Artificial signal peptides

Abstract

The present invention relates to nucleic acid constructs comprising a first polynucleotide encoding an artificial signal peptide and a second polynucleotide encoding a polypeptide having protease activity; expression vectors and host cells comprising said nucleic acid constructs; methods for producing polypeptides having protease activity; and fusion proteins comprising a protease polypeptide and an artificial signal peptide.

Description

[0001] ARTIFICIAL SIGNAL PEPTIDES

[0002] Reference to a Sequence Listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] Background of the Invention

[0005] Field of the Invention

[0006] The present invention relates to nucleic acid constructs comprising a first polynucleotide encoding an artificial signal peptide and a second polynucleotide encoding a polypeptide having protease activity; expression vectors and host cells comprising said nucleic acid constructs; methods for producing polypeptides having protease activity; and fusion proteins comprising a protease polypeptide and an artificial signal peptide.

[0007] Description of the Related Art

[0008] Product development in industrial biotechnology includes a continuous challenge to increase recombinant protein yields at large scale to reduce costs. Two major approaches have been used for this purpose in the last decades. The first one is based on classical mutagenesis and screening. Here, the specific genetic modification is not predefined, and the main requirement is a screening assay that is sensitive to detect increments in yield. High-throughput screening enables large numbers of mutants to be screened in search for the desired phenotype, i.e., higher recombinant protein yields. The second approach includes numerous strategies ranging from the use of stronger promoters and multi-copy strains to ensure high expression of the gene of interest to the use of codon-optimized gene sequences to aid translation. However, high-level production of a given protein may in turn trigger several bottlenecks in the cellular machinery for secretion of the enzyme of interest into the medium, emphasizing the need for further optimization strategies.

[0009] Signal peptides (SPs) are short amino acid sequences present in the amino terminus of many newly synthesized polypeptides that target these into or across cellular membranes, thereby aiding maturation and secretion. The amino acid sequence of the SP influences secretion efficiency and thereby the yield of the polypeptide manufacturing process. Bioinformatic tools such as SignalP and SignalP5 can predict SPs from amino acid sequences, but most cannot distinguish between various types of SPs (Armenteros et al., Nat. Biotechnol. 37: 420-423, 2019). Moreover, a large degree of redundancy in the amino acid sequence of SPs makes it difficult to predict the efficiency of any given SP for production of recombinant proteins at industrial scale. Hence, SP selection is an important step for manufacturing of recombinant proteins, but the optimal combination of signal peptide and mature protein is very context dependent and not easy to predict.

[0010] Thus, there is a need for identifying signal peptides which are related to increased protein yield.

[0011] Summary of the Invention

[0012] The present invention is based on the surprising and inventive finding that artificially created signal peptides, as well as codon-optimized signal peptides, outperform native signal peptides and / or their native codon variants.

[0013] In a first aspect, the present invention relates to a nucleic acid construct comprising: a first polynucleotide encoding a signal peptide having a sequence identity of at least 80% to SEQ ID NO: 254 or to any one of SEQ ID NO: 248 to 289; and a second polynucleotide encoding a polypeptide having a protease activity, wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion.

[0014] In a second aspect, the invention relates to an expression vector comprising a nucleic acid construct according to the first aspect.

[0015] In a third aspect, the invention relates to a bacterial host cell comprising in its genome: a) a nucleic acid construct according to the first aspect; and / or b) an expression vector according to the second aspect.

[0016] In a fourth aspect, the invention relates to a method of producing a polypeptide having protease activity, the method comprising: a) cultivating a host cell according to the third aspect under conditions conducive for production of the polypeptide; and optionally b) recovering the polypeptide.

[0017] In a fifth aspect, the invention relates to a fusion polypeptide, comprising: a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 254 or to any one of SEQ ID NO: 248 to SEQ ID NO: 289, and an polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Fig. 1 shows protease activities for signal peptides and control strains with aprL SP in a 1 copy strain.

[0020] Fig. 2 shows protease activities for signal peptides and control strains with aprL SP in a 3 copy strain.

[0021] SEQUENCE OVERVIEW

[0022] SEQ ID NOs:1 to 247 are DNA sequences encoding signal peptides.

[0023] SEQ ID NOs: 248 to 289 are amino acid sequences of signal peptides.

[0024] SEQ ID NO: 290 is an aprL control signal peptide (encoded by SEQ ID NO: 247).

[0025] SEQ ID NO: 292 is a protease amino acid sequence (encoded by SEQ ID NO: 291).

[0026] SEQ ID NO: 248 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 1- 10.

[0027] SEQ ID NO: 249 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 11-15.

[0028] SEQ ID NO: 252 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 32-41 .

[0029] SEQ ID NO: 253 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 42 - 52.

[0030] SEQ ID NO: 255 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 60-67.

[0031] SEQ ID NO: 256 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 68-74.

[0032] SEQ ID NO: 257 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 75-84. SEQ ID NO: 258 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 85-98.

[0033] SEQ ID NO: 264 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 116-129.

[0034] SEQ ID NO: 265 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 130-137.

[0035] SEQ ID NO: 266 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 138-147.

[0036] SEQ ID NO: 269 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 162-167.

[0037] SEQ ID NO: 274 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 184-193.

[0038] SEQ ID NO: 276 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 196-209.

[0039] SEQ ID NO: 278 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 217-221.

[0040] SEQ ID NO: 279 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 222-228.

[0041] SEQ ID NO: 280 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 229-235.

[0042] SEQ ID NO: 282 is a particular signal peptide, encoded by the codon variants of SEQ ID NOs: 237.

[0043] SEQ ID NO: 293 is a codon variant which encodes the signal peptide of SEQ ID NO: 259

[0044] SEQ ID NO: 294 is a codon variant which encodes the signal peptide of SEQ ID NO: 265

[0045] SEQ ID NO: 295 is a codon variant which encodes the signal peptide of SEQ ID NO: 266

[0046] Definitions

[0047] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0048] Protease: The term “protease” means a peptidease, such as a serine endopeptidase which hydrolyses N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide. Protease activity can be determined as described in the protease assay of the examples. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0049] Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0050] Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native ( / .e., from the same gene) or heterologous ( / .e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

[0051] Expression: The term “expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0052] Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0053] Extension: The term “extension” means an addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the “extended” polypeptide has protease activity. Persons skilled in the art will know that a polypeptide having a given amino acid sequence and enzymatic activity may be produced with one or a few additional amino acids at the N- and / or C-terminus, and that such a polypeptide can have essentially the same enzyme activity. Such extended polypeptides are intended to be encompassed by the present invention.

[0054] Fragment: The term “fragment” as used in the context of a polypeptide means a polypeptide having one or more amino acids absent from its amino and / or carboxyl terminus, wherein the fragment has protease activity. The fragment may be produced naturally during expression and / or purification of the polypeptide, or may be the result of expression of a modified nucleotide sequence expressing the fragment or of targeted removal of amino acids from the amino and / or carboxy terminus.

[0055] Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and / or the C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie etal., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0056] Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0057] Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., a protease) has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

[0058] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

[0059] Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

[0060] Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following translation and any post-translational modifications such as N-terminal processing (e.g. removal of signal peptide), C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides ( / .e., with a different C-terminal and / or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g. having a different C-terminal and / or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide. Mature polypeptides of the invention may therefore have slight differences at the N- and / or C-terminal due to such differentiated expression by the host cell. A mature polypeptide having one or more amino acids absent from the N- and / or C-terminal may be considered to be a “fragment” of the full-length polypeptide.

[0061] In one aspect the mature polypeptide is amino acids 1 to 380 of SEQ ID NO:292.

[0062] Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having protease activity.

[0063] In one aspect the mature polypeptide coding sequence at its N-terminal end comprises or consists of any one of the SP-coding sequences with SEQ ID NOs: 1 to 246 or SEQ ID NO: 293 to 295, followed by the protease-coding sequence of SEQ ID NO: 291 .

[0064] Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0065] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0066] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.

[0067] Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.

[0068] Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

[0069] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0070] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0071] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0072] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0073] (Identical Deoxyribonucleotides x 100) / (Length of Alignment- Total Number of Gaps in Alignment) Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0074] Subsequence: The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having protease activity.

[0075] Variant: The term “variant” means a polypeptide having protease activity, comprising a man-made mutation, i.e., a substitution, insertion (including extension), and / or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0076] Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

[0077] Detailed Description of the Invention

[0078] The present invention is based on the surprising and inventive finding that artificially created signal peptides, as well as codon-optimized signal peptides, outperform native signal peptides and / or their native codon variants.

[0079] Polynucleotides

[0080] The present invention also relates to polynucleotides encoding a polypeptide of the present invention, as described herein.

[0081] The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof.

[0082] In an embodiment, the polynucleotide is a subsequence encoding a fragment having protease activity. In one embodiment the polynucleotide encoding the signal peptide of the present invention is an artificial polynucleotide.

[0083] The polynucleotide may also be mutated by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.

[0084] In an aspect, the polynucleotide is isolated.

[0085] In another aspect, the polynucleotide is purified.

[0086] Nucleic Acid Constructs

[0087] The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0088] In a first aspect, the present invention relates to a nucleic acid construct comprising: a first polynucleotide encoding a signal peptide having a sequence identity of at least 80% to SEQ ID NO: 254 or to any one of SEQ ID NO: 248 to 289; and a second polynucleotide encoding a polypeptide having a protease activity, wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion.

[0089] In one embodiment, the second polynucleotide is located downstream from the first polynucleotide.

[0090] In one embodiment, the signal peptide is a naturally occurring signal peptide with an artificial codon-sequence.

[0091] In one embodiment, the signal peptide is an artificial signal peptide.

[0092] In one embodiment, the construct is further comprising a third polynucleotide downstream of the first polynucleotide and upstream of the second polynucleotide.

[0093] In one embodiment, the third polynucleotide is a non-coding intron.

[0094] In one embodiment, the signal peptide has a sequence identity of at least 85%, e.g. 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%, to any one of SEQ ID NO: 248 to 289. In one embodiment, the signal peptide has a sequence identity of at least 85%, e.g. 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%, to SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 269, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, to SEQ ID NO: 282, or SEQ ID NO: 283.

[0095] In one embodiment, the signal peptide comprises or consists of SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 269, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, to SEQ ID NO: 282, or SEQ ID NO: 283.

[0096] In one embodiment, the signal peptide has a sequence identity of at least 85%, e.g. 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%, to SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 254, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 269, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 282, or SEQ ID NO: 283.

[0097] In one embodiment, the signal peptide has a sequence identity of at least 85%, e.g. 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%, to SEQ ID NO: 248.

[0098] In one embodiment, the signal peptide comprises or consists of SEQ ID NO: 248.

[0099] In one embodiment, the signal peptide has a sequence identity of at least 85%, e.g. 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%, to SEQ ID NO: 279.

[0100] In one embodiment, the signal peptide comprises or consists of SEQ ID NO: 279.

[0101] In one embodiment, the signal peptide has a sequence identity of at least 85%, e.g. 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%, to SEQ ID NO: 254.

[0102] In one embodiment, the signal peptide comprises or consists of SEQ ID NO: 254.

[0103] In one embodiment, the signal peptide has a sequence identity of at least 85%, e.g. 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%, to SEQ ID NO: 256. In one embodiment, the signal peptide comprises or consists of SEQ ID NO: 256.

[0104] In one embodiment, the signal peptide has a sequence identity of at least 85%, e.g. 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%, to SEQ ID NO: 250.

[0105] In one embodiment, the signal peptide comprises or consists of SEQ ID NO: 250.

[0106] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 1 to 246, or SEQ ID NO: 293 to 295.

[0107] In one embodiment, the first polynucleotide comprises or consists of any one of SEQ ID NO: 1 to 246, or SEQ ID NO: 293 to 295.

[0108] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 17, 24, 33, 55, 57, 68, 89, 93, 94, 96, 221 , 225, 238, 293, 294, or 295.

[0109] In one embodiment, the first polynucleotide comprises or consists of any one of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, ,10, 17, 24, 33, 55, 57, 68, 89, 93, 94, 96, 221, 225, 238, 293, 294, or 295.

[0110] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO: 4

[0111] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 4.

[0112] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO: 17.

[0113] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 17.

[0114] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO: 57.

[0115] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 57. In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO: 10.

[0116] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 10.

[0117] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO: 293.

[0118] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 293.

[0119] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO: 238.

[0120] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 238.

[0121] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO: 8.

[0122] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 8.

[0123] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 1-10, SEQ ID NO: 11-15, SEQ ID NO: 16-26, SEQ ID NO: 32-41 , SEQ ID NO: 42 - 52, SEQ ID NO: 53-59, SEQ ID NO: 60-67, SEQ ID NO: 68-74, SEQ ID NO: 75-84, SEQ ID NO: 85-98, SEQ ID NO: 99-101 , SEQ ID NO: 293, SEQ ID NO: 116-129, SEQ ID NO: 130-137, SEQ ID NO: 295, SEQ ID NO: 138-147, SEQ ID NO: 295, SEQ ID NO: 162-167, SEQ ID NO: 184-193, SEQ ID NO: 196-209, SEQ ID NO: 210-216, SEQ ID NO: 217-221 , SEQ ID NO: 222-228, SEQ ID NO: 229- 235, or SEQ ID NO: 237.

[0124] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 1-10.

[0125] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 11- 15.

[0126] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 16- 26.

[0127] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 32- 41.

[0128] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 42 - 52.

[0129] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 53- 59.

[0130] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 60- 67.

[0131] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 68- 74.

[0132] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 75- 84.

[0133] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 85- 98. In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 99- 101 and 293.

[0134] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 116- 129.

[0135] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: ISO- 137 and 295.

[0136] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 138- 147 and SEQ ID NO: 295.

[0137] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 162- 167.

[0138] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 184- 193.

[0139] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 196- 209.

[0140] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 210- 216. In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 217- 221.

[0141] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 222- 228.

[0142] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 229- 235.

[0143] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 237.

[0144] In one embodiment, the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NO: 11 or SEQ ID NO: 13.

[0145] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 11 or SEQ ID NO: 13.

[0146] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 8.

[0147] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 4.

[0148] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 3.

[0149] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 6.

[0150] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 9.

[0151] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 10.

[0152] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 17.

[0153] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 57.

[0154] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 293.

[0155] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 238. In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 221.

[0156] In one embodiment, the first polynucleotide comprises or consists of SEQ ID NO: 294.

[0157] In one embodiment, the polypeptide having protease activity has a sequence identity of at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO: 292.

[0158] In one embodiment, the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, and the second polynucleotide, and optionally the third polynucleotide, are operably linked.

[0159] In one embodiment, the first polynucleotide encoding the signal peptide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 246 or SEQ ID NO: 293 to 295; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of any one of SEQ ID NO: 1 or SEQ ID NO: 246 or SEQ ID NO: 293 to 295.

[0160] In one embodiment, the first polynucleotide comprises, consists essentially of, or consists of any of any one of SEQ ID NO: 1 to SEQ ID NO: 246 or SEQ ID NO: 293 to 295.

[0161] In one embodiment, the signal peptide consists of the amino acid sequence of any of SEQ I D NO: 248 or SEQ I D NO: 289 with or without its C-terminal alanine, or a peptide fragment thereof that retains the ability to direct the polypeptide into or across a cell membrane.

[0162] In one embodiment, the N- and / or C-terminal end of the signal peptide has been extended by addition of one or more amino acids.

[0163] In one embodiment, the protease polypeptide comprises, consists essentially of, or consists of the mature polypeptide of SEQ ID NO: 292.

[0164] In one embodiment, the the N- and / or C-terminal end of the protease polypeptide has been extended by addition of one or more amino acids.

[0165] In one embodiment, the polynucleotide encoding the polypeptide having protease activity has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide coding sequence of SEQ ID NO: 291 ; most preferably the polynucleotide comprises, consists essentially of, or consists of the mature polypeptide coding sequence of SEQ ID NO: 291 .

[0166] In one embodiment, the polypeptide having protease activity is a bacterial polypeptide or variant thereof.

[0167] In one embodiment, the polypeptide having protease activity is derived from Alkalihalobacillus clausii.

[0168] It is expected that the invention will be just as effective when employing a signal peptide that is highly similar to the signal peptide disclosed in SEQ ID NO: 248 to 289, or a signal peptide encoded by SEQ ID NO: 1 to 246 or SEQ ID NO: 293 to 295. One or more non-essential amino acids may, for example, be altered. Non-essential amino acids in a signal peptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for signal peptide activity to identify amino acid residues that are critical to the activity of the molecule and residues that are non-essential. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The identity of essential and non-essential amino acids can also be inferred from an alignment with one or more related signal peptide.

[0169] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g. Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0170] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0171] In one aspect, the signal peptide is a variant ( / .e., functional variant) or fragment ( / .e., functional fragment) of the signal peptides of SEQ ID NOs: 248 to 289. In one aspect, the number of alterations in the signal peptide variant of the present invention is 1-10, e.g., 1-5, such as 1 , 2, 3, 4, or 5 alterations. Alterations includes substitutions, insertions, and / or deletions at one or more (e.g., several) positions compared to the parent. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0172] In a preferred embodiment, the signal peptide is a variant of the mature polypeptide of SEQ ID NOs: 248 to 289 comprising 1-10 alterations, e.g., 1-5, such as 1 , 2, 3, 4, or 5 alterations, compared to SEQ ID NO: 248 to 289, respectively.

[0173] The first and second polynucleotide are operably linked in translational fusion. In the context of the present invention, the term “operably linked in translation fusion” means that the signal peptide encoded by the first polynucleotide and the polypeptide encoded by the second polynucleotide are encoded in frame and translated together as a single polypeptide. Preferably, following translation, the signal peptide is removed to provide the mature protease polypeptide. Alternatively, the signal peptide is not removed, or only removed partly to provide the mature protease and comprising at least a fragment of the signal peptide.

[0174] The first and second polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a nucleic acid construct or expression vector may be desirable or necessary depending on the construct or vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0175] Besides a signal peptide, the nucleic acid constructs of the invention may be operably linked to one or more further control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0176] Promoters

[0177] The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0178] In one embodiment, the nucleic acid construct further comprises a heterologous promoter, and wherein said promoter, the first polynucleotide, and the second polynucleotide are operably linked. The promoter is orientated upstream of the first polynucleotide. Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, supra, and Song et al., 2016, PLOS One 11(7): e0158447. mRNA Stabilizers

[0179] The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

[0180] Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94 / 25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

[0181] Terminators

[0182] The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

[0183] Preferred terminators for bacterial host cells may be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0184] Propeptides

[0185] The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

[0186] Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence. Additionally or alternatively, when both signal peptide and propeptide sequences are present, the polypeptide may comprise only a part of the signal peptide sequence and / or only a part of the propeptide sequence. Alternatively, the final or isolated polypeptide may comprise a mixture of mature polypeptides and polypeptides which comprise, either partly or in full length, a propeptide sequence and / or a signal peptide sequence.

[0187] Regulatory Sequences

[0188] It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.

[0189] Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems.

[0190] Leader Sequences

[0191] The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

[0192] Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Blasi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.

[0193] Expression Vectors

[0194] In a second aspect, the present invention also relates to recombinant expression vectors comprising a nucleic acid construct according to the first aspect. The expression vectors comprise a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0195] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0196] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0197] The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0198] The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0199] For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).

[0200] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0201] More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0202] Host Cells

[0203] In a third aspect, the invention relates to bacterial host cells comprising in its genome: a) a nucleic acid construct according to the first aspect; and / or b) an expression vector according to the second aspect.

[0204] A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The polypeptide encoded by the introduced polynucleotide can be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may comprise a single copy, or at least two copies, e.g. three, four, five or more copies of the polynucleotide of the present invention.

[0205] In one embodiment, the host cell comprises two or more copies of the nucleic acid construct and / or the expression vector.

[0206] In one embodiment, the cell is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0207] In one embodiment, the cell is a Bacillus cell.

[0208] In one embodiment, the cell is a Bacillus licheniformis cell.

[0209] In one embodiment, the cell is a Bacillus subtilis cell.

[0210] In an aspect, the host cell is isolated.

[0211] In one embodiment, the host cell comprises at least two copies of the nucleic acid construct and / or the expression vector, such as two copies, three copies, four copies or more than four copies.

[0212] In one embodiment, the host cell comprises three copies of the nucleic acid construct and / or the expression vector.

[0213] In another aspect, the host cell is purified. Methods of Production

[0214] In a fourth aspect, the present invention also relates methods of producing a polypeptide having protease activity, the method comprising: a) cultivating a host cell according to the third aspect under conditions conducive for production of the polypeptide; and optionally b) recovering the polypeptide.

[0215] The host cell is cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state, and / or microcarrier-based fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and / or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0216] The polypeptide may be detected using methods known in the art that are specific for the polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an assay determining the relative or specific activity of the polypeptide.

[0217] The polypeptide may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the polypeptide is recovered. In another aspect, a cell-free fermentation broth comprising the polypeptide is recovered.

[0218] The polypeptide may be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science’, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0219] In an alternative aspect, the polypeptide having protease activity is not recovered. In one aspect the polypeptide having protease activity is not recovered, but rather a host cell of the present invention expressing the polypeptide having protease activity is used as a source of the variant.

[0220] Fusion Polypeptide

[0221] In a fifth aspect, the invention relates to a fusion polypeptide, comprising a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to any one of SEQ ID NO: 248 to SEQ ID NO: 289, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292, preferably the polypeptide has protease activity.

[0222] In one embodiment, the signal peptide is located upstream of the polypeptide, e.g., upstream of the polypeptide having protease activity.

[0223] In one embodiment, the signal peptide is located at the N-terminal end of the polypeptide, e.g. at the N-terminal end of the polypeptide having protease activity.

[0224] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 248, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0225] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 258, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0226] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 250, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0227] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 254, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0228] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 259, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0229] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 276, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0230] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 265, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0231] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 252, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0232] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 279, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0233] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 256, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0234] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 266, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0235] In one embodiment, the fusion polypeptide comprises a signal peptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 283, and a polypeptide having at least 60%, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

[0236] Fermentation Broth Formulations or Cell Compositions

[0237] The present invention also relates to a fermentation broth formulation or a cell composition comprising a polypeptide having protease activity. The fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the nucleic acid constructs of the present invention which are used to produce the polypeptide having protease activity), cell debris, biomass, fermentation media and / or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and / or cell debris, and culture medium.

[0238] The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and / or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., Bacillus cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or nonviable microbial cells.

[0239] In some embodiments, the fermentation broth formulation or the cell composition comprises a first organic acid component comprising at least one 1-5 carbon organic acid and / or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and / or a salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

[0240] In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and / or cell debris. In some embodiments, the killed cells and / or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.

[0241] The fermentation broth formulation or cell composition may further comprise a preservative and / or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0242] The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or cell composition contains the spent culture medium and cell debris present after the microbial cells (e.g., Bacillus cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains the spent cell culture medium, extracellular enzymes, and killed bacterial cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and / or lysed using methods known in the art.

[0243] A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and / or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition. The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90 / 15861 or WO 2010 / 096673.

[0244] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0245] Examples

[0246] Molecular biological methods

[0247] DNA manipulations and transformations were performed by standard molecular biology methods as described in:

[0248] • Sambrook et al. (1989): Molecular cloning: A laboratory manual. Cold Spring Harbor laboratory, Cold Spring Harbor, NY.

[0249] • Ausubel et al. (eds) (1995): Current pro7tocols in Molecular Biology. John Wiley and Sons.

[0250] • Harwood and Cutting (eds) (1990): Molecular Biological Methods for Bacillus. John Wiley and Sons.

[0251] Enzymes for DNA manipulation were obtained from New England Biolabs, Inc. and used essentially as recommended by the supplier.

[0252] Direct transformation into B. licheniformis was one as previously described in patent US 2019 / 0185847 A1. Conjugation into B. licheniformis was performed as described in WO 2018 / 077796 A1.

[0253] Genomic DNA was prepared by using the commercially available QIAamp DNA Blood Kit from Qiagen. The respective DNA fragments were amplified by PCR using the Phusion Hot Start DNA Polymerase system (Thermo Scientific). PCR amplification reaction mixtures contained 1 L (0,1 pg) of template DNA, 1 pL of sense primer (20pmol / pL), 1 pL of anti-sense primer (20pmol / pL), 10pL of 5X PCR buffer with 7,5mM MgCh, 8pL of dNTP mix (1 ,25mM each), 39pL water, and 0.5pL (2 U / ) DNA polymerase. A thermocycler was used to amplify the fragment. The PCR products were purified from a 1.2% agarose gel with 1x TBE buffer using the Qiagen QIAquick Gel Extraction Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's instructions.

[0254] The condition for POE-PCR is as follows: purified PCR products were used in a subsequent PCR reaction to create a single fragment using splice overlapping PCR (SOE) using the Phusion Hot Start DNA Polymerase system (Thermo Scientific) as follows. The very 5’ end fragment and the very 3’ end fragment have complementary end which will allow the SOE to concatemer into the POE PCR product. The PCR amplification reaction mixture contained 50 ng of each of the three gel purified PCR products. POE PCR was performed as described in (You, C et a / (2017) Methods Mol. Biol. 116, 183-92).

[0255] Media

[0256] Bacillus strains were grown on LB agar (10g / L Tryptone, 5g / L yeast extract, 5g / L NaCI, 15g / L agar) plates or in TY liquid medium (20g / L T ryptone, 5g / L yeast extract, 7mg / L FeCl2, 1 mg / L MnCl2, 15mg / L MgCy. To select for erythromycin resistance, agar and liquid media were supplemented with 5pg / ml erythromycin.

[0257] LB agar: 10 g / l peptone from casein; 5 g / l yeast extract, 10 g / l sodium chloride; 12 g / l Bacto-agar adjusted to pH 7.0 + / - 0.2. Premix from Merck was used (LB-agar (Miller) 110283)

[0258] Fermentation

[0259] Strains were fermented in microtiter plates with nutrient controlled media at 37°C, 1000 rpm.

[0260] Protease assay

[0261] The serine endopeptidase hydrolyses the substrate N-Succinyl-Ala-Ala-Pro-Phe p- nitroanilide. The reaction was performed at Room Temperature at pH 9.0. The release of pNA results in an increase of absorbance at 405 nm and this increase is proportional to the enzymatic activity measured against a standard.

[0262] Strains

[0263] SEQ ID NO: 248 (encoded by codon variants with SEQ ID NOs: 1 - 10),

[0264] SEQ ID NO: 249 (encoded by codon variants with SEQ ID NOs: 11 - 15),

[0265] SEQ ID NO: 250 (encoded by codon variants with SEQ ID NOs: 16 - 26)

[0266] SEQ ID NO: 252 (encoded by codon variants with SEQ ID NOs: 32 - 41),

[0267] SEQ ID NO: 253 (encoded by codon variants with SEQ ID NOs: 42 - 52), SEQ ID NO: 254 (encoded by codon variants with SEQ ID NOs: 53 - 59) SEQ ID NO: 255 (encoded by codon variants with SEQ ID NOs: 60 - 67), SEQ ID NO: 256 (encoded by codon variants with SEQ ID NOs: 68 - 74), SEQ ID NO: 257 (encoded by codon variants with SEQ ID NOs: 75 - 84), SEQ ID NO: 258 (encoded by codon variants with SEQ ID NOs: 85 - 98),

[0268] SEQ ID NO: 259 (encoded by codon variants with SEQ ID NOs: 99 - 101 , and SEQ ID NO:

[0269] 293)

[0270] SEQ ID NO: 264 (encoded by codon variants with SEQ ID NOs: 116 - 129),

[0271] SEQ ID NO: 265 (encoded by codon variants with SEQ ID NOs: 130 - 137, and SEQ ID NO:

[0272] 294),

[0273] SEQ ID NO: 266 (encoded by codon variants with SEQ ID NOs: 138 - 147, and SEQ ID NO:

[0274] 295),

[0275] SEQ ID NO: 269 (encoded by codon variants with SEQ ID NOs: 162 - 167), SEQ ID NO: 274 (encoded by codon variants with SEQ ID NOs: 184 - 193), SEQ ID NO: 276 (encoded by codon variants with SEQ ID NOs: 196 - 209), SEQ ID NO: 277 (encoded by codon variants with SEQ ID NOs: 210 - 216), SEQ ID NO: 278 (encoded by codon variants with SEQ ID NOs: 217 - 221), and SEQ ID NO: 280 (encoded by codon variants with SEQ ID NOs: 229 - 235).

[0276] Example 1 : Generated signal peptides and codon variants increase protease activities in 1 copy strains

[0277] With an Alkalihalobacillus clausii protease as input biological sequence and applying a model, 246 different signal peptide-encoding polynucleotides (SEQ ID NOs: 1 -246, and 293-295) were generated. These polynucleotides encode a total of 42 signal peptide amino acid sequences (SEQ ID NOs: 248 - 289). The aprL signal peptide (SEQ ID NO: 290) encoded by the polynucleotide with SEQ ID NO: 247 was used as control.

[0278] Synthetic DNA was ordered to contain the protease expression cassette under control of the triple promoter (as described in WO 99 / 43835) and fused to a polynucleotide sequence encoding one of the generated signal peptides. The protease expression cassette was combined with an upstream ara flanking region including the triple promoter and a downstream flanking region of the ara locus, including the ERM selection marker, in a POE PCR (patent US 2019 / 0185847 A1). The generated material was used for transformation into MOL3320 as described in patent US 2019 / 0185847 A1 , resulting in strains comprising one copy of the protease expression cassette. Selection was done on ERM. To evaluate the effect of the signal peptides on the activity of the protease, strains were fermented for app. 120 hours and protease activity was measured at the end of fermentation. For a subset of the generates signal peptides, the ranking of the clones by protease activity showed significant improvement of the protease activity compared to control strains with the aprL signal peptide (Figure 1).

[0279] For each signal peptide amino acid sequence, a plurality of DNA sequences with different codons was obtained (= codon variants), without changing the signal peptide’s amino acid sequence.

[0280] In Fig. 1 , the target performance was measured using a protease activity assay, which is a proxy for yield (Y-axis).

[0281] The aprL SP controls in Fig. 1 are shown with a black dot. The SP sequences ranked against the aprL SP are shown in blank dots. As can be seen in Fig. 1 , several SP sequences were identified which resulted in increased protease expression compared to aprL SP. As shown in Fig. 1 , the SP sequences associated with increased protease activities include the SP sequences of (highest protease activity listed first, i.e., in descending order) SEQ ID NO: 248 (encoded by SEQ ID NO: 8), SEQ ID NO: 248 (encoded by SEQ ID NO: 4), SEQ ID NO: 248 (encoded by SEQ ID NO: 3), SEQ ID NO: 248 (encoded by SEQ ID NO: 6), SEQ ID NO: 248 (encoded by SEQ ID NO: 9), SEQ ID NO: 258 (encoded by SEQ ID NO: 94), SEQ ID NO: 248 (encoded by SEQ ID NO: 5), SEQ ID NO: 258 (encoded by SEQ ID NO: 93), SEQ ID NO: 248 (encoded by SEQ ID NO: 7), SEQ ID NO: 254 (encoded by SEQ ID NO: 57), SEQ ID NO: 248(encoded by SEQ ID NO: 10), SEQ ID NO: 250 (encoded by SEQ ID NO: 24), SEQ ID NO: 258 (encoded by SEQ ID NO: 96), SEQ ID NO: 254 (encoded by SEQ ID NO: 55), and SEQ ID NO: 258 (encoded by SEQ ID NO: 89). The SP of SEQ ID NO: 248 resulted in the highest protease activity showing up to 100% increase in protease activity compared to the protease activity of the aprL SP. SPs of SEQ ID NO: 258 and 254 showed a protease activity increase of ca. 30% and 20% compared to the protease activity of the aprL SP, respectively.

[0282] Example 2: Generated signal peptides and codon variants increase protease activities in 3 copy strains

[0283] The same SP sequences and codon variants from Example 1 were investigated in Example 2. As in example 1 , the aprL signal peptide (SEQ ID NO: 290) encoded by the polynucleotide with SEQ ID NO: 247 was used as control.

[0284] The protease expression cassette was combined with an upstream flanking region for the three loci bgIC, xylA and lacA2 including the triple promoter (as described in WO 99 / 43835), the recombination sites FRT-F and FRT-F3 (as described in WO 18 / 077796) and a counter selection marker, in a POE PCR (patent US 2019 / 0185847 A1). POEs were generated by combining the different synthetic DNAs with plasmid elements needed to generate flp-FRT donor plasmids (WO 2018 / 077796 A1). The resulting plasmids were transformed into a Bacillus subtilis donor and conjugated into the Bacillus licheniformis strain BT15061 resulting in strains comprising 3 copies of the protease expression cassette. Selection was done using the counter selection marker.

[0285] To evaluate the effect of the signal peptides on the activity of the protease, strains were fermented for app. 120 hours and protease activity was measured at the end of fermentation. For a subset of the generated signal peptides, the ranking of the clones by protease activity showed significant improvement of the protease activity compared to control strains with the aprL signal peptide (Figure 2). The aprL SP controls in Fig. 2 are shown with a black dot. The SP sequences ranked against the aprL SP are shown in blank dots. As can be seen in Fig. 2, several SP sequences were identified which resulted in increased protease expression compared to aprL SP. The SP sequences associated with increased protease activities include the SP sequences of (highest protease activity listed first, i.e., in descending order) SEQ ID NO: 250 (encoded by SEQ ID NO: 17), SEQ ID NO: 254 (encoded by SEQ ID NO: 57), SEQ ID NO: 259 (encoded by SEQ ID NO: 293), SEQ ID NO: 283 (encoded by SEQ ID NO: 238), SEQ ID NO: 278 (encoded by SEQ ID NO: 221), SEQ ID NO: 265 (encoded by SEQ ID NO: 294), SEQ ID NO: 252 (encoded by SEQ ID NO: 33), SEQ ID NO: 248 (encoded by SEQ ID NO: 4), SEQ ID NO: 266 (encoded by SEQ ID NO: 295), SEQ ID NO: 256 (encoded by SEQ ID NO: 68), and SEQ ID NO: 279 (encoded by SEQ ID NO: 225). The SP of SEQ ID NO: 250 resulted in the highest protease activity, followed by the SP of SEQ ID NO: 254, which both showed at least ca. 25 % increase in protease activity compared to the protease activity of the aprL SP.

[0286] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

Claims1 . A nucleic acid construct comprising: a first polynucleotide encoding a signal peptide having a sequence identity of at least 80% to SEQ ID NO: 254 or any one of SEQ ID NOs: 248 to 289; and a second polynucleotide encoding a polypeptide having protease activity, wherein the first polynucleotide and the second polynucleotide are operably linked in translational fusion.

2. The nucleic acid construct according to claim 1 , wherein the signal peptide has a sequence identity of at least 85%, e.g., 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%, to any one of SEQ ID NO: 248 to 289, preferably to SEQ ID NO: 248, SEQ IDNO: 249, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ IDNO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ IDNO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 269, SEQ ID NO: 274, SEQ IDNO: 276, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, to SEQ ID NO: 282, or SEQ ID NO: 283.

3. The nucleic acid construct according to claim 1 or claim 2, wherein the signal peptide has a sequence identity of at least 85%, e.g. 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%, to any one of SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO:252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO:257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO:266, SEQ ID NO: 269, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:279, SEQ ID NO: 280, to SEQ ID NO: 282, or SEQ ID NO: 283.

4. The nucleic acid construct according to any preceding claims, wherein the first polynucleotide has a sequence identity of at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to any one of SEQ ID NOs: 1 to 246, or SEQ ID NOs: 293 to 295.

5. The nucleic acid construct according to any preceding claims, wherein the first polynucleotide has a sequence identity of at least 80%, e.g. at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to SEQ ID NO: 1-10, SEQ ID NO: 11-15, SEQID NO: 17, SEQ ID NO: 32-41 , SEQ ID NO: 42 - 52, SEQ ID NO: 57, SEQ ID NO: 60-67, SEQ ID NO: 68-74, SEQ ID NO: 75-84, SEQ ID NO: 85-98, SEQ ID NO: 116-129, SEQ ID NO: 130-137, SEQ ID NO: 138-147, SEQ ID NO: 162-167, SEQ ID NO: 184-193, SEQ ID NO: 196-209, SEQ ID NO: 217-221 , SEQ ID NO: 222-228, SEQ ID NO: 229-235, SEQ ID NO: 237, SEQ ID NO: 293, SEQ ID NO: 294, or to SEQ ID NO: 295; preferably to any one of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 17, or SEQ ID NO: 57.

6. The nucleic acid construct according to any preceding claims, wherein the polypeptide having protease activity has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the mature polypeptide of SEQ ID NO: 292.

7. An expression vector comprising a nucleic acid construct according to any of claims 1 to 6.

8. A bacterial host cell comprising in its genome: a) a nucleic acid construct according to any of claims 1 to 6; and / or b) an expression vector according to claim 7.

9. A method of producing a polypeptide having protease activity, the method comprising: a) cultivating a host cell according to claim 8 under conditions conducive for production of the polypeptide; and optionally b) recovering the polypeptide.

10. A fusion polypeptide, comprising: a signal peptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 254 or to any one of SEQ ID NO: 248 to SEQ ID NO: 289, and an polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to SEQ ID NO: 292.

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