Patent Description:
The new system includes vectors and new recombinant host strains, in particular for the production of Actinoallomurus endopeptidases.

Recombinant DNA (rDNA) technology offers a very potent set of technical platforms for the controlled and scalable production of polypeptides of interest by relatively inexpensive procedures. Recombinant proteins are typically obtained through recombinant DNA technology in Escherichia coli, Saccharomyces cerevisiae, in insect, hamster and mammalian cells. There is however a demand for improving the production of big quantities of recombinant proteins; furthermore, there are very strict requirements to be fulfilled when proteins are produced for human use, for instance for use as food supplements and/or as medicaments in the prevention and/or treatment of human diseases. Streptomycetes are regarded as a safe source of proteins for human alimentary use. Two examples of food enzymes sourced from Streptomyces spp are: glucose isomerases used for fructose syrup production (<NPL>), and the widely exploited transglutaminase from S. mobaraensis, used in food industry for its properties in improving the texture and overall quality of final food products, such as processed meat and fish products, as well as diary and baked food (<NPL>).

lividans cells are known to be efficient host cells for the production of recombinant proteins, since recombinant proteins expressed by said cells can be directly secreted and released in the culture medium. However, different proteins are obtained at very different yields in S. lividans, this result being unpredictable (<NPL>).

To improve the yield of heterologous proteins, a collection of derivative strains of S. lividans TK24 (TK24 Taxonomy ID: <NUM>) have been constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters. <NPL>) disclose efficient production of mithramycin A, with yields close to <NUM>/L, using this optimized collection of strains.

Still reproducibility of high yields with any polypeptide-encoding polynucleotide is not guaranteed; moreover, there is still room for further improvements in the provision of recombinant proteins production systems.

The present invention is aimed at providing improved systems for efficient manufacturing of recombinant proteins in Streptomyces, preferably Streptomyces lividans (S. lividans) host strains.

In particular, the system of the invention has been found to be particularly useful for the production of big quantities of a recombinant endopeptidase (endopeptidase <NUM> or E40) of the Actinoallomurus strain.

E40 native protein consists of <NUM> amino acid residues (see SEQ ID NO: <NUM>); the N-terminal signal peptide has been identified between positions <NUM> and <NUM>, and it has been predicted that the mature form of the protein (E40mat) is a <NUM> kDa polypeptide of sequence SEQ ID NO: <NUM>, starting from position <NUM> of the native protein (see <FIG>).

"E40pre-pro" is hereafter used to identify the native pre-pro-endopeptidase, including its native signal peptide, consisting of sequence SEQ ID NO: <NUM>, or derivatives thereof, such as tagged E40pre-pro having sequence SEQ ID NO:<NUM> (his-tagged E40pre-pro); "E40pre" is hereafter used to identify E40 pro-enzyme, without the signal peptide, consisting of aminoacidic sequence SEQ ID NO: <NUM>; "E40mat" is hereafter used to identify the mature endopeptidase of aminoacidic sequence consisting of SEQ ID NO: <NUM>, or derivatives thereof, such as tagged E40mat having aminoacidic sequence of SEQ ID NO: <NUM> (E40Hismat).

Said endopeptidase has been first disclosed in <CIT> and it has shown to provide very rapid and efficient degradation of gluten peptides into non-toxic peptides, being active at the whole pH range of the gastric and intestinal environment (enzymatic activity is shown in the range of pH <NUM>-<NUM> with optimum at pH <NUM>) and being moreover resistant to degradation by gastrointestinal endogenous enzymes. This makes said Actinoallomurus endopeptidases very suitable for being used in the treatment and/or prevention of Celiac Disease (CD) and CD-associated disorders. There is therefore a strong interest in developing efficient and inexpensive methods of manufacturing said Actinoallomurus endopeptidase.

<CIT> discloses a method for manufacturing recombinant Actinoallomurus endopeptidases in a S. lividans host cell, wherein expression of E40 is preferably driven in S. lividans TK24 strain by a high-copy replicative expression vector (pIJ86, SEQ ID NO:<NUM>).

Several replicative plasmids have been successfully used, especially for their high-copy number, for overproduction of many biotechnologically relevant heterologous proteins in Streptomycetes. Replicative vectors have however some drawbacks; for instance, they can only be transferred to Streptomycetes by laborious protoplast transformation and they need a permanent antibiotic selection for stable maintenance in Streptomycetes.

Integrating vectors containing a DNA segment encoding attachment/integration functions (Att/Int) for site-specific integration into specific sites of the Streptomyces genome (such as an attB site for specific integrase) have also been employed for expressing recombinant proteins in Streptomyces. Most of the site-specific integration systems are based on various actinomycete temperate bacteriophages (actinophages) identified in Streptomyces species. Temperate bacteriophages (and integration vectors prepared using their functional elements) are integrated into the host chromosome at a specific site by a recombination process that requires specialized attachment sites, attP in the phage (or in the integration vector) and attB in the host chromosome.

Despite several benefits, these Att/Int systems also have some limitations, such as the fact that they integrate the entire integration plasmid with the E. coli replicon, the integrase gene and the resistance marker gene. This can be a significant biotechnological obstacle, as the stability of the constructs can be affected. In fact, the instability of several Att/Int systems has been reported in several Streptomyces strains (<NPL>). Therefore, these integrating vectors method have been considered unsuitable for the construction of stable biotechnological production strains.

Moreover, several other elements also participate in the efficiency of a recombinant protein production system, such as the regulatory elements in the expression cassettes cloned into the expression vector.

Therefore, the achievement of a satisfying yield of protein and the avoidance of undesired drawbacks is never an easy and clear-cut task in the field of recombinant proteins production.

The present invention provides a surprisingly improved system for producing recombinant proteins, and preferably recombinant Actinoallomurus endopeptidases, in Streptomyces, preferably S. lividans, host cells.

The system of the invention for the production of recombinant proteins in Streptomyces host cells, preferably of S. lividans strains, includes new recombinant vectors bearing an expression cassette comprising a polynucleotide encoding for the recombinant protein of interest under the regulation of a strong promoter, which is an engineered kasO promoter (kasOp*), of sequence SEQ ID NO: <NUM>, and a regulatory element, which is a synthetic variant SR40 ribosome-binding site (RBS) based on PhiC31 capsid protein gene (<NPL>) of sequence SEQ ID NO: <NUM>. The recombinant vectors of the invention are single-site integrating vectors based on the phage PhiBT1, preferably integrating at its attB site in Streptomyces genomes.

The polynucleotide encoding for the recombinant protein of interest encodes for a recombinant pre-protein that includes a signal peptide. In preferred aspects, said signal peptide is a heterologous signal peptide, more preferably signal peptide vsi of subtilisin inhibitor of Streptomyces venezuelae (<NPL>), having sequence SEQ ID NO: <NUM>.

Preferably, the recombinant protein of interest is an Actinoallomurus endopeptidase, preferably Actinoallomurus endopeptidases <NUM> (E40), a biologically active fragment, a variant, or a derivative thereof.

The systems of the invention also include new recombinant host strains comprising the new vectors. The present invention is also directed to the use of the new systems of the invention for the production of recombinant proteins by secretion of the same in the culture medium of the host cell, in particular for the production of Actinoallomurus endopeptidases, and to methods of production of the recombinant protein by means of the new systems of the invention.

Surprisingly, despite the use of a single copy vector, the system of the invention provides expression of recombinant protein that is stable and in high amounts.

The present invention is first directed to a recombinant vector bearing an expression cassette for heterologous expression of a recombinant protein in a Streptomyces host cell, said cassette comprising: a promoter, a regulatory element down-stream said promoter, and a polynucleotide encoding a recombinant protein, down-stream said promoter and said regulatory element, and operably linked to the same.

The terms "vector", "expression vector" and "plasmid" are used herein interchangeably. The expression vector for heterologous expression of a recombinant protein in a Streptomyces host cell of the invention is a single-site integrating vector containing the attP site and integrase gene from PhiBT1 phage, more preferably a vector integrating at its attB site of Streptomyces, having sequence SEQ ID NO: <NUM>.

Examples of single-site integrating vectors according to the present invention include those described in <NPL>.

In preferred embodiments, the single-site integrating vector is a vector having the backbone of pMU1 vector (see <NPL>).

The promoter of said expression cassette is an engineered kasO promoter (kasOp*) of sequence SEQ ID NO: <NUM>; the regulatory element of the expression cassette is a synthetic ribosome-binding site (SR40 RBS) of sequence SEQ ID NO: <NUM>; and the polynucleotide encoding for the recombinant protein of interest is a polynucleotide encoding for a recombinant protein that includes the signal peptide.

The expression cassette of the invention is particularly suitable for heterologous expression of recombinant proteins in Streptomyces host cells, wherein the recombinant protein is expressed by the host cell, cultured in a medium under suitable conditions for the growth of the host cell, and it is secreted in said culture medium. The signal peptide drives secretion of the protein in the medium.

Optionally the signal peptide is a heterologous signal peptide, more preferably Vsi signal peptide of subtilisin inhibitor of Streptomyces venezuelae having sequence SEQ ID NO: <NUM>. Surprisingly, the use of a heterologous signal peptide fused to the protein of interest, according to the invention, provides secretion of the recombinant protein as good as the protein's native signal peptide.

In preferred embodiments of the present invention, the recombinant protein encoded by the polynucleotide of the expression cassette is a recombinant Actinoallomurus endopeptidase with glutenase activity; more preferably said endopeptidase is endopeptidase <NUM> (E40) of sequence comprising SEQ ID NO: <NUM>, a biologically active fragment of E40, a naturally occurring allelic variant of E40; or an endopeptidase of sequence having at least <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% of identity to SEQ ID NO: <NUM>. SEQ ID NO: <NUM> is the polypeptide sequence of the mature form of E40.

Therefore, preferably the expression cassette comprises a polynucleotide encoding for the endopeptidase E40 of sequence comprising SEQ ID NO: <NUM>, for a biologically active fragment of E40, for a naturally occurring allelic variant of E40, or for an endopeptidase of sequence having at least <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% of identity to SEQ ID NO: <NUM>. The term "biologically active fragment" of E40 refers to portions of the endopeptidase which maintain its specific glutenase activity. A polynucleotide encoding for a "biologically-active fragment" of E40 can be identified as disclosed by <CIT>. The polynucleotide encoding for the recombinant protein of interest can have sequence that differs from the annotated nucleic acid sequence due to degeneracy of the genetic code and thus it encodes the same protein encoded by a polynucleotide having the annotated nucleic acid sequence.

The terms "identity" or "homology" when referred to a nucleotide or aminoacidic sequence are herein used interchangeably and refer to the degree to which two polynucleotide or polypeptide sequences are identical or homologous on a residue-by-residue basis over a particular region of comparison. The alignment and the percent 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 (<NPL>). Preferred programs include the GCG Pileup program, FASTA (<NPL>), and BLAST (<NPL>).

The term "allelic variant" denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered aminoacidic sequence. The term allelic variant refers also to a protein encoded by an allelic variant of a gene.

The polynucleotide encoding for the recombinant protein of interest can encode for a protein that is operatively-fused to another polypeptide, for instance a tag, such as a histidine-tag. For instance, said polynucleotide can be a polynucleotide encoding for a tagged protein, such as for the tagged E40 of sequence comprising or consisting of SEQ ID NO: <NUM>. In preferred embodiments, said polynucleotide encoding for the protein of interest is a polynucleotide of sequence comprising or consisting of sequence SEQ ID NO: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> and the expression cassette is of sequence SEQ ID NO: <NUM>, <NUM>, <NUM> or <NUM>.

Therefore, in preferred embodiment, the present invention is directed to single-site integrating vector, more preferably a vector integrating at attB site for PhiBT1 phage of Streptomyces, bearing an expression cassette for the expression in Streptomyces host cells, preferably of S. lividans strains, of the Actinoallomurus endopeptidase, preferably of the Actinoallomurus endopeptidase <NUM> (E40), a biologically active fragment, a variant, or a derivative thereof, under the control of an engineered kasO promoter (kasOp*) of sequence SEQ ID NO: <NUM> and a ribosome-binding site (RBS) of sequence SEQ ID NO: <NUM> (SR40 RBS). Optionally the recombinant protein is the Actinoallomurus endopeptidase whose signal peptide is substituted with a Vsi signal peptide.

More preferably the single-site integrating vector bearing an expression cassette for the expression of the Actinoallomurus endopeptidase <NUM> (E40) is a pMU1 vector of sequence SEQ ID NO: <NUM>, <NUM>, <NUM> or <NUM>.

The present invention is also directed to a host cell for heterologous expression of a recombinant protein comprising an expression vector according to the invention, preferably being a recombinant Streptomyces host cell, more preferably a S. lividans host cell. The host cell can be a wild-type S. lividans host cell or a mutant S. lividans host cell, such as a S. lividans host cell of strain S. lividans RedStrep <NUM> (Δact, Δred, Δcda), S. lividans RedStrep <NUM> (Δact, Δred, Δcda, Δmel), or S. lividans RedStrep <NUM> (Δact, Δred, Δcda, Δmel, ΔmatAB) (Novakova et al.

Preferably, said host cell is a cell of strain DSM <NUM> (S. lividans <NUM> conjugated with pMU1s-e40His) DSM <NUM> (S. lividans <NUM> conjugated with pMU1s-e40His), deposited on <NUM> June <NUM> with the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7B <NUM> Braunschweig - GERMANY, under the provision of the Budapest Treaty.

The present invention is then directed also the use of the recombinant vectors or of the host cells of the invention for the production of a recombinant protein, which is secreted in the culture medium of the host cell.

Advantageously, the system of the invention provides production of the protein of interest, which is stable and in high amounts. Moreover, the protein is easily recoverable from the host cell culture medium wherein it is secreted.

In fact, the systems of the invention is also directed to a method for producing a recombinant protein in a Streptomyces host cell, comprising in series: culturing a recombinant Streptomyces host cell, preferably a S. lividans host cell, more preferably of the TK24 strain, in a culture medium under fermentation conditions, said recombinant host cell comprising a recombinant expression vector according to the invention; recovering the supernatant of the culture medium and purifying from said supernatant a preparation comprising the recombinant protein.

For "fermentation conditions" it is meant conditions of cultivation of the host cell strain (medium composition, stirring parameters, aeration and temperature) suitable for the strain to grow and to produce the compound of interest (the recombinant endopeptidase). Suitable fermentation conditions can be for instance those described in <CIT> or those described in the following examples.

It should be understood that all the possible combinations of the preferred aspects of the present invention s are also described, and therefore similarly preferred.

Examples of embodiments of the present invention are given below, provided for illustrative and non-limiting purposes.

Plasmids that have been prepared in the following examples <NUM>-<NUM> are summarized in Table <NUM>. The promoter, RBS, signal peptide, gene and vector's backbone are reported for each plasmid.

lividans wild-type strain TK24 (Taxonomy ID: <NUM>) and S. lividans mutant strains RedStrep <NUM>, <NUM>, <NUM> (Novakova et al. <NUM>) have been employed in examples <NUM>-<NUM> to test production of recombinant protein with different plasmids.

pIJ86/e40 and pIJ86/e40His (<FIG>) are pIJ86 high copy replicative plasmids of the prior art (see <CIT>), including the gene encoding for e40 (pre-proe40) under the ermEp* promoter (comprised of weaker ermEp2 and stronger mutated ermEp1*). These have been obtained from Fondazione Istituto Insubrico di Ricerche per la Vita (FIIRV).

pMU1s-kasOpSR40gusA(<FIG>) is a PhiBT1-based integrative vector pMU1s (<NPL>), bearing an expression cassette comprising gusA reporter gene (<NPL>) under the control of the strong kasOp* promoter and with optimized synthetic strong SR40 RBS (<NPL>). Activity of this combination was <NUM> U/g of GUS, <NUM>-fold higher than the same expression cassette under ermEp* promoter.

A polynucleotide encoding for a fusion polypeptide of vsi signal peptide sequence with red fluorescent protein (mRFP) reporter gene, with NheI site inserted between the signal peptide sequence and mRFP, was prepared. The whole vsi-mRFP cassette, as an <NUM>-bp NdeI-NotI DNA fragment, was used to replace gusA in pMU1s-kasOpSR40gusAof Example <NUM>, resulting in pMU1s-kasOpSR40vsi3mRFPA vector (<FIG>). It similarly contained the strong kasOp* promoter, optimized synthetic strong SR40 RBS and Vsi signal peptide. This construct had strong secretion of mRFP (<NUM><NUM> FU). SDS-PAGE revealed correct band of secreted mRFP in rich TSB medium (<FIG>).

A <NUM>-bp KpnI-NotI expression cassette, containing the kasOp* promoter, optimized SR40 RBS, with inserted single NdeI site in ATG codon of the vsi-mRFP reporter fusion gene, was cloned in the standard E. coli cloning vector pBluescript II SK (Stratagene) digested with the same enzymes, resulting in pBS-kasOpSR40vsi3mRFP. This fragment was subsequently cloned from this plasmid as a <NUM>-bp KpnI-SacI fragment in the standard E. coli cloning vector LITMUS <NUM> (New England BioLabs) digested with the same enzymes, resulting in pLit-kasOpSR40vsi3mRFP (<FIG>). The plasmid was verified by nucleotide sequencing and used as a vector for cloning of all e40 genes into vectors according to the invention. The entire e40 gene from its ATG initiation codon together with its signal peptide sequence (e40 pre-pro), with and without C-terminally located <NUM> x His tag, was PCR amplified from pIJ86/e40His plasmid, inserting an NdeI site in ATG initiation codon, and SpeI and HindIII sites after e40 stop codon. The PCR amplified and purified <NUM>-bp DNA fragments were digested with NdeI and SpeI and cloned in pLit-kasOpSR40vsi3mRFP digested with AvrII and NdeI, resulting in pLit-e40 or pLit-e40His, respectively (<FIG>). Several positive clones were analyzed by restriction mapping and subsequently verified by nucleotide sequencing.

To prepare a vsi-e40pre gene fusion containing fusion of Vsi with a pro enzyme E40pre, we PCR amplified the e40pre gene (with and without C-terminally located <NUM> xHis tag), with pIJ86/e40His plasmid as a template and proof-reading Pfu DNA polymerase, using primers inserting an NheI site in Ala34 codon, and SpeI and HindIII sites after e40 stop codon. The PCR amplified and purified <NUM>-bp DNA fragments were digested with NheI and HindIII and cloned in pLitkasOpSR40vsi3mRFP digested with NheI and HindIII, resulting in pLit-vsie40pre or pLit-vsi40Hispre, respectively (<FIG>). Several positive clones were analyzed by restriction mapping and subsequently verified by nucleotide sequencing. Sequencing of the clones revealed one clone where synthesized primer produced mutation replacing the His38 CAC codon to the Gln38 CAA codon. The plasmid was also taken for further analysis and labelled as pLitvsi40HQHispre.

To prepare a vsi-e40mat gene fusion containing fusion of Vsi with a mature enzyme E40mat, we PCR amplified the e40mat gene (with and without C-terminally located <NUM> x His tag), with pIJ86/e40His plasmid as a template and proof-reading Pfu DNA polymerase, using primers that inserted an NheI site in Ala73 codon with changing further Ala74 codon to Ser, and SpeI and HindIII sites after e40 stop codon. The PCR amplified and purified <NUM>-bp DNA fragments were digested with NheI and HindIII and cloned in pLit-kasOpSR40vsi3mRFP digested with NheI and HindIII, resulting in pLit-vsie40mat or pLit-vsi40Hismat, respectively (<FIG>). Several positive clones were analyzed by restriction mapping and subsequently verified by nucleotide sequencing.

All the cassettes containing the kasOp* promoter, RBS, and e40 (with its original signal peptide or under Vsi signal peptide, with and without <NUM> x His tag at the end), have been cloned as <NUM>-bp KpnI-EcoRV DNA fragments from pLit-e40, pLit-e40His, pLit-vsie40pre, pLit-e40Hispre, pLitvsie40HQHispre, pLit-vsie40mat, pLit-vsie40Hismat (<FIG>, <FIG>) into pMU1skasOpSR40gusA (<FIG>), digested with KpnI and EcoRV, resulting in pMU1s-e40, pMU1s-e40His, pMU1s-vsie40pre, pMU1s-vsie40Hispre, pMU1s-vsie40HQHispre, pMU1s-vsie40mat, pMU1s-vsie40Hismat vectors (<FIG>). Several positive clones were analyzed by restriction mapping and subsequently verified by nucleotide sequencing. All the constructs can integrate into a single position of the chromosome of S. lividans TK24 using the phage PhiBT1-based integrative system after conjugation of the plasmids into S. lividans TK24 with apramycin resistance (AprR) selection. The final constructs are stable and no AprR selection is required.

All the cassettes containing the kasOp* promoter, RBS, and e40 (with its original signal peptide or under Vsi signal peptide, with and without <NUM> x His tag at the end), have been cloned as <NUM>-bp KpnI-EcoRV DNA fragments from pLit-e40, pLit-e40His, pLit-vsie40pre, pLit-e40Hispre, pLitvsie40HQHispre, pLit-vsie40mat, pLit-vsie40Hismat (<FIG>, <FIG>) to pIJ86, digested with KpnI and HindIII, resulting in pIJ86-kasOpe40, pIJ86-kasOpe40His, pIJ86-vsie40pre, pIJ86-vsie40Hispre, pIJ86-vsie40HQHispre, pIJ86-vsie40mat, pIJ86-vsie40Hismat vectors (<FIG>). Several positive clones were analyzed by restriction mapping and subsequently verified by nucleotide sequencing. All the constructs can replicate in S. lividans TK24 in high-copy number (about <NUM>-<NUM> copies) after conjugation of the plasmids into S. lividans TK24 with AprR selection. However, permanent AprR selection is required and some instability has been reported.

Last, a <NUM>-bp XbaI (filled with Klenow) - HindIII fragment from the high-copy number shuttle plasmid pIJ86/e40His was cloned into pMU1s-vsiRFPB vector cut with EcoRV and HindIII, resulting in pMU1s-ermEe40 (<FIG>). The plasmid was verified by sequencing.

E40 activity with the prior art strain S. lividans/pIJ86/e40His was determined. First, we prepared a spore stock after its sporulation on solid Bennet and Apramycin (Apr) medium. Mixed spores and spores from five independent sporulated colonies were tested for stability. Spore suspension from <NUM> x <NUM> sporulated region from confluent mixed spores (or whole <NUM> large single sporulated colonies) were inoculated in Erlenmeyer flasks (<NUM>) containing <NUM> of Medium V (glucose <NUM>/L, yeast extract Difco <NUM>/L, soy peptone Sigma Aldrich <NUM>/L, NaCl <NUM>/L), with Apr added to final <NUM> ug/ml and incubated on a rotatory shaker at <NUM> rpm at <NUM> for <NUM> day. <NUM> beads (<NUM>) were added, then cultivation continued for another day. <NUM> of such seed culture was inoculated in Erlenmeyer flask (<NUM>) containing <NUM> of the Medium P (Sucrose <NUM>/L, Glucose10 g/L, Yeast extract <NUM>/L, Soy peptone <NUM>/L, Malt extract <NUM>/L), with Apr added to final <NUM>µg/ml, and incubated in the same conditions. At 2d, 5d, 6d, 7d, 8d, <NUM> of culture into Eppendorf tube was taken, centrifuged (<NUM> at <NUM> rpm), supernatant transferred into new tube and stored at - <NUM>. E40 activity was measured by SSA protocol (Standard Activity Assay, SSA, performed in <NUM> wells transparent microtiter plates and measured in Biotec Microplate reader at A405) with <NUM> x dilution of the samples. Comparable E40 activities of about <NUM> AU/ml was determined in all five independent colonies and mixed culture. Samples were also analyzed by SDS-PAGE, all showing the E40 <NUM> kDa band (not shown).

E40 activity was tested with two S. lividans strains, wild-type TK24 and mutant S. lividans RedStrep <NUM>, conjugated with the prior art plasmids pIJ86, pIJ86/e40, and pIJ86/e40His. The sporulated culture was inoculated in Erlenmeyer flasks (<NUM>) containing <NUM> Niedercorn medium (<NUM>% sucrose, <NUM>% corn steep liquor, <NUM>% ammonium sulfate, <NUM>% CaCO3, pH <NUM>, in distilled water), with Apr to final <NUM> ug/ml and incubated on a rotatory shaker at <NUM> rpm at <NUM> for <NUM> days.

<NUM> of such seed culture was inoculated in Erlenmeyer flask (<NUM>) containing <NUM> of the Medium P. E40 activity was measured by SSA protocol as above with <NUM> x dilution of samples consisting of the supernatant of cultures taken at <NUM>, <NUM>, <NUM>, <NUM>, <NUM> days. In the case of vector pIJ86 alone, only low background protease activity was shown in both strains. With both constructs pIJ86/e40 and pIJ86/e40His E40 activity was of about <NUM> AU/ml in TK24 strain and of about <NUM> AU/ml in RedStrep <NUM> strain (<FIG>).

E40 activity was measured in wild-type S. lividans TK24 strain and in three mutant strains, S. lividans RedStrep <NUM>; <NUM>; <NUM> using the control plasmid pMU1s-ermEe40 (<FIG>). Cultures were prepared as in Example <NUM>.

With single copy chromosomally-integrated construct pMU1s-ermEe40, E40 activity was much lower compared to the activity with high copy number pIJ86 vectors of Example <NUM>: <NUM> AU/ml with pMU1s-ermEe40 in wild-type S. lividans TK24 and about <NUM>-fold higher in RedStrep <NUM> and <NUM> strains (<FIG>).

E40 activity was measured in wild-type S. lividans TK24 strain conjugated with four clones of the single-site integrating plasmids pMU1s-e40 and pMU1s-e40His, containing the kasOp* promoter, strong SR40 RBS, and e40 with its original signal peptide (without and with C-terminal x His tag). Cultures were prepared as in Example <NUM>.

In three clones (No. <NUM>, <NUM>, <NUM>) with the plasmid pMU1s-e40, there was comparable E40 activity of about <NUM> AU/ml, however, in the clone No.<NUM>, the activity was much higher (<NUM> AU/ml) (<FIG>. Similarly, in three clones (No. <NUM>, <NUM>, <NUM>) with the plasmid pMU1s-e40His, there was comparable E40 activity of about <NUM> AU/ml, however, in the clone No.<NUM>, the activity was much higher (<NUM> AU/ml) (<FIG>). Based on the comparison of the three similar clones with the single-copy integrative plasmid pMU1s-ermEe40 of Example <NUM>, pMU1s-e40 and pMU1s-e40His show about <NUM>-fold and <NUM>-fold increase of the E40 activity, respectively.

E40 activity was analyzed in four independent clones from the wild-type S. lividans TK24 strain conjugated with high copy vector pIJ86 containing the kasOp* promoter, strong SR40 RBS, and e40 with its original signal peptide or with vsi signal peptide, without and with a C-terminal His tag (pIJ86-kasOpe40, pIJ86-kasOpe40His, pIJ86-vsie40pre, pIJ86-vsie40Hispre, pIJ86-vsie40HQHispre, pIJ86-vsie40mat, pIJ86-vsie40Hismat). The results indicated high instability of this high-copy number vector containing cloned cassettes kasOp* promoter, strong SR40 RBS and e40, either with its original signal peptide (<FIG>) or fused to Vsi signal peptide (<FIG>), without and with C-terminal x His tag. In fact, in the case of four clones of pIJ86-kasOpe40, one out of four clones had quite high E40 activity (<NUM> AU/ml), one had E40 activity of <NUM> AU/ml and two other clones had zero activity (<FIG>. Similarly, in the case of four clones of pIJ86-kasOpe40His, one clone had quite high E40 activity (<NUM> AU/ml), another clone had E40 activity of <NUM> AU/ml and two other clones had zero activity (<FIG>.

Similarly, in the case of four clones of plasmids pIJ86-vsie40pre, pIJ86- vsie40Hispre, pIJ86-vsie40HQHispre, pIJ86-vsie40mat, pIJ86-vsie40Hismat in S. lividans TK24, the E40 activities were very low (between <NUM> and <NUM> AU/ml) in all the clones tested (<FIG>). These results were surprising, since their single copy chromosomally integrated counterparts, pMU1s-e40 and pMU1s-e40His, had stable and high E40 activity (<FIG>).

High E40 activity was found in all four clones with the plasmids pMU1s-vsie40pre and pMU1s-vsie40Hispre, containing the kasOp* promoter, strong SR40 RBS, and pro-enzyme part of e40 fused to Vsi signal peptide (<FIG>). In the case of the plasmid pMU1s-vsie40pre, there was E40 activity of about <NUM> AU/ml, and up to <NUM> AU/ml in one clone (<FIG>). Similarly, with the plasmid pMU1s-vsie40Hispre, there was comparable E40 activity of about <NUM> AU/ml in three clones, and <NUM> AU/ml in one clone (<FIG>). Based on the comparison of these E40 activities to the ones of E40 from cells conjugated with control single-copy integrative plasmid pMU1s-ermEe40 of Example <NUM>, there is about <NUM>-fold increase of the E40 activity for the construct pMU1s-vsie40pre (<NUM>-fold in the highest activity clone) and about <NUM>- fold increase of the E40 activity for the construct pMU1s-vsie40Hispre (even <NUM>-fold in the highest activity clone). Comparing these clones to the clones with high-copy number plasmid pIJ86/e40His in TK24, the two best clones show a <NUM>-fold higher E40 activity. Moreover, these clones show high stability.

Plasmid pMU1s-vsie40HisHQpre, containing the kasOp* promoter, strong RBS, and pro-enzyme part of e40 with 38His/Gln mutation fused to Vsi signal peptide showed E40 activity partially decreased compared to its wild-type variant (about <NUM> AU/ml).

pMU1s-e40 and pMUIs-e40His were conjugated in the S. lividans mutant strains RedStrep1. <NUM>, <NUM> and <NUM>. E40 activity was analyzed in four independent clones together with the four previously analyzed clones of example <NUM> in the wild-type S. lividans TK24 strain. In the case of pMU1s-e40, the E40 activities were clearly higher in RedStrep <NUM> (<NUM> ± <NUM> AU/ml) than in TK24 (<NUM> ± <NUM> AU/ml). The E40 activities were also higher in RedStrep <NUM> (<NUM> ± <NUM> AU/ml), but the E40 activities in RedStrep <NUM> were similar to TK24 (<NUM> ± <NUM> AU/ml) (<FIG>). In the case of pMUIs-e40His E40 activities were similar in all four strains; in WT TK24 (<NUM> ± <NUM> AU/ml), in RedStrep <NUM> (<NUM> ± <NUM> AU/ml), in RedStrep <NUM> (<NUM> ± <NUM> AU/ml) and in RedStrep <NUM> (<NUM> ± <NUM> AU/ml) (<FIG>).

pMU1s-vsie40pre and pMU1s-vsie40Hispre (containing the kasOp* promoter, strong SR40 RBS, and pro-enzyme part of e40 fused to Vsi signal peptide), were conjugated into RedStrep1. <NUM>, <NUM> and <NUM> mutant strains and E40 activity was analyzed in four independent clones together with the four previously analyzed clones of example <NUM> in the wild-type S. lividans TK24 strain. Recombinant cells were grown as in example <NUM>.

In the case of pMU1s-vsie40pre vector, E40 activities were higher in RedStrep <NUM> (<NUM> ± <NUM> AU/ml) than in TK24 (<NUM> ± <NUM> AU/ml). The E40 activities in RedStrep <NUM> (<NUM> ± <NUM> AU/ml) were similar to WT TK24 strain. In the case of pMU1s-vsie40Hispre vector, E40 activities were again higher in RedStrep <NUM> (<NUM> ± <NUM> AU/ml), and similar in RedStrep <NUM> (<NUM> ± <NUM> AU/ml), in RedStrep <NUM> (<NUM> ± <NUM> AU/ml), but lower in RedStrep <NUM> (<NUM> ± <NUM> AU/ml) (<FIG>).

Claim 1:
A recombinant vector for heterologous expression of a recombinant protein in a Streptomyces host cell bearing an expression cassette comprising:
a promoter, a regulatory element, and a polynucleotide encoding a recombinant protein operably linked to said promoter and regulatory element; wherein:
- said promoter is an engineered kasO promoter (kasOp*) having sequence SEQ ID NO: <NUM>;
- said regulatory element is an optimized synthetic SR40 ribosome-binding site (RBS) having sequence SEQ ID NO: <NUM>;
- the polynucleotide encoding for the recombinant protein of interest encodes for a recombinant protein that includes a N-terminal signal peptide; and
- the recombinant vector is a single-site integrating vector.