillus cells, it is also possible to inactivate or inhibit the metalloproteinase itself. Inactivation of the metalloproteinase can be achieved, for example, by contacting the metalloproteinase with an inhibitor of the metalloproteinase. However, the inventors have found that it is also possible to inactivate the metalloproteinase by adding a chelating agent to a composition containing the metalloproteinase in an amount and time that effectively inactivates the metalloproteinase. Without being bound by theory, the inventors believe that the chelating agent binds to the metal ions required by the metalloproteinase to maintain its proteolytic activity. Therefore, in another embodiment, the present invention relates to the use of a chelating agent for inactivating the metalloproteinase described herein. Inactivation of the metalloproteinase during the production of the protein of interest or in a composition containing the metalloproteinase and the protein of interest results in a reduction in the degradation of the protein of interest by the metalloproteinase. Therefore, the present invention also relates to the use of a chelating agent for inactivating the metalloproteinase described herein in order to increase the yield of the protein of interest in a method for producing the protein of interest by means of Bacillus cells expressing the metalloproteinase.

[0126] Therefore, in another embodiment, the present invention relates to a method for inactivating metalloproteinases, wherein the method includes the following steps: I. Provide metalloproteinases selected from the following groups: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07, and II. Contact the metalloproteinase with a chelating agent, preferably EDTA, MGDA, or EDDS, for a sufficient amount and time to inactivate the metalloproteinase.

[0127] In another embodiment, the present invention relates to a method for inactivating a metalloproteinase comprising, preferably, a composition comprising, and preferably, a liquid composition, a protein of interest, preferably a mannanase and a metalloproteinase as described herein, wherein the metalloproteinase is selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. The protein of interest is sensitive to the metalloproteinase, and the method includes the following steps: contacting the composition containing the protein of interest and the metalloproteinase with a chelating agent, preferably EDTA, MGDA or EDDS, in an amount and for a time sufficient to inactivate the metalloproteinase.

[0128] Therefore, in another embodiment, the present invention relates to a method for producing a protein of interest, preferably an enzyme as described herein, preferably a mannanase as described herein, the method comprising the following steps: I. Providing Bacillus cells with reduced expression of genes encoding metalloproteinases selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07, and II. An expression cassette encoding the protein of interest, preferably the enzyme, that is sensitive to the metalloproteinase is introduced into the Bacillus cell. III. The Bacillus cells are cultured under conditions that allow for the expression of both the protein of interest and the metalloproteinase, thereby forming a fermentation broth containing both the protein of interest and the metalloproteinase. IV. Optionally, the protein of interest is isolated from the culture medium to form a solution containing the protein of interest and the metalloproteinase, and V. Contact the fermentation broth from step III and / or the solution from step IV with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a duration that effectively inactivates the metalloproteinase.

[0129] The concentration of the chelating agent used to inactivate the mannanase is preferably from 1 mmol / L to 150 mmol / L, more preferably from 50 mol / L to 120 mol / L.

[0130] Preferably, the protein of interest is mannanase as described herein.

[0131] In another embodiment, the present invention relates to the use of a chelating agent for improving the stability of a protein of interest in a composition comprising a metalloproteinase and a protein of interest, preferably a mannanase as described herein, wherein the composition comprising the protein of interest and the metalloproteinase, preferably in a liquid composition, is selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. The protein of interest is sensitive to the metalloproteinase, and the method includes the following steps: contacting the composition containing the protein of interest and the metalloproteinase with a chelating agent, preferably EDTA, MGDA or EDDS, in an amount and for a time sufficient to inactivate the metalloproteinase.

[0132] Therefore, the present invention also relates to the use of a chelating agent, preferably EDTA, MGDA, or EDDS, for improving the yield and / or stability of the protein of interest in a method for producing a protein of interest, preferably an enzyme, preferably a mannanase as described herein, via Bacillus cells, wherein the Bacillus cells express a metalloproteinase selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. The protein of interest is sensitive to this metalloproteinase.

[0133] In another embodiment, the present invention relates to the use of a chelating agent, preferably EDTA, MGDA, or EDDS, for improving the yield and / or stability of a protein of interest, preferably an enzyme, preferably a mannanase as described herein, in a method for producing a protein of interest via Bacillus cells, wherein the Bacillus cells express a metalloproteinase selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. The protein of interest is sensitive to this metalloproteinase. The method includes the following steps: I. Provides a composition, preferably a liquid composition, comprising a protein of interest and a metalloproteinase selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07, and II. Contact the composition from step I with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time sufficient to inactivate the metalloproteinase. Preferably, the composition is a fermentation broth or a solution obtained by purifying the protein of interest from a fermentation broth, wherein the fermentation broth is obtained by a method comprising the following steps: aa. An expression cassette encoding a protein of interest, preferably an enzyme, sensitive to this metalloproteinase is introduced into the Bacillus cell. bb. The Bacillus cells were cultured under conditions that allowed for the expression of both the protein of interest and the metalloproteinase, thereby forming a fermentation broth containing both the protein of interest and the metalloproteinase. cc. Optionally, the protein of interest is isolated from the culture medium to form a solution containing the protein of interest and the metalloproteinase.

[0134] Preferably, the solution to which the chelating agent is added, comprising the protein of interest, preferably mannanase as described herein, and the metalloproteinase, is a non-complex formulation (e.g., a liquid enzyme formulation) or a complex formulation (e.g., a liquid detergent formulation). In a preferred embodiment, the solution is a non-complex formulation, preferably a liquid enzyme formulation, which preferably comprises one or more additional compounds selected from the group consisting of solvents, salts, pH adjusters, preservatives, enzyme stabilizers, and thickeners. Preferably, the liquid enzyme formulation is surfactant-free. The solvent may be water and / or an organic solvent. The liquid enzyme formulation may contain more than 30% by weight, more than 40% by weight, more than about 50% by weight, more than about 60% by weight, more than about 70% by weight, or more than about 80% by weight of an organic solvent, all amounts relative to the total weight of the enzyme formulation. The organic solvent may be a water-miscible solvent. The organic solvent may be one or more selected from the group consisting of glycerol, propylene glycol, polypropylene glycol, and polyethylene glycol. Preferably, the liquid enzyme preparation comprises or consists of the protein of interest as described herein, the metalloproteinase as described herein, a chelating agent and a solvent, an enzyme stabilizing system and optionally a preservative; preferably, the liquid enzyme preparation is free of surfactants. The chelating agent is preferably contained in the liquid enzyme preparation in an amount of 1 mmol / L to 150 mmol / L, preferably 50 mol / L to 120 mol / L.

[0135] In an alternative embodiment, the solution to which the chelating agent is added, comprising the protein of interest, preferably mannanase as described herein, and the metalloproteinase, is a complex formulation, preferably a liquid detergent formulation. The detergent formulation comprises one or more detergent components, preferably selected from the group consisting of surfactants, defoamers, polymers, bleaching systems (bleach), rheology modifiers, water-soluble additives, softeners, desiccants, brighteners, buffers, preservatives, anti-corrosion additives, dyes, fragrances, and detergent enzymes, different from the protein of interest. Preferably, at least one detergent component is selected from the group consisting of surfactants, polymers, preservatives, and detergent enzymes, different from the protein of interest. Preferably, one or more detergent components, preferably surfactants and / or additives, are biodegradable and / or bio-based. Preferably, the liquid detergent formulation comprises or consists of the protein of interest as described herein, the metalloproteinase as described herein, the chelating agent, and surfactants, polymers, preservatives, and detergent enzymes, different from the protein of interest.

[0136] Various publications have been referenced in this application. The disclosures of all such publications and the references cited in them are hereby incorporated, in their entirety, by reference in order to provide a more complete description of the state of the art to which this invention pertains.

[0137] Preferred implementation scheme 1. A Bacillus cell, said Bacillus cell having reduced expression of a gene encoding a metalloproteinase, said metalloproteinase being selected from the group consisting of: I. Metalloproteinases having at least 55%, at least 60%, at least 65%, preferably at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and preferably having at least 55%, at least 60%, at least 65% identity with SEQ ID NO: 08. II. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07.

[0138] 2. The Bacillus cells according to embodiment 1, wherein the Bacillus cells are recombinant Bacillus cells.

[0139] 3. Bacillus cells according to any one of embodiments 1 or 2, wherein the Bacillus cells are metalloproteinase knockout cells.

[0140] 4. A Bacillus cell according to any one of embodiments 1 to 3, wherein the Bacillus cell has reduced expression of a gene encoding a metalloproteinase, the metalloproteinase having an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with SEQ ID NO: 08, preferably having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or 100% identity with SEQ ID NO: 08, most preferably having 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%, at least 99% or 100% identity with SEQ ID NO: 08.

[0141] 5. A Bacillus cell according to any one of embodiments 1 to 4, wherein the Bacillus cell expresses a protein of interest sensitive to the metalloproteinase, the protein of interest preferably comprising or consisting of two or more domains connected via a flexible linker, preferably the flexible linker being sensitive to the metalloproteinase, and preferably wherein the protein of interest sensitive to the metalloproteinase is an enzyme.

[0142] 6. The Bacillus cell according to embodiment 5, wherein the protein of interest is heterologous to the cell, preferably wherein the protein of interest is a heterologous enzyme.

[0143] 7. Bacillus cells according to any one of embodiments 5 or 6, wherein the enzyme is selected from the group consisting of: mannanase, amylase, pullulanase, protease, lipase, keratinase, acyltransferase, cellulase, endoglucanase, glucosidase, glucosaminidase, cellobiase, lactase, xylanase, xyloglucantransferase, xylosidase, xanthan gum lyase, DNase, dispersin, phytase, phosphatase, xylose isomerase, glucose isomerase, acetolactate decarboxylase, pectinase, pectic acid lyase, pectin methyl esterase, polygalacturonidase, lyase, pectic acid lyase, arabinose, arabinofuranase, galactanase, laccase, peroxidase, oxidoreductase and asparaginase, preferably mannanase.

[0144] 8. Bacillus cells according to any one of embodiments 5 to 7, wherein the protein of interest is heteromannanase.

[0145] 9. A Bacillus cell according to any one of embodiments 5 to 8, wherein the enzyme is a mannanase, the mannanase preferably comprising or consisting of two or more domains linked via a flexible linker, preferably the flexible linker being sensitive to the metalloproteinase, the mannanase preferably comprising or consisting of a catalytic domain and a carbohydrate-binding domain, preferably the catalytic domain and the carbohydrate domain of the mannanase being linked via a flexible linker, preferably the flexible linker being sensitive to the proteolytic cleavage of the metalloproteinase, preferably wherein the mannanase is a variant of the parental mannanase (i.e., a mannanase variant), wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 12 and with SEQ ID NO: 12 The amino acid sequences numbered 59, 66, 89, 234, 259, 282, 318, 319 and 322 having at least 55%, at least 60%, at least 65%, %, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%, but less than 100% identity.

[0146] 10. Bacillus cells according to any one of embodiments 7 to 9, wherein the mannanase is a variant of the parental mannanase, wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 12 and having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identity with SEQ ID NO: 12, preferably with SEQ ID NO: 12 The amino acid sequences numbered 59, 66, 89, 234, 259, 282, 318, 319 and 322 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% identity.

[0147] 11. Bacillus cells according to any one of embodiments 7 to 10, wherein the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions selected from the group consisting of: X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G as indicated by the numbers in SEQ ID NO: 12.

[0148] 12. Bacillus cells according to any one of embodiments 7 to 11, wherein the mannanase variant comprises all amino acid substitutions selected from the group consisting of: X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G and X322G according to the numbers stated in SEQ ID NO: 12.

[0149] 13. Bacillus cells according to any one of embodiments 7 to 12, wherein the mannanase variant comprises or consists of the following: SEQ ID NO: 12 having amino acid substitutions of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G and X322G according to the numbering specified in SEQ ID NO: 12 and optionally having 1 to 10, preferably 1 to 5, conservative substitutions.

[0150] 14. A Bacillus cell according to any one of embodiments 5 to 13, wherein the protein of interest is secreted by the cell.

[0151] 15. The Bacillus cell according to any one of the foregoing embodiments, wherein the Bacillus cell further has reduced expression of at least one other gene, wherein the at least one other gene is preferably selected from the group consisting of: a gene encoding a protease different from the metalloproteinase, a gene encoding a sporulation factor, a gene encoding a secretory enzyme different from the protease, a gene encoding a protein involved in the formation of the extracellular matrix, or a gene encoding an autolysin, preferably a gene encoding a protease different from the metalloproteinase.

[0152] 16. The Bacillus cell according to any one of the foregoing embodiments, wherein the Bacillus cell further has reduced expression of a gene encoding a protease different from the metalloproteinase, the gene being selected from the group consisting of protease-encoding genes composed of aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, more preferably selected from aprE, mpr, bpr, vpr, and ispA, most preferably all aprE, mpr, bpr, vpr, and ispA, and preferably, the Bacillus cell contains a functional wprA and / or epr gene.

[0153] 17. The Bacillus cell according to any one of the foregoing embodiments, wherein the Bacillus cell further has reduced expression of at least one other gene selected from the group consisting of sigF, pga and amyB, preferably all of sigF, pga and amyB.

[0154] 18. The Bacillus cell according to any one of the foregoing embodiments, wherein the Bacillus cell further comprises reduced expression of at least one other gene selected from the group consisting of sigF, pga, amyB, aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, preferably selected from sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, most preferably having reduced expression of all sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, and preferably, the Bacillus cell comprises a functional wprA and / or epr gene.

[0155] 19. The Bacillus cells according to any one of the foregoing embodiments, wherein the Bacillus cells are selected from the group consisting of: Bacillus hygroscopicus, Bacillus amyloliquefaciens, Bacillus atrophicus, Bacillus spp., Bacillus spp., Bacillus hesperidinus, Bacillus licheniformis, Bacillus nakamura, Bacillus basilaceus, Bacillus paralicheniformis, Bacillus pumilus, Bacillus saforgensis, Bacillus sonora, Bacillus stratosphericus, Bacillus belleus, and Bacillus cladosporium, preferably selected from the group consisting of: Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens and Bacillus belleus, more preferably Bacillus licheniformis and Bacillus pumilus, and most preferably Bacillus licheniformis.

[0156] 20. The Bacillus cell according to any one of the foregoing embodiments, wherein the Bacillus cell is a Bacillus licheniformis cell.

[0157] 21. A method for producing a protein of interest, preferably an enzyme, said method comprising the steps of: I. Provide Bacillus cells according to embodiment 1, II. Introducing an expression cassette encoding a protein of interest, preferably an enzyme, sensitive to the metalloproteinase into the Bacillus cells. III. The Bacillus cells are cultured under conditions that allow for the expression of the protein of interest, thereby forming a fermentation broth containing the protein of interest, and IV. Optionally, the protein of interest is isolated from the fermentation broth of step III.

[0158] 22. The method according to embodiment 21, wherein the Bacillus cells have reduced expression of a gene encoding a metalloproteinase, the metalloproteinase having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or 100% identity with SEQ ID NO: 08, preferably having 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%, at least 99% or 100% identity with SEQ ID NO: 08.

[0159] 23. The method according to any one of embodiments 21 or 22, wherein the Bacillus cell expresses a protein of interest sensitive to the metalloproteinase, the protein of interest preferably comprising or consisting of two or more domains connected via a flexible linker, preferably the flexible linker being sensitive to the metalloproteinase, and preferably wherein the protein of interest sensitive to the metalloproteinase is an enzyme.

[0160] 24. The method according to any one of embodiments 21 to 23, wherein the protein of interest is secreted by the cell.

[0161] 25. The method according to any one of embodiments 21 to 24, wherein the protein of interest is a mannanase, the mannanase preferably comprising or consisting of two or more domains connected via a flexible linker, preferably the flexible linker being sensitive to the metalloproteinase.

[0162] 26. The method according to embodiment 25, wherein the mannanase is a variant of the parental mannanase, wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 12 and having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identity with SEQ ID NO: 12, preferably with SEQ ID NO: 12. NO:12 has amino acid sequences numbered 59, 66, 89, 234, 259, 282, 318, 319 and 322 that have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% identity.

[0163] 27. The method according to any one of embodiments 25 or 26, wherein the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions selected from the group consisting of: X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G as indicated by the numbers in SEQ ID NO: 12.

[0164] 28. The method according to any one of embodiments 25 to 27, wherein the mannanase variant comprises all amino acid substitutions selected from the group consisting of: X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G and X322G according to the numbers stated in SEQ ID NO: 12.

[0165] 29. The method according to any one of embodiments 25 to 28, wherein the mannanase variant comprises or consists of SEQ ID NO: 12 having amino acid substitutions of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12 and optionally having 1-10, preferably 1-5, conservative substitutions, wherein preferably the mannanase variant comprises or consists of SEQ ID NO: 12 having amino acid substitutions of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12, wherein the mannanase variant comprises or consists of SEQ ID NO: 12 having amino acid substitutions of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12.

[0166] 30. The method according to any one of embodiments 21 to 29, wherein the Bacillus cells further have reduced expression of at least one other gene, said at least one other gene preferably selected from the group consisting of: a gene encoding a protease different from said metalloproteinase, a gene encoding a sporulation factor, a gene encoding a secretory enzyme different from said protease, a gene encoding a protein involved in the formation of the extracellular matrix, or a gene encoding an autolysin, preferably a gene encoding a protease different from said metalloproteinase.

[0167] 31. The method according to any one of embodiments 21 to 30, wherein the Bacillus cell further has reduced expression of a gene encoding a protease different from the metalloproteinase, the gene being selected from the group consisting of protease genes aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, more preferably selected from aprE, mpr, bpr, vpr, and ispA, and the Bacillus cell most preferably has reduced expression of all aprE, mpr, bpr, vpr, and ispA.

[0168] 32. The method according to any one of embodiments 21 to 31, wherein the Bacillus cells further have reduced expression of at least one other gene selected from the group consisting of sigF, pga and amyB, preferably having reduced expression of all sigF, pga and amyB.

[0169] 33. The method according to any one of embodiments 21 to 32, wherein the Bacillus cell further comprises reduced expression of at least one other gene selected from the group consisting of sigF, pga, amyB, aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, preferably selected from sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, most preferably having reduced expression of all sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, and preferably, the Bacillus cell comprises a functional wprA and / or epr gene.

[0170] 34. A method for producing a protein of interest, preferably an enzyme, said method comprising the following steps: I. Providing Bacillus cells expressing a metalloproteinase, wherein the metalloproteinase is selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. II. Introducing an expression cassette encoding a protein of interest, preferably an enzyme, sensitive to the metalloproteinase into the Bacillus cells. III. The Bacillus cells are cultured under conditions that allow for the expression of the protein of interest and also allow for the expression of the metalloproteinase, thereby forming a fermentation broth containing the protein of interest and the metalloproteinase. IV. Optionally, the protein of interest is isolated from the culture medium to form a solution containing the protein of interest and the metalloproteinase, and V. Contact the fermentation broth of step III and / or the solution of step IV with a chelating agent, preferably EDTA, MGDA or EDDS, in an amount and for a time that effectively inactivates the metalloproteinase.

[0171] 35. Use of a chelating agent, preferably EDTA, MGDA, or EDDS, for increasing the yield of the protein of interest, preferably an enzyme, in a method for producing a protein of interest from Bacillus cells containing a metalloproteinase, wherein the metalloproteinase is selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07. The protein of interest is sensitive to the metalloproteinase.

[0172] 36. The method according to embodiment 34 or the use according to embodiment 35, wherein the metalloproteinase comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or 100% identity with SEQ ID NO: 08, preferably having 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%, at least 99% or 100% identity with SEQ ID NO: 08.

[0173] 37. The method or use according to embodiment 36, wherein the Bacillus cell is a Bacillus licheniformis cell.

[0174] 38. The method or use according to embodiment 37, wherein the protein of interest is a mannanase, the mannanase preferably comprising or consisting of two or more domains linked via a flexible linker, preferably the flexible linker being sensitive to the metalloproteinase, the mannanase preferably comprising or consisting of a carbohydrate-binding domain, preferably the catalytic domain and the carbohydrate domain of the mannanase being linked via a flexible linker, preferably the flexible linker being sensitive to the proteolytic cleavage of the metalloproteinase, preferably wherein the mannanase is a variant of the parental mannanase, wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 12 and with SEQ ID NO: 12 The amino acid sequences numbered 59, 66, 89, 234, 259, 282, 318, 319 and 322 having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%, but less than 100% identity.

[0175] 39. The method or use according to embodiment 38, wherein the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions selected from the group consisting of: X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G as indicated by the numbers in SEQ ID NO: 12.

[0176] 40. The method or use according to embodiment 39, wherein the mannanase variant comprises or consists of SEQ ID NO:12 having amino acid substitutions of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering in SEQ ID NO:12 and optionally having 1-10, preferably 1-5, conservative substitutions, wherein the mannanase variant comprises or consists of SEQ ID NO:12 having amino acid substitutions of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering in SEQ ID NO:12.

[0177] 41. A method for inactivating metalloproteinases, wherein the method comprises the following steps: I. Provide metalloproteinases selected from the following groups: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07, and II. Contact the metalloproteinase with a chelating agent, preferably EDTA, MGDA, or EDDS, for a sufficient amount and time to inactivate the metalloproteinase.

[0178] 42. The method according to embodiment 41, wherein the metalloproteinase comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99% or 100% identity with SEQ ID NO: 08, preferably having 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%, at least 99% or 100% identity with SEQ ID NO: 08.

[0179] 43. The method according to any one of embodiments 41 or 42, wherein contacting the metalloproteinase with the chelating agent is carried out in a formulation containing the protein of interest, preferably containing an enzyme.

[0180] 44. The method according to any one of embodiments 41 to 43, wherein the contacting of the metalloproteinase with the chelating agent is carried out in a non-compound formulation, preferably a liquid enzyme formulation or a compound formulation, preferably a liquid detergent formulation.

[0181] 45. The method according to embodiment 44, wherein the protein of interest is a mannanase, the mannanase preferably comprising or consisting of two or more domains linked via a flexible linker, preferably the flexible linker being sensitive to the metalloproteinase, the mannanase preferably comprising or consisting of a catalytic domain and a carbohydrate-binding domain, preferably the catalytic domain and the carbohydrate domain of the mannanase being linked via a flexible linker, preferably the flexible linker being sensitive to the proteolytic cleavage of the metalloproteinase, preferably wherein the mannanase is a variant of the parental mannanase, wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 12 and with SEQ ID NO: 12 The amino acid sequences numbered 59, 66, 89, 234, 259, 282, 318, 319 and 322 having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%, but less than 100% identity.

[0182] Example Materials and methods The following examples are for illustrative purposes only. Many possible variations, which will be apparent to those skilled in the art, also fall within the scope of this invention.

[0183] Unless otherwise stated, the following experiments were performed using standard equipment, methods, chemicals, and biochemicals used in the production of compounds through genetic engineering, molecular biology, and microbial culture and fermentation. See also Sambrook et al. (Sambrook, J. and Russell, DW. Molecular cloning. A laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001).

[0184] Electroporation of competent Bacillus licheniformis cells and electroporation DNA was transformed into Bacillus licheniformis strain Bli#008 (WO2022018260) via electroporation, which contained deletions of the additional mpr, vpr, bpr, and ispA genes (referred to herein as Bli#099). The preparation of electroporated competent Bacillus licheniformis cells and the transformation of DNA were performed essentially as described by Bridi et al. (Brigidi, P., Mateuzzi, D. (1991). Biotechnol. Techniques 5, 5), with the following modifications: after DNA transformation, cells were recovered in 1 ml of LB 5-PSG buffer and incubated at 37°C for 60 min after being plated on selective LB-agar plates (Vehmaanperä J., 1989, FEMS Microbio. Lett., 61: 165-170).

[0185] To overcome the Bacillus licheniformis-specific restriction modification system of Bacillus licheniformis strains, plasmid DNA was isolated from Ec#098 cells or Bacillus subtilis Bs#056 cells as described below.

[0186] plasmid separation Plasmid DNA was isolated from Bacillus and Escherichia coli cells using standard molecular biology methods described below: (Sambrook, J. and Russell, DW Molecular cloning. A laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001) or alkaline lysis (Birnboim, HC, Doly, J. (1979). Nucleic Acids Res 7(6): 1513-1523). Bacillus cells were compared with Escherichia coli treated with 10 mg / ml lysozyme at 37°C for 30 minutes prior to cell lysis.

[0187] plasmid plasmid pEC194RS The Bacillus temperature-sensitive deletion plasmid (WO2022018260) was used to clone gene deletion and gene integration constructs.

[0188] pDel045-wprA deletion plasmid A gene deletion plasmid for the wprA gene (SEQ ID NO: 01) encoding the protease WprA (SEQ ID NO: 02) of *Bacillus licheniformis* was constructed using plasmid pEC194RS and gene synthesis construct SEQ ID NO: 03. This gene synthesis construct included the 5' and 3' genomic regions of the wprA gene flanked by a BsaI site compatible with pEC194RS. Type II assembly of the restriction endonuclease BsaI was performed as described (Radeck et al., 2017), and the reaction mixture was subsequently transformed into *E. coli* DH10B cells (Life Technologies). The transformants were spread out and incubated overnight at 37°C on LB agar plates containing 100 µg / ml ampicillin. Plasmid DNA was isolated from individual clones and its correctness was analyzed by restriction enzyme digestion. The resulting wprA deletion plasmid was named pDel045.

[0189] pDel046-epr deletion plasmid For example, a gene deletion plasmid for the epr gene (SEQ ID NO: 04) encoding the protease Epr (SEQ ID NO: 05) of Bacillus licheniformis was constructed as described in pDel045. However, a gene synthesis construct (SEQ ID NO: 06) was used, which includes the 5' and 3' genomic regions of the epr gene flanked by a BsaI site compatible with pEC194RS. The resulting epr deletion plasmid was named pDel046.

[0190] pDel047-metalloproteinase BL03917 deletion plasmid For example, a gene deletion plasmid for the BL03917 gene (SEQ ID NO: 07) encoding peptidase M84 (SEQ ID NO: 08) of Bacillus licheniformis was constructed as described in pDel045. However, a gene synthesis construct (SEQ ID NO: 09) was used, which includes the 5' and 3' genomic regions of the BL03917 gene flanked by a BsaI site compatible with pEC194RS. The resulting BL03917 gene deletion plasmid was named pDel047.

[0191] Plasmid pUK57: Type II assembly of the target Bacillus plasmid Plasmid pUK57 (described in WO2022018260) is a derivative of plasmid pUB110, which contains a type II cloning cassette for assembling gene expression vectors.

[0192] Mannanase expression plasmid The mannanase expression plasmid consists of three genetic elements: a pUK57 plasmid backbone, a plasmid derived from Bacillus licheniformis, and a... aprEThe promoter of the gene (SEQ ID NO:16), the signal peptide for the mannanase gene, and the coding sequence of the mannanase gene, respectively. As described (Radeck et al., 2017; Sci.Rep.7: 14134), in an in vitro type II assembly reaction, the pUK57 vector, promoter fragment (SEQ ID NO:17), signal peptide gene fragment, and mannanase gene fragment, each containing a compatible type II restriction endonuclease BpiI site (see Table 1), were assembled using the restriction endonuclease BpiI. The reaction mixture was then transformed into Bacillus subtilis Bs#056 cells to make them competent after being plated on LB agar plates containing 20 µg / ml kanamycin, according to Spizizen's method (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, 741-746). The correct cloning of the final mannanase plasmid was analyzed by restriction enzyme digestion and sequencing. Table 1 summarizes the mannanase expression plasmids.

[0193] Table 1: Mannanase expression plasmids

[0194] strain Escherichia coli strain Ec#098 Escherichia coli strain Ec#098 is an Escherichia coli INV110 strain (Life technologies) carrying a DNA-methyltransferase encoding the expression plasmid pMDS003 WO2019016051.

[0195] Bacillus subtilis strain Bs#056 Prototrophic Bacillus subtilis strain KO-7S (BGSCID: 1S145; Zeigler DR) was made competent using the following method: Spizizen (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, 741-746.) and transformed with the linearized DNA-methyltransferase expression plasmid pMIS012 for the integration of the DNA-methyltransferase into the amyE gene, as described in WO2019 / 016051 for the production of Bacillus subtilis Bs#053. Cells were spread out and incubated overnight at 37°C on LB agar plates containing 10 µg / ml chloramphenicol. After overnight incubation at 37°C, grown colonies were picked and gently rubbed onto LB agar plates containing 10 µg / ml chloramphenicol and LB agar plates containing 10 µg / ml chloramphenicol and 0.5% soluble starch (Sigma). Starch plates were covered with iodine-containing Lugols solution, and positively integrated clones were identified using negative amylase activity. Genomic DNA from positive clones was isolated by standard phenol / chloroform extraction after treatment with lysozyme (10 mg / ml) at 3°C ​​for 30 minutes, followed by PCR analysis to confirm proper integration of the MTase expression cassette. The resulting Bacillus subtilis strain was named Bs#056.

[0196] Mannanase activity assay To determine mannanase activity, stained insoluble polysaccharide (AZCL-galactomannan obtained from Megazymes, Ireland) was used. For the activity assay, the enzyme solution was diluted in 100 mM Tris-buffered saline (pH 8.6, 30°C) supplemented with 0.1% Brij 35 and 0.1% xanthan gum. Using a 96-well plate, 900 μL of 0.2% AZCL-galactomannan (dissolved in 100 mM Tris-buffered saline (pH 8.6, 30°C) supplemented with 0.1% Brij 35 and 0.1% xanthan gum) was added to 100 μL of the diluted enzyme solution. After incubation at 40°C for 30 min, the plate was centrifuged at 4500 g for 5 min to stop the reaction and separate the insoluble fragment from the dissolved polysaccharide. 200 μL of the supernatant was carefully removed, and the stained water-soluble fragment was measured at 590 nm. The values ​​of the stored samples were normalized relative to the corresponding samples kept frozen at -80°C.

[0197] Example 1: Construction of a protease gene knockout in Bacillus licheniformis Using Bacillus licheniformis Bli#099, the gene encoding the protease shown in Table 2 was deleted, resulting in the strains outlined in Table 3 below.

[0198] For gene deletions in *Bacillus licheniformis* strains, after selection on LB agar plates containing 100 µg / ml ampicillin and 30 µg / ml chloramphenicol at 37°C, the gene deletion plasmid (see the "Plasmid" section) was transformed into *Escherichia coli* strain Ec#098 to make the strain competent, according to Chung's method (Chung, CT, Niemela, SL, and Miller, RH (1989). One-step preparation of competent *Escherichia coli*: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172-2175). Plasmid DNA was isolated from individual clones and used for subsequent transfer to *Bacillus licheniformis* strains. The isolated plasmid DNA correspondingly carried the DNA methylation pattern of the *Bacillus licheniformis* strain and was protected from degradation after transfer to *Bacillus licheniformis*. Gene deletion was performed according to the procedure outlined in the following sections.

[0199] Bacillus licheniformis cells carrying a gene deletion plasmid were grown on LB agar plates containing 5 µg / ml erythromycin at 45°C, thereby driving the integration of the deletion plasmid into a chromosome via Campbell recombination. This chromosome possessed one of the homologous regions present on the deletion plasmid that was homologous to the 5' or 3' sequence of the desired target gene to be deleted. Clones were picked and plated on LB agar plates containing 5 µg / ml erythromycin at 30°C, and then incubated in LB medium without selection pressure at 45°C for 6 hours. Individual clones were picked and analyzed by colony PCR, wherein oligonucleotides were located at least 100 bp in the 5' and 3' homologous regions to confirm successful deletion of the target gene. Appropriate selection of oligonucleotides is well known to those skilled in the art.

[0200] Selected single clones that were presumed to be deletion-positive and incubated twice overnight at 45°C in antibiotic-free LB medium to solidify the plasmid. The plasmids were then plated onto LB agar plates and incubated overnight at 30°C. The single clones were streaked again onto LB agar plates containing 5 µg / ml erythromycin and analyzed by colony PCR to confirm successful deletion of the target gene. Single erythromycin-sensitive clones with the correctly deleted target gene were isolated. The following proteases were selected for gene deletion in Bacillus licheniformis.

[0201] Table 2: Deleted proteases in Bacillus licheniformis

[0202] Table 3 summarizes the genotypes of Bacillus licheniformis gene-deleted strains.

[0203] Table 3: Genotypes of Bacillus licheniformis strains with protease gene deletion

[0204] * D indicator missing Example 2: Construction of Bacillus licheniformis mannanase expression strain As described above, the *Bacillus licheniformis* strains listed in Table 3 were made competent. The mannanase expression plasmid pMan117, carrying a *Bacillus licheniformis*-specific DNA methylation pattern, was isolated from *Bacillus subtilis* Bs#056 and transformed into *Bacillus licheniformis* strains with gene deletions of the protease genes given in Table 3. The transformed strains were plated on LB agar plates containing 20 µg / µl kanamycin. The correctness of the plasmid DNA from individual clones was analyzed by restriction digestion and Sanger sequencing. The resulting *Bacillus licheniformis* expression strains are listed in Table 4.

[0205] Table 4: Mannanase Expression Strains

[0206] * D indicator missing Example 3: Cultivating mannanase expression strains The Bacillus licheniformis mannanase-expressing strains listed in Table 4 were cultured in a fed-batch process based on microtiter plates (Habicher et al., 2019 Biotechnol J.; 15(2)). Each strain was cultured in six replicates.

[0207] All cultures were performed in a 25 mm diameter orbital shaker at 30 °C and 400 rpm (Innova 42, NewBrunswick Scientific, Eppendorf AG; Hamburg, Germany). The strain was cultured in two subsequent pre-cultures in flower discs (MTP-48-OFF, m2p-labs GmbH) for synchronized growth. The first pre-culture was performed in 800 µl TB medium inoculated with fresh single colonies of the strain streaked on LB agar plates. After 20 hours at 30 °C, a second pre-culture containing 800 µl V3 basal medium (Meissner et al., 2015, Journal of Industrial Microbiology & Biotechnology 42 (9): 1203–1215) was inoculated with 8 µl of the first pre-culture and cultured at 30 °C for 24 hours. Feed-batch master cultures were performed using 48-well circular and deep-well microtiter plates, with each well containing a glucose-containing polymer (FeedPlate, trade number: SMFP08004, Kuhner Shaker GmbH; Herzogenrath, Germany) at the bottom. 700 µl of glucose-free V3-FP basal medium was inoculated with 70 µl of a second preculture. The master cultures were incubated at 30 °C for 72 h. The precultures were covered with sterile, breathable sealing foil (AeraSeal film, Sigma-Aldrich) to prevent contamination. The feed plate was sealed with sterile, breathable, anti-evaporation foil (F-GPR48-10, m2p-labs GmbH) to reduce evaporation and prevent contamination.

[0208] At the end of the feed-and-dose process based on microtiter plates, the cultured samples were removed and supernatants were prepared by centrifugation and aseptic filtration through a 0.2 µm filter. The concentrations of full-length mannanase and mannnanase degradation products were analyzed and quantified using a capillary electrophoresis system (Labchip GX Touch HT, Perkin Elmer) with a suitable protein assay kit (Protein Express assay kit). Therefore, samples were diluted to approximately 1 mg / mL of total protein and prepared for electrophoresis according to the manufacturer's instructions. Electrophoretic patterns were analyzed using Labchip GX Reviewer software (version 5.5.2312.0). The amounts of full-length and truncated mannanase were quantified and calculated as a percentage of total mannanase content (full-length and truncated mannanase). LC-MS / MS analysis showed that the truncated mannanase was a fragment of the full-length mannanase.

[0209] The results are shown in Table 5. For strains lacking protease BL03917 (SEQ ID NO: 08), no degradation of mannanase was detected (<2.0%), and the full-length mannanase product remained stable. Surprisingly, the absence of proteases WprA and Epr did not cause a reduction in the amount of truncated mannanase.

[0210] Table 5: Percentage of full-length mannanase and truncated mannanase

[0211] * D indicator missing Example 4: Storage test of mannanases derived from strains with different protease backgrounds Mannanase aqueous solution is produced via microbial fermentation as described above. The enzyme is recovered by removing cells and concentrated using ultrafiltration.

[0212] A final enzyme protein concentration of 0.5% w / w was prepared by mixing an aqueous mannanase solution with 25% w / w glycerol and 25% w / w sorbitol at pH 6.0. Three samples were prepared using a reference strain (BES#334). One sample was prepared without any chelating agent, while the other two samples were prepared by adding 0.25% EDTA (ethylenediaminetetraacetic acid) to the mannanase solution. The pH was adjusted accordingly if necessary. Additional samples were prepared using the strain according to the invention (BES#336) without the addition of a chelating agent.

[0213] The diluted mannanase solution was incubated in sealed vials at -80°C and 45°C for four weeks. The mannanase activity in the stored samples was measured, and the mannanase activity in the samples stored at -80°C was used as a reference to calculate the residual activity.

[0214] Table 6: Residual activity of mannanase after four weeks of storage The data in Table 6 show that the strain according to the invention reduces enzyme degradation. After four weeks of storage, the residual mannanase activity of the mannanase preparation of the strain according to the invention is 77% higher than that of the reference strain (44%). It can also be seen from Table 6 that, alternatively, inactivation of metalloproteinases can be achieved by adding a chelating agent, thereby reducing enzyme degradation; therefore, the residual mannanase activity is increased after four weeks of storage compared to the enzyme preparation of the reference strain without added chelating agent.

Claims

1. A Bacillus cell, said Bacillus cell having reduced expression of a gene encoding a metalloproteinase, said metalloproteinase being selected from the group consisting of: I. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and II. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO:

07.

2. The Bacillus cell according to claim 1, wherein the Bacillus cell is a metalloproteinase knockout cell.

3. The Bacillus cell according to any one of claims 1 or 2, wherein the Bacillus cell expresses a protein of interest sensitive to the metalloproteinase, wherein the protein of interest is heterologous to the cell, preferably wherein the protein of interest is an enzyme.

4. The Bacillus cell according to claim 3, wherein the enzyme is selected from the group consisting of: mannanase, protease, amylase, lipase, cellulase, hemicellulase, phospholipase, esterase, keratinase, pectinase, lactase, peroxidase, xylanase, pectic acid lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannic acid enzyme, pentosanase, melaninase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, nuclease, DNase, phosphodiesterase, phytase, glycosylase, galactanase, xanthan gum. The enzyme comprises, but is not limited to, xyloglucanase, oxidoreductase, hydrolase, aminopeptidase, asparaginase, glycosylase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertase, ribonuclease, transglutaminase, and dispersin. Preferably, the enzyme is selected from the group consisting of: protease, amylase, lipase, cellulase, mannanase, xylanase, DNase, dispersin, pectinase, pectic acid lyase, glycosidase, and oxidoreductase. Most preferably, the enzyme is mannanase.

5. The Bacillus cell of claim 4, wherein the enzyme is a mannanase, wherein the mannanase preferably comprises or consists of two or more domains linked via a flexible linker, preferably the flexible linker is sensitive to the metalloproteinase, wherein the mannanase preferably comprises or consists of a catalytic domain and a carbohydrate-binding domain, preferably the catalytic domain and the carbohydrate domain of the mannanase are linked via a flexible linker, preferably the flexible linker is sensitive to the proteolytic cleavage of the metalloproteinase, preferably wherein the mannanase is a variant of the parental mannanase, wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 12 and with SEQ ID NO: 12 The amino acid sequences numbered 59, 66, 89, 234, 259, 282, 318, 319 and 322 having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%, but less than 100% identity.

6. The Bacillus cell of claim 5, wherein the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions selected from the group consisting of: X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G as indicated in SEQ ID NO:

12.

7. The Bacillus cell according to any one of claims 3 to 6, wherein the protein of interest is secreted by the cell.

8. The Bacillus cell according to any one of the preceding claims, wherein the Bacillus cell further has reduced expression of at least one other gene, said at least one other gene preferably selected from the group consisting of: a gene encoding a protease different from said metalloproteinase, a gene encoding a sporulation factor, a gene encoding a secretory enzyme different from said protease, a gene encoding a protein involved in the formation of the extracellular matrix, or a gene encoding an autolysin, preferably a gene encoding a protease different from said metalloproteinase, said gene preferably selected from the group consisting of: protease encoding genes composed of aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, more preferably selected from aprE, mpr, bpr, vpr, and ispA.

9. The Bacillus cell according to any one of the preceding claims, wherein the Bacillus cell is selected from the group consisting of: Bacillus hygroscopicus, Bacillus amyloliquefaciens, Bacillus atrophicus, Bacillus spp., Bacillus spp., Bacillus hesperidinus, Bacillus licheniformis, Bacillus nakamura, Bacillus basilaceus, Bacillus paralicheniformis, Bacillus pumilus, Bacillus saforgensis, Bacillus sonora, Bacillus stratosphericus, Bacillus belleus, and Bacillus cladosporium, preferably selected from the group consisting of: Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus belleus, more preferably Bacillus licheniformis and Bacillus pumilus, and most preferably Bacillus licheniformis.

10. A method for producing a protein of interest, preferably an enzyme, the method comprising the following steps: I. Providing Bacillus cells according to claim 1, II. Introducing an expression cassette encoding a protein of interest, preferably an enzyme, sensitive to the metalloproteinase into the Bacillus cells. III. The Bacillus cells are cultured under conditions that allow for the expression of the protein of interest, thereby forming a fermentation broth containing the protein of interest. IV. Optionally, the protein of interest is isolated from the fermentation broth of step III.

11. A method for producing a protein of interest, preferably an enzyme, said method comprising the following steps: I. Providing Bacillus cells expressing a gene encoding a metalloproteinase, wherein the metalloproteinase is selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO:

07. II. Introducing an expression cassette encoding a protein of interest, preferably an enzyme, sensitive to the metalloproteinase into the Bacillus cells. III. The Bacillus cells are cultured under conditions that allow for the expression of the protein of interest and also allow for the expression of the metalloproteinase, thereby forming a fermentation broth containing the protein of interest and the metalloproteinase. IV. Optionally, the protein of interest is isolated from the culture medium to form a solution containing the protein of interest and the metalloproteinase, and V. Contact the fermentation broth of step III and / or the solution of step IV with a chelating agent, preferably EDTA, MGDA or EDDS, in an amount and for a time that effectively inactivates the metalloproteinase.

12. Use of a chelating agent, preferably EDTA, MGDA, or EDDS, for increasing the yield of the protein of interest in a method for producing a protein of interest via Bacillus cells, wherein the Bacillus cells express a gene encoding a metalloproteinase selected from the group consisting of: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO:

07. The protein of interest is sensitive to the metalloproteinase.

13. The method of any one of claims 10 or 11 or the use according to claim 12, wherein the protein of interest is a mannanase, wherein the mannanase preferably comprises a catalytic domain and a carbohydrate-binding domain, wherein the catalytic domain and the carbohydrate domain of the mannanase are connected via a flexible linker, wherein the flexible linker is sensitive to proteolytic cleavage by the metalloproteinase, wherein the mannanase is preferably a variant of a parental mannanase, wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of: 59, 66, 89, 234, 259, 282, 318, 319, and 322 according to SEQ ID NO: 12 and having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, but less than 100% identity with SEQ ID NO:

12.

14. The method or use according to claim 13, wherein the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions selected from the group consisting of: X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbers stated in SEQ ID NO:

12.

15. A method for inactivating metalloproteinases, wherein the method comprises the following steps: I. Provide metalloproteinases selected from the following groups: a. Metalloproteinases having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. A metalloproteinase encoded by a polynucleotide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 07, and II. Contact the metalloproteinase with a chelating agent, preferably EDTA, MGDA, or EDDS, for a sufficient amount and time to inactivate the metalloproteinase.

Images