Improved bacillus cell with inactivated metalloprotease

Abstract

The present invention provides an improved Bacillus cell having reduced expression of a metalloprotease gene. Inactivation of the metalloprotease gene reduces proteolytic degradation of a protein of interest produced by said Bacillus cell and thus, improves the yield of the protein of interest in such production method. The present invention is also directed to a method of inactivating the metalloprotease and the application of this method of inactivation when producing a protein of interest.

Description

[0001] Improved Bacillus cell with inactivated metalloprotease

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an improved Bacillus cell for the production of a protein of interest. Specifically, the present invention relates to a Bacillus cell having reduced expression of a gene encoding a metalloprotease comprising an amino acid having a least 55% identity to SEQ ID NO: 08.

[0004] BACKGROUND

[0005] Microorganisms of the Bacillus genus are widely applied as industrial workhorses for the production of valuable compounds, such as chemicals, polymers and proteins, in particular proteins like washing- and / or cleaning-active enzymes or enzymes used for feed and food applications. The biotechnological production of these enzymes is conducted via fermentation of such Bacillus species and subsequent purification of the product. Bacillus species are capable of secreting significant amounts of protein to the fermentation broth. This allows a simple product purification process of the protein of interest compared to intracellular production and explains the success of Bacillus in industrial application. Thus, the continuous optimization of Bacillus cells for increased production of these proteins is of high relevance. In particular, in large-scale industrial production settings, even small improvements have a great impact on production costs.

[0006] Bacillus cells naturally secrete various extracellular proteins and enzymes so that the protein or enzyme of interest is not the only protein in the supernatant. Most often these cell proteins are neglected due to their low abundance compared to the protein of interest. However, due to their ability to degrade other proteins, in particular cell proteases might not be desired in case either the protein of interest is proteolytically instable or in applications the protein products must be devoid of any proteolytic activity. The latter is for example the case in dairy applications, where milk derived casein shall not be degraded by minor traces of proteases or in detergent formulations wherein other co-formu- lated enzymes would be degraded.

[0007] Preventing degradation of heterologous proteins by deletion of cell proteases has been recognized already by Kawa- mura et al. (Karamura and Doi, Journal of Bacteriology Oct. 1984, Vol. 160, No. 1 , p. 442-444). Here two major host proteases have been inactivated (NprE and AprA (also referred to as AprE)) in a Bacillus subtilis parent host resulting in the Bacillus subtilis strain DB104. It was already here discussed that this mutant strain being deficient of an extracellular neutral and an extracellular alkaline protease can be useful to produce foreign proteins and peptides. Further host proteases were deleted by Wu et al. (Wu et al., Journal of Bacteriology Aug. 1991 , Vol. 173, No. 16, p. 4952-4958). Here a stain already devoid of four host proteases (Bacillus subtilis DB428 with inactive genes encoding NprE, AprE, Epr and Bpf (also referred to as Bpr)) was used as template to construct the Bacillus subtilis WB600 with inactive genes encoding NprE, AprE, Epr, Bpr and further deletion of the genes encoding Mpr and NprB. Wu et al. does also describe the utilization of such a strain for the improved production of the heterologous protein beta-lac- tamase. Here the proteolytic sensitive beta-lactamase has a significantly higher yield as a result of reduced loss due to degradation after prolonged cultivation time. WB600 was optimized by the same group with the deletion of the genes encoding two additional proteases (WprA and Vpr) allowing the successful secretion of a single chain antibody fragment (Wu et al., Applied and Environmental Microbiology July. 2002, Vol. 68, No. 7, p. 3261-3269). This strain is called Bacillus subtilis WB800 with the following protease genes inactivated: nprE, aprE, epr, bpr, mpr, nprB, vpr, wprA. Similarly, W009022162 even further develops the multi-protease knockout strain with additional deletions in the genes encoding the quality control proteases htrA and hrtB resulting in increased yield and purity of the secreted recombinant protein.

[0008] The concept of deleting host proteases to improve the overexpression of proteins of interests is not only described for Bacillus subtilis but also for other Bacillus species. Schallmey et al. (Schallmey et al, Canadian Journal of Microbiology 2004 Vol. 50; p. 1-17; doi: 10.1139 / W03-07) describes generally that the production and secretion of high yields of recombinant proteins in Bacillus hosts is hampered by the degradation of the products by the host proteases.

[0009] Vehmannapera et al. (Vehmannapera et al.; Journal of Biotechnology, 1991, Vol 19, p. 221-240) describe for example the improved secretion of amylases and glucanases in a Bacillus amyloliquefaciens devoid of functional nprE and aprE genes even in complex media requiring basal proteolytic activity for media degradation. With cultivation techniques using chemically defined media one might assume here an even higher effect.

[0010] WO2014206829 describes a Bacillus licheniformis strain with deletions of several genes encoding proteases useful for the production of heterologoues enzymes (aprL, mprL, bprAB, epr, vpr, wprA, and additionally ispA). The latter gene is remarkable as it encodes for the intracellular serine protease IspA. This underlines that not only extracellular proteases need to be considered to reduce proteolytic activity in the supernatant, but also intracellular proteases. Likewise, EP1829959 describes the inactivation of the intracellular protease AprX alone or in combination of the above-mentioned proteases for increasing the yield of recombinant proteins.

[0011] However, proteolytic sensitive proteins of interest might still be degraded by host protease not known to the person skilled in the art.

[0012] Identification of host protease proteases imposes a significant challenge as more than 100 genes are described for proteolytic function for example in Bacillus licheniformis DSM 13. A search for proteases and peptidases of Bacillus licheniformis via the UniProt database (www.uniprot.org; UniProt Consortium. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021 Jan 8;49(D1):D480-D489) with 'protease' or 'peptidase' and ‘Bacillus licheniformis' (Organism ID 279010 with Bacillus licheniformis strains ATCC 145801 DSM 13 / JCM 25051 CCUG 74221 NBRC 122001 NCI MB 93751 NCTC 10341 1 NRRL NRS-12641 Gibson 4) resulted in 177 entries (date of search: May 02, 2023).

[0013] As the inventors revealed herein, selecting genes for inactivation remains very challenging as no certainty is given that deletion of a particular gene encoding a protein with proteolytic activity is effective for reducing the degradation of the protein of interest.

[0014] Herein, the present inventors have surprisingly discovered that the inactivation of a gene encoding a putative metalloprotease in a Bacillus cell results in an improved yield of a protein of interest produced with said Bacillus cell. BRIEF SUMMARY OF THE INVENTION

[0015] Thus, the present invention is directed to a Bacillus cell having reduced expression of a gene encoding a metalloprotease selected from the group consisting of:

[0016] I. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and

[0017] II. a metalloprotease 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 to SEQ ID NO: 07.

[0018] Moreover, the present invention is directed to a method of producing a protein of interest, preferably an enzyme, comprising the steps of

[0019] I. providing a Bacillus cell having reduced expression of a gene encoding a metalloprotease selected from the group consisting of: a) a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b) a metalloprotease 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 to SEQ ID NO: 07,

[0020] II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metalloprotease,

[0021] III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest, thereby forming a fermentation broth comprising the protein of interest, and

[0022] IV. optionally isolating the protein of interest from the fermentation broth of step III.

[0023] In a further embodiment, the present invention is directed to a method of inactivating a metal loprotease, wherein the method comprises the steps of:

[0024] I. providing a metalloprotease selected from the group consisting of: a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, and

[0025] II. contacting the metalloprotease with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time sufficient to inactivate the metalloprotease.

[0026] Thus, in an alternative embodiment, the present invention is directed to a method of producing a protein of interest, preferably an enzyme, comprising the steps of I. providing a Bacillus cell expressing a metal loprotease selected from the group consisting of a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07,

[0027] II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metalloprotease,

[0028] III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest and which also allow expression of the metalloprotease, thereby forming a fermentation broth comprising the protein of interest and the metalloprotease,

[0029] IV. optionally isolating the protein of interest from the cultivation medium, thereby forming a solution comprising the protein of interest and also the metalloprotease, and

[0030] V. contacting the fermentation broth of step III or the solution of step IV with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time effective to inactivate the metal loprotease.

[0031] Hence, the present invention is also directed to the use of a chelating agent, preferably EDTA, MGDA, or EDDS, for increasing the yield of a protein of interest and / or the stability of the protein of interest, preferably an enzyme, in a method of producing the protein of interest by a Bacillus cell, wherein the Bacillus cell expresses a metal loprotease selected from the group consisting of a. a metalloprotease having a 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

[0032] 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, wherein the protein of interest is sensitive towards the metalloprotease.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention may be understood more easily by reference to the following definitions, detailed description of the embodiments of the invention and the examples included herein. Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.

[0035] Definitions

[0036] Unless otherwise noted, the terms used herein are to be understood according to conventional use by those of ordinary skill in the relevant art.

[0037] Before describing exemplary embodiments of the present invention in detail, definitions important for understanding the present invention are provided. Unless stated otherwise or apparent from the nature of the definition, the definitions apply to all compounds, methods and uses described herein. It is to be understood that as used in the specification and in the claims, "a” or "an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell” can mean that at least one cell can be utilized.

[0038] Further, it will be understood that the term "at least one” as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one feed solution shall be used this may be understood as one feed solution or more than one feed solutions, i.e. two, three, four, five or any other number of feed solutions. Depending on the item the term refers to the skilled person understands as to what upper limit the term may refer, if any.

[0039] The term "about” as used herein means that with respect to any number recited after said term an interval accuracy exists within in which a technical effect can be achieved. Accordingly, about as referred to herein, preferably, refers to the precise numerical value or a range around said precise numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, and even more preferably ±5 %.

[0040] The term "comprising” as used herein shall not be understood in a limiting sense. The term rather indicates that more than the actual items referred to may be present, e.g., if it refers to a method comprising certain steps, the presence of further steps shall not be excluded. However, the term "comprising” also encompasses embodiments where only the items referred to are present, i.e. in the sense of "consisting of”.

[0041] The terms "coding for" and "encoding” are used interchangeably herein. Typically, the terms refer to the property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein, if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.

[0042] "Variant” proteins differ from "parent” proteins by certain amino acid alterations, preferably amino acid substitutions at one or more amino acid positions.

[0043] In describing the polypeptides and variants of the present invention, the abbreviations for single amino acids used are according to the accepted IUPAC single letter or three letter amino acid abbreviation.

[0044] "Amino acid alteration” as used herein refers to amino acid substitution, deletion, or insertion.

[0045] "Substitutions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence followed by the amino acid, which substitutes the original amino acid. For example, the substitution of histidine at position 120 with alanine is designated as “His120Ala” or "H120A”. Substitutions can also be described by merely naming the resulting amino acid without specifying the initial amino acid at this position, e.g., "X120A” or "120A” or "Xaa120Ala” or “120Ala”. Positions of substitutions can be described by merely providing the number of the position within the amino acid sequence.

[0046] "Deletions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence followed by *. Accordingly, the deletion of glycine at position 150 is designated as "Gly150*” or "G150*”. Alternatively, deletions are indicated by, e.g., "deletion of G184”.

[0047] "Insertions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence followed by the original amino acid and the additional amino acid. For example, an insertion at position 180 of lysine next to glycine is designated as “Gly 180GlyLys” or "G180GK”. When more than one amino acid residue is inserted, such as, e.g., a Lys and an Ala after Gly 180 this may be indicated as: “Gly 180Gly LysAla” or "G195GKA”.

[0048] In cases where a substitution and an insertion occur at the same position, this may be indicated as "S99A+S99SD” or in short "S99AD”. Sequences comprising multiple alterations are separated by “+”, e.g., "Arg 170Tyr+Gly 195Glu”, "R170Y+G195E” or "X170Y+X195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple alterations may be separated by space or a comma, e.g., "R170Y G195E” or "R170Y, G195E” respectively. Where different alternative alterations can be introduced at a position, the different alterations are separated by a comma, e.g., "Arg170Tyr, Glu” and "R170T, E”, respectively, represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Alternative substitutions at a particular position can also be indicated as "X120A,G,H”, "120A,G, H”, "X120A / G / H”, or “120A / G / H”. Alternatively, different alterations or optional substitutions may be indicated in brackets, e.g., “Arg170 [Tyr, Gly]” or “Arg170 {Tyr, Gly}” or in short "R170 [Y, G]” or "R170 {Y, G}”.

[0049] Variants of a parent protein may be defined by their sequence identity when compared to a parent protein. Sequence identity usually is provided as "% sequence identity” or "% identity”. To determine the percent-identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e., a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1970) 48, p. 443- 453), preferably by using the program "NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EBLOSUM62). The preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.

[0050] After aligning the two sequences, in a second step, an identity value shall be determined from the alignment. Therefore, according to the present invention the following calculation of percent-identity applies:

[0051] %-identity = (identical residues I length of the alignment region which is showing the respective sequence of this invention over its complete length) *100. Thus, sequence identity in relation to 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 which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give "%-identity”.

[0052] For calculating the percent identity of two DNA sequences the same applies as for the calculation of percent identity of two amino acid sequences with some specifications. For DNA sequences encoding a protein the pairwise alignment shall be made over the complete length of the coding region from start to stop codon excluding introns. For non-protein-coding DNA sequences the pairwise alignment shall be made over the complete length of the sequence of this invention, so the complete sequence of this invention is compared to another sequence, or regions out of another sequence. Moreover, the preferred alignment program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453) is "NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EDNAFULL). Herein, the exchange of one amino acid with a similar amino acid may be called "conservative substitution”. Similar amino acids according to the invention are defined as follows:

[0053] Amino acid A is similar to amino acids S

[0054] Amino acid D is similar to amino acids E; N

[0055] Amino acid E is similar to amino acids D; K; Q

[0056] Amino acid F is similar to amino acids W; Y

[0057] Amino acid H is similar to amino acids N; Y

[0058] Amino acid I is similar to amino acids L; M; V

[0059] Amino acid K is similar to amino acids E; Q; R

[0060] Amino acid L is similar to amino acids I; M; V

[0061] Amino acid M is similar to amino acids I; L; V

[0062] Amino acid N is similar to amino acids D; H; S

[0063] Amino acid Q is similar to amino acids E; K; R

[0064] Amino acid R is similar to amino acids K; Q

[0065] Amino acid S is similar to amino acids A; N; T

[0066] Amino acid T is similar to amino acids S

[0067] Amino acid V is similar to amino acids I; L; M

[0068] Amino acid W is similar to amino acids F; Y

[0069] Amino acid Y is similar to amino acids F; H; W.

[0070] A "homologue of a protein” is defined herein as a protein with sequence similarity and functional similarity to another protein due to shared ancestry. The corresponding meaning applies for a "homologue of a gene”.

[0071] The term "native” (or naturally or wildtype or endogenous) cell or organism or polynucleotide or polypeptide refers to the cell or organism or polynucleotide or polypeptide as found in nature (i.e. , without there being any human intervention).

[0072] The term "isolated” molecule, e. g. polypeptide or polynucleotide, is defined herein as a molecule, which has been separated from its natural environment.

[0073] The term "heterologous polypeptide” (or exogenous or foreign polypeptide) is defined herein as a polypeptide, which is naturally not expressed by a cell. The term "heterologous nucleotide” (or exogenous or foreign polynucleotide) is defined herein as a polynucleotide, which is naturally not contained in a cell.

[0074] For the purpose of the invention, "recombinant" (or non-native or non-naturally) with regards to a cell or an organism means that the cell or organism contains a polynucleotide, which is introduced using gene technology or that a polynucleotide has been removed from the cell or organism using gene technology or a combination of both. Recombinant with regards to a polynucleotide or a polypeptide means that the polynucleotide or polypeptide has been newly combined or rearranged in terms of its genetic environment by using recombinant DNA techniques. Thus, recombinant polynucleotides or polypeptides include a polypeptide or polynucleotide native to the cell, whose expression is quantitatively altered or whose expression is directed from a genomic location different from the native cell as a result of manipulation of the DNA of the cell by recombinant DNA techniques, e.g., a stronger promoter. Recombinant polynucleotides or polypeptides can also be heterologous, meaning they can be foreign sequences, but they can also originate from the same organism that they are introduced to.

[0075] With respect to the relation between two or more polynucleotides or the relation between two or more polypeptides, the term "recombinant” is used to characterize that the two or more polynucleotides or two or more polypeptides are naturally not occurring in the specific combination with each other.

[0076] "Modified recombinant” polynucleotides or polypeptides means recombinant polynucleotides or recombinant polypeptides that have been modified by introducing alterations, e.g., deletions, substitutions, and / or insertions, using recombinant DNA techniques to alter the native polypeptide or native polynucleotide.

[0077] A "synthetic" compound is obtained by in vitro chemical and / or enzymatic synthesis.

[0078] "Genetic construct” or "expression cassette” as used herein, is a nucleic acid molecule composed of at least one sequence of interest to be expressed, operably linked to one or more control sequences (at least to a promoter) as described herein.

[0079] The term "vector” as used herein comprises any kind of construct suitable to carry polynucleotide sequences for transfer to a cell, or for stable or transient expression within a given cell. This encompasses any kind of cloning vehicles, such as but not limited to plasmids, phagemids, viral vectors (e.g., phages), bacteriophage, baculoviruses, cos- mids, fosmids, artificial chromosomes, and any other vectors specific for specific hosts of interest. Foreign polynucleotide sequences usually comprise a sequence encoding a protein of interest, which may be referred to herein as "gene of interest”.

[0080] The term "introduction of a polynucleotide” or "transformation of a polynucleotide” as referred to herein encompasses the transfer of a polynucleotide outside a cell into a cell, irrespective of the method used for transfer. That is, the term "transformation of a polynucleotide” as used herein is independent from vector, shuttle system, or cell, and it not only relates to the polynucleotide transfer method of transformation as known in the art (cf., 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 encompasses any further kind of polynucleotide transfer methods such as, but not limited to, transduction or transfection.

[0081] The term "expression” or "gene expression” means the transcription of a specific gene or specific genes or specific nucleic acid construct. The term "expression” or "gene expression” in particular means the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product. The term "expression of a protein" specifically means the transcription of a gene or genes or genetic construct into mRNA with subsequent translation of the latter into a protein.

[0082] Cells expressing recombinant polynucleotides or polypeptides may exhibit "increased” or "reduced” expression when compared to the respective parent cell. The term "increased expression”, "enhanced expression” or "overexpression” as used herein means any form of expression that is additional to the expression level in the parent cell (which can be absence of expression or immeasurable expression as well). Reference herein to "increased expression”, "enhanced expression” or "overexpression” is taken to mean an increase in gene expression and / or, as far as referring to polypeptides, increased polypeptide levels and / or increased polypeptide activity, relative to control organisms. The increase in expression 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 compared to that of the control organism.

[0083] The term "reduced” expression in connection with a gene means that the level of gene expression is decreased compared to the level of gene expression in the parent cell, so that the gene whose expression level is reduced produces no active protein or reduced amounts of active protein. The expression level may be reduced by e.g., 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 by 100%, compared to that of the parent cell. Preferably, the cell produces no active protein. A cell having a reduced expression level such that no active protein is produced is called a "knockout” cell.

[0084] The term "reduction of expression of a protein" or "reduced expression of a protein” shall mean a reduced amount of the protein being produced by the cell, for instance, by a reduced transcription or reduced translation. The term "nonnative reduction of expression of a protein" shall mean a reduced amount of the protein being produced by the cell achieved by any kind genetic modification by human intervention, in particular recombinant DNA techniques, applied to the cell compared to the cell not subject to the genetic modification, i.e. , the respective parent cell.

[0085] A "parent cell” is a cell, which differs from the cell of the present invention only in that the gene expression has not been reduced, i.e., a corresponding host cell with a functional gene. Reduction of gene expression is preferably achieved by deletion of the gene. Unless otherwise specified "deletion” of a gene encompasses partial or full-length deletion of the gene resulting in no expression of active protein from said gene. Deletions of genes can be indicated by a “D” in front of the gene name.

[0086] The term "inactivation" (also called herein "functional inactivation") in connection with a protein means that the functionality of said protein has been reduced compared to the functionality of the protein not subject to the inactivation, so that the protein is inactive or reduced in its activity, preferably, wherein the protein is inactivated.

[0087] Proteins or genes can be inactivated, or their expression levels can be reduced by any kind of modification by human intervention, i.e., a non-native inactivation, in particular by using recombinant DNA techniques, applied to the gene or protein compared to the gene or protein not subject to the inactivation resulting in an inactivation. The deletion (complete or partial) of a gene shall mean a non-native deletion by means of any kind of genetic modification by human intervention, in particular recombinant DNA techniques, applied to the cell compared to the cell not subject to the genetic modification resulting in a complete for partial removal of the gene.

[0088] The term "purification” or "purifying” refers to a process in which at least one component, e.g., a protein of interest, is separated from at least another component, e.g., a particulate matter of a fermentation broth, and transferred into a different compartment or phase, wherein the different compartments or phases do not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filtration membrane or cloth, i.e., filtrate and retentate; examples of such different phases are pellet and supernatant or cake and filtrate, respectively. The resulting solution after purifying the enzyme of interest from the fermentation broth is called herein "purified enzyme solution”.

[0089] The term "metalloprotease” refers to an enzyme with proteolytic activity comprising one or more metal ions bound to the enzyme, wherein the association of the metal ions to the enzyme is necessary for full enzymatic activity. In particular, the metalloprotease described herein belongs to the class EC 3.4.24, which characterizes zinc-dependent metalloproteases belonging to the metallopeptidase family M12.

[0090] A "chelating agent” is a chemical compound that can form stable complexes, called chelates, with metal ions by coordinating with them through multiple sites.

[0091] The term "sensitive towards the metalloprotease” with respect to a protein shall refer to the property of said protein to be proteolytically degraded by the metalloprotease in the presence of the metalloprotease under conditions under which the metalloprotease shows proteolytic activity, i.e., the protein is prone to proteolytic degradation by the metalloprotease. "Degradation” in this context shall refer to partial degradation, i.e., truncation with remaining one or more larger protein fragments, as well as complete degradation, i.e., digestion into only small peptides and / or single amino acids.

[0092] A "carbohydrate binding domain” (CBD) or "carbohydrate-binding module” (CBM) is understood herein as a protein domain found in carbohydrate-active enzymes (for example glycoside hydrolases, in particular mannanases), which has carbohydrate-binding activity.

[0093] Bacillus cell with inactivated metalloprotease

[0094] In one embodiment, the present invention is directed to a Bacillus cell having reduced expression of a gene encoding a metalloprotease selected from the group consisting of:

[0095] I. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and

[0096] II. a metalloprotease encoded by a polynucleotide having at least at least 55%, at least 56%, at least 57%, at least

[0097] 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least

[0098] 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least

[0099] 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least

[0100] 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

[0101] 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0102] 98%, at least 99%, or 100% identity to SEQ ID NO: 07.

[0103] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence with at least 55% identity to SEQ ID NO: 08. Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence with 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 to SEQ ID NO: 08.

[0104] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence with 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 to SEQ ID NO: 08. More preferably, the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence 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%, at least 99%, or 100% identity to SEQ ID NO: 08. Most preferably, the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence with 100% identity to SEQ ID NO: 08. It is well within the skilled persons knowledge that the Bacillus species use alternative start codons, with atg coding for methionine, gtg coding for valine and ttg coding or leucine. These sequence variants are within the scope of this invention.

[0105] Preferably, the Bacillus cell has reduced expression of a 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 to SEQ ID NO: 07 encoding a metalloprotease. More preferably, the Bacillus cell has reduced expression of a 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 to SEQ ID NO: 07 encoding a metalloprotease. Most preferably, the Bacillus cell has reduced expression of a gene with 100% identity to SEQ ID NO: 07 encoding a metalloprotease. It is well within the skilled persons knowledge that the Bacillus species use alternative start codons, with atg coding for methionine, gtg coding for valine and ttg coding or leucine. These sequence variants are within the scope of this invention.

[0106] The Bacillus cell according to the present invention is a recombinant Bacillus cell.

[0107] Preferably, the reduction of expression of the gene encoding the metalloprotease is obtained by deletion of the gene (in part or full-length, preferably full-length deletion). Preferably, the Bacillus cell is a metalloprotease knockout cell, wherein the expression of the gene encoding the metal loprotease is reduced such that no active metalloprotease is produced.

[0108] In one embodiment, the invention is directed to a Bacillus cell comprising a knockout of a gene encoding a metalloprotease selected from the group consisting of:

[0109] I. a metalloprotease 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 to

[0110] SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and

[0111] II. a metalloprotease encoded by a polynucleotide having at least 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

[0112] 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least

[0113] 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least

[0114] 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

[0115] 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0116] 98%, at least 99%, or 100% identity to SEQ ID NO: 07.

[0117] Preferably, the invention is directed to a Bacillus cell comprising a knockout of a gene encoding a metalloprotease 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 to SEQ ID NO: 08.

[0118] The Bacillus cell can be selected from the group consisting of Bacillus altitudinis, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus australimaris, Bacillus glycinifermentans, Bacillus haynesii, Bacillus licheniformis, Bacillus naka- murai, Bacillus pakistanensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus safensis, Bacillus sonorensis, Bacillus stratosphericus, Bacillus velezensis, Virgi baci Hus, preferably selected from the group consisting of Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus velezensis, more preferably, Bacillus licheniformis and Bacillus pumilus. Most preferably, the Bacillus cell is a Bacillus licheniformis cell. Preferably, the Bacillus licheniformis cell is of the strain Bacillus licheniformis ATCC 14580, ATCC 31972, ATCC 53757, ATCC 53926, ATCC 55768, DSM 13, DSM 394, DSM 641, DSM 1913, DSM 11259, or DSM 26543.

[0119] Preferably, the Bacillus cell is a Bacillus licheniformis cell having reduced expression of a gene encoding a metalloprotease having an amino acid sequence with 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 to SEQ ID NO: 08. Most preferably, the Bacillus cell is a Bacillus licheniformis cell having reduced expression of a gene encoding a metalloprotease having an amino acid sequence with 100% identity to SEQ ID NO: 08.

[0120] Preferably, the Bacillus cell is a Bacillus licheniformis cell having reduced expression of a gene with 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 to SEQ ID NO: 07 encoding a metalloprotease. Most preferably, the Bacillus cell is a Bacillus licheniformis cell having reduced expression of a gene with 100% identity to SEQ ID NO: 07 encoding a metalloprotease.

[0121] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloprotease, wherein the metalloprotease is a homologue to a metalloprotease with an amino acid sequence shown in SEQ ID NO: 08. Preferably the homologue of the metal loprotease with an amino acid sequence shown in SEQ ID NO: 08 comprises an amino acid sequence with at least 55% identity to SEQ ID NO: 08. The sequence identity of homologues of the metal loprotease depicted in SEQ ID NO: 08 are shown in the table below.

[0122] Thus, in one embodiment the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence with 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 to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus licheniformis, to SEQ ID NO: 18, wherein the Bacillus cell is Bacillus altitudinis, to SEQ ID NO: 19, wherein the Bacillus cell is Bacillus amyloliquefaciens, to SEQ ID NO: 20, wherein the Bacillus cell is Bacillus atrophaeus, to SEQ ID NO: 21, wherein the Bacillus cell is Bacillus australimaris, to SEQ ID NO: 22, wherein the Bacillus cell is Bacillus glycinifermentans, to SEQ ID NO: 23, wherein the Bacillus cell is Bacillus haynesii, to SEQ ID NO: 24, wherein the Bacillus cell is Bacillus nakamurai, to SEQ ID NO: 25, wherein the Bacillus cell is Bacillus paki- stanensis, to SEQ ID NO: 26, wherein the Bacillus cell is Bacillus paralicheniformis, to SEQ ID NO: 27, wherein the Bacillus cell is Bacillus pumilus, to SEQ ID NO: 28, wherein the Bacillus cell is Bacillus safensis, to SEQ ID NO: 29, wherein the Bacillus cell is Bacillus siamensis, to SEQ ID NO: 30, wherein the Bacillus cell is Bacillus sonorensis, to SEQ ID NO: 31, wherein the Bacillus cell is Bacillus stratosphericus, to SEQ ID NO: 32, wherein the Bacillus cell is Bacillus velezensis, or to SEQ ID NO: 33, wherein the Bacillus cell is VI rgibacil lus sp, 7505.

[0123] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence with 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 to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus licheniformis, to SEQ ID NO: 19, wherein the Bacillus cell is Bacillus amyloliquefaciens, to SEQ ID NO: 27, wherein the Bacillus cell is Bacillus pumilus, or to SEQ ID NO: 32, wherein the Bacillus cell is Bacillus velezensis. In one embodiment the Bacillus cell has reduced expression of a gene encoding a metal loprotease having an amino acid sequence with at least 70% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus altitudinis, with at least 69% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus amyloliquefaciens, with at least 72% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus atrophaeus, with at least 69% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus australimaris, with at least 86% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus glycinifermentans, with at least 91% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus haynesii, with at least 68% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus nakamurai, with at least 55% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus pakistanensis, with at least 86% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus paralicheniformis, with at least 68% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus pumilus, with at least 69% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus safensis, with at least 69% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus siamensis, with at least 80% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus sonorensis, with at least 70% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus stratosphericus, with at least 67% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus velezensis, or with at least 56% identity to SEQ ID NO: 08, wherein the Bacillus cell is VI rgibacil lus sp, 7505.

[0124] Preferably, the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence with at least 78% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus amyloliquefaciens, with at least 76% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus pumilus, or with at least 76% identity to SEQ ID NO: 08, wherein the Bacillus cell is Bacillus velezensis.

[0125] In one embodiment, the Bacillus cell comprises additional genetic modifications. Preferably, the Bacillus cell additionally has reduced expression levels of at least one further gene, preferably reduced expression of at least one further endogenous gene. Preferably, the Bacillus cell additionally has reduced gene expression (such as deletion) of at least one further gene, preferably endogenous gene, selected from the group consisting of a gene coding for a protease different to the metalloprotease, a gene coding for a sporulation factor, a gene coding for a secreted enzyme different to a protease, a gene coding for a protein involved in the formation of the extracellular matrix, or a gene coding for an autolysin, preferably a gene coding for a protease different to the metalloprotease. Preferably, the Bacillus cell additionally has reduced gene expression of at least one further gene selected from the group consisting of a gene coding for a protease different to the metalloprotease, a gene coding for a sporulation factor, a gene coding for a secreted enzyme different to a protease, and a gene coding for a protein involved in the formation of the extracellular matrix. Preferably, the Bacillus cell additionally has reduced gene expression of a gene coding for a protease different to the metalloprotease, which is inactivated in the Bacillus cell as described herein. Most preferred, the Bacillus cell additionally has reduced gene expression of a gene coding for a protease different to the metal loprotease, a gene coding for a sporulation factor, a gene coding for a secreted enzyme different to a protease, and a gene coding for a protein involved in the formation of the extracellular matrix.

[0126] Expression levels of genes, which code for proteases different to the metalloprotease that can additionally be reduced (such as deleted) are preferably selected from the group of protease-encoding genes consisting of aprE, mpr, epr, bpr, vpr, wprA, aprX, and IspA, more preferably selected from aprE, mpr, bpr, vpr, and IspA. Thus, 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 comprises a functional wprA and / or epr gene. Thus, most preferably the Bacillus cell has reduced gene expression of all of the protease-encoding genes aprE, mpr, bpr, vpr, and ispA. Preferably, the Bacillus cell comprises a functional wprA and / or epr gene.

[0127] In one embodiment, the Bacillus cell has reduced gene expression (such as deleted) of a gene coding for a protein involved in the formation of the extracellular matrix. Preferably, it is envisaged that the modified cell as set forth herein does not produce poly-gamma-glutamate (pga) or produces a reduced amount of pga. Accordingly, preferably the Bacillus cell has reduced gene expression of at least one gene involved in pga production. Preferably, the at least one gene involved in poly-gamma-glutamate is at least one gene selected from ywsC (pgsB), ywtA (pgsC), ywtB (pgsA) and ywtC (pgsE). Thus, 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 aforementioned genes, i.e. ywsC (pgsB), ywtA (pgsC), ywtB (pgsA), and ywtC (pgsE), have been inactivated (such as deleted). When reference is made herein to the inactivation of a pga gene any or several or all of the above listed genes can be inactivated (such as deleted).

[0128] Preferably, the Bacillus cell additionally has reduced gene expression (such as deletion) of at least one further gene coding for a sporulation factor. Thus, in one embodiment, it is envisaged that the modified cell is not capable to spor- ulate. This may be achieved by inactivating (such as deleting) at least one gene involved in sporulation. Genes involved in sporulation are well known in the art (e.g., EP1391502) and comprise but are not limited to sigE, sig F, spol IGA, spoil E, sigG, spoIVCB, and yqfD. Thus, preferably the Bacillus cell further has reduced gene expression of at least one gene selected from the group consisting of sigE, sig F, spol I GA, spol I E, sigG, spoIVCB, and yqfD. In a preferred embodiment, the sigF gene is deleted.

[0129] Preferably, the Bacillus cell additionally has reduced gene expression (such as deletion) of at least one further gene coding for a secreted enzyme different to a protease. In a preferred embodiment, at least one gene coding for a secreted enzyme different to a protease selected from the group consisting of amylase, cellulase, xylanase, phospha- tase, and pullulanase is inactivated. In particular, it is envisaged that the modified cell is reduced in glycosidase activities. This can be achieved by inactivating (such as deleting) at least one gene encoding glycosidases comprising but not limited to alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2) and glucan 1,4-alpha-maltohydrolase (EC 3.2.1.133)), a cellulase (EC 3.2.1.4), an endo-1,3-beta-xylanase (EC 3.2.1.32), an endo-1,4-beta-xylanase (EC 3.2.1.8), a lactase (EC 3.2.1.108), a galactosidase (EC 3.2.1.23 and EC 3.2.1.24), and a mannanase (EC 3.2.1.24 and EC 3.2.1.25). In a preferred embodiment, at least one of gene encoding an endogenous alpha-amylase polypeptide is inactivated. Thus, preferably, the Bacillus cell further has reduced gene expression (such as deletion) of the amyB gene (also referred to as amyL gene).

[0130] Preferably, the Bacillus cell additionally has reduced gene expression (such as deletion) of at least one gene coding for an autolysin. Thus, in one embodiment, it is envisaged that the modified cell is not capable to lyse or has a reduced rate of lysis. This may be achieved by inactivating (such as deleting) at least one gene involved in cell lysis. Genes involved in cell lysis comprise but are not limited to the genes selected from the group consisting of lytC, lytD, lytE, lytF cwID, cwlE, and cwIJ. Thus, preferably the Bacillus cell further has reduced gene expression of at least one gene selected from the group consisting of lytC, lytD, lytE, lytF cwID, cwlE, and cwIJ.

[0131] Preferably, the Bacillus cell described herein additionally has reduced gene expression (such as deletion) of at least one further gene selected from the group consisting of sig F, pga, and amyB, preferably all of sigF, pga, and amyB.

[0132] Most preferably, the Bacillus cell described herein additionally has reduced gene expression (such as deletion) of at least one further gene selected from the group consisting of sigF, pga, amyB, aprE, mpr, epr, bpr, vpr, wprA, aprX, and IspA, even more preferably selected from sigF, pga, amyB, aprE, mpr, bpr, vpr, and IspA, most preferably having reduced expression of all of the sigF, pga, amyB, aprE, mpr, bpr, vpr, and IspA genes. Preferably, the Bacillus cell comprises a functional wprA and / or epr gene.

[0133] Even more preferably, the Bacillus cell is a Bacillus licheniformis cell having reduced gene expression of a gene encoding a metalloprotease having an amino acid sequence with 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 to SEQ ID NO: 08 that additionally has reduced gene expression (such as deletion) of at least one further gene selected from the group consisting of sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, most preferably having reduced gene expression of all of the sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA genes, preferably comprising a functional wprA and / or epr gene. Most preferably, the Bacillus cell is a Bacillus licheniformis cell having reduced gene expression of a gene encoding a metalloprotease having an amino acid sequence with 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 to SEQ ID NO: 08 additionally having reduced gene expression (such as deletion) of at least one further gene selected from the group consisting of sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, most preferably having reduced gene expression of all of the sig F, pga, amyB, aprE, mpr, bpr, vpr, and IspA genes and comprising a functional wprA and / or epr gene, preferably a functional wprA and epr gene.

[0134] The present invention also refers to a method of producing a Bacillus cell as described herein. In one embodiment, the present invention is directed to a method of producing a Bacillus cell, wherein the method comprises the steps of:

[0135] I. providing a Bacillus cell comprising a gene encoding a metalloprotease selected from the group consisting of a) a metal loprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b) a metal loprotease encoded by a polynucleotide having at least 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 to SEQ ID NO: 07, and

[0136] II. inactivating the metal loprotease gene.

[0137] Methods of inactivating gene are well-known to the person skilled in the art. Inactivation of a gene can be achieved by partial or full deletion of the coding region or of the complete gene including coding region and regulatory sequences, by modification or deletion of the promoter region, by introduction of point mutations that lead to an inactivated metalloprotease (e.g., by the CRISPR / Cas method), or by gene silencing.

[0138] Gene inactivation can also be achieved by homologous recombination, i.e. an incoming DNA molecule comprises sequences that are homologous to the 5' and 3' flanking sequences of the target sequence on the chromosome of the host cell (e.g. Bacillus) to be inactivated. Subsequently the sequence between said flanking sequences is replaced by the homologous sequences of the incoming DNA molecule in the process of homologous recombination, i.e. the sequence is deleted from the chromosome. Likewise, "gene integration”, i.e. a DNA sequence such as a gene expression cassette with or without a selectable marker, can be integrated into the chromosome of the bacterial host cell by homologous recombination. Hence, the DNA sequence to be integrated is flanked by DNA sequences that are homologous to the 5' and 3' flanking sequences on the chromosome. It is understood in terms of the invention that gene integration can also combine gene integration and gene deletion in one step, i.e. a DNA sequence on the chromosome is replaced by the incoming DNA sequence for gene integration. Homologous recombination can be achieved by two different methods known in the art: By two consecutive rounds of homologous recombination (Campbell recombination) with circular plasmid DNA, e.g. based on the well-known temperature sensitive plasmid pE194 (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). Alternatively, a non-replicative 'suicide' plasmid can be used forcing the integration by selection on the selectable marker. Only cells that have integrated the plasmid into the genome by homologous recombination are able to grow under the selective conditions. Plasmid removal / excision from the chromosome is achieved with a second homologous recombination which is forced by the activation of a counterselection marker present on the plasmid.

[0139] The second method of homologous recombination refers to two homologous recombination events simultaneously taking place, also known as "double crossing over” or "double homologous recombination”. The incoming DNA sequence is linear and can be obtained by PCR, linearization of plasmid DNA or preparation of chromosomal DNA which inevitable results in fragmented linear DNA. W00308125 uses linear DNA constructs (either linearized plasmids or PCR fragments) comprising a selectable marker flanked by the 5' and 3' homologous regions which are used for genomic integration via double crossing over homologous recombination. It is well understood that next to the selectable marker additional DNA, such as gene expression cassettes, when flanked by said homologous region are integrated into the chromosome of the bacterial host cell.

[0140] Homologous recombination requires DNA sequences homologous to the 5' and 3' flanking sequences of the target sequence on the chromosome of the host cell of sufficient size, hence should contain a sufficient number of nucleic acid 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 have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination (Dubnau, 1993, Genetic exchange and homologous recombination. In Bacillus subtilis and Other Gram-positive Bacteria, p. 555-584. Edited by A. I. Sonenshein, J.A. Hoch & R. Losick, Washington DC, American Society for Microbiology; Michel and Ehrlich, 1984, The EMBO Journal, vol. 3, pp. 2879-2884).

[0141] Gene inactivation by deletion I insertion I substitution can also be achieved by CRISPR / Cas9 genome editing technologies where the CRISPR cutting properties could be used to disrupt genes in almost any organism's genome with unprecedented ease (Mali P, et al (2013) Science. 339(6121):819-823; Cong L, et al (2013) Science 339(6121)). Recently it became clear that providing a template for repair, e.g. homologous regions, allowed for editing the genome with nearly any desired sequence at nearly any site, transforming CRISPR into a powerful gene editing tool (WC2014 / 150624, WC2014 / 204728).

[0142] CRISPR-based genome editing systems for application in gram positive organisms have been well described such as the Bacillus species based single-plasmid system approach, i.e. comprising the Cas9 endonuclease, the gRNA (e.g. sgRNA or crRNA / tracrRNA), repair homology sequences (donor DNA) on one single E. coli -Bacill us 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 system or with Cas9 endonuclease integrated into the Bacillus genome as described e.g. in WC2020 / 206202 and WC2020 / 206197.

[0143] Alternatively to "directed” methods of inactivation it is understood in the scope of the invention that whole-cell mutagenesis by applying mutagenizing conditions such as exposure of the cells to UV radiation, or chemical mutagenizing chemicals such as NTG (N-methyl-N'-nitro-N-nitrosoguanidine) or EMS (ethyl-methane-sulfonate), in combination with screening and / or selection of the desired property, e.g. reduced lipase / esterase activity is a well-known approach to achieve functional inactivation.

[0144] Further, a gene may have been inactivated by gene silencing. Gene silencing can be achieved by introducing into said bacterial host cell antisense expression constructs that result in antisense RNAs complementary to the mRNA of the gene, thereby inhibiting expression of said genes. Alternatively, the expression of said gene can be inhibited by blocking transcriptional initiation or transcriptional elongation through the mechanism of CRISPR-inhibition (WO18 / 009520).

[0145] Protein of interest

[0146] Preferably, the Bacillus cell having reduced expression of a gene encoding a metalloprotease as described herein expresses a protein of interest, preferably, a protein of interest sensitive towards the metal loprotease. Preferably, the protein of interest is secreted by the Bacillus cell, preferably into the extracellular space. Preferably, the protein of interest is heterologous to the Bacillus cell.

[0147] Preferably, the protein of interest is an enzyme. Thus, particularly preferred the protein of interest is sensitive towards the metalloprotease, secreted by the Bacillus cell, preferably into the extracellular space and is an enzyme heterologous to the Bacillus cell.

[0148] Preferably, the protein of interest, preferably the enzyme, comprises or consists of two or more domains which are connected via flexible linkers (preferably at least two domains connected via one flexible linker), preferably the flexible linkers are sensitive towards the metalloprotease.

[0149] Preferably, the enzyme is selected from the group consisting of mannanase, amylase, pullulanase, protease, lipase, cutinase, acyl transferase, cellulase, endoglucanase, glucosidase, glucoamylase, cellubiohydrolase, lactase, xy- lanase, xyloglucantransferase, xylosidase, xanthan lyase, DNase, dispersin, phytase, phosphatase, xylose isomerase, glucoase isomerase, acetolactate decarboxylase, pectinase, pectate lyase, pectin methylesterase, polygalac- turonidase, lyase, pectate lyase, arabinase, arabinofuranosidase, galactanase, laccase, peroxidase, oxidoreductase, and asparaginase.

[0150] 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. The mannanases according to the present invention have mannan degrading activity and are of the enzyme class EC 3.2.1.78. In one embodiment, mannan degrading activity relates to degradation of at least one galactomannan. Preferably, at least one galactomannan is characterized by the ratio mannose:galactose of about 1 :1, about 2:1, about 3: 1 , about 4: 1 , and / or 5: 1 .

[0151] Mannan degrading activity or mannanase activity may be tested according to standard test procedures known in the art. For example: a mannanase to be tested may be applied to 4 mm diameter holes punched out in agar plates comprising 0.2% AZCL galactomannan (carob), i.e. substrate for the assay of endo-1,4-beta-D-mannanase available as l-AZGMA from the company Megazyme (Megazyme's Internet address: http: / / www. megazyme. com / Purchase / in- dex. html). Mannan degrading activity may also be tested in a liquid assay using carob galactomannan dyed with Remazol Brilliant Blue as described in McCleary, B. V. (1978). Carbohydrate Research, 67(1), 213-221. Another method for testing mannan degrading activity uses detection of reducing sugars when incubated with substrate such as guar gum or locust bean gum - for reference see Miller, G. L. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars. Analytical Chemistry 1959; 31 : 426-428.

[0152] Preferably, the mannanase comprises or consists of two or more domains which are connected via flexible linkers (preferably at least two domains connected via one flexible linker), preferably the flexible linkers are sensitive towards the metalloprotease. 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, preferably the flexible linker being sensitive towards proteolytic cleavage by the metalloprotease.

[0153] The mannanase may be selected from mannanases originating from Bacillus organisms, such as described in JP- 0304706, JP-63056289, JP-63036774, JP-08051975, WO 97 / 11164, WO 91 / 18974, WO 97 / 11164, WO 2014 / 100018. Suitable mannanases are also described in WO 99 / 064619. The mannanase may be selected from mannanases originating from Trichoderma organisms, such as disclosed in WO 93 / 24622. The mannanase may be selected from a commercially available mannanase such as Mannaway® (Novozymes A / S) or Preferenz® (M100) (DuPont).

[0154] Preferably, the mannanase is a variant of a parent mannanase (i.e., mannanase variant). The parent mannanase can be any mannanase. Preferably the parent 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 59, 66, 89, 234, 259, 282, 318, 319, and 322, according to the numbering of SEQ ID NO: 12, and an amino acid sequence which 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

[0155] 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% identical to SEQ ID NO: 12.

[0156] Preferably, the mannanase is a variant of a parent mannanase, preferably of a parent mannanase as shown in SEQ ID NO: 12, 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 the numbering of SEQ ID NO: 12, and an amino acid sequence which 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%, or at least 99%, but less than 100% identical to SEQ ID NO: 12, preferably 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% identical to SEQ ID NO: 12.

[0157] 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 amino acid substitutions selected from the group consisting of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12, preferably 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% identical to SEQ ID NO: 12. More preferably, the man- nanase 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 numbering of SEQ ID NO: 12. Most preferably, the mannanase variant comprises or consists of SEQ ID NO: 12 with the amino acid substitutions X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12, and optionally with 1-10, preferably 1-5 conservative substitutions.

[0158] In one embodiment, the protein of interest is a lipase. "Lipases”, "lipolytic enzyme”, "lipid esterase”, all refer to an enzyme of EC class 3.1.1 ("carboxylic ester hydrolase”). Lipase means active protein having lipase activity (or lipolytic activity; triacyl-glycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; enzymes having cutinase activity may be called cutinase herein), 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 the follow-ing: lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanugino- sus) as described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500. In one embodiment, lipase is selected from fungal triacylglycerol lipase (EC class 3.1.1.3). Thermomyces lanuginosa lipase variants may be selected from variants having lipolytic activity which are 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 when compared to the full- length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438, preferably comprising one or more, preferably all of the following amino acid substitutions when compared to amino acids 1-269 of SEQ ID NO: 2 of US5869438: T231R, N233R, Q4V, V60S, A150G, L227G, P256K. Thermomyces lanuginosa lipase may also be selected from variants comprising at least one, preferably more than one, more preferably all of the following substitutions N11 K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q within the polypeptide se-quence of amino acids 1-269 of SEQ ID NO: 1 of WQ2015 / 010009 and are at least 95%, at least 96%, or at least 97% similar when compared to the full length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WQ2015 / 010009. In one embodiment, at least one lipase is selected from commercially available lipases which include but are not limited to products sold under the trade names Lipolase™, Lipex™, Lipolex™ and Lipoclean™ (Novozymes A / S), Lumafast (origi-nally from Genencor), Preferenz L (DuPont), and Lipomax (Gist-Brocades / now DSM).

[0159] In one embodiment, the protein of interest is a cellulase. "Cellulases" are enzymes capable of hydrolysing of cellulose. Cellulases may be selected from cellobiohydrolase (1,4-P-D-glucan cellobiohydrolase, EC 3.2.1.91), endo-ss- 1,4-glucanase (EC 3.2.1.4) and ss-glucosidase (EC 3.2.1.21). Endoglucanases of EC class 3.2.1.4 may be named endoglucanase, endo-1,4-ss-D-glucan 4-glucano hydrolase, endo-1,4-beta-glucanase, carboxymethyl cellulase, and beta-1, 4-glucanase. The cellulase may be a Humicola insolens DSM 1800 cellulase complex having endoglucanase, cellobiohydrolase and beta-glucosidase activity. The cellulase may be a Humicola insolens DSM 1800 endoglucanase (EC 3.2.1.4), preferably having the polypeptide sequence according to position 21-435 of SEQ ID NO:2 as disclosed in WO 2018 / 224544 or variants with least 80% identity thereto. In one embodiment, at least one cellulases is selected from commercially available cellulases which include but are not limited to products sold under the trade names Renozyme®, Celluzyme®, Celluclean®, Endolase® and Carezyme® (Novozymes A / S), Clazinase™, and Puradax HA™ (Genencor Int. Inc.), and KAC-500(B)™ (Kao Corporation). In one embodiment, the protein of interest is a protease different to the metal loprotease described herein. Preferably the protease is a subtilisin protease 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 or variants thereof. Suitable examples of protease variants comprise proteases derived from SEQ ID NO:22 as described in EP 1921147 with amino acid substitutions in 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 the BPN' numbering), which have proteolytic activity. Preferably, a subtilisin variant is at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and is characterized by an amino acid substitution at position 101, such as R101 E or R101D, 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' numbering) and has proteolytic activity. A subtilisin variant may have an amino acid sequence being at least 80% identical to SEQ ID NO:22 as described in EP 1921147 and being further characterized by comprising R101 E, 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, G61 D,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.

[0160] In one embodiment, the protein of interest is an amylase. Amylases maybe bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, amylases are selected from the group of alpha-amylases (EC 3.2.1.1). Amylases maybe from Bacillus licheniformis having SEQ ID NO:2 as described in WO 95 / 10603 and variants at least 95% thereto. Suitable variants are described in WO 95 / 10603 comprising one or more substitutions in 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 amylolytic activity. Variants are described in WO 94 / 02597, WO 94 / 018314, WO 97 / 043424 and SEQ ID NO:4 of WO 99 / 019467. Amylases further maybe from B. stearothermophi- lus having SEQ ID NO:6 as disclosed in WO 02 / 10355 or an amylase with optionally having a C-terminal truncation over the wildtype sequence. Suitable variants of SEQ ID NO:6 include those comprising a deletion in positions 179 and / or 181 and / or 182 and / or a substitution in position 193. Amylases further maybe from Bacillus sp.707 having SEQ ID NO:6 as disclosed in WO 99 / 19467 and variants thereof, preferably those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, 1206, E212, E216 and K269. Amylases may have SEQ ID NO:12 as described in WO 2006 / 002643 or amylase variants thereof comprising the substitutions Y295F and M202LITV within said SEQ ID NO: 12. Amylases may have SEQ ID NO:2 as described in WO 2013 / 001087 or amylase variants comprising a deletion of positions 181+182, or 182+183, or 183+184, within said SEQ ID NO:2, optionally comprising one or two or more modifications in any of positions 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. Hybrid amylases may be according to WO 2021 / 032881 comprising an A and B domain originating from the alpha amylase originating from Bacillus sp. A 7-7 (DSM 12368) and a C domain originating from the alpha-amylase from Bacillus cereus; preferably, the A and B domain are at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and a C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44 - both sequences as disclosed in WO 2021 / 032881; more preferably, the hybrid amylase is at least 80% identical to SEQ ID NO:54 as disclosed in WO 2021 / 032881 . In one embodiment, at least one amylase is selected from commercially available amylases which include but are not limited to products sold under the trade names Du- ramyl™, Termamyl™, Fun-gamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™, Amplify™, Amplify Prime™ (from Novozymes A / S), and Rapidase™, Purastar™, PoweraseTM, Effectenz™ (M100 from DuPont), Preferenz™ (S1000, S110 and F1000; from DuPont), PrimaGreen™ (ALL; DuPont), Optisize™ (DuPont).

[0161] Method for producing a protein of interest

[0162] In another embodiment, the present invention is directed to a method of producing a protein of interest, preferably an enzyme as described herein, comprising the steps of

[0163] I. providing a Bacillus cell having reduced expression of a gene encoding a metalloprotease selected from the group consisting of a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, and

[0164] II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metalloprotease,

[0165] III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest, thereby forming a fermentation broth comprising the protein of interest, and

[0166] IV. optionally isolating the protein of interest from the fermentation broth of step III.

[0167] Preferably, the protein of interest is a mannanase as described herein.

[0168] In preferred embodiment, the present invention is directed to a method of producing a protein of interest, preferably an enzyme as described herein, comprising the steps of

[0169] II. providing a Bacillus cell having reduced expression of a gene encoding a metalloprotease 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 to SEQ ID NO: 08,

[0170] II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metalloprotease,

[0171] III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest, thereby forming a fermentation broth comprising the protein of interest, and

[0172] IV. optionally isolating the protein of interest from the fermentation broth of step III.

[0173] Preferably, the protein of interest is a mannanase as described herein. Preferably, the Bacillus cell is a Bacillus II- cheniformis cell. Typically, the expression cassette comprises three elements: a promoter sequence (including 5'UTR), an open reading frame, and a 3' untranslated region. Additional regulatory elements may include transcriptional as well as translational enhancers. The expression cassette may be part of a vector or may be integrated into the genome of a host cell and replicated together with the genome of its host cell. The expression vector can be a low copy number vector or high copy number vector. A vector as used herein may provide segments for transcription and translation of a foreign polynucleotide upon transformation into a host cell. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication that is required for its maintenance and / or replication in a specific cell type, one or more selectable markers, a polyadenylation signal, a suitable site for the insertion of foreign coding sequences such as a multiple cloning site etc. Non-limiting examples of suitable origins of replication include the f1 -ori and colE1 . A vector may replicate without integrating into the genome of a host cell, e.g., as a plasmid in a bacterial host cell, or it may integrate part or all of its DNA into the genome of the host cell and thus lead to replication and expression of its DNA. The polynucleotide encoding the protein of interest may be introduced into a vector by means of standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide comprising a coding sequence may be suitable to be introduced (transformed, transduced, transfected, etc.) into a host cell. A cloning vector may be chosen suitable for expression of the polynucleotide sequence in the host cell.

[0174] The polynucleotide encoding the protein of interest as described herein may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid or cosmid. Usually, stable transformation is due to integration of the nucleic acid comprising a foreign coding sequence into the chromosome. Usually, transient transformation is due to nucleic acid comprising a foreign nucleic acid sequence is not integrated into the chromosome. Methods of introducing a nucleic acid into a host cell are well-known to the person skilled in the art. The introduction of nucleic acid into a host cell may, for instance, but not limited thereto, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., 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, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271 -5278).

[0175] After introduction into the Bacillus cell the expression cassette encoding a protein of interest the Bacillus cell is cultivated under conditions which allow for the expression of the protein of interest, thereby forming a fermentation broth comprising the protein of interest. The term "cultivating” as used herein refers to keeping alive and / or propagating the modified cell comprised in a culture at least for a predetermined time. The term encompasses phases of exponential cell growth at the beginning of growth after inoculation as well as phases of stationary growth. The cultivation conditions shall allow for the expression, i.e. the production, of the protein of interest. Such conditions can be chosen by the skilled person without further ado. Exemplary conditions for the cultivation of the modified cell are described in W02020169564 A1 or in Example 3 in the Examples section provided herein. In an embodiment of the method of the present invention, the step of cultivating the Bacillus cell is carried out as fed batch cultivation. The method of the present invention, if applied, allows for increasing the production, of the protein of interest. Preferably, production is increased as compared to the expression in an unmodified control cell, preferably a Bacillus cell, preferably a Bacillus licheniformis, control cell which does not have an inactivation of the metal loprotease as described herein. In a preferred embodiment, the production of the protein of interest is increased by at least 20%, such as by at least 50%, in particular, by at least 100% or at least 200% as compared to the expression in the control cell. For example, the production of the protein of interest may be increased by 20% to 300%, such as by 100% to 300% as compared to the control cell. The expression can be measured by determining the amount of the protein of interest in the cell and / or in the cultivation medium. Further, the production may be assessed by determining the enzymatic activity. For example, an enzyme assay may be used to determine the activity of the protein of interest.

[0176] The protein of interest described herein may be secreted (into the liquid fraction of the fermentation broth) or may not be secreted from the microbial cells (and therefore is comprised in 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 cell lysates. Preferably, the protein of interest is secreted from the cell into the fermentation broth, preferably by means of a secretion signal peptide added to the terminus of the amino acid sequence of the protein of interest.

[0177] The protein of interest may be isolated from the fermentation broth by methods known in the art. For example, the protein of interest may be isolated from the fermentation broth by 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 binds at least in part to the particulate matter of the fermentation broth additional treatment steps might be needed to release the protein of interest from the biomass and / or to solubilize crystals and precipitates of the protein of interest. W00043502A1, W02008110498 A1, and WO2017097869A1 describe methods for recovering a protein of interest, which precipitates and / or crystallizes during fermentation, from the fermentation broth. In case the desired protein is comprised in the cells of the fermentation broth release of the product of interest from the cells might be needed. Release from the cells can be achieved for instance, but not being limited thereto, by cell lysis with techniques well known to the skilled person, e.g., lysozyme treatment, ultrasonic treatment, French press or combinations thereof.

[0178] The isolated protein of interest may then be further purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). The purified protein of interest may then be concentrated by procedures known in the art including, but not limited to, ultrafiltration and evaporation.

[0179] Method of inactivating a metalloprotease

[0180] In an alternative embodiment to reducing the expression of a gene encoding the metalloprotease in a Bacillus cell it is also possible to inactive or inhibit the metal loprotease itself. Inactivating the metalloprotease can be achieved for instance by contacting the metalloprotease with an inhibitor of the metalloprotease. However, the present inventors discovered that it is also possible to inactivate the metalloprotease by adding a chelating agent to a composition comprising the metalloprotease in an amount and for a time effective to inactivate the metalloprotease. Without being bound by theory, the inventors believe that the chelating agent binds the metal ions the metal loprotease needs to assert its proteolytic activity. Thus, in a further embodiment, the present invention is directed to the use of a chelating agent to inactivate the metalloprotease described herein. Inactivating the metalloprotease during the production of a protein of interest or inactivating the metalloprotease in a composition comprising the metal loprotease and a protein of interest leads to a reduced degradation of the protein of interest by the metalloprotease. The present invention is thus also directed to the use of a chelating agent to inactivate the metalloprotease described herein, in order to increase the yield of a protein of interest in a method of producing the protein of interest by a Bacillus cell that expresses the metalloprotease.

[0181] Therefore, in a further embodiment, the present invention is directed to a method of inactivating a metal loprotease, wherein the method comprises the steps of:

[0182] I. providing a metalloprotease selected from the group consisting of: a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, and

[0183] II. contacting the metalloprotease with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time sufficient to inactivate the metalloprotease.

[0184] In another embodiment, the present invention is directed to a method for inactivating a metal loprotease selected from the group consisting of a. a metalloprotease having a 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

[0185] 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, in a composition, preferably a liquid composition, comprising a protein of interest, preferably a mannanase as described herein, and the metalloprotease, wherein the protein of interest is sensitive towards the metalloprotease, comprising the step of contacting the composition comprising the protein of interest and the metalloprotease with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time sufficient to inactivate the metalloprotease.

[0186] Thus, in a further embodiment the present invention is directed to a method of producing a protein of interest, preferably an enzyme as described herein, preferably a mannanase as described herein, comprising the steps of I. providing a Bacillus cell having reduced expression of a gene encoding a metal loprotease selected from the group consisting of a. a metal loprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metal loprotease 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 to SEQ ID NO: 07, and

[0187] II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metalloprotease,

[0188] III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest and which also allow expression of the metal loprotease, thereby forming a fermentation broth comprising the protein of interest and the metalloprotease,

[0189] IV. optionally isolating the protein of interest from the cultivation medium, thereby forming a solution comprising the protein of interest and also the metalloprotease, and

[0190] V. contacting 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 effective to inactivate the metalloprotease.

[0191] The concentration of chelating agent to inactivate the mannanase is preferably in an amount of 1 mmol / l to 150 mmol / l, preferably 50-120 mol / l.

[0192] Preferably, the protein of interest is a mannanase as described herein.

[0193] In another embodiment, the present invention is directed to the use of a chelating agent for improving the stability of a protein of interest, preferably a mannanase as described herein, in a composition comprising a metal loprotease and a protein of interest, wherein the metalloprotease is selected from the group consisting of a. a metalloprotease having a 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

[0194] 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, in a composition, preferably a liquid composition, comprising a protein of interest and the metalloprotease, wherein the protein of interest is sensitive towards the metalloprotease, comprising the step of contacting the composition comprising the protein of interest and the metalloprotease with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time sufficient to inactivate the metalloprotease.

[0195] Hence, the present invention is also directed to the use of a chelating agent, preferably EDTA, MGDA, or EDDS, for increasing the yield of a protein of interest and / or the stability of the protein of interest, preferably an enzyme, preferably a mannanase as described herein, in a method of producing the protein of interest by a Bacillus cell, wherein the Bacillus cell expresses a metalloprotease selected from the group consisting of a. a metalloprotease having a 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

[0196] 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, wherein the protein of interest is sensitive towards the metalloprotease. In another embodiment, the present invention is directed to the use of a chelating agent, preferably EDTA, MGDA, or EDDS, for increasing the yield of a protein of interest and / or the stability of the protein of interest, preferably an enzyme, preferably a mannanase as described herein, in a method of producing the protein of interest by a Bacillus cell, wherein the Bacillus cell expresses a metalloprotease selected from the group consisting of a. a metalloprotease having a 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,

[0197] 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, wherein the protein of interest is sensitive towards the metalloprotease, wherein the method of comprises the steps of

[0198] I. providing a composition, preferably a liquid composition, comprising a protein of interest and a metal loprotease selected from the group consisting of: a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, and

[0199] II. contacting the composition of step I. with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time sufficient to inactivate the metalloprotease, wherein preferably the composition is a fermentation broth or a solution obtained from purifying a protein of interest from a fermentation broth, wherein the fermentation broth is obtained by a method comprising the steps of aa. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metalloprotease, bb. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest and which also allow expression of the metalloprotease, thereby forming a fermentation broth comprising the protein of interest and the metalloprotease, cc. optionally isolating the protein of interest from the cultivation medium, thereby forming a solution comprising the protein of interest and also the metalloprotease.

[0200] Preferably, the solution comprising the protein of interest, preferably the mannanase as described herein, and the metalloprotease, to which the chelating agent is added, is a non-complex formulation, e.g., a liquid enzyme preparation, or a complex formulation, e.g., a liquid detergent formulation. In a preferred embodiment, the solution is a noncomplex formulation, preferably a liquid enzyme formulation, preferably comprising one or more additional compounds selected from the group consisting of solvent, salt, pH regulator, preservative, enzyme stabilizer, and thickening agent. Preferably, the liquid enzyme formulation is devoid of surfactants. The solvent may be water and / or an organic solvent. The liquid enzyme formulation may comprise an organic solvent in amounts of more than 30%, more than 40%, 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, all 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, propanediol, polypropylene glycol, and polyethylene glycol. Preferably, the liquid enzyme formulation comprises or consists of a protein of interest as described herein, the metalloprotease as described herein, a chelating agent and a solvent, an enzyme stabilizing system, and optionally a preservative, preferably the liquid enzyme formulation is devoid of surfactants. The chelating agent is contained in the liquid enzyme formulation preferably in an amount of 1 mmol / l to 150 mmol / l, preferably 50-120 mol / l.

[0201] In an alternative embodiment, the solution comprising the protein of interest, preferably the mannanase as described herein, and the metal loprotease, to which the chelating agent is added, is a complex formulation, preferably, a liquid detergent formulation. The detergent formulation comprises one or more detergent component, preferably selected from the group consisting of surfactant, defoamer, polymer, bleaching system (bleach), rheology modifier, hydrotrope, softening agent, desiccant, whitening agent, buffer, preservative, anti-corrosion additive, dyestuff, fragrance, and detergent enzyme different to the protein of interest. Preferably, at least one detergent component is selected from the group consisting of surfactant, polymer, preservative, and detergent enzyme different to the protein of interest. Preferably one or more of the detergent component, preferably the surfactant and / or the builder, is bio-de- gradable and / or bio-based. Preferably, the liquid detergent formulation comprises or consists of a protein of interest as described herein, the metalloprotease as described herein, a chelating agent and surfactant, polymer, preservative, and detergent enzyme different to the protein of interest.

[0202] Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0203] Preferred embodiments

[0204] 1 . A Bacillus cell having reduced expression of a gene encoding a metalloprotease selected from the group consisting of:

[0205] I. a metal loprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, preferably at least 55%, at least 60%, at least 65% identity to SEQ ID NO: 08, and

[0206] II. a metal loprotease 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 to SEQ ID NO: 07.

[0207] 2. The Bacillus cell of embodiment 1 , wherein the Bacillus cell is a recombinant Bacillus cell.

[0208] 3. The Bacillus cell of any of embodiments 1 or 2, wherein the Bacillus cell is a metalloprotease knockout cell.

[0209] 4. The Bacillus cell of any of embodiments 1 to 3, wherein the Bacillus cell has reduced expression of a gene encoding a metalloprotease having an amino acid sequence with 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 to SEQ ID NO: 08, preferably 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 to SEQ ID NO: 08, most preferably 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 to SEQ ID NO: 08.

[0210] 5. The Bacillus cell of any of embodiments 1 to 4, wherein the Bacillus cell expresses a protein of interest sensitive towards the metalloprotease, preferably comprising or consisting of two or more domains which are connected via flexible linkers, preferably the flexible linkers are sensitive towards the metal loprotease, preferably wherein the protein of interest sensitive towards the metalloprotease is an enzyme.

[0211] 6. The Bacillus cell of embodiment 5, wherein the protein of interest is heterologous to the cell, preferably wherein the protein of interest is a heterologous enzyme.

[0212] 7. The Bacillus cell of any of embodiments 5 or 6, wherein the enzyme is selected from the group consisting of mannanase, amylase, pullulanase, protease, lipase, cutinase, acyl transferase, cellulase, endoglucanase, glucosidase, glucoamylase, cellubiohydrolase, lactase, xylanase, xyloglucantransferase, xylosidase, xanthan lyase, DNase, dispersin, phytase, phosphatase, xylose isomerase, glucoase isomerase, acetolactate decarboxylase, pectinase, pectate lyase, pectin methylesterase, polygalacturonidase, lyase, pectate lyase, arabinase, arabinofuranosidase, galactanase, laccase, peroxidase, oxidoreductase, and asparaginase, preferably mannanase.

[0213] 8. The Bacillus cell of any of embodiments 5-7, wherein the protein of interest is a heterologous mannanase.

[0214] 9. The Bacillus cell of any of embodiments 5-8, wherein the enzyme is a mannanase, preferably comprising or consisting of two or more domains which are connected via flexible linkers, preferably the flexible linkers are sensitive towards the metalloprotease, preferably comprising or consisting 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, preferably the flexible linker being sensitive towards proteolytic cleavage by the metalloprotease, preferably wherein the mannanase is a variant of a parent mannanase (i.e., mannanase variant), 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 the numbering of SEQ ID NO: 12, and an amino acid sequence which is 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% identical to SEQ ID NO: 12.

[0215] 10. The Bacillus cell of any of embodiments 7-9, wherein the mannanase is a variant of a parent 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 the numbering of SEQ ID NO: 12, and an amino acid sequence which 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%, or at least 99%, but less than 100% identical to SEQ ID NO: 12, preferably 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% identical to SEQ ID

[0216] NO: 12.

[0217] 11 . The Bacillus cell of any of embodiments 7-10, 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 amino acid substitutions selected from the group consisting of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12.

[0218] 12. The Bacillus cell of any of embodiments 7-11, 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 numbering of SEQ ID NO: 12.

[0219] 13. The Bacillus cell of any of embodiments 7-12, wherein the mannanase variant comprises or consists of SEQ ID NO: 12 with the amino acid substitutions X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12, and optionally with 1-10, preferably 1-5 conservative substitutions.

[0220] 14. The Bacillus cell of any of embodiments 5-13, wherein the protein of interest is secreted by the cell.

[0221] 15. The Bacillus cell of any of the preceding embodiments additionally having reduced expression of at least one further gene, preferably selected from the group consisting of a gene coding for a protease different to the metalloprotease, a gene coding for a sporulation factor, a gene coding for a secreted enzyme different to a protease, a gene coding for a protein involved in the formation of the extracellular matrix, or a gene coding for an autolysin. preferably a gene coding for a protease different to the metalloprotease.

[0222] 16. The Bacillus cell of any of the preceding embodiments additionally having reduced expression of a gene coding for a protease different to the metal loprotease selected from the group of protease-encoding genes consisting of aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, more preferably selected from aprE, mpr, bpr, vpr, and ispA, most preferably all of aprE, mpr, bpr, vpr, and ispA, preferably, the Bacillus cell comprises a functional wprA and / or epr gene.

[0223] 17. The Bacillus cell of any of the preceding embodiments additionally having reduced expression of at least one further gene selected from the group consisting of sig F, pga, and amyB, preferably all of sig F, pga, and amyB.

[0224] 18. The Bacillus cell according to any of the preceding embodiments additionally having reduced expression of at least one further 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 of sigF, pga, amyB, aprE, mpr, bpr, vpr, and ispA, preferably, the Bacillus cell comprises a functional wprA and / or epr gene.

[0225] 19. The Bacillus cell of any of the preceding embodiments, wherein the Bacillus cell is selected from the group consisting of Bacillus altitudinis, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus australimaris, Bacillus glycinifermentans, Bacillus haynesii, Bacillus licheniformis, Bacillus nakamurai, Bacillus pakistanensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus safensis, Bacillus sonorensis, Bacillus stratosphericus, Bacillus ve- lezensis, Virgibacillus, preferably selected from the group consisting of Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus velezensis, more preferably, Bacillus licheniformis and Bacillus pu- milus, most preferably Bacillus licheniformis.

[0226] 20. The Bacillus cell of any of the preceding embodiments, wherein the Bacillus cell is a Bacillus licheniformis cell.

[0227] 21 . A method of producing a protein of interest, preferably an enzyme, comprising the steps of

[0228] I. providing a Bacillus cell according to embodiment 1,

[0229] II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metalloprotease,

[0230] III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest, thereby forming a fermentation broth comprising the protein of interest, and

[0231] IV. optionally isolating the protein of interest from the fermentation broth of step III.

[0232] 22. The method of embodiment 21 , wherein the Bacillus cell has reduced expression of a gene encoding a metalloprotease with 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 to SEQ ID NO: 08, preferably 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 to SEQ ID NO: 08.

[0233] 23. The method of any of embodiments 21 or 22, wherein the Bacillus cell expresses a protein of interest sensitive towards the metalloprotease, preferably comprising or consisting of two or more domains which are connected via flexible linkers, preferably the flexible linkers are sensitive towards the metal loprotease, preferably wherein the protein of interest being sensitive towards the metalloprotease is an enzyme.

[0234] 24. The method of any of embodiments 21-23, wherein the protein of interest is secreted by the cell.

[0235] 25. The method of any of embodiments 21-24, wherein the protein of interest is a mannanase, preferably comprising or consisting of two or more domains which are connected via flexible linkers, preferably the flexible linkers are sensitive towards the metalloprotease.

[0236] 26. The method of embodiment 25, wherein the mannanase is a variant of a parent 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 the numbering of SEQ ID NO: 12, and an amino acid sequence which 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%, or at least 99%, but less than 100% identical to SEQ ID NO: 12, preferably 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% identical to SEQ ID NO: 12.

[0237] 27. The method of any 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 amino acid substitutions selected from the group consisting of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12. 28. The method of any of embodiments 25-27, 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 numbering of SEQ ID NO: 12.

[0238] 29. The method of any of embodiments 25-28, wherein the mannanase variant comprises or consists of SEQ ID NO: 12 with the amino acid substitutions X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12, and optionally with 1-10, preferably 1-5 conservative substitutions, preferably wherein the mannanase variant comprises or consists of SEQ ID NO: 12 with the amino acid substitutions X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12.

[0239] 30. The method of any of embodiments 21-29, wherein the Bacillus cell additionally has reduced expression of at least one further gene, preferably selected from the group consisting of a gene coding for a protease different to the metalloprotease, a gene coding for a sporulation factor, a gene coding for a secreted enzyme different to a protease, a gene coding for a protein involved in the formation of the extracellular matrix, or a gene coding for an autolysin, preferably a gene coding for a protease different to the metalloprotease.

[0240] 31. The method of any of embodiments 21-30, wherein the Bacillus cell additionally has reduced expression of a gene coding for a protease different to the metalloprotease selected from the group of protease genes consisting of aprE, mpr, epr, bpr, vpr, wprA, aprX, and IspA, more preferably selected from aprE, mpr, bpr, vpr, and IspA, most preferably reduced expression of all of aprE, mpr, bpr, vpr, and IspA

[0241] 32. The method of any of embodiments 21-31, wherein the Bacillus cell additionally has reduced expression of at least one further gene selected from the group consisting of sigF, pga, and amyB, preferably a reduced expression of all of sig F, pga, and amyB.

[0242] 33. The method of any of embodiments 21-32, wherein the Bacillus cell additionally has reduced expression of at least one further 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 reduced expression of all of sigF, pga, amyB, aprE, mpr, bpr, vpr, and IspA, preferably, the Bacillus cell comprises a functional wprA and / or epr gene.

[0243] 34. A method of producing a protein of interest, preferably an enzyme, comprising the steps of

[0244] I. providing a Bacillus cell expressing a metalloprotease selected from the group consisting of a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07,

[0245] II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metalloprotease, III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest and which also allow expression of the metalloprotease, thereby forming a fermentation broth comprising the protein of interest and the metal loprotease,

[0246] IV. optionally isolating the protein of interest from the cultivation medium, thereby forming a solution comprising the protein of interest and also the metal loprotease, and

[0247] V. contacting the fermentation broth of step III or the solution of step IV with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time effective to inactivate the metalloprotease.

[0248] 35. Use of a chelating agent, preferably EDTA, MGDA, or EDDS, for increasing the yield of a protein of interest, preferably an enzyme, in a method of producing the protein of interest by a Bacillus cell comprising of a metalloprotease selected from the group consisting of a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, wherein the protein of interest is sensitive towards the metalloprotease.

[0249] 36. The method of embodiment 34 or the use of embodiment 35, wherein the metal loprotease comprises and amino acid sequence with 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 to SEQ ID NO: 08, preferably 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 to SEQ ID NO: 08.

[0250] 37. The method or the use of embodiment 36, wherein the Bacillus cell is a Bacillus licheniformis cell.

[0251] 38. The method or the use of embodiment 37, wherein the protein of interest is a mannanase, preferably comprising or consisting of two or more domains which are connected via flexible linkers, preferably the flexible linkers are sensitive towards the metal loprotease, preferably comprising or consisting of a carbohydrate binding domain, preferably the catalytic domain and the carbohydrate domain of the mannanase are connected via a flexible linker, preferably the flexible linker being sensitive towards proteolytic cleavage by the metalloprotease, preferably wherein the mannanase is a variant of a parent 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 the numbering of SEQ ID NO: 12, and an amino acid sequence which is 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% identical to SEQ ID NO: 12.

[0252] 39. The method or the use of 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 amino acid substitutions selected from the group consisting of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12.

[0253] 40. The method or the use of embodiment 39, wherein the mannanase variant comprises or consists of SEQ ID NO: 12 with the amino acid substitutions X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12, and optionally with 1-10, preferably 1-5 conservative substitutions, wherein the mannanase variant comprises or consists of SEQ ID NO: 12 with the amino acid substitutions X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12.

[0254] 41 . A method of inactivating a metalloprotease, wherein the method comprises the steps of:

[0255] I. providing a metal loprotease selected from the group consisting of: a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, and

[0256] II. contacting the metal loprotease with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time sufficient to inactivate the metalloprotease.

[0257] 42. The method of embodiment 41 , wherein the metal loprotease comprises an amino acid sequence with at least

[0258] 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

[0259] 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

[0260] 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 08, preferably 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 to SEQ ID NO: 08.

[0261] 43. The method of any of embodiment 41 or 42, wherein contacting the metal loprotease with a chelating agent is in a formulation comprising a protein of interest, preferably comprising an enzyme.

[0262] 44. The method of any of embodiments 41-43, wherein contacting the metalloprotease with a chelating agent is in a non-complex formulation, preferably, a liquid enzyme preparation, or a complex formulation, preferably, a liquid detergent formulation.

[0263] 45. The method of embodiment 44, wherein the protein of interest is a mannanase, preferably comprising or consisting of two or more domains which are connected via flexible linkers, preferably the flexible linkers are sensitive towards the metalloprotease, preferably comprising or consisting 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, preferably the flexible linker being sensitive towards proteolytic cleavage by the metalloprotease, preferably wherein the mannanase is a variant of a parent 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 the numbering of SEQ ID NO: 12, and an amino acid sequence which is 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% identical to SEQ ID NO: 12.

[0264] Examples

[0265] Materials and Methods

[0266] The following examples only serve to illustrate the invention. The numerous possible variations that are obvious to a person skilled in the art also fall within the scope of the invention.

[0267] Unless otherwise stated the following experiments have been performed by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering, molecular biology and fermentative production of chemical compounds by cultivation of microorganisms. See also Sambrook et al. (Sambrook.J. and Russell, D.W. Molecular cloning. A laboratory manual, 3rded, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001).

[0268] Electrocompetent Bacillus licheniformis cells and electroporation

[0269] Transformation of DNA into Bacillus licheniformis strain Bli#008 (WC2022018260) comprising additional deletions in the mpr, vpr, bpr, and IspA genes (herein named Bli#099) was performed via electroporation. Preparation of electro- competent Bacillus licheniformis cells and transformation of DNA was performed as essentially described by Brigidi et al (Brigidi, P., Mateuzzi.D. (1991). Biotechnol. Techniques 5, 5) with the following modification: Upon transformation of DNA, cells are recovered in 1 ml LBSPG buffer and incubated for 60 min at 37°C (Vehmaanpera J., 1989, FEMS Microbio. Lett., 61 : 165-170) following plating on selective LB-agar plates.

[0270] In order to overcome the Bacillus licheniformis specific restriction modification system of Bacillus licheniformis strains, plasmid DNA was isolated from Ec#098 cells or B. subtilis Bs#056 cells as described below.

[0271] Plasmid Isolation

[0272] Plasmid DNA was isolated from Bacillus and E. coll cells by standard molecular biology methods described in (Sambrook.J. and Russell, D.W. Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001) or the alkaline lysis method (Birnboim, H. C., Doly, J. (1979). Nucleic Acids Res 7(6): 1513-1523). Bacillus cells were in comparison to E. coll treated with 10 mg / ml lysozyme for 30 min at 37°C prior to cell lysis.

[0273] Plasmids

[0274] Plasmid pEC194RS

[0275] Bacillus temperature sensitive deletion plasmid (WC2022018260) was used for the cloning of gene deletion and gene integration constructs. pDelO45 -wprA deletion plasmid

[0276] The gene deletion plasmid for the wprA gene (SEQ ID NO: 01) of Bacillus licheniformis encoding the protease WprA (SEQ ID NO: 02) was constructed with plasmid pEC194RS and the gene synthesis construct SEQ ID NO: 03 comprising the genomic regions 5' and 3' of the wprA gene flanked by Bsal sites compatible to pEC194RS. The type-ll-assem- bly with restriction endonuclease Bsal was performed as described (Radeck et al., 2017) and the reaction mixture subsequently transformed into E. coll DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37°C on LB-agar plates containing 10Opig / ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest. The resulting wprA deletion plasmid was named pDelO45. pDe!046 -epr deletion plasmid

[0277] The gene deletion plasmid for the epr gene (SEQ ID NO: 04) of Bacillus licheniformis encoding the protease Epr (SEQ ID NO: 05) was constructed as described for pDelO45, however the gene synthesis construct SEQ ID NO: 06 comprising the genomic regions 5' and 3' of the epr gene flanked by Bsal sites compatible to pEC194RS was used. The resulting epr deletion plasmid was named pDelO46. pDe!047 -Metalloprotease BL03917 deletion plasmid

[0278] The gene deletion plasmid for the BL03917 gene (SEQ ID NO: 07) of Bacillus licheniformis encoding a Peptidase M84 (SEQ ID NO: 08) was constructed as described for pDelO45, however the gene synthesis construct SEQ ID NO: 09 comprising the genomic regions 5' and 3' of the BL03917 gene flanked by Bsal sites compatible to pEC194RS was used. The resulting BL03917 gene deletion plasmid was named pDelO47.

[0279] Plasmid pUK57: Type-ll-assembly destination Bacillus plasmid

[0280] Plasmid pUK57 (described in WQ2022018260) is a derivative of plasmid pUB110 comprising a type-ll cloning cassette for assembly of gene expression vectors.

[0281] Mannanase expression plasmid

[0282] The mannanase expression plasmids was composed of 3 genetic elements - the plasmid backbone of pU K57, the promoter of the aprE gene from Bacillus licheniformis (SEQ ID NO: 16), the coding sequences for the signal peptide of the mannanase gene and the mannanase gene respectively. The pUK57 vector, the promoter fragment (SEQ ID NO: 17), the signal peptide gene fragment and the mannanase gene fragment each comprising compatible type-ll restriction endonuclease Bpil sites (see Table 1) were assembled in an in vitro type-ll-assembly reaction with restriction endonuclease Bpil as described (Radeck et al., 2017; Sci. Rep. 7: 14134) and the reaction mixture subsequently transformed into B. subtilis Bs#056 cells made competent according to the method of Spizizen (Anagnos- topoulos.C. and Spizizen, J. (1961). J. Bacteriol. 81 , 741-746) following plating on LB-agar plates with 20 pig / ml kana- mycin. Correct clones of final mannanase plasmid wereanalyzed by restriction enzyme digest and sequencing. Table 1 summarizes the mannanase expression plasmid.

[0283] Table 1 : Mannanase expression plasmid

[0284] Strains

[0285] E. coli strain Ec#098

[0286] E. coli strain Ec#098 is an E. coli I NV110 strain (Life technologies) carrying the DNA-methyltransferase encoding expression plasmid pMDS003 WO2019016051.

[0287] B. subtilis strain Bs#056

[0288] The prototrophic Bacillus subtilis strain KO-7S (BGSCID: 1S145; Zeigler D.R.) was made competent according to the method of Spizizen (Anagnostopoulos.C. and Spizizen.J. (1961). J. Bacteriol. 81, 741-746.) and transformed with the linearized DNA-methyltransferase expression plasmid pMIS012 for integration of the DNA-methyltransferase into the amyE gene as described for the generation of B. subtilis Bs#053 in WO2019 / 016051 . Cells were spread and incubated overnight at 37°C on LB-agar plates containing 10 pig / ml chloramphenicol. Grown colonies were picked and stroke on both LB-agar plates containing 10pig / ml chloramphenicol and LB-agar plates containing 10pig / ml chloramphenicol and 0.5% soluble starch (Sigma) following incubation overnight at 37°C. The starch plates were covered with iodine containing Lugols solution and positive integration clones identified with negative amylase activity. Genomic DNA of positive clones was isolated by standard phenol / chloroform extraction methods after 30 min treatment with lysozyme (10 mg / ml) at 3°C, following analysis of correct integration of the MTase expression cassette by PCR. The resulting B. subtilis strain was named Bs#056.

[0289] Mannanase activity assay

[0290] For the determination of the mannanase activity a dyed insoluble polysaccharide was used (AZCL-Galactomannan obtained from Megazymes, Ireland). For the activity assay the enzyme solution was diluted in 100mM Tris-Buffer (pH8.6 at 30°C) supplemented with 0,1% Brij 35 and 0,1% Xanthan. To 100 piL of the diluted enzyme solution 900 piL of 0,2% AZCL-Galactomannan (dissolved in 100mM Tris-Buffer (pH8.6 at 30°C) supplemented with 0,1% Brij 35 and 0,1% Xanthan) was added using a 96 deep well plate. After incubation for 30 min at 40°C the plate was centrifuged for 5 min at 4500 g and thereby stopping the reaction and also separating insoluble fragments from solubilized polysaccharides. 200 piL of the supernatant was carefully removed and the dyed water-soluble fragments are measured at a wavelength of 590nm. The value for the stored sample was normalized to the corresponding sample kept frozen at -80°C.

[0291] Example 1: Construction of protease gene knockouts in Bacillus licheniformis

[0292] Bacillus licheniformis Bli#099 was used to delete genes encoding the proteases shown in Table 2 resulting in strains as outlined in Table 3 below.

[0293] For gene deletion in Bacillus licheniformis strains gene deletion plasmids (see ‘plasmids' section) were transformed into E. coli strain Ec#098 made competent according to the method of Chung (Chung, C.T., Niemela.S.L, and Miller, R.H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. U. S. A 86, 2172-2175), following selection on LB-agar plates containing 10O g / ml ampicillin and 30pg / ml chloramphenicol at 37°C. Plasmid DNA was isolated from individual clones and used for subsequent transfer into Bacillus licheniformis strains. The isolated plasmid DNA carries the DNA methylation pattern of Bacillus licheniformis strains respectively and was protected from degradation upon transfer into Bacillus licheniformis. The gene deletions were conducted according to the procedure as outlined in the following section. Gene deletion plasmid carrying Bacillus licheniformis cells were grown on LB-agar plates with 5 pig / ml erythromycin at 45°C forcing integration of the deletion plasmid via Campbell recombination into the chromosome with one of the homology regions present on the deletion plasmid which is homologous to the sequences 5' or 3' of the desired target gene to be deleted. Clones were picked and cultivated in LB-media without selection pressure at 45°C for 6 hours, following plating on LB-agar plates with 5 pig / ml erythromycin at 30°C. Individual clones were picked and analyzed by colony-PCR with oligonucleotides located at least 100 bp 5' and 3' of the homology regions to confirm successful deletion of the target gene. The appropriate selection of oligonucleotides is well known to the person skilled in the art. Putative deletion positive individual clones were picked and taken through two consecutive overnight incubation in LB media without antibiotics at 45°C to cure the plasmid and plated on LB-agar plates for overnight incubation at 30°C. Single clones were again restroke on LB-agar plates with 5pig / ml erythromycin and analyzed by colony PCR for successful deletion of the target gene. A single erythromycin-sensitive clone with the correct deleted target gene was isolated. The following proteases were selected for gene deletion in B. licheniformis.

[0294] Table 2: Deleted proteases of B. licheniformis

[0295] Table 3 summarizes the genotypes of the B. licheniformis gene deletion strains. Table 3: Genotypes of B. licheniformis protease gene deletion strains

[0296] * D denotes deletion

[0297] Example 2: Construction of Bacillus licheniformis mannanase expression strains

[0298] Bacillus licheniformis strains as listed in Table 3 were made competent as described above. The mannanase expression plasmid pManl 17 was isolated from B. subtilis Bs#056 to carry the B. licheniformis specific DNA methylation pattern and transformed into B. licheniformis strains with gene deletions of the protease genes as given in Table 3. The transformed strains were plated on LB-agar plates with 20pig / pil kanamycin. Individual clones were analyzed for correctness of the plasmid DNA by restriction digest and Sanger sequencing. The resulting B. licheniformis expression strains are listed in Table 4.

[0299] Table 4: Mannanase expression strains

[0300] * D denotes deletion

[0301] Example 3: Cultivation of mannanase expression strains

[0302] Bacillus licheniformis mannanase expression strains as listed in Table 4 were cultivated in a microtiter plate-based fed-batch process (Habicher et al., 2019 Biotechnol J.; 15(2)). Each strain was cultivated with six replicates.

[0303] All cultivations were conducted in an orbital shaker with a diameter of 25 mm (Innova 42, New Brunswick Scientific, Eppendorf AG; Hamburg, Germany) at 400 rpm. Strains were cultivated in two subsequent precultures in Flower- Plates (MTP-48-OFF, m2p-labs GmbH) for synchronization of growth. The first preculture was carried out in 800 l TB medium inoculated with a fresh single colony from the strain streaked onto LB agar plates. After 20 h at 30 °C, the second preculture containing 800 l V3 minimal medium (Meissner et al., 2015, Journal of industrial microbiology & biotechnology 42 (9): 1203-1215) was inoculated with 8 l of the first preculture and cultivated for 24 h at 30 °C. Microtiter plate-based fed-batch main cultivations were conducted using 48-well round- and deep-well-microtiter plates with glucose-containing polymer on the bottom of each well (FeedPlate, article number: SMFP08004, Kuhner Shaker GmbH; Herzogenrath, Germany). 70 l of the second preculture were used to inoculate 700 l V3-FP minimal medium without glucose. Main cultures were incubated for 72 h at 30 °C. Precultures were covered with a sterile gas-permeable sealing foil (AeraSeal film, Sigma-Aldrich) to avoid contamination. FeedPlates were sealed with a sterile gas-permeable, evaporation reducing foil (F-GPR48-10, m2p-labs GmbH) to reduce evaporation and to avoid contamination.

[0304] At the end of the microtiter plate-based fed-batch process, cultivation samples were withdrawn and supernatants were prepared by centrifugation and sterile filtration with a 0.2 m filter. The content of full-length mannanase and degradation product of Mannnanase were analyzed and quantified using a capillary electrophoresis system (Labchip GX Touch HT, Perkin Elmer) with a suitable kit for proteins (Protein Express assay kit). Samples were herefore diluted to a total protein content of approximately 1 mg / mL and prepared for electrophoresis according to the instructions of the manufacturer. The electropherogramms were analyzed using the Labchip GX Reviewer Software (Version 5.5.2312.0). The amount of the full-length mannanase and the amount of the truncated mannanase were quantified and are calculated as percentage of total mannanase content (full-length and truncated mannanase). The truncated mannanase was shown by LC-MS / MS analysis to be a fragment of the full-length mannanase.

[0305] The results are shown in Table 5. For strains lacking the Protease BL03917 (SEQ ID NO: 08) no degradation (< 2.0%) of the mannanase was detected and the full-length mannanase product remained stable. Surprisingly, deletion of the proteases WprA and Epr did not result in a reduction of the amount of the truncated mannanase.

[0306] Table 5: Percentage of full-length mannanase and truncated mannanase

[0307] * D denotes deletion

[0308] Example 4: Storage tests with mannanase derived from strains with different protease backgrounds

[0309] An aqueous mannanase solution was produced via fermentation of microorganisms as described above. The enzyme was recovered by removal of cells and concentrated using ultrafiltration.

[0310] The aqueous mannanase solution was formulated with 25% w / w glycerol and 25% w / w sorbitol at pH 6.0 to a final enzyme protein concentration of 0.5% w / w. Three samples were prepared using the reference strain (BES#334. One sample without any addition of chelating agent and two samples by adding 0.25% EDTA (Ethylenediaminetetraacetic acid) to the mannanase solution. If required, the pH was adjusted accordingly. An additional sample was prepared using a strain according to the invention (BES#336 without addition of a chelating agent. The diluted mannanase solutions were incubated in closed vials at -80°C and 45°C for four weeks. The mannanase activity was measured in the stored samples and residual activity calculated using mannanase activity in samples stored at -80 °C as reference. Table 6: Residual activity of mannanase after four weeks storage

[0311] The data in Table 6 show that the strain according to the invention reduces the degradation of the enzyme. The residual mannanase activity of the mannanase enzyme preparation of the strain according to the invention is with 77% higher compared to the reference strain (44%) after four weeks of storage. As can also be derived from Table 6, al- ternatively, inactivation of the metalloprotease can be achieved by adding a chelating agent, which leads to a reduction of the degradation of the enzyme, and thus, to an increase in mannanase residual activity after four weeks of storage compared to the enzyme preparation of the reference strain without the addition of a chelating agent.

Claims

Claims1 . A Bacillus cell having reduced expression of a gene encoding a metal loprotease selected from the group consisting of:I. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, andII. a metalloprotease 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 to SEQ ID NO: 07.

2. The Bacillus cell of claim 1 , wherein the Bacillus cell is a metalloprotease knockout cell.

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

4. The Bacillus cell of claim 3, wherein the enzyme is selected from the group consisting of mannanase, protease, amylase, lipase, cellulase, hemicellulase, phospholipase, esterase, cutinase, pectinase, lactase, peroxidase, xylanase, pectate lyase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninass, pullu- lanase, tannase, pentosanase, malanase, beta-glucanase, arabi nosid ase, hyaluronidase, chondroitinase, laccase, nuclease, DNase, phosphodiesterase, phytase, carbohydrase, galactanase, xanthanase, xyloglu- canase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, ribonuclease, transglutaminase, and dispersin, preferably, the enzyme is selected from the group consisting of protease, amylase, lipase, cellulases, mannanase, xylanase, DNase, dispersin, pectinases, pectate lyase, glycosidase, and oxidoreductase, most preferably the enzyme is a mannanase.

5. The Bacillus cell of claim 4, wherein the enzyme is a mannanase, preferably comprising or consisting out of two or more domains which are connected via flexible linkers, preferably the flexible linkers are sensitive towards the metalloprotease, preferably comprising or consisting 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, preferably the flexible linker being sensitive towards proteolytic cleavage by the metalloprotease, preferably wherein the mannanase is a variant of a parent 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 the numbering of SEQ ID NO: 12, and an amino acid sequencewhich is at least 55%, at least 60%, at least 65%, 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% identical to SEQ ID NO: 12.

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 amino acid substitutions selected from the group consisting of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12.

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

8. The Bacillus cell of any of the preceding claims additionally having reduced expression of at least one further gene, preferably selected from the group consisting of a gene coding for a protease different to the metalloprotease, a gene coding for a sporulation factor, a gene coding for a secreted enzyme different to a protease, a gene coding for a protein involved in the formation of the extracellular matrix, or a gene coding for an autoly- sin, preferably a gene coding for a protease different to the metalloprotease, preferably selected from the group of protease encoding genes consisting of aprE, mpr, epr, bpr, vpr, wprA, aprX, and ispA, more preferably selected from aprE, mpr, bpr, vpr, and ispA.

9. The Bacillus cell of any of the preceding claims, wherein the Bacillus cell is selected from the group consisting of Bacillus altitudinis, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus australimaris, Bacillus glycini- fermentans, Bacillus haynesii, Bacillus licheniformis, Bacillus nakamurai, Bacillus pakistanensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus safensis, Bacillus sonorensis, Bacillus stratosphericus, Bacillus velezensis, Virgibacillus, preferably selected from the group consisting of Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus velezensis, more preferably, Bacillus licheniformis and Bacillus pumilus, most preferably Bacillus licheniformis.

10. A method of producing a protein of interest, preferably an enzyme, comprising the steps ofI. providing a Bacillus cell according to claim 1,II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metal loprotease,III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest, thereby forming a fermentation broth comprising the protein of interest, andIV. optionally isolating the protein of interest from the fermentation broth of step III.

11. A method of producing a protein of interest, preferably an enzyme, comprising the steps ofI. providing a Bacillus cell expressing a gene encoding a metalloprotease selected from the group consisting of a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07,II. introducing into the Bacillus cell an expression cassette encoding a protein of interest, preferably an enzyme, sensitive towards the metal loprotease,III. cultivating the Bacillus cell under conditions which allow for the expression of the protein of interest and which also allow expression of the metalloprotease, thereby forming a fermentation broth comprising the protein of interest and the metal loprotease,IV. optionally isolating the protein of interest from the cultivation medium, thereby forming a solution comprising the protein of interest and also the metalloprotease, andV. contacting 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 effective to inactivate the metalloprotease.

12. Use of a chelating agent, preferably EDTA, MGDA, or EDDS, for increasing the yield of a protein of interest, preferably an enzyme, in a method of producing the protein of interest by a Bacillus cell, wherein the Bacillus cell expresses a gene encoding a metalloprotease selected from the group consisting of a. a metalloprotease having a 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 to SEQ ID NO: 08, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, wherein the protein of interest is sensitive towards the metalloprotease.

13. The method of any of claims 10 or 11 or the use of claim 12, wherein the protein of interest is a mannanase, preferably comprising 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, preferably the flexible linker being sensitive towards proteolytic cleavage by the metal loprotease, preferably wherein the mannanase is a variant of a parent 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 the numbering of SEQ ID NO: 12, and an amino acid sequence which is 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% identical to SEQ ID NO: 12.

14. The method or use of 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 amino acid substitutions selected from the group consisting of X59V, X66D, X89H, X234Q, X259M, X282Y, X318N, X319G, and X322G according to the numbering of SEQ ID NO: 12.

15. A method of inactivating a metalloprotease, wherein the method comprises the steps of:I. providing a metalloprotease selected from the group consisting of: a. a metalloprotease 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 to SEQ ID NO: 08, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33, and b. a metalloprotease 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 to SEQ ID NO: 07, andII. contacting the metal loprotease with a chelating agent, preferably EDTA, MGDA, or EDDS, in an amount and for a time sufficient to inactivate the metalloprotease.

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