阅读说明：本技术 改善蛋白酶表达的手段和方法 (Means and methods for improving protease expression ) 是由 A.Q.加努扎 M.D.拉斯穆森 A.K.尼尔森 于 2020-03-04 设计创作，主要内容包括：本发明涉及通过与折叠酶共表达来改善蛋白酶表达的手段和方法。(The present invention relates to means and methods for improving protease expression by co-expression with a folding enzyme.)

1. A nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9.

2. The nucleic acid construct according to claim 1, wherein the first heterologous promoter and the second heterologous promoter are the same or different promoters; preferably, the first heterologous promoter and the second heterologous promoter are identical copies of the same promoter.

3. The nucleic acid construct according to any of the preceding claims, wherein the protease is a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamine protease, a metalloprotease, or an asparagine peptide cleaving enzyme.

4. The nucleic acid construct according to claim 3, wherein the protease is a serine protease; subtilases are preferred, subtilisins being most preferred.

5. The nucleic acid construct according to any of the preceding claims, wherein the protease comprises a C-terminal propeptide or an N-terminal propeptide and/or an N-terminal signal peptide; or wherein the protease is a mature protease.

6. The nucleic acid construct according to any of the preceding claims, wherein the protease has at least 80% sequence identity with SEQ ID No. 3.

7. The nucleic acid construct according to any of the preceding claims, wherein the protease comprises or consists of SEQ ID No. 3.

8. The nucleic acid construct according to any of the preceding claims, wherein the protease is a bacillus clausii alkaline protease (AprH) or a variant thereof.

9. The nucleic acid construct according to any of the preceding claims, wherein the at least one protease-encoding polynucleotide has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID No. 1.

10. The nucleic acid construct according to any of the preceding claims, wherein the at least one protease-encoding polynucleotide comprises or consists of SEQ ID No. 1.

11. The nucleic acid construct according to any of the preceding claims, wherein the at least one polynucleotide encoding a foldase has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID No. 4 or SEQ ID No. 7.

12. The nucleic acid construct according to any of the preceding claims, wherein the at least one polynucleotide encoding a foldase comprises or consists of SEQ ID No. 4 or SEQ ID No. 7.

13. An expression vector comprising the nucleic acid construct according to any one of the preceding claims.

14. A gram-positive host cell comprising in its genome the nucleic acid construct according to any one of claims 1-12 and/or the expression vector according to claim 13.

15. The gram-positive host cell according to claim 14, wherein the gram-positive host cell is a bacillus host cell; preferably, the bacillus host cell is selected from the group consisting of: bacillus alkalophilus, Bacillus altivelis, Bacillus amyloliquefaciens subspecies, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus saffron, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

16. A method for producing a protease, the method comprising:

a) providing a gram-positive host cell according to any one of claims 14-15;

b) culturing said gram-positive host cell under conditions conducive to the expression of the protease and the folding enzyme; and, optionally

c) Recovering the protease.

17. The method according to claim 16, wherein the gram-positive host cell is a bacillus host cell; preferably, the bacillus host cell is selected from the group consisting of: bacillus alkalophilus, Bacillus altivelis, Bacillus amyloliquefaciens subspecies, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus saffron, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

Technical Field

The present invention relates to means and methods for improving protease expression by co-expression with a folding enzyme.

Background

In industrial biotechnology, there is a continuing need to increase production yields and thereby increase profit margins in the production of enzymes and other industrially relevant proteins. One successful strategy is to use a production host cell that overexpresses the gene encoding the target protein, for example by using a multicopy strain containing several gene copies or by modifying its control sequences to enhance the activity of the gene. To take full advantage of the beneficial effects of gene overexpression, it is desirable to increase the secretory capacity of the production host cell to overcome any bottlenecks in the secretory machinery.

Folding enzymes are proteins that assist in folding of other proteins. Overexpression of one or more molecular folding enzymes in a production host cell may result in enhanced folding of a given target protein, which in turn may lead to enhanced secretion of the correctly folded protein, thereby increasing production yield.

PrsA is an extracytoplasmic folding enzyme found in a variety of gram-positive bacteria including industrially relevant B.licheniformis. PrsA is a dimeric lipoprotein anchored in the outer leaflet of the cell membrane (outer leaf), where it contributes to the folding of proteins secreted through the conserved SecA-YEG pathway.

Overexpression of native PrsA has been shown to improve expression of polypeptides in gram-positive bacteria (WO 1994/019471).

We have observed that the co-expression of bacillus clausii alkaline protease (AprH) with certain bacterial foldases can significantly improve the expression of AprH. Based on this finding, we propose that these specific folding enzymes can be used to improve the expression of proteases in general.

Disclosure of Invention

The present invention relates to the surprising and inventive discovery that the co-expression of B.clausii alkaline protease (AprH) and certain bacterial foldases increases the yield of AprH expression.

In a first aspect, the present invention relates to a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9.

In a second aspect, the present invention relates to expression vectors comprising the nucleic acid construct according to the first aspect.

In a third aspect, the present invention relates to gram-positive host cells comprising in their genome the nucleic acid construct according to the first aspect and/or the expression vector according to the second aspect.

In a fourth aspect, the present invention relates to methods for producing a protease, the methods comprising:

a) providing a gram-positive host cell according to the third aspect;

b) culturing the host cell under conditions conducive to the expression of the protease; and, optionally

c) Recovering the protease.

Drawings

Figure 1 shows a phylogenetic tree depicting the interrelationship between different PrsA homologs obtained from various gram-positive species. The branch length is proportional to the divergence of the amino acid sequence. PrsA from Bacillus species (Bacillus sp.) has sequence number 25, and 47.7% sequence identity with PrsA from Bacillus subtilis. PrsA from Thermolignin-degrading bacteria (Geobacillus caldoxylosylyticus) has sequence number 26, and 53.3% sequence identity with PrsA from Bacillus subtilis.

FIG. 2 shows a schematic representation of the linear DNA product used to integrate the prsA gene in Bacillus subtilis strain AN 2.

FIG. 3 shows a schematic representation of the linear DNA products used to integrate the aprH gene in Bacillus subtilis strains AN2, AN2406 and AN 2407.

Definition of

Folding enzyme: the term "foldase" means an enzyme having foldase activity. Folding enzymes are proteins that promote folding of a polypeptide into a functional three-dimensional structure and/or prevent aggregation of unfolded polypeptides into non-functional structures and any subsequent degradation of the protein. PrsA is an example of a folding enzyme in gram-positive bacteria. PrsA is a dimer consisting of two monomers forming two domains; the peptidyl-prolyl isomerase (PPIase, E.C.5.2.1.8) domain serves for interconversion of cis and trans isomers of peptidyl-prolyl bonds, while the foldase domain assists in polypeptide folding (Jakob et al 2015, J.biol. chem. [ J.Biol.290 (6): 3278) 3292). Jakob et al (supra) provide the crystal structure of PrsA from Bacillus subtilis.

Folding enzyme activity: the term "foldase activity" means a PPIase activity and/or a foldase activity and is determined as the expression yield or activity yield of a polypeptide of interest (e.g.a protease) when co-expressed with the foldase in a suitable host cell.

Allelic variants: the term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

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 of mRNA that is processed through a series of steps, including splicing, and then rendered into mature spliced mRNA.

Clade evolution: the term "clade" means a group of polypeptides that are grouped together based on homology features traced back to a common ancestor. A polypeptide clade can be viewed as a phylogenetic tree, and a clade is a group of polypeptides consisting of a common ancestor and all its ancestral descendants. A group of polypeptides formed within the clade (sub-clade) of the phylogenetic tree may also share common properties and be more closely related to other polypeptides in the clade.

A coding sequence: the term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

And (3) control sequence: the term "control sequences" means the nucleic acid sequences necessary for the expression of the protease-encoding polynucleotides and foldase-encoding polynucleotides of the invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the protease and/or the polynucleotide encoding the foldase, or native or foreign with respect to one another. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotides of the invention. The term "heterologous promoter" means a promoter that is foreign (i.e., from a different gene) to the polynucleotide to which it is operably linked.

Expressing: the term "expression" includes any step involving the production of a protease, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide of the present invention and operably linked to control sequences that provide for their expression.

Host cell: the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Separating: the term "isolated" means a substance in a form or environment not found in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more or all of the naturally occurring components associated with its property; (3) any substance that is modified by man relative to substances found in nature; or (4) any substance that is modified by increasing the amount of the substance relative to other components that are intrinsically associated with the substance (e.g., multiple copies of a gene encoding the substance; using a promoter that is stronger than the promoter intrinsically associated with the gene encoding the substance). The isolated material may be present in a sample of fermentation broth.

Mature polypeptide: the term "mature polypeptide" means a polypeptide that is in its final form after translation and any post-translational modifications (e.g., N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). It is known in the art that host cells can produce a mixture of two of a plurality of different mature polypeptides (i.e., having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide.

Mature polypeptide coding sequence: the term "mature polypeptide coding sequence" means, depending on the context, a polynucleotide encoding a mature protease or a polynucleotide encoding a mature folding enzyme.

Nucleic acid construct: the term "nucleic acid construct" means a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain segments of nucleic acids in a manner not otherwise found in nature, or that is synthetic, that contains one or more control sequences.

Operatively connected to: the term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For The purposes of The present invention, The sequence identity between two amino acid sequences is determined using The Needman-Wunsch algorithm (Needleman and Wunsch,1970, J.Mol.biol. [ J.M.Biol ]48: 443-. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The output of the "longest identity" of the nidel label (obtained using the non-reduced option) is used as a percentage of identity and is calculated as follows:

(same residue x 100)/(alignment Length-total number of vacancies in alignment)

For The purposes of The present invention, The sequence identity between two deoxyribonucleotide sequences is determined using The Needman-Weng algorithm (Needleman and Wunsch,1970, supra) as implemented in The Nidel program of The EMBOSS Software package (EMBOSS: The European Molecular Biology Open Software Suite), Rice et al, 2000, supra (preferably version 5.0.0 or later). The parameters used are gap open penalty of 10, gap extension penalty of 0.5 and the EDNAFULL (EMBOSS version of NCBI NUC 4.4) substitution matrix. The output of the "longest identity" of the nidel label (obtained using the non-reduced option) is used as a percentage of identity and is calculated as follows:

(identical deoxyribonucleotides x 100)/(alignment length-total number of vacancies in alignment)

Variants: the term "variant" means a polypeptide having protease activity comprising an alteration (i.e., a substitution, insertion, and/or deletion) at one or more (e.g., several) positions. Substitution means the substitution of an amino acid occupying a position with a different amino acid; deletion means the removal of an amino acid occupying a position; and insertion means the addition of one or more (e.g., a few) amino acids (e.g., 1-5 amino acids) adjacent to and immediately following the amino acid occupying a position.

Sequence listing

1, SEQ ID NO: a polynucleotide sequence of a bacillus clausii alkaline protease (AprH).

2, SEQ ID NO: a polypeptide sequence of bacillus clausii alkaline protease (AprH), comprising a signal peptide.

3, SEQ ID NO: mature polypeptide sequence of bacillus clausii alkaline protease (AprH).

4, SEQ ID NO: a polynucleotide sequence encoding PrsA, consisting of a signal peptide of bacillus subtilis PrsA and a mature polypeptide of bacillus species PrsA.

5, SEQ ID NO: a polypeptide sequence of PrsA, consisting of a signal peptide of bacillus subtilis PrsA and a mature polypeptide of bacillus species PrsA.

6 of SEQ ID NO: a mature polypeptide sequence of a bacillus species PrsA.

7, SEQ ID NO: a polynucleotide sequence encoding PrsA, consisting of a signal peptide of bacillus subtilis PrsA and a mature polypeptide of high temperature lignin-degrading bacteria PrsA.

8, SEQ ID NO: the polypeptide sequence of PrsA consists of a signal peptide of the PrsA of the bacillus subtilis and a mature polypeptide of the PrsA of the high-temperature lignin degrading bacteria.

9 of SEQ ID NO: mature polypeptide sequence of high temperature lignin degrading bacterium PrsA.

10, SEQ ID NO: polynucleotide sequence of sigF gene.

11, SEQ ID NO: a polynucleotide sequence of sigF Δ 297 bp.

12, SEQ ID NO: SOE PCR product for integration of the prsA gene from Bacillus species into AN 2.

13 in SEQ ID NO: SOE PCR product for integration of prsA gene from Thermolignin-degrading bacteria into AN 2.

14, SEQ ID NO: SOE PCR products for integration of the aprH gene from B.clausii into AN2, AN2406 and AN 2407.

Detailed Description

The present invention relates to the surprising and inventive discovery that the co-expression of B.clausii alkaline protease (AprH) and certain bacterial foldases increases the yield of AprH expression. The Bacillus subtilis strain co-expressing AprH (SEQ ID NO:3) is co-cultured with Bacillus species PrsA (SEQ ID NO:6) or high temperature lignin degrading bacteria PrsA (SEQ ID NO:9), and the expression yield of AprH is respectively improved by 14% and 23%. Furthermore, phylogenetic analyses showed that these specific folding enzymes are closely related (fig. 1), suggesting that other structurally related folding enzymes (e.g., folding enzymes belonging to the same phylogenetic clade) will have similar beneficial effects on AprH expression.

Based on this finding, we propose that the bacillus species PrsA and the thermophilic lignin degrading bacteria PrsA, as well as closely related folding enzymes, can be used to improve the expression of proteases in general, in particular serine proteases, such as subtilisin (subtilisin).

Nucleic acid constructs

In a first aspect, the present invention relates to a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9.

The first and second heterologous promoters may be any heterologous promoter suitable for expression of the protease and the foldase, respectively. In one embodiment, the first heterologous promoter and the second heterologous promoter are the same or different promoters; preferably, the first heterologous promoter and the second heterologous promoter are identical copies of the same promoter.

The nucleic acid constructs of the invention comprise at least one (i.e., one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) polynucleotide encoding a protease. In some embodiments, a nucleic acid construct of the invention comprises two or more polynucleotides encoding two or more proteases, wherein the two or more proteases are the same or different proteases.

The protease may be any protease, such as a microbial protease, a plant protease, an animal protease or a human protease. Preferably, the protease is secreted. Preferably, the protease is a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamine protease, a metalloprotease, or an asparagine peptide cleaving enzyme. More preferably, the protease is a serine protease; even more preferably subtilases; subtilisin is most preferred.

In some embodiments, the protease comprises a C-terminal propeptide or an N-terminal propeptide and/or an N-terminal signal peptide. In some embodiments, the protease is a mature protease.

In preferred embodiments, the protease has 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% sequence identity to SEQ ID No. 3. More preferably, the protease comprises or consists of SEQ ID NO. 3. Most preferably, the protease is a bacillus clausii alkaline protease (AprH) or a variant thereof.

Preferably, the at least one protease-encoding polynucleotide has 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% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 1. More preferably, the at least one protease-encoding polynucleotide comprises or consists of SEQ ID NO. 1.

The nucleic acid construct of the invention further comprises at least one (i.e., one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) polynucleotide encoding a folding enzyme. The foldase has 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% sequence identity to SEQ ID NO 6 or SEQ ID NO 9. Preferably, the folding enzyme comprises or consists of SEQ ID NO 6 or SEQ ID NO 9. Most preferably, the folding enzyme is a bacillus species PrsA or variant thereof, or a thermophilic lignin degrading bacterium PrsA or variant thereof.

Preferably, the at least one polynucleotide encoding a foldase has 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% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 4 or SEQ ID NO. 7. Preferably, the at least one polynucleotide encoding a foldase comprises or consists of SEQ ID NO 4 or SEQ ID NO 7.

In some embodiments, the nucleic acid constructs of the invention comprise two or more polynucleotides encoding two or more folding enzymes, wherein the two or more folding enzymes are the same or different folding enzymes, i.e., Bacillus species PrsA (SEQ ID NO:6) and/or high temperature lignin-degrading bacteria PrsA (SEQ ID NO: 9). Thus, in some embodiments, two or more polynucleotides encoding two or more folding enzymes have 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% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 4 and/or SEQ ID NO. 7.

The at least one protease-encoding polynucleotide and the at least one foldase-encoding polynucleotide (i.e., the polynucleotides of the invention) are 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. The polynucleotide may be manipulated in a variety of ways to provide for expression of the protease and/or the folding enzyme. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

The control sequence may be a promoter, i.e., a polynucleotide, which is recognized by a host cell for expression of the polynucleotide. Preferably, the promoter is a heterologous promoter. The promoter contains transcriptional control sequences that mediate the expression of proteases and/or foldases. 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.

Examples of suitable promoters for directing transcription of the nucleic acid construct of the invention in a bacterial host cell are promoters obtained from the following genes: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase gene (amyQ), Bacillus licheniformis (Bacillus licheniformis) alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus (Bacillus stearothermophilus) maltogenic amylase gene (amyM), bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus,1994, Molecular Microbiology [ Molecular Microbiology ]13:97-107), Streptomyces coelicolor agarolytase gene (dagA) and prokaryotic beta-lactamase gene (Villa-Kamaroff et al, 1978, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci.USA ]75: 3727) and tac promoter (DeBoer et al, 1983, Proc. Natl. Acad. Sci. USA [ national academy of sciences ]80: 21-25). Other promoters are described in Gilbert et al, 1980, Scientific American [ Scientific Americans ]242:74-94, "Useful proteins from recombinant bacteria ]; and Sambrook et al, 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

The control sequence may also be a transcription terminator which is recognized by a host cell to terminate transcription. A terminator sequence is operably linked to the 3' -terminus of the polynucleotide of the invention. Any terminator which is functional in the host cell may be used.

Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH) and Bacillus licheniformis alpha-amylase (amyL).

The control sequence may also be an mRNA stability region downstream of the promoter and upstream of the coding sequence of the gene, which increases the expression of the gene.

Examples of suitable mRNA stability regions are obtained from: bacillus thuringiensis cryIIIA gene (WO 94/25612) and Bacillus subtilis SP82 gene (Hue et al, 1995, Journal of Bacteriology 177: 3465-.

The control sequence may also be a signal peptide coding region that codes for a signal peptide linked to the N-terminus of the protease and/or foldase and directs the protease and/or foldase into the cell's secretory pathway. The 5' end of the coding sequence of the polynucleotide of the invention may itself contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the protease and/or the foldase. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence in order to enhance secretion of the protease and/or foldase. However, any signal peptide coding sequence that directs the expressed protease and/or foldase into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for use in bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Other signal peptides are described by Simonen and Palva,1993, Microbiological Reviews [ Microbiological review ]57:109- & 137.

The control sequence may also be a propeptide coding sequence that codes for a propeptide positioned N-terminally of a protease and/or a foldase. The resulting polypeptide is called a pro-enzyme (proenzyme) or propolypeptide (or 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 gene for Bacillus subtilis alkaline protease (aprE) or Bacillus subtilis neutral protease (nprT).

In the case where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned next to the N-terminus of a protease and/or a folding enzyme and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate the expression of proteases and/or foldases associated with the growth of the host cell. Examples of regulatory systems are those that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.

Polynucleotide

The present invention also relates to polynucleotides encoding proteases and polynucleotides encoding folding enzymes of the present invention. In one embodiment, the polynucleotides have been isolated.

Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA or a combination thereof. Cloning of polynucleotides from genomic DNA can be performed, for example, by detecting cloned DNA fragments with shared structural features using the well-known Polymerase Chain Reaction (PCR) or antibody screening of expression libraries. See, e.g., Innis et al, 1990, PCR: A Guide to Methods and Application [ PCR: method and application guide ], Academic Press, New York. Other nucleic acid amplification procedures such as Ligase Chain Reaction (LCR), Ligation Activated Transcription (LAT) and polynucleotide-based amplification (NASBA) can be used. These polynucleotides may be cloned from a strain of Bacillus or a related organism and thus, for example, may be allelic or species variants of the polypeptide coding region of the polynucleotides of the invention.

Expression vector

In a second aspect, the present invention also relates to a recombinant expression vector comprising a nucleic acid construct comprising:

(a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease;

(b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9.

The expression vectors of the invention also comprise additional control sequences, such as transcription and translation termination signals.

Multiple polynucleotides and control sequences may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of a polynucleotide of the invention at such sites. Alternatively, the polynucleotide of the present invention 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 such that the coding sequence is operably linked with the appropriate control sequences for expression.

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 the 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.

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

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

Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance).

The vector preferably contains one or more elements that allow the vector to integrate into the genome of the host cell or the vector to replicate autonomously in the cell, independently of the genome.

For integration into the host cell genome, the vector may rely on the coding sequence of a polynucleotide of the invention or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the host cell genome at one or more precise locations in one or more chromosomes. To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, e.g., 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. Alternatively, the vector may be integrated into the genome of the host cell by non-homologous recombination.

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 replicon mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pUB110, pE194, pTA1060 and pAM β 1, which are allowed to replicate in Bacillus.

More than one copy of a polynucleotide of the invention may be inserted into a host cell to increase production of the protease and/or the folding enzyme. Increased 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, wherein cells comprising amplified copies of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected for by culturing the cells in the presence of the appropriate selectable agent.

Procedures for ligating the elements described above to construct the recombinant expression vectors of the invention are well known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).

Host cell

In a third aspect, the present invention also relates to gram-positive host cells comprising in their genome:

(i) a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9; and/or

(ii) An expression vector comprising the nucleic acid construct.

The nucleic acid construct and/or expression vector comprising the polynucleotide of the invention is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector, as described previously.

The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will to a large extent depend on the gene encoding the protease and its source.

The prokaryotic host cell may be any gram-positive cell for recombinant production of proteases. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, enterococcus, Geobacillus, Lactobacillus, lactococcus, Paenibacillus, Staphylococcus, Streptococcus, and Streptomyces.

The gram-positive host cell may be any Bacillus cell, including but not limited to, 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, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equisimilis (Streptococcus equisimilis), Streptococcus pyogenes (Streptococcus pyogenenes), Streptococcus uberis (Streptococcus uberis) and Streptococcus equi subsp.

The bacterial host cell may also be any streptomyces cell, including but not limited to: streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

Introduction of DNA into bacillus cells can be achieved by: protoplast transformation (see, e.g., Chang and Cohen,1979, mol.Gen. Genet. [ molecular and general genetics ]168: 111-.

The introduction of DNA into Streptomyces cells can be achieved by: protoplast transformation, electroporation (see, e.g., Gong et al, 2004, Folia Microbiol. (Praha) [ leaf-line microbiology (Bragg) ]49: 399-.

The introduction of DNA into Streptococcus cells can be achieved by: natural competence (natural competence) (see, e.g., Perry and Kuramitsu,1981, infection. Immun. [ infection and immunity ]32: 1295-.

However, any method known in the art for introducing DNA into a host cell may be used.

Production method

In a fourth aspect, the present invention also relates to a method of producing a protease, the method comprising:

I) providing a gram-positive host cell comprising:

i) a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9; and/or

ii) an expression vector comprising said nucleic acid construct;

II) culturing said gram-positive host cell under conditions conducive to the expression of the protease and the folding enzyme; and, optionally

III) recovering the protease.

Preferably, the gram-positive host cell is a bacillus host cell; preferably, the bacillus host cell is selected from the group consisting of: bacillus alkalophilus, Bacillus altivelis, Bacillus amyloliquefaciens subspecies, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus saffron, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

Gram-positive host cells are cultured in a nutrient medium suitable for the production of the protease using methods known in the art. For example, the cell may be cultured by shake flask culture, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the protease to be expressed and/or isolated. Culturing occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions, for example, in catalogues of the American Type Culture Collection. If the protease is secreted into the nutrient medium, the protease can be recovered directly from the medium. If the protease is not secreted, it can be recovered from the cell lysate.

Proteases can be detected using methods known in the art that are specific for proteases. These detection methods include, but are not limited to: the use of specific antibodies, the formation of enzyme products or the disappearance of enzyme substrates. For example, an enzymatic assay can be used to determine the activity of a protease.

The protease may be recovered using methods known in the art. For example, the protease may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

Proteases can be purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focus chromatography, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing electrophoresis), differential solubilization (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden editors, VCH Publishers [ VCH Publishers ], New York, 1989) to obtain substantially pure proteases.

In an alternative aspect, the protease is not recovered, but rather the gram-positive host cell of the invention expressing the protease is used as a source of the variant.

Fermentation broth formulations or cell compositions

The invention also relates to fermentation broth formulations or cell compositions comprising the protease of the invention and optionally a folding enzyme. The fermentation broth product further comprises additional components used in the fermentation process, such as, for example, cells (including gram-positive host cells containing polynucleotides encoding the proteases and foldases of the invention used to produce the proteases), cell debris, biomass, fermentation medium, and/or fermentation product. In some embodiments, the composition is a cell-killed whole broth comprising one or more organic acids, killed cells and/or cell debris, and culture medium.

The term "fermentation broth" as used herein means a preparation produced by fermentation of a cell that has not undergone or has undergone minimal recovery and/or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation by incubation under carbon-limited conditions that allow protein synthesis (e.g., expression of an enzyme by a host cell) and secretion of the protein into the cell culture medium. The fermentation broth may contain an unfractionated or fractionated content of the fermented material obtained at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises spent culture medium and cell debris present after removal of microbial cells (e.g., filamentous fungal cells), e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or non-viable microbial cells.

In one embodiment, the fermentation broth formulation and cell composition comprises a first organic acid component (comprising at least one organic acid of 1-5 carbons and/or salt thereof) and a second organic acid component (comprising at least one organic acid of 6 or more carbons and/or salt thereof). In particular 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, salts thereof, or mixtures of two or more of the foregoing.

In one aspect, the composition contains one or more organic acids and optionally further contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from the cell-killed whole broth to provide a composition free of these components.

The fermentation broth formulations or cell compositions may further comprise preservatives and/or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.

The cell-killed whole broth or composition may contain unfractionated contents of the fermented material obtained at the end of the fermentation. Typically, the cell-killing whole broth or composition contains spent medium and cell debris present after incubating gram-positive host cells to saturate their growth under carbon-limiting conditions that allow protein synthesis. In some embodiments, the cell-killed whole culture broth or composition contains spent cell culture medium, extracellular enzymes, and killed gram-positive cells. In some embodiments, methods known in the art can be used to permeabilize and/or lyse gram-positive host cells present in a cell-killed whole culture fluid or composition.

The whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, media components, and/or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

The whole broth formulations and cell compositions of the invention may be produced by the methods described in WO 1990/15861 or WO 2010/096673.

Enzyme composition

The invention also relates to compositions comprising a protease of the invention and optionally a folding enzyme.

These compositions may comprise the protease of the invention as the main enzyme component, e.g. a one-component composition. Alternatively, the compositions may comprise enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of: a hydrolase, isomerase, ligase, lyase, oxidoreductase or transferase; more preferably, the enzyme is an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, asparaginase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, green fluorescent protein, glucanotransferase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The composition may be prepared according to methods known in the art, and may be in the form of a liquid or dry composition. The composition may be stabilized according to methods known in the art.

Preferred embodiments

1) A nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9.

2) The nucleic acid construct of embodiment 1, wherein the first heterologous promoter and the second heterologous promoter are the same or different promoters; preferably, the first heterologous promoter and the second heterologous promoter are identical copies of the same promoter.

3) The nucleic acid construct according to any of the preceding embodiments, wherein the protease is a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamic protease, a metalloprotease, or an asparagine peptide cleaving enzyme.

4) The nucleic acid construct according to embodiment 3, wherein the protease is a serine protease; subtilases are preferred, subtilisins being most preferred.

5) The nucleic acid construct according to any of the preceding embodiments, wherein the protease comprises a C-terminal propeptide or an N-terminal propeptide and/or an N-terminal signal peptide; or wherein the protease is a mature protease.

6) The nucleic acid construct according to any of the preceding embodiments, wherein the protease has at least 80% sequence identity with SEQ ID No. 3.

7) The nucleic acid construct according to any of the preceding embodiments, wherein the protease comprises or consists of SEQ ID No. 3.

8) The nucleic acid construct according to any of the preceding embodiments, wherein the protease is a bacillus clausii alkaline protease (AprH) or a variant thereof.

9) The nucleic acid construct according to any of the preceding embodiments, wherein the at least one protease-encoding polynucleotide has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID No. 1.

10) The nucleic acid construct according to any of the preceding embodiments, wherein the at least one protease-encoding polynucleotide comprises or consists of SEQ ID No. 1.

11) The nucleic acid construct according to any of the preceding embodiments, wherein the at least one polynucleotide encoding a foldase has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 4 or SEQ ID NO. 7.

12) The nucleic acid construct according to any of the preceding embodiments, wherein the at least one polynucleotide encoding a foldase comprises or consists of SEQ ID NO 4 or SEQ ID NO 7.

13) An expression vector comprising a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9.

14) The expression vector according to embodiment 13, wherein the first heterologous promoter and the second heterologous promoter are the same or different promoters; preferably, the first heterologous promoter and the second heterologous promoter are identical copies of the same promoter.

15) The expression vector according to any one of embodiments 13-14, wherein the protease is a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamine protease, a metalloprotease, or an asparagine peptide cleaving enzyme.

16) The expression vector according to embodiment 15, wherein the protease is a serine protease; subtilases are preferred, subtilisins being most preferred.

17) The expression vector according to any one of embodiments 13-16, wherein the protease comprises a C-terminal propeptide or an N-terminal propeptide and/or an N-terminal signal peptide; or wherein the protease is a mature protease.

18) The expression vector according to any one of embodiments 13-17, wherein the protease has at least 80% sequence identity to SEQ ID No. 3.

19) The expression vector according to any one of embodiments 13-18, wherein the protease comprises or consists of SEQ ID No. 3.

20) The expression vector according to any one of embodiments 13-19, wherein the protease is a bacillus clausii alkaline protease (AprH) or a variant thereof.

21) The expression vector according to any one of embodiments 13-20, wherein the at least one protease-encoding polynucleotide has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID No. 1.

22) The expression vector according to any one of embodiments 13-21, wherein the at least one protease-encoding polynucleotide comprises or consists of SEQ ID No. 1.

23) The expression vector according to any one of embodiments 13-22, wherein the at least one polynucleotide encoding a foldase has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID No. 4 or SEQ ID No. 7.

24) The expression vector according to any one of the preceding embodiments, wherein the at least one polynucleotide encoding a foldase comprises or consists of SEQ ID NO 4 or SEQ ID NO 7.

25) A gram-positive host cell comprising in the genome of the gram-positive host cell:

(i) a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9; and/or

(ii) An expression vector comprising the nucleic acid construct.

26) The gram-positive host cell according to embodiment 25, wherein the gram-positive host cell is a bacillus host cell; preferably, the bacillus host cell is selected from the group consisting of: bacillus alkalophilus, Bacillus altivelis, Bacillus amyloliquefaciens subspecies, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus saffron, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

27) The gram-positive host cell according to any one of embodiments 25-26, wherein the first heterologous promoter and the second heterologous promoter are the same or different promoters; preferably, the first heterologous promoter and the second heterologous promoter are identical copies of the same promoter.

28) The gram-positive host cell according to any one of embodiments 25-27, wherein the protease is a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamine protease, a metalloprotease, or an asparagine peptide cleaving enzyme.

29) The gram-positive host cell according to embodiment 28, wherein the protease is a serine protease; subtilases are preferred, subtilisins being most preferred.

30) The gram-positive host cell according to any one of embodiments 25 to 29, wherein the protease comprises a C-terminal propeptide or an N-terminal propeptide and/or an N-terminal signal peptide; or wherein the protease is a mature protease.

31) The gram-positive host cell according to any one of embodiments 25 to 30, wherein the protease has at least 80% sequence identity to SEQ ID No. 3.

32) The gram-positive host cell according to any one of embodiments 25 to 31, wherein the protease comprises or consists of SEQ ID No. 3.

33) The gram-positive host cell according to any one of embodiments 25 to 32, wherein the protease is bacillus clausii alkaline protease (AprH) or a variant thereof.

34) The gram-positive host cell according to any one of embodiments 25 to 33, wherein the at least one polynucleotide encoding a protease has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

35) The gram-positive host cell according to any one of embodiments 25 to 34, wherein the at least one protease-encoding polynucleotide comprises or consists of SEQ ID No. 1.

36) The gram-positive host cell according to any one of claims 25 to 35, wherein the at least one polynucleotide encoding a foldase has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID No. 4 or SEQ ID No. 7.

37) The gram-positive host cell according to any one of claims 25 to 36, wherein the at least one polynucleotide encoding a foldase comprises or consists of SEQ ID No. 4 or SEQ ID No. 7.

38) A method for producing a protease, the method comprising:

I) providing a gram-positive host cell comprising in the genome thereof:

i) a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a protease; and

b) a second heterologous promoter operably linked to at least one polynucleotide encoding a foldase;

wherein the foldase has at least 80% sequence identity to SEQ ID NO 6 or SEQ ID NO 9; and/or

ii) an expression vector comprising said nucleic acid construct;

II) culturing said gram-positive host cell under conditions conducive to the expression of the protease and the folding enzyme; and, optionally

III) recovering the protease.

39) The method according to embodiment 38, wherein the gram-positive host cell is a bacillus host cell; preferably, the bacillus host cell is selected from the group consisting of: bacillus alkalophilus, Bacillus altivelis, Bacillus amyloliquefaciens subspecies, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus saffron, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

40) The method according to any one of embodiments 38-39, wherein the first heterologous promoter and the second heterologous promoter are the same or different promoters; preferably, the first heterologous promoter and the second heterologous promoter are identical copies of the same promoter.

41) The method according to any one of embodiments 38-40, wherein the protease is a serine protease, a cysteine protease, a threonine protease, an aspartic protease, a glutamine protease, a metalloprotease, or an asparagine peptide cleaving enzyme.

42) The method of embodiment 41, wherein the protease is a serine protease; subtilases are preferred, subtilisins being most preferred.

43) The method according to any one of embodiments 38-42, wherein the protease comprises a C-terminal propeptide or an N-terminal propeptide and/or an N-terminal signal peptide; or wherein the protease is a mature protease.

44) The method according to any one of embodiments 38-43, wherein the protease has at least 80% sequence identity to SEQ ID NO 3.

45) The method according to any one of embodiments 38-44, wherein the protease comprises or consists of SEQ ID NO 3.

46) The method according to any one of embodiments 38-45, wherein the protease is a Bacillus clausii alkaline protease (AprH) or a variant thereof.

47) The method according to any one of embodiments 38-46, wherein the at least one polynucleotide encoding a protease has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 1.

48) The method according to any one of embodiments 38-47, wherein the at least one protease-encoding polynucleotide comprises or consists of SEQ ID NO: 1.

49) The method according to any one of embodiments 38-48, wherein the at least one polynucleotide encoding a foldase has at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO. 4 or SEQ ID NO. 7.

50) The method according to any one of embodiments 38-49, wherein the at least one polynucleotide encoding a foldase comprises or consists of SEQ ID NO 4 or SEQ ID NO 7.

The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

Examples of the invention

Materials and methods

The chemicals used as buffers and substrates are commercial products of at least reagent grade.

PCR amplification was performed using a standard textbook program using a commercial thermal cycler and Ready-To-Go PCR beads, Phusion polymerase or RED-TAQ polymerase from commercial suppliers.

LB agar: see EP 0506780.

LBPSG agar plates contain LB agar, supplemented with phosphate (0.01M K)₃PO₄) Glucose (0.4%) and starch (0.5%); see EP 0805867B 1.

TY (liquid broth): see WO 94/14968, page 16.

Oligonucleotide primers were obtained from the continental group (Eurofins) (olhuss, denmark). DNA manipulations (plasmid and genomic DNA preparation, restriction digestion, purification, ligation, DNA sequencing) were performed using standard textbook procedures with commercially available kits and reagents.

DNA was introduced into B.subtilis rendered naturally competent cells using either a two-step procedure (Yasbin et al, 1975, J.Bacteriol. [ J.Bacteriol ]121: 296. multidot. 304.) or a one-step procedure in which the cell material from the agar plates was resuspended in Spizisen 1 medium (12ml) (WO 2014/052630), DNA was added to 400 microliter aliquots with shaking at 200rpm for about 4 hours at 37 ℃ and these aliquots were shaken at 150rpm for an additional 1 hour at the desired temperature prior to plating on selective agar plates.

All constructs described in the examples were assembled from synthetic DNA fragments ordered from genetic arts-seemer flying siell science, GeneArt-ThermoFisher Scientific. As described in the examples below, fragments are assembled by Sequence Overlap Extension (SOE).

Genomic DNA was prepared from all relevant isolates using a commercially available QIAamp DNA blood kit from Qiagen (Qiagen).

Standard microplate batch fermentation

The PrsA library strain was grown in biological triplicate in 500mL LB medium in a 96 deep well plate (CR1496b, enzyme screening (Enzyscreen)) covered with Sandwich lid (Sandwich Covers) (CR1296, enzyme screening). Cultures were grown in a clamping system (Clamp Systems) (CR1700, enzyme screening Co.) at 37 ℃ and 300rpm for 24 hours (1). After 24 hours, a sample was taken for enzyme activity measurement.

All assays were performed in 96-well microtiter plates and each sample was measured simultaneously at 2 different dilutions using bacillus clausii alkaline protease (AprH) as standard. The assay was performed on a Biomek Fx liquid handler and absorbance readings were measured on a Spectramax plate reader (Molecular Devices).

Samples were diluted in dilution buffer (Tris pH 9.0+ 0.01% Triton X). Mu.l of the diluted sample was mixed with a substrate solution (0.6mg/ml Suc-ala-ala-pro-phe-pNA, Baheng (Bachem)) in a dilution buffer. The kinetic absorbance at 405nM was measured immediately for 5 minutes and the results were extrapolated from the corresponding standard curve.

Bacterial strains

Construction of phylogenetic trees

The phylogenetic tree shown in FIG. 1 is constructed by: ClustalW alignment (Bioconductor 3.8, "msa" package, R) was performed, followed by construction of identity matrices (discrimination function in R [ R ], W.M.Fitch, s.l.: J.mol.biol. [ J.mol.biol. ], Vol.16, pages 9-16) and adjacency tree estimates (adjacency function in nj function in R [ R ], N.Saitou and M.Nei, s.l.: Molecular Biology and Evolution [ Molecular Biology and Evolution ], Vol.4, page 406 425). This tree is drawn as a rootless tree.

EXAMPLE 1 construction of the Bacillus subtilis host AN2

As described in the examples below, bacillus subtilis AN2 was used as the host strain for expression of the prsA and protease genes. Strain AN2 is a sporulation-deficient derivative of Bacillus subtilis 168, since 297bp is deleted in the sigF gene (the complete sigF gene sequence is provided as SEQ ID NO:10 and the inactive version comprising the deletion is provided as SEQ ID NO: 11).

Example 2 construction of the prsA Gene expression cassette and chromosomal integration of these genes in B.subtilis AN2

Bacillus subtilis strain AN2 was used as the host strain for insertion of the expression cassette for the prsA gene copy. The PrsA expression cassette is integrated into the pel locus by the synthetic promoter P_consSDHeel prsA gene and natural prsA of Bacillus subtilisAnd (3) a terminator. The DNA for integration can be assembled by PCR amplification of synthetic DNA consisting of the following DNA components: pel 5 'region + ermC (resistance to erythromycin) + synthetic consensus promoter with SD sequence (PconsSD) + prsA open reading frame with terminator + pel 3' region. The purified PCR products were used in subsequent PCR reactions to generate single linear DNAs using the overlap extension gene Splicing (SOE) method (Horton RM 1989) and Phusion hot start DNA polymerase system (Thermo Scientific) as shown below: the PCR amplification reaction mixture contained 50ng each of the gel-purified PCR products, and DNA was assembled and amplified using a thermal cycler. The resulting SOE product was used directly for transformation of the Bacillus subtilis host AN 2. Homologous recombination promotes chromosomal integration and selects for cells that undergo double crossover events on LB agar plates containing 1. mu.g/ml erythromycin. A schematic of the linear DNA product used to integrate the prsA gene into AN2 is shown in FIG. 2, and the DNA sequence used to integrate the prsA gene from Bacillus species into AN2 to produce strain AN2406 is provided as SEQ ID NO. 12. A strain expressing the prsA gene from Thermoligninolytic bacteria (DNA sequence provided as SEQ ID NO: 13) was constructed in a similar manner and designated AN 2407.

Example 3 construction of the Bacillus clausii alkaline protease Gene (aprH) expression cassette and chromosomal integration of the Bacillus clausii alkaline protease Gene in Bacillus subtilis strains AN2, AN2406 and AN2407

The aprH expression cassette, consisting of the synthetic promoter P, was integrated into the amyE locus of Bacillus subtilis AN2, AN2406 and AN2407_consSDThe heel aprH gene and the Bacillus amyloliquefaciens amyQ terminator. The amyE gene was inactivated during this process. The DNA for integration can be assembled by PCR amplification of synthetic DNA consisting of the following DNA components: amyE 5 'region + synthetic consensus promoter with SD sequence (PconsSD) + aprH open reading frame + Bacillus amyloliquefaciens amyQ terminator + cat gene (resistance to chloramphenicol) + amyE 3' region. The purified PCR product was used in subsequent PCR reactions to generate a single linear DNA using the SOE method described in example 2. The SOE product obtained was used directly for transformation of Bacillus subtilis strain AN2 (to produceThe strain AQG88, the strain AN2406 (strain AQG825 was produced) and the strain AN2407 (strain AQG812 was produced). Homologous recombination promotes chromosomal integration and cells undergoing double crossover events were selected on LB agar plates containing 6. mu.g/ml chloramphenicol. Strain AQG88 expresses Bacillus Clausensis AprH (SEQ ID NO:3) from the amyE locus and contains the native pel locus. Strain AQG825 expresses PrsA (SEQ ID NO:6) from a Bacillus species from the pel locus and AprH from the amyE locus. Strain AQG812 expresses PrsA (SEQ ID NO:9) from thermophilic lignin-degrading bacteria from the pel locus and AprH from the amyE locus. A schematic of the linear DNA product used to integrate the aprH gene in Bacillus subtilis strains AN2, AN2406 and AN2407 is shown in FIG. 3, and the DNA sequence is provided as SEQ ID NO. 14.

Example 4 expression of proteases in batch cultures with Bacillus subtilis AQG88, AQG825 and AQG812

The bacillus subtilis strain constructed in example 3 was tested for protease productivity in standard microplate batch cultures as described above. The culture was incubated in biological triplicate for 24 hours, as described above, after which the supernatant was collected for subsequent protease activity measurements. In this example, we obtained greater than 14% protease activity from cultures containing AQG825 expressing Bacillus species PrsA (SEQ ID NO:6) and greater than 23% protease activity from cultures expressing AQG812 of Thermolignin-degrading bacteria PrsA (SEQ ID NO:9) compared to cultures with AQG88 not expressing any heterologous prsA gene.