Novel system for producing recombinant proteins

Abstract

The present invention relates to a novel system for producing recombinant proteins in Streptomyces host strains, including vectors and novel recombinant host strains, in particular for the production of Actinoallomurus endopeptidase.

Description

[Technical Field]

[0001] This invention relates to a novel system for producing recombinant proteins in Streptomyces host strains.

[0002] This novel system includes, in particular, vectors and novel recombinant host strains for the production of Actinoallomurus endopeptidase. [Background technology]

[0003] Recombinant DNA (rDNA) technology provides a very powerful set of technological platforms for controlling and scalably producing polypeptides of interest using relatively inexpensive procedures. Recombinant proteins are typically obtained by recombinant DNA technology in E. coli, budding yeast, insects, hamsters, and mammalian cells. However, there is a demand for improved mass production of recombinant proteins. Furthermore, when proteins are produced for human use (e.g., as food supplements, and / or as pharmaceuticals in the prevention and / or treatment of human diseases), there are very stringent requirements that must be met. Actinomycetes are considered a safe source of protein for human consumption. Two examples of food enzymes derived from the genus Streptomyces are glucose isomerase, used in the production of fructose syrup (Non-Patent Literature 1), and the widely used transglutaminase from S. mobaraensis, which are used in the food industry for their properties that improve the texture and overall quality of finished foods, such as processed meats and fish products, and everyday goods and baked goods (Non-Patent Literature 2).

[0004] S. lividans cells are known to be efficient host cells for recombinant protein production because recombinant proteins expressed in these cells can be directly secreted and released into the culture medium. However, various proteins can be obtained in very different yields in S. lividans, and this result is unpredictable (Non-Patent Literature 1).

[0005] To improve the yield of heterologous proteins, a collection of derivative strains of S. lividance TK24 (TK24 taxonomy ID: 457428) was constructed by sequentially deleting known potentially interfering secondary metabolite gene clusters. Non-patent document 3 discloses the efficient production of mitramycin A in yields close to 3 g / L using this optimized strain collection. Nevertheless, the reproducibility of high yields is not guaranteed with any polynucleotide encoding polypeptide. In addition, there is still room for further improvement in providing recombinant protein production systems.

[0006] The present invention aims to provide an improved system for the efficient production of recombinant proteins in Streptomyces, preferably Streptomyces lividans, host strains.

[0007] In particular, the system of the present invention has been found to be especially useful for the mass production of recombinant endopeptidase (endopeptidase 40 or E40) of the Actinoaromurus strain.

[0008] The native E40 protein consists of 398 amino acid residues (see SEQ ID NO: 3), with the N-terminal signal peptide identified between positions 1 and 27. The mature form of the protein (E40mat) is predicted to be a 32.5 kDa polypeptide of the sequence SEQ ID NO: 1, starting at position 74 of the native protein (see Figure 1).

[0009] In the following, "E40 prepro" is used to identify the natural prepro endopeptidase containing its natural signal peptide or a derivative thereof, consisting of the sequence of SEQ ID NO: 3 (e.g., labeled E40 prepro having the sequence of SEQ ID NO: 4 (his-labeled E40 prepro)). In the following, "E40pre" is used to identify the E40 proenzyme that does not contain the signal peptide consisting of the amino acid sequence of SEQ ID NO: 9. In the following, "E40mat" is used to identify the mature endopeptidase or a derivative thereof, consisting of the amino acid sequence of SEQ ID NO: 1 (e.g., labeled E40mat having the amino acid sequence of SEQ ID NO: 2 (E40Hismat)).

[0010] The aforementioned endopeptidase was first disclosed in Patent Document 1 and has been shown to decompose gluten peptides into non-toxic peptides very rapidly and efficiently, be active across the entire pH range of the gastric and intestinal environment (enzyme activity is shown in the pH range of pH 3 to 6, optimal at pH 5), and be resistant to degradation by endogenous gastrointestinal enzymes. For these reasons, the actinoaromulus endopeptidase is highly suitable for use in the treatment and / or prevention of celiac disease (CD) and CD-related diseases. Therefore, there is strong interest in developing an efficient and inexpensive method for producing the actinoaromulus endopeptidase.

[0011] Patent Document 2 discloses a method for producing recombinant actinoalomulus endopeptidase in S. lividance host cells. Here, E40 expression is preferably carried out in S. lividance TK24 strain using a high-copy replication expression vector (pIJ86, SEQ ID NO: 7).

[0012] Some replication plasmids, particularly those with high copy numbers, have been successfully used for the overproduction of heterologous proteins related to many biotechnologies in actinomycetes. However, replication vectors have several drawbacks. For example, they can only be introduced into actinomycetes through laborious protoplast transformation, and their stable maintenance within the actinomycetes requires the selection of permanent antibiotics.

[0013] Integration vectors containing DNA segments encoding binding / integration function (Att / Int) for site-specific integration into specific sites of the Streptomyces genome (e.g., the attB site for a particular integrase) have also been employed to express recombinant proteins in Streptomyces. Most site-specific integration systems are based on temperate bacteriophages (actinophages) from various actinomycetes identified in Streptomyces species. Temperate bacteriophages (and integration vectors prepared using their functional elements) are integrated into the host chromosome at specific sites by a recombination process that requires a specific binding site between the phage (or integration vector) attP and the host chromosome attB.

[0014] Despite some advantages, these Att / Int systems also have some limitations, such as integrating the entire embedded plasmid with the E. coli replicon, integrase gene, and resistance marker gene. This can be a significant biotechnological obstacle, as it may affect the stability of the construct. In fact, instability in some Att / Int systems has been reported in several Streptomyces strains (Non-Patent Literature 4). Therefore, these embedded vector methods have been considered unsuitable for constructing stable biotechnology-production strains.

[0015] Furthermore, several other elements (e.g., regulatory elements of expression cassettes cloned into expression vectors) also contribute to the efficiency of recombinant protein production systems.

[0016] Therefore, achieving satisfactory protein yields and avoiding undesirable drawbacks in the field of recombinant protein production is by no means an easy or straightforward challenge.

[0017] The present invention provides a surprisingly improved system for producing a recombinant protein, preferably a recombinant Actinoallomurus endopeptidase, in a host cell of Streptomyces, preferably S. lividans.

PRIOR ART DOCUMENTS

PATENT DOCUMENTS

[0018]

PATENT DOCUMENT 1

PATENT DOCUMENT 2

NON-PATENT DOCUMENTS

[0019]

NON-PATENT DOCUMENT 1

NON-PATENT DOCUMENT 2

NON-PATENT DOCUMENT 3

NON-PATENT DOCUMENT 4

SUMMARY OF THE INVENTION

[0020] The system of the present invention for the production of a recombinant protein in a Streptomyces host cell, preferably an S. lividans strain, comprises a strong promoter [the artificial kasO promoter having the sequence of SEQ ID NO: 10 (kasOp *The present invention comprises a novel recombinant vector having an expression cassette containing a polynucleotide encoding the target recombinant protein under the control of (), and a regulatory element which is the SR40 ribosome-binding site (RBS), a synthetic variant of the sequence of Sequence ID No. 11 based on the PhiC31 capsid protein gene (Bai et al. 2015, Proc Natl Acad Sci USA 112, 12181-12186). The recombinant vector of the present invention is a single-site integration vector based on the phage PhiBT1, and is preferably integrated into the attB site in the Streptomyces genome.

[0021] The polynucleotide encoding the target recombinant protein encodes a recombinant preprotein containing a signal peptide. In a preferred embodiment, the signal peptide is a heterologous signal peptide, more preferably a signal peptide vsi of the subtilisin inhibitor of Streptomyces venezuelae having the sequence of SEQ ID NO: 12 (Lammertyn et al. 1997 Appl Environ Microbiol 63, 1808-1813).

[0022] Preferably, the recombinant protein of interest is actinoaromulus endopeptidase, preferably actinoaromulus endopeptidase 40 (E40), a biologically active fragment, variant, or derivative thereof.

[0023] The system of the present invention also includes a novel recombinant host strain containing a novel vector. The present invention also relates to the use of the novel system of the present invention for the production of recombinant proteins by secretion in the culture medium of host cells, particularly for the production of actinoaromulus endopeptidase, and to a method for producing recombinant proteins using the novel system of the present invention.

[0024] Remarkably, despite using a single-copy vector, the system of the present invention provides stable and high-volume expression of recombinant protein. [Brief explanation of the drawing]

[0025] [Figure 1] Sequence of the natural E40 prepro enzyme (E40 prepro). Its signal peptide is shown in bold, and the mature enzyme is underlined. [Figure 2] A scheme of the prior art recombinant vector pIJ86 / e40, containing the e40 gene (with and without 6xHis labeling) encoding the E40pre endopeptidase, inserted between the BamHI and HindIII sites of the cloning vector pIJ86. The sequences of the promoter's -10 and -35 regions (bold and underlined), TSS (bold, underlined and highlighted), and the ermEp* promoter region (SEQ ID NO: 30) containing the BamHI / BclI fusion with e40 are shown below the plasmid scheme. The amino acids of E40 are located at the second position of each codon. [Figure 3] Scheme of the expression vector pMU1s-kasOpSR40vsi3mRFPA. Below the plasmid scheme, the sequence of the vsi-mRFP fusion with the restriction site (bold and underlined) inserted is shown. The amino acids of the Vsi signal peptide (bold) and mRFP (red) are located at the second position of each codon. [Figure 4] 13.5% SDS-PAGE of 20 μl culture medium samples (two separate clones) from S. lividance TK24 containing pMU1s-kasOpSR40vsi3mRFPA, grown in TSB medium to various time points, after mRFP secretion. Lane: 1 = LMW standard; 2-5 = TK24 pMU1s-kasOpSR40vsi3mRFPA cultured for 24 hours, 48 ​​hours, 72 hours, and 146 hours, respectively; 10 = TK24 cultured for 48 hours. [Figure 5] A scheme for the cloning vector pLit-kasOpSR40vsi3mRFP, which contains a robust kasOp* promoter, an optimized robust SR40 RBS, and a single NdeI site inserted into the ATG codon of the vsi-mRFP reporter fusion gene. [Figure 6]Schemes of recombinant plasmids pLit-e40 and pLit-e40His containing the entire e40 gene with the original signal peptide sequence (6xHis labeled and unlabeled, respectively) inserted between the NdeI and AvrII sites of the cloning vector pLit kasOpSR40vsi3mRFP. The sequences of the kasOp* promoter, including the -10 and -35 regions of the promoter (bold and underlined), its TSS (bold, underlined and highlighted), RBS (bold and underlined), and the NdeI fusion with e40, are shown below the plasmid scheme. The amino acids of E40 are located at the second position of each codon. [Figure 7] A scheme of the cloning expression vector pMU1s-kasOpSR40gusA, which contains a robust kasOp* promoter, an optimized robust SR40 RBS, and a single NdeI site inserted into the ATG codon of the gusA reporter gene. The sequences of this promoter and RBS region, including the -10 and -35 regions of the promoter (bold and underlined), its TSS (bold, underlined and highlighted), and the RBS (bold and underlined), are shown below the plasmid scheme. [Figure 8] Schemes of recombinant plasmids pLit-vsie40pre, pLit-e40Hispre, pLitvsie40HQHispre, pLit-vsie40mat, and pLit-vsie40Hismat, containing the vsi-e40pre gene fusion of Vsi and the proenzyme E40pre, and the vsi-e40mat gene fusion of Vsi and the mature enzyme E40mat (each with and without 6xHis labeling), inserted between the NheI and HindIII sites of the cloning vector pLit-kasOpSR40vsi3mRFP. The sequences of the kasOp* promoter, including the -10 and -35 regions of the promoter (bold and underlined), its TSS (bold, underlined and highlighted), RBS (bold and underlined), and the NdeI and NheI of the vsi-E40 fusion site, are shown below the plasmid scheme. [Figure 9]Schemes for recombinant plasmids pMU1s-e40, pMU1s-e40His, pMU1s-vsie40pre, pMU1s-vsie40Hispre, pMU1s-vsie40HQHispre, pMU1s-vsie40mat, and pMU1s-vsie40Hismat containing all forms of the e40 gene fusion as KpnI EcoRV DNA fragments from a pLit plasmid (see Figures 6 and 8) inserted between the KpnI and EcoRV sites of the cloning vector pMU1s-kasOpSR40gusA (Figure 7). [Figure 10] Schemes for recombinant plasmids pIJ86-kasOpe40, pIJ86-kasOpe40His, pIJ86-vsie40pre, pIJ86-vsie40Hispre, pIJ86-vsie40HQHispre, pIJ86-vsie40mat, and pIJ86-vsie40Hismat, containing all forms of the e40 gene fusion (shown in Figures 6 and 8) (with and without 6xHis labeling, respectively) as KpnI-EcoRV DNA fragments from a pLit plasmid (Figures 6 and 8) inserted between the KpnI and HindIII sites of the cloning vector pIJ86 (Figure 2). [Figure 11] A scheme for recombinant plasmid pMU1s-ermEe40, containing an ermEp*-e40His fusion from plasmid pIJ86 / e40His, which contains the e40 gene with 6xHis labeling. [Figure 12] E40 activity in wild-type and mutant S. lividan RedStrep1.3 strains conjugated to prior art high-copy replication plasmids. [Figure 13] E40 activity in wild-type and mutant S. lividance RedStrep strains conjugated to a single-site embedded plasmid expressing native E40 under the ermEp* promoter. [Figure 14A] E40 activity in wild-type S. lividance strain conjugated to a single-site embedded plasmid expressing native E40 under the kasOp* promoter and optimized potent SR40 RBS according to the present invention. [Figure 14B]E40 activity in wild-type S. lividance strain conjugated to a single-site embedded plasmid expressing His-labeled E40 under the kasOp* promoter and optimized potent SR40 RBS according to the present invention. [Figure 15A] E40 activity in wild-type S. lividance strains conjugated to a high-copy pIJ86 plasmid expressing E40 under the kasOp* promoter and optimized potent SR40 RBS. [Figure 15B] E40 activity in wild-type S. lividance strains conjugated to a high-copy pIJ86 plasmid expressing E40His label under the kasOp* promoter and optimized potent SR40 RBS. [Figure 16A] E40 activity in wild-type S. lividance strain conjugated to the high-copy pIJ86 plasmid pIJ86 containing vsi-e40pre under the kasOp* promoter and optimized potent SR40 RBS. [Figure 16B] E40 activity in wild-type S. lividance strain conjugated to the high-copy pIJ86 plasmid pIJ86 containing His-labeled vsi-e40pre under the kasOp* promoter and optimized potent SR40 RBS. [Figure 16C] E40 activity in wild-type S. lividance strain conjugated to the high-copy pIJ86 plasmid pIJ86 containing vsi-e40mat under the kasOp* promoter and optimized potent SR40 RBS. [Figure 16D] E40 activity in wild-type S. lividance strain conjugated to the high-copy pIJ86 plasmid pIJ86 containing the His-labeled vsi-e40mat fusion gene under the kasOp* promoter and optimized potent SR40 RBS. [Figure 17A] E40 activity in wild-type S. lividance strains conjugated to a single-site embedded plasmid containing vsi-e40pre under the kasOp* promoter and optimized potent SR40 RBS. [Figure 17B]E40 activity in wild-type S. lividance strain conjugated to a single-site embedded plasmid containing His-labeled vsi-e40pre under the kasOp* promoter and optimized potent SR40 RBS. [Figure 18A] E40 activity in wild-type or mutant S. lividance RedStrep strains conjugated to a single-site embedded plasmid expressing native E40 under the kasOp* promoter and optimized potent SR40 RBS. [Figure 18B] E40 activity in wild-type or mutant S. lividance RedStrep strains conjugated to a single-site embedded plasmid expressing the native E40His label under the kasOp* promoter and optimized potent SR40 RBS. [Figure 19A] E40 activity in wild-type or mutant S. lividance RedStrep strains conjugated to a single-site embedded plasmid expressing vsi-e40pre under the kasOp* promoter and optimized potent SR40 RBS. [Figure 19B] E40 activity in wild-type or mutant S. lividance RedStrep strains conjugated to a single-site embedded plasmid expressing His-labeled vsi-e40pre under the kasOp* promoter and optimized potent SR40 RBS. [Modes for carrying out the invention]

[0026] First, the present invention relates to a recombinant vector having an expression cassette for heterologous expression of recombinant proteins in Streptomyces host cells. The cassette comprises a promoter, a regulatory element downstream of the promoter, and a polynucleotide encoding a recombinant protein, located downstream of the promoter and the regulatory element and operably linked thereto.

[0027] The terms “vector,” “expression vector,” and “plasmid” are used interchangeably in this specification.

[0028] The expression vector for heterologous expression of recombinant proteins in Streptomyces host cells of the present invention is a single-site integration vector containing an attP site and an integrase gene derived from the PhiBT1 phage, and more preferably a vector having the sequence of SEQ ID NO: 13, which is integrated into the attB site of Streptomyces.

[0029] An example of a single-site embedded vector according to the present invention is the one described in Kormanec J. et al., Applied Microbiology and Biotechnology (2019) 103:5463-5482.

[0030] In a preferred embodiment, the single-site embedded vector is a vector having a pMU1 vector backbone (Craney et al. 2007, Nucleic Acids Res 35, e46).

[0031] The promoter of the expression cassette is an artificial kasO promoter (kasOp) of the sequence of sequence number 10. * ) and the regulatory element of the expression cassette is the synthetic ribosome binding site (SR40 RBS) of the sequence of SEQ ID NO: 11; and the polynucleotide encoding the recombinant protein of interest is a polynucleotide encoding a recombinant protein containing a signal peptide.

[0032] The expression cassette of the present invention is particularly suitable for heterologous expression of recombinant proteins in Streptomyces host cells. Recombinant proteins are expressed by host cells cultured in a medium under conditions suitable for host cell proliferation and secreted into the medium. The signal peptide promotes the secretion of proteins into the medium.

[0033] In some cases, the signal peptide is a heterologous signal peptide, more preferably a Vsi signal peptide of the subtilisin inhibitor of Streptomyces venezuelae having the sequence of SEQ ID NO: 12. Surprisingly, the use of a heterologous signal peptide fused to the target protein according to the present invention provides good recombinant protein secretion as well as the protein's natural signal peptide.

[0034] In a preferred embodiment of the present invention, the recombinant protein encoded by the polynucleotide of the expression cassette is a recombinant actinoaromulus endopeptidase having glutenase activity. More preferably, the endopeptidase is endopeptidase 40 (E40) of a sequence containing SEQ ID NO: 1, a biologically active fragment of E40, a naturally occurring allelic variant of E40; or an endopeptidase of a sequence having at least 60%, 70%, 80%, 90%, or 95% identity with SEQ ID NO: 1.

[0035] Sequence ID 1 is the mature polypeptide sequence of E40.

[0036] Therefore, preferably, the expression cassette comprises an endopeptidase E40 of a sequence containing SEQ ID NO: 1, a biologically active fragment of E40, a naturally occurring allelic variant of E40, or a polynucleotide encoding an endopeptidase of a sequence having at least 60%, 70%, 80%, 90%, or 95% identity with SEQ ID NO: 1.

[0037] The term "biologically active fragment" of E40 refers to the portion that maintains the specific glutenase activity of the endopeptidase. The polynucleotide encoding the "biologically active fragment" of E40 can be identified as disclosed in International Publication No. 2021 / 013553.

[0038] Because the polynucleotide encoding the target recombinant protein may have a different sequence from the annotated nucleic acid sequence due to the degeneracy of the genetic code, it will encode the same protein as the polynucleotide with the annotated nucleic acid sequence.

[0039] In this specification, the terms “identity” or “homology” when referring to nucleotide or amino acid sequences are used interchangeably and refer to the degree to which two polynucleotide or polypeptide sequences are identical or homologous residue by residue across a particular region being compared. Sequences and percentages of identity or homology can be determined using any suitable software program known in the Art, for example, those described in Current Protocols in Molecular Biology (Ausubel FM et al., “Commercially Available Software”, Current Protocols in Molecular, 1987, Supplement 30, Section 7.7.18, Table 7.7.1). Preferred programs include the GCG Pileup program, FASTA (Pearson R. and Lipman DJ, "Improved Tools for Biological Sequence Analysis," Proc. Natl., Acad. Sci. USA, 1988, 85, 2444-2448), and BLAST (Altschul SF, Gish W., Miller W., Myers EW, Lipman DJ, "Basic local alignment search tool," J. Mol. Biol., 1990, 215, 403-410).

[0040] The term "allele variant" refers to one of two or more alternative forms of a gene that occupies the same chromosomal locus. Allele mutations can occur spontaneously through spontaneous mutation and can also result in phenotypic polymorphism within a population. Gene mutations can be silent (the encoded polypeptide remains unchanged) or they can encode a polypeptide with altered amino acid sequences. The term allele variant also refers to the protein encoded by an allele variant of a gene.

[0041] The polynucleotide encoding the target recombinant protein may encode a protein operably fused to another polypeptide, such as a histidine label. For example, the polynucleotide may encode a labeled protein, such as a polynucleotide encoding a label E40 containing a sequence of sequence number 2 or sequence consisting of sequence number 2. In a preferred embodiment, the polynucleotide encoding the target protein is a polynucleotide containing a sequence of sequence number 5, 6, 15, 16, 17, or 18 or sequence consisting of sequence number 5, 6, 15, 16, 17, or 18, and the expression cassette is the sequence of sequence number 20, 21, 22, or 23.

[0042] Therefore, in a preferred embodiment, the present invention relates to a single-site embedded vector, more preferably in Streptomyces host cells (preferably S. lividance strain), in which actinoaromulus endopeptidase, preferably actinoaromulus endopeptidase 40 (E40), a biologically active fragment, variant, or derivative thereof is embedded in an artificial kasO promoter (kasOp) of the sequence of SEQ ID NO: 10. * The present invention relates to a vector for embedding into the attB site for the PhiBT1 phage of Streptomyces, having an expression cassette for expression under the control of the ribosome-binding site (RBS) (SR40 RBS) of the sequence of SEQ ID NO: 11. In some cases, the recombinant protein is actinoalomulus endopeptidase in which the signal peptide is replaced with the Vsi signal peptide.

[0043] More preferably, a single-site embedded vector having an expression cassette for the expression of actinoalomulus endopeptidase 40 (E40) is a pMU1 vector of sequence number 25, 26, 27, or 28.

[0044] The present invention also relates to host cells for heterologous expression of recombinant proteins containing the expression vector according to the present invention, preferably recombinant Streptomyces host cells, more preferably S. lividance host cells. The host cells may be wild-type S. lividance host cells or mutant S. lividance host cells, for example, S. lividance RedStrep1.3 strain (Δact, Δred, Δcda), S. lividance RedStrep1.6 strain (Δact, Δred, Δcda, Δmel), or S. lividance RedStrep1.9 strain (Δact, Δred, Δcda, Δmel, ΔmatAB) (Novakova et al., 2018).

[0045] Preferably, the host cells are cells of strain DSM33930 (S. lividance 1.3 conjugated with pMU1s-e40His) and strain DSM33931 (S. lividance 1.9 conjugated with pMU1s-e40His) deposited with the German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B 38124 Braunschweig-GERMANY, Leibniz Institute DSMZ on June 24, 2021, in accordance with the provisions of the Budapest Convention.

[0046] Next, the present invention also relates to the use of recombinant vectors or host cells of the present invention for the production of recombinant proteins secreted into the culture medium of host cells.

[0047] Advantageously, the system of the present invention provides stable and large-scale production of the target protein. Furthermore, the protein can be easily recovered from the host cell culture medium from which it is secreted.

[0048] In fact, the system of the present invention also relates to a method for producing recombinant proteins in Streptomyces host cells, comprising the following steps in sequence: culturing recombinant Streptomyces host cells, preferably S. lividance host cells, more preferably strain TK24, in a culture medium under fermentation conditions; the recombinant host cells comprising the recombinant expression vector according to the present invention; collecting the supernatant of the culture medium; and purifying a preparation containing recombinant proteins from the supernatant.

[0049] "Fermentation conditions" refers to the host cell line culture conditions (culture medium composition, stirring parameters, aeration, and temperature) suitable for the growth of the host cell line and the production of the target compound (recombinant endopeptidase). Suitable fermentation conditions may be those described in, for example, International Publication No. 2021 / 013553, or those described in the following examples.

[0050] All possible combinations of preferred embodiments of the present invention are also described and should therefore be understood to be equally preferred.

[0051] Examples of embodiments of the present invention are shown below, provided for illustrative and non-limiting purposes. [Examples]

[0052] plasmid Table 1 summarizes the plasmids prepared in Examples 1-6 below. For each plasmid, the promoter, RBS, signal peptide, gene, and vector backbone are reported.

[0053] [Table 1]

[0054] host cell In Examples 7-14, the wild-type strain TK24 (Taxonomy ID: 457428) and the S. lividance mutant strains RedStrep 1.3, 1.6, and 1.9 (Novakova et al. 2018) were used to test the production of recombinant proteins using various plasmids.

[0055] Example 1 pIJ86 / e40 and pIJ86 / e40His (Figure 2) are prior art (see International Publication No. 2021 / 013553) pIJ86 high-copy replication plasmids, and ermEp * Promoter (relatively weak ermEp2 and relatively strong mutant ermEp1) * (including) the gene encoding e40 (preproe40). These were obtained from Fondazione Istituto Insubrico di Ricerche per la Vita (FIIRV).

[0056] Example 2 pMU1s-kasOpSR40gusA (Figure 7) is a PhiBT1-based embedded vector pMU1s (Craney et al. 2007, Nucleic Acids Res 35, e46) and is potent kasOp * It has an expression cassette containing a gusA reporter gene under promoter control (Myronovskyi et al. 2011, Appl Environ Microbiol 77, 5370-5383) and an optimized synthetic potent SR40 RBS (Bai et al. 2015, Proc Natl Acad Sci USA 112, 12181-12186). The activity of this combination is 1897 U / g of GUS, and ermEp * The expression level was 47 times higher than that of the same expression cassette under the promoter.

[0057] Example 3 A polynucleotide encoding a fusion polypeptide of the vsi signal peptide sequence with an NheI site inserted between the signal peptide sequence and mRFP, and the red fluorescent protein (mRFP) reporter gene was prepared. As an 800 bp NdeI-NotI DNA fragment, this entire vsi-mRFP cassette was used to replace the gusA of pMU1s-kasOpSR40gusA from Example 2, to obtain the pMU1s-kasOpSR40vsi3mRFPA vector (Figure 3). This similarly potent kasOp * promoter, optimized synthetic strong SR40 RBS and Vsi signal peptide. This construct potently secreted mRFP (27 886 FU). SDS-PAGE revealed the correct band of mRFP secreted into the rich TSB medium (Figure 4).

[0058] Example 4 kasOp *A 900bp KpnI-NotI expression cassette containing a promoter, an optimized SR40 RBS, and a vsi-mRFP reporter fusion gene with a single NdeI site inserted into the ATG codon was cloned into the standard E. coli cloning vector pBluescript II SK (Stratagene) digested with the same enzyme to obtain pBS-kasOpSR40vsi3mRFP. Subsequently, this fragment was cloned from the plasmid as a 900bp KpnI-SacI fragment in the standard E. coli cloning vector LITMUS 28 (New England BioLabs) digested with the same enzyme to obtain pLit-kasOpSR40vsi3mRFP (Figure 5). This plasmid was confirmed by nucleotide sequencing and used as a vector to clone all e40 genes into the vector according to the present invention. The entire e40 gene from the ATG start codon and its signal peptide sequence (e40 prepro) (with and without 6xHis labeling at the C-terminus) were extracted from the pIJ86 / e40His plasmid by inserting an NdeI site at the ATG start codon and SpeI and HindIII sites after the e40 stop codon, and then amplified by PCR. The 1200 bp DNA fragments amplified and purified by PCR were digested with NdeI and SpeI, and cloned into pLit-kasOpSR40vsi3mRFP digested with AvrII and NdeI to obtain pLit-e40 or pLit-e40His, respectively (Figure 6). Several positive clones were analyzed by restriction enzyme mapping and subsequently validated by nucleotide sequencing.

[0059] To prepare the vsi-e40pre gene fusion containing the fusion of Vsi and the proenzyme E40pre, the e40pre gene (with and without 6xHis labeling at the C-terminus) was PCR amplified using proofreading Pfu DNA polymerase with pIJ86 / e40His plasmid as a template. This was done using primers with the NheI site inserted at the Ala34 codon and primers with the SpeI and HindIII sites inserted after the e40 stop codon. The 1100 bp DNA fragments amplified and purified by PCR were digested with NheI and HindIII, and cloned into pLitkasOpSR40vsi3mRFP digested with NheI and HindIII to obtain pLit-vsie40pre or pLit-vsi40Hispre, respectively (Figure 8). Several positive clones were analyzed by restriction enzyme mapping and subsequently validated by nucleotide sequencing. Clone sequencing revealed one clone in which the synthesized primer generated a mutation that replaced the His38 CAC codon with the Gln38 CAA codon. This plasmid was isolated for further analysis and labeled with pLitvsi40HQHispre.

[0060] To prepare the vsi-e40mat gene fusion containing the fusion of Vsi and the maturation enzyme E40mat, the e40mat gene (with and without 6xHis labeling at the C-terminus) was PCR amplified using proofreading Pfu DNA polymerase with pIJ86 / e40His plasmid as a template. This was done using primers in which the NheI site was inserted at the Ala73 codon and the Ala74 codon was changed to Ser, and primers in which the SpeI and HindIII sites were inserted after the e40 stop codon. The 1000 bp DNA fragments amplified and purified by PCR were digested with NheI and HindIII, and cloned into pLit-kasOpSR40vsi3mRFP digested with NheI and HindIII to obtain pLit-vsie40mat or pLit-vsi40Hismat, respectively (Figure 8). Several positive clones were analyzed by restriction enzyme mapping and subsequently validated by nucleotide sequencing.

[0061] Example 5 kasOp * All cassettes containing the promoter, RBS, and e40 (with and without 6xHis labeling at the terminal, below its original signal peptide or Vsi signal peptide) are 1400 bp of KpnI-EcoRV. DNA fragments pLit-e40, pLit-e40His, pLit-vsie40pre, pLit-e40Hispre, pLitvsie40HQHispre, pLit-vsie40mat, and pLit-vsie40Hismat (Figures 6 and 8) were cloned into pMU1skasOpSR40gusA (Figure 7), which was digested with KpnI and EcoRV, to obtain the pMU1s-e40, pMU1s-e40His, pMU1s-vsie40pre, pMU1s-vsie40Hispre, pMU1s-vsie40HQHispre, pMU1s-vsie40mat, and pMU1s-vsie40Hismat vectors (Figure 9). Several positive clones were analyzed by restriction enzyme mapping and subsequently verified by nucleotide sequencing. All constructs can be integrated into a single chromosomal position of S. lividance TK24 using a phage PhiBT1-based integration system after the plasmid has been conjugated to S. lividance TK24 via apramycin resistance (AprR) selection. The final constructs are stable and do not require AprR selection.

[0062] Example 6 kasOp *All cassettes containing the promoter, RBS, and e40 (with and without 6xHis labeling at the terminal, below its original signal peptide or Vsi signal peptide) are 1400 bp of KpnI-EcoRV. DNA fragments pLit-e40, pLit-e40His, pLit-vsie40pre, pLit-e40Hispre, pLitvsie40HQHispre, pLit-vsie40mat, and pLit-vsie40Hismat (Figures 6 and 8) were cloned into pIJ86 digested with KpnI and HindIII to obtain the pIJ86-kasOpe40, pIJ86-kasOpe40His, pIJ86-vsie40pre, pIJ86-vsie40Hispre, pIJ86-vsie40HQHispre, pIJ86-vsie40mat, and pIJ86-vsie40Hismat vectors (Figure 10). Several positive clones were analyzed by restriction enzyme mapping and subsequently validated by nucleotide sequencing. All constructs can be replicated on S. lividance TK24 at high copy numbers (approximately 100-300 copies) after conjugating the plasmid to S. lividance TK24 with AprR selection. However, persistent AprR selection is required, and some instability has been reported.

[0063] Finally, a 1500 bp XbaI (Krenow-packed)-HindIII fragment from the high-copy-number shuttle plasmid pIJ86 / e40His was cloned into a pMU1s-vsiRFPB vector cleaved with EcoRV and HindIII to obtain pMU1s-ermEe40 (Figure 11). The plasmid was validated by sequencing.

[0064] Example 7 E40 activity was measured using the prior art strain S. lividance / pIJ86 / e40His. First, spore stocks were prepared after spore formation on solid Bennett and Apramycin (Apr) medium. The stability of spores from mixed spores and five independent spore-forming colonies was tested. Spore suspensions from 1 × 1 cm spore-forming regions from confluent mixed spores (or entire colonies of five large single-spore-forming colonies) were inoculated into 100 ml Erlenmeyer flasks containing 10 ml of medium V (20 g / L glucose, 5 g / L Difco yeast extract, 10 g / L Sigma Aldrich soy peptone, 1.5 g / L NaCl). Apr was added until the final concentration reached 50 μg / ml, and the cultures were incubated on a rotary shaker at 200 rpm and 30°C for 1 day. Five beads (3.5 mm) were added, and the cultures were continued for another day. Two ml of this seed culture was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of medium P (340 g / L sucrose, 10 g / L glucose, 3 g / L yeast extract, 5 g / L soy peptone, 3 g / L malt extract), and Apr was added until the final concentration reached 50 μg / ml. The culture was then incubated under the same conditions. On days 2, 5, 6, 7, and 8, 1 ml of medium was transferred to an Eppendorf tube, centrifuged (13,000 rpm for 10 minutes), and the supernatant was transferred to a new tube and stored at -20°C. E40 activity was measured by diluting the sample 40-fold and using the SSA protocol (Standard Activity Assay, SSA, performed on a 96-well clear microtiter plate and measured with a Biotec microplate reader (A405)). Similar E40 activity of approximately 20 AU / ml was measured in all five independent colonies and the mixed culture. The samples were also analyzed by SDS-PAGE, and all showed an E40 38kDa band (not shown).

[0065] Example 8 E40 activity was measured using two S. lividance strains conjugated with prior art plasmids pIJ86, pIJ86 / e40, and pIJ86 / e40His: wild-type TK24 and mutant S. lividance RedStrep1.3. Spore-forming cultures were inoculated into 100 ml Erlenmeyer flasks containing 10 ml of Niedercorn medium (3% sucrose, 2% corn steep liquor, 0.2% ammonium sulfate, 0.7% CaCO3, pH 7, distilled water), Apr was added until the final concentration reached 50 μg / ml, and the cultures were incubated on a rotary shaker at 200 rpm and 30°C for 2 days.

[0066] Two ml of this seed culture was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of medium P. E40 activity was measured using a 40-fold dilution of a sample consisting of the supernatant of the cultures collected on days 3, 4, 5, 6, and 7, according to the SSA protocol described above. When the vector pIJ86 alone was used, only low background protease activity was observed in both strains. The E40 activity of both constructs, pIJ86 / e40 and pIJ86 / e40His, was approximately 15 AU / ml in the TK24 strain and approximately 20 AU / ml in the RedStrep1.3 strain (Figure 12).

[0067] Example 9 The E40 activity of wild-type S. lividance TK24 strain and three mutant S. lividance RedStrep1.3;1.6;1.9 strains was measured using the control plasmid pMU1s-ermEe40 (Figure 11). Cultures were prepared in the same manner as in Example 8.

[0068] In the single-copy chromosome integration construct pMU1s-ermEe40, E40 activity was significantly lower compared to the activity with the high-copy-number pIJ86 vector in Example 8: 1.13 AU / ml in wild-type S. lividance TK24 with pMU1s-ermEe40, and approximately three times higher in RedStrep strains 1.6 and 1.9 (Figure 13).

[0069] Example 10 E40 activity, kasOp *Measurements were taken using wild-type S. lividance TK24 strain conjugated to four clones of the single-site embedded plasmid pMU1s-e40 and pMU1s-e40His, which contain the promoter, potent SR40 RBS, and e40 (with and without C-terminal xHis labeling) having its original signal peptide. Cultures were prepared in the same manner as in Example 8.

[0070] In three clones (No. 1, 2, and 3) containing plasmid pMU1s-e40, E40 activity was similar at approximately 55 AU / ml, but clone No. 4 showed significantly higher activity (260 AU / ml) (Figure 14A). Similarly, in three clones (No. 1, 2, and 4) containing plasmid pMU1s-e40His, E40 activity was similar at approximately 45 AU / ml, but clone No. 3 showed significantly higher activity (180 AU / ml) (Figure 14B). Based on a comparison of the three similar clones with the single-copy embedded plasmid pMU1s-ermEe40 from Example 6, pMU1s-e40 and pMU1s-e40His show approximately 50-fold and 40-fold increases in E40 activity, respectively.

[0071] Example 11 kasOp * E40 activity was analyzed in four independent clones from wild-type S. lividance TK24 strain conjugated to the high-copy vector pIJ86 containing the promoter, potent SR40 RBS, and e40 (with and without C-terminal His labeling) having its original signal peptide or vsi signal peptide (pIJ86-kasOpe40, pIJ86-kasOpe40His, pIJ86-vsie40pre, pIJ86-vsie40Hispre, pIJ86-vsie40HQHispre, pIJ86-vsie40mat, pIJ86-vsie40Hismat). As a result, the cloning cassette kasOpe *This high copy number vector, containing the promoter, potent SR40 RBS, and e40, was shown to be highly unstable whether it contained the original signal peptide (Figure 15) or fused with the Vsi signal peptide (Figure 16) (with and without C-terminal xHis labeling). In fact, for the four clones of pIJ86-kasOpe40, one of the four clones had very high E40 activity (150 AU / ml), one had E40 activity of 40 AU / ml, and the other two clones had zero activity (Figure 15A). Similarly, for the four clones of pIJ86-kasOpe40His, one clone had fairly high E40 activity (51 AU / ml), another had E40 activity of 38 AU / ml, and the other two clones had zero activity (Figure 15B).

[0072] Similarly, in the case of the four clones of the S. lividance TK24 plasmid pIJ86-vsie40pre, pIJ86-vsie40Hispre, pIJ86-vsie40HQHispre, pIJ86-vsie40mat, and pIJ86-vsie40Hismat, E40 activity was very low in all clones tested (0.5–2.5 AU / ml) (Figure 16A–D). These results were surprising because the single-copy chromosomal integrated counterparts, pMU1s-e40 and pMU1s-e40His, exhibited stable and high E40 activity (Figure 14).

[0073] Example 12 kasOp *High E40 activity was observed in all four clones using plasmids pMU1s-vsie40pre and pMU1s-vsie40Hispre, which contain a promoter, a potent SR40 RBS, and a proenzyme moiety of e40 fused to a Vsi signal peptide (Figure 17). Plasmid pMU1s-vsie40pre showed an E40 activity of approximately 50 AU / ml, with one clone reaching a maximum of 85 AU / ml (Figure 17A). Similarly, plasmid pMU1s-vsie40Hispre showed comparable E40 activity of approximately 30 AU / ml in three clones and 84 AU / ml in one clone (Figure 17B). Based on a comparison of these E40 activities with those from cells bound to the control single-copy embedded plasmid pMU1s-ermEe40 in Example 9, the E40 activity of construct pMU1s-vsie40pre increased by approximately 45-fold (75-fold in the most active clone), and the E40 activity of construct pMU1s-vsie40Hispre increased by approximately 26-fold (up to 75-fold in the most active clone). When these clones were compared to clones with the high-copy-number TK24 plasmid pIJ86 / e40His, the two best clones showed 5.6-fold higher E40 activity. Furthermore, these clones exhibited high stability.

[0074] kasOp * Plasmid pMU1s-vsie40HisHQpre, containing the proenzyme moiety of e40 with a 38His / Gln mutation fused to the promoter, potent RBS, and Vsi signal peptide, showed partially reduced E40 activity compared to its wild-type variant (approximately 27 AU / ml).

[0075] Example 13 pMU1s-e40 and pMU1s-e40His were conjugated to S. lividance mutants RedStrep 1.3, 1.6, and 1.9. E40 activity was analyzed in four independent clones of the wild-type S. lividance TK24 strain, along with the four clones analyzed in Example 10. For pMU1s-e40, E40 activity was clearly higher in RedStrep 1.3 (75±13AU / ml) than in TK24 (56±7AU / ml). E40 activity was also high in RedStrep 1.6 (67±24AU / ml), but the E40 activity of RedStrep 1.9 was similar to that of TK24 (52±7AU / ml) (Figure 18A). In the case of pMU1s-e40His, E40 activity was similar in all four strains: WT TK24 (33±7 AU / ml), RedStrep1.3 (29±6 AU / ml), RedStrep1.6 (31±7 AU / ml), RedStrep1.9 (31±7 AU / ml) (Figure 18B).

[0076] Example 14 pMU1s-vsie40pre and pMU1s-vsie40Hispre(kasOp * The promoter, potent SR40 RBS, and the proenzyme moiety of e40 fused to the Vsi signal peptide were conjugated to RedStrep 1.3, 1.6, and 1.9 mutants, and E40 activity was analyzed in four independent clones of the wild-type S. lividance TK24 strain, along with the four clones analyzed previously in Example 12. Recombinant cells were grown as in Example 8.

[0077] In the case of the pMU1s-vsie40pre vector, E40 activity was higher in RedStrep1.3 (40±13.3AU / ml) than in TK24 (28.75±6.2AU / ml). The E40 activity of RedStrep1.6 (29.75±3.8AU / ml) was similar to that of the WT TK24 strain (Figure 19A). In the case of the pMU1s-vsie40Hispre vector, E40 activity was again high in RedStrep1.3 (30±3.5AU / ml), similar in RedStrep1.6 (24.75±0.9AU / ml) and RedStrep1.6 (31±7AU / ml), but lower in RedStrep1.9 (22±2.9AU / ml) (Figure 19B).

[0078] The experimental data demonstrate that the embedded vectors based on PhiBT1 phage integrase according to the present invention (pMU1s-e40, pMU1s-e40His, pMU1s-vsie40pre, pMU1s-vsie40Hispre, pMU1s-vsie40HHQHispre, pMU1s-vsie40mat, pMU1s-vsie40Hismat) provide stable and high production of E40. The pMU1s-e40 and pMU1s-e40His vectors are kasOp * This is the best vector containing a promoter, an optimally strong SR40 RBS, the original signal peptide sequence, followed by the original e40 or e40His, producing an average of 50 AU / ml or 35 AU / ml of enzyme, respectively. Furthermore, the optimal host cell line for E40 production is S. lividance RedStrep 1.3.

Claims

1. A recombinant vector for heterologous expression of recombinant proteins in Streptomyces host cells, The expression cassette comprises a promoter, a regulatory element, and a polynucleotide encoding a recombinant protein operably linked to the promoter and the regulatory element. - The promoter is an artificial kasO promoter (kasOp) having the sequence of Sequence ID No.

10. ＊ ) and; - The regulatory element is an optimized synthetic SR40 ribosome binding site (RBS) having the sequence of sequence number 11; - The polynucleotide encoding the target recombinant protein encodes a recombinant protein including an N-terminal signal peptide; and, - The recombinant vector is a single-site embedded vector. Recombinant vector.

2. The recombinant vector according to claim 1, wherein the recombinant protein is an actinoaromulus endopeptidase of a sequence containing SEQ ID NO: 1 or a sequence consisting of SEQ ID NO: 1; a biologically active fragment of E40; a naturally occurring allelic variant of E40; or an endopeptidase of a sequence having at least 60%, 70%, 80%, 90%, or 95% identity with SEQ ID NO:

1.

3. The recombinant vector according to claim 2, wherein the recombinant protein is a histidine-labeled actinoaromulus endopeptidase having a sequence containing SEQ ID NO:

2.

4. The recombinant vector according to claim 1, wherein the signal peptide is a Vsi signal peptide of a subtilisin inhibitor of Streptomyces venezuelae having the sequence of SEQ ID NO:

12.

5. The recombinant vector according to claim 2, wherein the signal peptide is a Vsi signal peptide of a subtilisin inhibitor of Streptomyces venezuelae having the sequence of SEQ ID NO:

12.

6. The recombinant vector according to any one of claims 1 to 5, wherein the polynucleotide encoding the recombinant protein is a sequence comprising the sequence of SEQ ID NOs. 5, 6, 15, 16, 17, or 18.

7. The recombinant vector according to any one of claims 1 to 5, wherein the expression cassette has the sequence of sequence number 20, 21, 22, or 23.

8. A recombinant vector according to any one of claims 1 to 5, which is a vector incorporated into the attB site of Streptomyces.

9. The recombinant vector according to claim 8, which is a vector of the sequence sequence 25, 26, 27, or 28.

10. Streptomyces host cells for heterologous expression of recombinant proteins comprising the expression vectors described in claims 1 to 5.

11. S. lividance host cells for heterologous expression of recombinant proteins comprising the expression vectors described in Claims 1 to 5.

12. The host cell according to claim 10, which is a TK24 strain or a RedStrep 1.3, 1.6, or 1.9 strain of S. lividance host cell.

13. The host cell according to claim 10, which is a S. lividance host cell of strain DSM33930 or strain DSM33931.

14. Use of a recombinant vector according to any one of claims 1 to 5 for producing a recombinant protein, wherein the recombinant protein is secreted into the culture medium of a host cell.

15. A use of a host cell according to claim 10 for producing a recombinant protein, wherein the recombinant protein is secreted into the culture medium of the host cell.

16. A method for producing recombinant protein, comprising the following steps: i) A step of providing a recombinant host cell comprising a recombinant expression vector according to any one of claims 1 to 5; ii) A step of culturing recombinant host cells in a culture medium under conditions suitable for proliferation; iii) A step of recovering recombinant proteins expressed by recombinant host cells from the culture medium.

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