Bacterial strain for the release of a recombinant protein in a fermentation process

Description

[0001] The invention relates to a process for the fermentative production of recombinant proteins as described in the attached set of claims.

[0002] Recombinant proteins can be produced cost-effectively in bacteria compared to mammalian cell cultures due to their short generation time and ease of handling. The Gram-negative enterobacterium is particularly well-studied due to its genetics and physiology. Escherichia coli Currently, this is the most frequently used organism for the production of recombinant proteins. Production methods for recombinant proteins are particularly attractive in... E. coli, in which the target protein is released directly into the fermentation medium in high yield and in the correct folding, as this avoids complex cell disruption and protein folding processes.

[0003] In the literature, a number of E . E. coli-strains revealed that achieve the release of recombinant proteins into the culture medium. These are mutants of E. coli, These strains exhibit a permanent defect in the outer membrane and therefore partially release periplasmic proteins into the culture medium. This is a non-specific mechanism. An example of such "leaky" mutants are strains with defects in the Tol-Pal complex, e.g., tol- and pal-deletion mutants. It is known that tol- and pal-deletion mutants release endogenous periplasmic proteins into the culture medium. Such strains are extremely sensitive to EDTA, various detergents, and antibiotics, and exhibit growth defects (Clavel et al. 1998, Mol. Microbiol. 29, pp. 359–367; Chen et al. 2014, Microb. Biotechnol. 7, pp. 360–370). Therefore, they are only of limited use for cultivation at high cell densities and over extended periods.

[0004] For this reason, it is preferable to use strains that can release proteins into the culture medium without exhibiting permanent defects. The inducible release of recombinant proteins at a specific time point in a production process can be achieved by weakening the peptidoglycan layer, which is an essential stabilizing factor of the bacterial cell wall.

[0005] The peptidoglycan or murein layer is a cross-linked macromolecule found in the cell wall of Gram-positive and Gram-negative bacteria, providing structural rigidity. Peptidoglycan consists of strands of linked N-acetylglucosamine and N-acetylmuramic acid molecules, forming a linear backbone. The N-acetylmuramic acid residues of adjacent strands are linked together via an oligopeptide (Silhavy et al. 2010, Cold Spring Harb. Perspect. Biol. 2(5)).

[0006] Peptidoglycan hydrolases are a group of enzymes that can cleave covalent bonds in the peptidoglycan layer (Vollmer, FEMS Microbiol Rev. 32, 2008, pp. 259-286). Depending on the type of bond cleaved, peptidoglycan hydrolases are divided into different classes. Glycosidases (N-acetylglucosaminidases, lytic transglycosylases), which include lysozymes, cleave the glycosidic bonds of the peptidoglycan backbone, which is composed of N-acetylmuramic acid and N-acetylglucosamine units. Amidases (N-acetylmuramyl-L-alanine amidases) cleave the amide bond between the N-acetylmuramic acid of the peptidoglycan backbone and the L-alanine of the peptide crosslink. Peptidases (carboxypeptidases and endopeptidases), on the other hand, cleave the peptide bonds within the peptide crosslink.

[0007] A well-known example of the use of peptidoglycan hydrolase in the prior art is the addition of lysozyme to cells after harvesting a bacterial culture in order to break them down (Pierce et al 1997, J Biotechnol 58, p. 1-11).

[0008] Thus, in patent specification CN 104762285 B ClyN, a fusion of lysozyme and peptidoglycan binding domain, is inducibly produced and in this way recombinantly produced cytoplasmic green fluorescent protein (GFP) is released from the cells into the culture medium.

[0009] Another well-known principle is the co-transformation of E . E. coli with a vector for the production of a recombinant protein and a vector for the production of lysozyme from the T4 phage for the release of the recombinant protein such as cytoplasmic β-glucuronidase (Morita et al 2001, Biotechnol. Prog. 17, pp. 573-576) or an anti-CD18 antibody fragment or IGF-I (EP 1 124 941).

[0010] The production of lysozyme leads to cell lysis; therefore, these approaches have the major disadvantage that the release of recombinant proteins is accompanied by the release of other intracellular components, such as host cell proteins and DNA. This contaminates the culture supernatant, causing difficulties in purification, particularly due to increased viscosity.

[0011] This adverse effect is described in CN 104762285 B for the release of GFP by ClyN, where the viable cell count decreases to 0.2% after induction of ClyN production compared to a culture without induction of ClyN production. The optical density of the E . E. coliIn cultures expressing ClyN, the optical density decreases by approximately two-thirds of its pre-ClyN production value within 30 minutes due to cell lysis. After 5 hours, the optical density of the ClyN-expressing culture is only about 20% of that of a control culture without ClyN production. The release of β-glucuronidase by T4 phage lysozyme in Morita et al. is associated with a significantly lower optical density of the culture.

[0012] The same adverse effect is reported in EP 1 124 941 for the release of the anti-CD18 antibody fragment, where the overproduction of T4 phage lysozyme led to a decrease in cell density to 30% of the maximum value. A sharp decrease in cell density due to lysis was also observed with the release of IGF-I by T4 phage lysozyme, and microscopic analysis revealed only a few intact cells.

[0013] Peptidoglycan peptidases affect cell growth and the viability of bacterial cells (Singh et al., 2012, Mol. Microbiol., 86, pp. 1036-1051). WO2013 / 007388 describes an antibody-producing E. coli strain that expresses a non-functional Spr mutant. Furthermore, E. coli strains are known to express the peptidoglycan-cleaving D,D-carboxypeptidases DacA or DacB together with a recombinant protein lacking a signal sequence (Yang et al., 2018, Appl. Microbiol. Biotech, 103, pp. 793-806). Also known is an E. coli strain that expresses a fusion protein consisting of a membrane protein and GPF, as well as a Spr peptidoglycan peptidase (Ojima et al., 2017, Biotech. Progr., 34, S51-57). Finally, a number of other proteins are known that lead to permeabilization and destabilization of the bacterial cell envelope and to an increased release of recombinantly expressed proteins (Kleiner-Grote et al., 2018, Eng. Life Sci., 18, pp. 532-550).

[0014] The object of the present invention is to provide a method in which a recombinant protein is released into the culture medium in increased yield compared to a wild-type host cell, without causing severe cell lysis of the host cells.

[0015] This problem is solved by a process for the fermentative production of recombinant proteins as described in the attached set of claims.

[0016] The bacterial strain is characterized by the fact that it is a strain of the species Escherichia It concerns E. coli.

[0017] The term wild-type gene refers to the form of the gene that arose naturally through evolution and is present in the wild-type bacterial genome.

[0018] The gene expresses the precursor protein, which contains the amino acid sequence of the entire protein, including the amino acid sequence for the signal sequence, and carries no post-translational modifications.

[0019] The term mature protein refers to the protein that comprises the amino acid sequence of the entire protein from which the signal sequence has been removed and in which any post-translational modifications are present. One such post-translational modification is, for example, the addition of a membrane anchor.

[0020] Within the scope of this invention, the term full-length protein refers to the mature protein.

[0021] According to the invention, the additional open reading frame (ORF) contained in the bacterial strain encodes a signal peptide and a peptidoglycan peptidase, which belongs to the peptidoglycan hydrolases. Hereinafter, the term "peptidoglycan peptidase" always refers to the additional protein expressed by this additional ORF in its mature form, i.e., after cleavage of the signal peptide, and not to the protein encoded by the gene present and expressed in the bacterial wild-type genome. Examples of genes encoding peptidoglycan peptidases in E. coli are spr, ydhO, yebA, dacA, dacC, dacD, yfeW, pbpG, mepA, dacB, ampH and ldcA. 1. In a preferred embodiment, the peptidoglycan peptidase encoded by the additional ORF is a wild-type full-length protein that cleaves the bond between the D-alanine and the meso-diaminopimelic acid of the peptide side chain, such as Spr (Uniprot entry P0AFV4) or YdhO (Uniprot entry P76190) with the amino acid sequence known from the specified database entries; 2. in an alternatively preferred embodiment, the peptidoglycan peptidase encoded by the additional ORF is a mutated form of a wild-type full-length protein with a sequence identity of at least 30%, preferably at least 70%, to the amino acid sequence of the corresponding wild-type full-length protein; 3. in a further preferred embodiment, the peptidoglycan peptidase encoded by the additional ORF is a fragment of a wild-type full-length protein; 4.In a further preferred embodiment, the peptidoglycan peptidase encoded by the additional ORF is a mutated version of a fragment of a wild-type protein with a sequence identity of at least 30%, preferably at least 70%, to the amino acid sequence of the corresponding fragment of a wild-type full-length protein. in all cases 1-4, the protein encoded by the additional ORF and selected as a peptidoglycan peptidase exhibits peptidoglycan peptidase activity. Methods for detecting this activity are known to those skilled in the art from the literature ( Gonzalez-Leiza et al 2011, J Bacteriol 193, pp. 6887-94 ).

[0022] The mutations at the amino acid level, as described, for example, under points 2 and 4, may involve substitutions, deletions and / or insertions compared to the wild-type peptidoglycan peptidase full-length protein or fragments thereof.

[0023] Examples of peptidoglycan peptidases are Spr and YdhO, which cleave the bond between D-alanine and meso-diaminopimelic acid in the peptide cross-links of the bacterial peptidoglycan layer (Singh et al. 2012, Mol. Microbiol. 86, pp. 1036-1051), thus enabling the incorporation of new strands into the existing peptidoglycan network and thereby cell growth.

[0024] The spr gene from E. coliThe encodes a Spr protein with 188 amino acids (Uniprot P0AFV4), wherein amino acids 1-26 form the signal sequence for transport from the cytosol to the periplasm. In the mature Spr protein, the signal sequence is proteolytically cleaved, and the cysteine ​​residue at position 1 of the mature protein (position 27 of the overall sequence) is additionally modified with a membrane anchor, by which the Spr protein is anchored in the outer membrane. The mature Spr protein accordingly consists of 162 amino acids and is referred to in this invention as the full-length Spr protein.

[0025] The ydhO The gene encodes a YdhO protein with 271 amino acids (Uniprot P76190). Amino acids 1-27 are predicted to be periplasmic export signal peptides. The mature YdhO protein therefore consists of 244 amino acids and is referred to as full-length YdhO protein within the scope of this invention.

[0026] The DNA sequence encoding the Spr precursor protein is given in SEQ ID No. 4, and the DNA sequence encoding the YdhO precursor protein is given in SEQ ID No. 6. The amino acid sequence of the Spr precursor protein is given in SEQ ID No. 5, where amino acids 1-26 form the signal sequence and amino acids 27-188 form the mature Spr protein (equivalent to the full-length Spr protein). The amino acid sequence of the YdhO precursor protein is given in SEQ ID No. 7, where amino acids 1-27 form the signal sequence and amino acids 28-271 form the mature YdhO protein (equivalent to the full-length YdhO protein).

[0027] The peptidoglycan peptidase is (i) Spr, (ii) YdhO, or (iii) Spr and YdhO. Spr and YdhO are the wild-type full-length protein specified in the database sequence, a mutated form of the wild-type full-length protein with a sequence identity of at least 30%, preferably at least 70%, to the amino acid sequence of the wild-type full-length protein, a fragment of the wild-type full-length protein, or a mutated version of a fragment of the wild-type full-length protein with a sequence identity of at least 30%, preferably at least 70%, to the amino acid sequence of a fragment of the wild-type full-length protein, wherein the selected protein exhibits murein DD endopeptidase activity. In particular, Spr is preferably the sequence specified in SEQ ID No. 5 of amino acids 27-188 and YdhO is preferably the sequence specified in SEQ ID No. 7 of amino acids 28-271.

[0028] In the preferred embodiment, in which the peptidoglycan peptidase is Spr, it is particularly preferred that the Spr protein interacts with the outer membrane.

[0029] The terms DNS and DNA are used synonymously within the scope of this invention.

[0030] The DNA sequences encoding the peptidoglycan peptidase according to the invention are preferably a DNA sequence consisting of Escherichia coli originates.

[0031] As an open reading frame ( open reading frame, The ORF (coding region) is the region of DNA or RNA located between a start codon and a stop codon that codes for the amino acid sequence of a protein. The ORF is also referred to as the coding region.

[0032] ORFs are surrounded by non-coding regions. A gene is the DNA segment that contains all the basic information for producing a biologically active RNA. A gene contains the DNA segment from which a single-stranded RNA copy is produced by transcription and the expression signals involved in regulating this copying process. Expression signals include, for example, at least one promoter, one transcription start site, one translation start site, and one ribosome binding site. Furthermore, a terminator and one or more operators are possible expression signals.

[0033] For a functional promoter, the ORF under the regulation of this promoter is transcribed into an RNS.

[0034] A messenger RNA (mRNA) that contains only one open reading frame is called monocistronic.

[0035] An operon is a functional unit of DNA that includes multiple ORFs, a promoter, and possibly other expression signals.

[0036] The bacterial strain can contain (1) one ORF encoding a signal peptide and a recombinant protein, (2) multiple ORFs encoding the same signal peptide and recombinant protein, or (3) multiple ORFs encoding different signal peptides and recombinant proteins. While the second option is used, for example, to increase the expression and yield of the recombinant protein, the third option is particularly useful for expressing proteins composed of multiple polypeptides (subunits). An example is the expression of antibody fragments, which are then assembled after transport from the cytoplasm.

[0037] Each of these ORFs can be located in a separate gene, or several ORFs can be organized in one operon, i.e., regulated by common expression signals.

[0038] In the case that the recombinant protein is composed of several polypeptide chains (subunits), it is preferred that the ORFs of the individual peptide chains (subunits), each linked to a signal peptide, are organized in an operon.

[0039] Each operon can also contain an ORF encoding a signal peptide and a peptidoglycan peptidase.

[0040] It is preferred that the ORF encodes a signal peptide and that the peptidoglycan peptidase is transcribed as a monocistronic messenger RNA. That is, that the peptidoglycan peptidase is expressed by a separate, dedicated gene. In the preferred embodiment, where the peptidoglycan peptidase is Spr or YdhO, the corresponding gene is referred to as the spr gene or ydhO -Gene is designated.

[0041] The ORF(s) encoding the signal peptide(s) and recombinant protein(s), and the signal peptide(s) and peptidoglycan peptidase(s), can be expressed chromosomally or by a plasmid. If separate genes are involved, they can also be expressed by separate plasmids that are compatible with each other with respect to origin of replication and selection markers. Plasmid-encoded ORFs are preferred.

[0042] The origin of the DNA sequence of the ORF according to the invention encoding a peptidoglycan peptidase is not limited to the bacterial strain used for the production of the recombinant protein, as long as the corresponding peptidoglycan peptidase is functional in the bacterial strain used for the production of the recombinant protein, i.e., exhibits murein DD endopeptidase activity (so). Preferably, the sequence of the ORF encoding the peptidoglycan peptidase protein is a DNA sequence that originates from the same organism that is also used for the production of the recombinant proteins.

[0043] The terms "an ORF encoding a signal peptide and a recombinant protein" and "a recombinant protein," used in the singular within the scope of this invention, can also refer to multiple ORFs and / or multiple different recombinant proteins, each prefixed by a signal peptide. Preferably, these are one to three different recombinant proteins, and particularly preferably one or two different recombinant proteins. The cloning and expression of recombinant proteins in bacteria is carried out as described in the prior art. The recombinant protein possesses, for example, a signal sequence for transport into the periplasm.

[0044] The recombinant protein is a protein that is either not expressed at all or expressed in a different amount by the wild-type bacterial genome. The bacterium serves to produce the recombinant protein, which, according to the invention, is released into the culture medium.

[0045] The fermentation process according to the invention has the advantage that the bacterial strain used expresses a peptidoglycan peptidase as an additional protein along with the recombinant protein. The term "additional" refers to the fact that the bacterial strain contains a further or "additional" open reading frame encoding a peptidoglycan peptidase in addition to the wild-type gene. Preferably, this additional open reading frame encoding a peptidoglycan peptidase is located on a plasmid.

[0046] Due to its signal sequence, peptidoglycan peptidase is localized in the periplasm and, by cleaving peptide cross-links, destabilizes the peptidoglycan layer. Despite the destabilization of the bacterial cell wall, no increased cell lysis was observed, and in the experiments, the dry cell weight of the bacterial strain expressing one recombinant protein and the peptidoglycan peptidase levels remained the same compared to the bacterial strain expressing only the recombinant protein. The destabilization of the bacterial cell wall allows recombinant proteins to be released from the cell's periplasm and isolated from the culture supernatant in increased yield. In this way, higher product yields in the culture supernatant can be achieved compared to the prior art, without significant bacterial cell death.Increased yield means that with the bacterial strain according to the invention, at least 110%, particularly preferably at least 150%, and especially preferably at least 200% of the amount of recombinant protein is released into the culture medium, compared to what can be produced according to the current state of the art with a wild-type bacterial strain containing the same gene for a recombinant protein. That is, the yield of recombinant protein released into the culture medium is at least 1.1 times, particularly preferably at least 1.5 times, and especially preferably at least 2 times higher than the yield that can be achieved with corresponding bacterial strains without overproduction of a peptidoglycan peptidase.

[0047] In the present invention, CGTase was used as an example of a recombinant protein. A comparison of the CGTase yield in Example 1 shows that the CGTase yield, measured in U / ml, is increased by almost 10-fold when using the W3110 / pCGT-Spr bacterial strain compared to the W3110 / pCGT bacterial strain (see Table 1). Neither the cell dry weight (CDW) nor the viable cell count was reduced by the additional production of the Spr protein compared to the control strain. The W3110 / pCGT-YdhO strain achieved almost the same CGTase yield as the W3110 / pCGT-Spr strain (see Table 2), without significantly affecting the cell dry weight compared to the control culture using the W3110 / pCGT bacterial strain.

[0048] Example 2 also confirms, for the production of a Fab antibody fragment as a recombinant protein, in this case the CD154 Fab fragment, that a significantly higher yield of recombinant protein could be achieved with the strain W3110 / pJF118ut-CD154-Spr, which expresses the peptidoglycan peptidase Spr, compared to the strain W3110 / pJF118ut-CD154, in this case approximately 200 times higher (Table 3). At the same time, the cell dry mass decreased only slightly (Table 4).

[0049] OD 600 denotes the optical density of the cell culture determined using a spectrophotometer at 600 nm and is a measure of the number of cells per unit volume (cell concentration, cell density), which in turn is a measure of cell division activity as well as a measure of cell lysis, whereby cell lysis leads to a decrease in cell density.

[0050] CN 104762285 B, on the other hand, shows in Fig. 2, that the OD600 value, i.e. the cell density, drops significantly after induction of ClyN expression, which suggests strong cell lysis.

[0051] Since strong cell lysis leads to the release of host cell proteins, the recombinant protein becomes contaminated with host cell proteins. Furthermore, the release of DNA leads to an increase in the viscosity of the culture medium, which complicates further processing. Because the bacterial strain according to the invention does not exhibit an increased cell lysis rate, the use of this strain for the production of recombinant proteins does not result in contamination of the recombinant protein with host cell proteins. This is confirmed by the analysis of the protein content of the culture supernatant by SDS-PAGE in Example 1 ( Fig. 5), which shows that when using the bacterial strain according to the invention, no significant contamination of the recombinant protein by host cell proteins occurred. The additionally expressed peptidoglycan peptidase was also not released in increased amounts.

[0052] To achieve the release of the recombinant protein into the culture medium, it is necessary that both the recombinant protein and the precursor peptidoglycan peptidase are transported into the periplasm after protein biosynthesis in the cytosol. For transport into the periplasm, the 5' end of the coding DNA sequence of the recombinant protein and the 5' end of the coding DNA sequence of the peptidoglycan peptidase must be ligated. in frameto link to the 3' end of a signal sequence for protein export. In principle, all signal sequences are suitable that enable translocation of the target protein in the bacterial strain used, via the Sec or Tat apparatus. Various signal sequences are described in the prior art, such as the signal sequences of the following genes: phoA, ompA, pelB, ompF, ompT, lamB, malE, Staphylococcal protein A, StII, and others (Choi and Lee 2004, Appl. Microbiol. Biotechnol. 64, pp. 625-635). According to the invention, the signal sequence of the phoA or ompA gene is preferably used for recombinant proteins. E. coli or the signal sequence for a cyclodextrin glycosyltransferase (α-CGTase) from Klebsiella pneumoniaeM5a1 or derived signal sequences disclosed in US 2008 / 0076157 as SEQ ID Nos. 1 and 3. Preferably, the peptidoglycan peptidase in its precursor form carries the signal sequence native to the respective protein. If the peptidoglycan peptidase is Spr or YdhO, the precursor form of the respective peptidoglycan peptidase is the sequence specified in SEQ ID Nos. 5 and 7, respectively. Preferably, the recombinant protein and the peptidoglycan peptidase carry different signal sequences, both of which induced protein export.

[0053] The expression of the ORFs encoding peptidoglycan peptidase and the recombinant protein can be controlled by one, two identical, or two different promoters. It is preferred that the ORFs for peptidoglycan peptidase and for the recombinant protein are controlled by two different promoters.

[0054] The DNA segment encoding the peptidoglycan peptidase can first be isolated by PCR using oligonucleotides as primers and a DNA template encoding the peptidoglycan peptidase, for example, genomic DNA from E. coli,The signal peptide is amplified and then linked, using standard molecular biology techniques, to the DNA molecule containing the sequence of a signal peptide, which was generated analogously, in such a way as to create an in-frame fusion. Alternatively, the entire DNA molecule can be produced via gene synthesis. This DNA molecule, consisting of the respective signal sequence and the coding sequence for the mature protein, can then either be inserted into a vector, e.g., a plasmid, or integrated directly into the chromosome of the bacterial strain using known methods. Preferably, the DNA molecule is inserted into a plasmid, such as a derivative of known expression vectors like pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3, or pET. Plasmids are introduced into the bacterial cells using methods known to those skilled in the art (transformation).

[0055] The plasmids used can carry selection markers. Suitable selection markers are genes that encode resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, kanamycin, or others. Preferably, the plasmid contains a gene whose expression mediates tetracycline resistance. Furthermore, auxotrophy markers are suitable as selection markers; these must encode an essential gene that is deleted in the respective bacterial strain containing the plasmid. If two plasmids are transformed into the cells, selection for the presence of both plasmids is preferentially performed using two different antibiotic resistances, one antibiotic resistance and one auxotrophy marker, or two different auxotrophy markers.

[0056] Suitable promoters include all promoters known to those skilled in the art, such as constitutive promoters like the GAPDH promoter, or inducible promoters like the lac, tac, trc, lambda PL, ara, cumate, or tet promoters, or sequences derived from them. The ORFs encoding the recombinant protein and the ORF encoding the peptidoglycan peptidase can be controlled as operons by a single promoter or by multiple promoters. Preferably, the functional promoter that controls the expression of the open reading frame encoding the peptidoglycan peptidase is an inducible promoter.

[0057] Preferably, the ORFs encoding the recombinant protein and the ORF encoding the peptidoglycan peptidase are controlled by different inducible promoters. Particularly preferred is the expression of the recombinant protein under the control of the tac promoter and the expression of the peptidoglycan peptidase under the control of the ara (arabinose) promoter.

[0058] Preferably, the recombinant protein is a heterologous protein. Heterologous proteins are defined as proteins that do not belong to the proteome, i.e., the entire natural protein makeup of the bacterial strain, preferably of a particular strain. E . E. coli K12 strain, belong. All naturally occurring in the bacterial strain used, e.g. in E. coli K12 strain proteins can be derived from known genome sequences (e.g., from the gene bank entry under accession no. NC_000913 for E. coli K12).

[0059] Eukaryotic proteins containing one or more disulfide bridges are particularly preferred as heterologous proteins. Eukaryotic proteins that exist in their functional form as dimers or multimers are especially preferred.

[0060] The most important heterologous protein classes include antibodies and their fragments, cytokines, growth factors, protein kinases, protein hormones, lipocalins, anticalins, enzymes, binding proteins and molecular scaffolds and proteins derived from them. Examples of these protein classes include heavy-chain antibodies and their fragments (e.g., nanobodies), single-chain antibodies, interferons, interleukins, interleukin receptors, interleukin receptor antagonists, G-CSF, GM-CSF, M-CSF, leukemia inhibitors, stem cell growth factors, tumor necrosis factors, growth hormones, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, transforming growth factors, hepatocyte growth factors, bone morphogenetic factors, nerve growth factors, brain-derived neurotrophic factors (BDNF), glial cell line-derived neurotrophic factors, angiogenesis inhibitors, and tissue plasminogen activators.Blood coagulation factors, trypsin inhibitors, elastase inhibitors, complement components, hypoxia-induced stress proteins, proto-oncogene products, transcription factors, virus constitutive proteins, proinsulin, prourokinase, erythropoietin, thrombopoietin, neurotrophin, protein C, glucocerebrosidase, superoxide dismutase, renin, lysozyme, P450, prochymosin, lipocortin, reptin, serum albumin, streptokinase, tenecteplase, CNTF and cyclodextrin glycosyltransferases.

[0061] Examples of proteins derived from molecular scaffolds include evibodies (derived from CTLA-4) and affibodies (protein A of CTLA-4). S. aureus), Avimers (of human A-domain family), Transbodies (of transferrin), DARPins (of ankyrin repeat protein), Adnectin (of fibronectin III), Peptide Aptamers (of thioredoxin), Microbodies (of microprotein), Affilins (of ubiquitin), α-Crystallin, Charybdotoxin, Tetranectin, PDZ domain of the RAS-binding protein AF-6, Kunitz-type domain of protein inhibitors.

[0062] Within the scope of this invention, any selection marker used is not considered a gene encoding a recombinant protein. The term selection marker refers to a gene that, together with the "gene of interest"—that is, the desired gene encoding the recombinant protein—is introduced into the modified organism to identify individuals with successful gene modification. Commonly used selection markers include antibiotic resistance or auxotrophies. Under certain conditions, these marker genes confer a survival advantage to a cell or organism. For example, the expression of β-lactamase from the ampicillin resistance gene, acting as a marker gene for the cell, enables survival in ampicillin-containing medium. Recombinant proteins, on the other hand, are produced using the bacterial strain and isolated after a certain culture period.

[0063] Compared to the chromosomal integration of ORFs encoding the recombinant protein and the peptidoglycan peptidase, the advantage of using plasmids lies in the fact that the plasmid-bearing cells of the bacterial strain have a selective advantage and can be selected using standard methods. Furthermore, it is particularly advantageous that plasmids can be present in high copy numbers within the cells of the bacterial strain.

[0064] The invention relates to a process for the fermentative production of recombinant proteins, characterized in that a bacterial strain is cultivated in a fermentation medium, the fermentation medium is separated from the cells after fermentation, and recombinant proteins are isolated from the fermentation medium.

[0065] The cultivation of the cells of the bacterial strain, transformed by chromosomal integration or with one or two expression plasmids, is carried out according to usual procedures known to those skilled in the art in a shake flask or in a bioreactor (fermenter).

[0066] In principle, all common media known to experts for cultivating bacteria are suitable as fermentation media (nutrient media, culture media). Complex media or minimal salt media, to which a specific proportion of complex components such as peptone, tryptone, yeast extract, molasses, or corn steep liquor are added, can be used. Furthermore, additional components such as vitamins, salts, amino acids, and trace elements can be added to the medium to improve cell growth.

[0067] The fermentation preferably takes place in a conventional bioreactor, for example a stirred tank fermenter, a bubble column fermenter, or an airlift fermenter. A stirred tank fermenter is particularly preferred.

[0068] During fermentation, the cells of the protein-producing strain are cultivated in a nutrient medium, with various parameters, such as nutrient supply, oxygen partial pressure, pH, and culture temperature, being continuously monitored and precisely controlled. The cultivation period is preferably 24–65 hours.

[0069] In principle, any sugars, sugar alcohols, or organic acids or their salts that can be utilized by the cells can be used as the primary carbon source for fermentation. Glucose, lactose, arabinose, or glycerol are preferred. Glucose and arabinose are particularly preferred. A combined feeding of several different carbon sources is also possible. The carbon source can be completely added to the fermentation medium at the beginning of the fermentation, or none or only a portion of the carbon source can be added initially, with the carbon source being added during the course of the fermentation. A particularly preferred embodiment involves adding part of the carbon source initially and adding part as needed.Glucose is particularly preferred as the carbon source, initially presented at a concentration of 10-30 g / l. Feeding is started when the concentration has dropped below 5 g / l during fermentation and is designed to keep the concentration below 5 g / l.

[0070] If the expression of the recombinant protein and / or peptidoglycan peptidase is controlled by an inducible promoter, expression is induced by adding the appropriate inducer to the fermentation mixture. Suitable inducers include, for example, arabinose, lactose, IPTG, tetracycline, or cumates. The inducer can be added at any time during fermentation, either as a single dose or multiple doses. Alternatively, the inducer can be added continuously. Preferably, the expression of the recombinant protein is induced by adding IPTG, and the expression of the peptidoglycan peptidase is induced by adding arabinose. Particularly preferably, IPTG is added as a single dose.The induction of peptidoglycan peptidase expression is preferably designed such that a mixture of glucose and arabinose is added at a time after the glucose concentration has fallen below 5 g / l, or the arabinose is administered as a single dose.

[0071] The induction of peptidoglycan peptidase expression preferentially occurs during the cultivation phase when few or no cell divisions take place, i.e., shortly before or at the beginning of the stationary phase of the growth curve. This point in time is determined by the fact that the cell density, measured as the OD 600 or CDW value (su), increases only slightly or not at all.

[0072] Preferably, the medium in the fermenter is stirred before inoculation and aerated with sterile compressed air. The oxygen content is calibrated to 100% saturation, and a target value for the O₂ saturation during fermentation is selected, which is between 10 and 70%, preferably between 20 and 60%, and particularly preferably at 30% of this value. After the O₂ saturation drops below the target value, a regulatory cascade is initiated to bring the O₂ saturation back up to the target value, whereby the gas supply and stirring speed can be adjusted.

[0073] The pH of the culture is preferably between pH 6 and pH 8. Preferably, a pH between 6.5 and 7.5 is set, and particularly preferably, the pH of the culture is maintained between 6.8 and 7.2.

[0074] The temperature of the culture is preferably between 15 and 45 °C. A temperature range between 20 and 40 °C is preferred, a temperature range between 25 and 35 °C is particularly preferred, and a temperature of 30 °C is most preferably preferred.

[0075] According to the invention, the fermentation medium is separated from the cells after fermentation, and recombinant proteins are isolated from the fermentation medium. This can be done using conventional methods known in the prior art. Typically, in a first step, the cells are separated from the recombinant proteins released into the culture medium by separation methods such as centrifugation or filtration. The recombinant proteins can then be concentrated, for example, by ultrafiltration.

[0076] Expression of peptidoglycan peptidase improves the release of recombinant proteins into the culture supernatant. This improved release allows for the isolation of recombinant proteins in increased yield.

[0077] The above definition applies to the term "increased yield".

[0078] Using the co-expression of peptidoglycan peptidase in a bacterial strain, preferably in E. coli,Fab antibody fragments can also be produced extracellularly. In this process, the bacterial cell must simultaneously synthesize the corresponding fragments of the light chain, comprising domains VL and CL, and the heavy chain, comprising domains VH and CH1, of the antibody and then secrete them into the periplasm. Outside the cytoplasm, the two chains are then assembled to form the functional Fab fragment. The corresponding ORFs can be located in different genes. It is preferred that the ORFs encoding antibody fragments of the light and heavy chains are organized in a single operon. Through the simultaneous production of the peptidoglycan peptidase according to the invention, the heavy and light chains of the antibody are released into the fermentation medium in increased concentrations.

[0079] This method has the major advantage of being suitable for the production of recombinant proteins requiring long culture times, as well as for fermentation at high cell densities of > 40 g / l. This allows for the production of complex, eukaryotic proteins as recombinant proteins.

[0080] Preferably, the process is characterized in that, after the fermentation medium has been separated, the recombinant proteins are purified from the fermentation medium.

[0081] Recombinant proteins can be further purified using standard methods such as precipitation or chromatography. Methods such as affinity chromatography, which utilizes the protein's already correctly folded native conformation, are particularly preferred.

[0082] The method is preferably characterized by the induction of peptidoglycan peptidase expression. This means that either only the expression of peptidoglycan peptidase or the expression of both the recombinant protein and peptidoglycan peptidase is induced. In this preferred embodiment, the expression of peptidoglycan peptidase is always under the control of an inducible promoter. In contrast, the expression of the recombinant protein can be under the control of either a constitutive or an inducible promoter. Preferably, both the ORF encoding the signal peptide and the recombinant protein, as well as the ORF encoding the signal peptide and the peptidoglycan peptidase, are under the control of an inducible promoter, particularly preferably by promoters with different inducibility. Therefore, the expression of both peptidoglycan peptidase and the recombinant protein is preferably inducible.

[0083] Since the promoters of the genes encoding the recombinant protein and the peptidoglycan peptidase can be preferentially induced independently of each other, the optimal time can be chosen independently for the expression of the recombinant protein and for the expression of the peptidoglycan peptidase.

[0084] By using inducible promoters, the expression of the corresponding proteins can be induced at any desired time point during fermentation. Preferably, the expression of peptidoglycan peptidase is induced after the induction of the expression of the recombinant protein. Particularly preferably, the expression of peptidoglycan peptidase is induced at least 1 hour, more preferably at least 2 hours, and more preferably 15 to 40 hours, and especially preferably 20, 27, or 38 hours after the induction of the expression of the recombinant protein.

[0085] The induction of expression of the recombinant protein and the peptidoglycan peptidase is triggered by adding the inducer to the culture medium, the inducer being selected according to the gene used. In the preferred embodiment, where the peptidoglycan peptidase is under the control of the arabinose (ara) promoter, expression of the peptidoglycan peptidase is induced by adding the inducer arabinose to the culture. In the preferred embodiment, where the recombinant gene contains a tac promoter, expression of the recombinant protein is induced by adding the inducer lactose or the lactose analog IPTG to the culture.

[0086] According to the invention, even after induction of peptidoglycan peptidase expression and further cell culture, the cell density of the strain is comparable to that of a bacterial strain that does not express additional peptidoglycan peptidase. In particular, there is no significant decrease in cell dry mass due to cell lysis.

[0087] The invention has the advantage that recombinant proteins are released into the culture medium without causing excessive lysis of the bacterial cells. The process according to the invention is therefore characterized by its suitability both for the production of complex eukaryotic proteins with correspondingly long culture times and for fermentation at high cell densities >40 g / l.

[0088] A long cultivation period means that the cultivation time is at least 24 h, preferably at least 48 h and most preferably at least 65 h.

[0089] Within the scope of the invention, reduced (or only slightly increased or no significant) cell lysis means that the CDW value, OD 600 value, or the viable cell count of the bacterial strain culture, determined after a culture period of preferably 7-17 h following induction of peptidoglycan peptidase expression, is only slightly or not at all reduced compared to the wild-type bacterial strain, which does not produce additional peptidoglycan peptidase. In particular, for the culture of bacterial strains expressing peptidoglycan glycosidases, whose cell lysis is massively increased, as shown by Morita et al. (2001, so) and in patent specifications CN 104762285 and EP 1 124 941 for cells expressing T4 phage lysozyme or the ClyN protein, respectively, bacterial strains expressing a peptidoglycan peptidase have a significant advantage.

[0090] Preferably, the method is characterized in that, 5 to 24 hours after induction of the expression of the additional peptidoglycan peptidase, the cell dry mass differs by a maximum of 20% from the cell dry mass of a cell culture at the same time from a fermentation process of a bacterial strain that does not express the additional peptidoglycan peptidase.A cell culture produced according to the fermentation process according to the invention is preferably characterized in that, 5 to 24 hours, particularly preferably 7 hours or 17 hours after induction of the expression of the additional peptidoglycan peptidase, it has a cell dry mass that is at most 20%, particularly preferably at most 15%, and particularly preferably at most 10% lower than the cell dry mass of a cell culture at the same time from a fermentation process which differs from the process according to the invention only in that a bacterial strain is cultivated which differs from the bacterial strain according to the invention only in that it does not contain an ORF-encoding peptidoglycan peptidase. The cell dry mass serves as a measure of cell density and thus of the number of unlysed cells.Cell dry mass is determined by isolating a defined amount of culture by filtration or centrifugation, preferably centrifugation, then drying and weighing it. Fig. 1 shows the plasmid map of the plasmid pCGT-Spr. Fig. 2 shows the plasmid map of the plasmid pCGT-YdhO. Fig. 3 shows the plasmid chart of the plasmid pJF118ut-CD154. Fig. 4 shows the plasmid card of the plasmid pJF118ut-CD154-Spr. Fig. 5 This shows the analysis of the culture supernatants by SDS-PAGE, as described in Example 1. The following sample designations apply: S: SeeBlue Prestained Protein Standard, 1: Supernatant of W3110 / pCGT after 65 h, 2: Supernatant of W3110 / pCGT-Spr without arabinose addition after 65 h, 3: Supernatant of W3110 / pCGT-Spr with arabinose addition after 65 h.

[0091] The abbreviations used in the figures represent DNA regions that encode the following functions: tac p / o: tac promoter / operator pBAD p / o: arabinose promoter / operator bla: β-lactamase gene (ampicillin resistance) TcR: tetracycline resistance lacIq: repressor of the tac promoter cgt-SP: signal peptide of CGTase CGTase: cyclodextrin glycosyltransferase ColE1: origin of replication phoA-SP: phoA signal peptide AFA-SP: derivative of the signal peptide of CGTase rrnB terminator: terminator region of the rrnB gene trpA terminator: terminator region of the trpA gene Spr: peptidoglycan peptidase Spr Spr-SP: signal peptide of Spr YdhO: peptidoglycan peptidase YdhO HisTag: histidine tag light chain: antibody fragment comprising the domains VL and CL heavy chain: Antibody fragment encompassing the VH and CH1 domains BamHI / MauBI / EcoRI: Interfaces of the corresponding restriction endonucleases Examples

[0092] The invention will be described in more detail below using exemplary embodiments, without being limited by this.

[0093] All molecular biological procedures used, such as polymerase chain reaction (PCR), gene synthesis, isolation and purification of DNA, modification of DNA by restriction enzymes and ligase, transformation, etc., were carried out in a manner known to those skilled in the art, described in the literature or recommended by the respective manufacturers. Description of the plasmids: pCGT:

[0094] The preparation of the pCGT plasmid is described in Example 4 of US 2008 / 0254511 A1, the plasmid chart in Fig. 4 as specified by US 2008 / 0254511 A1.

[0095] Essentially, in addition to the gene for resistance to tetracycline, the plasmid also contains the structural gene for cyclodextrin glycosyltransferase (CGTase). Klebsiella pneumoniae M5a1 including the native CGTase signaling sequence. The expression of the CGTase gene is controlled by the tac promoter. pCGT-Spr:

[0096] To pCGT-Spr (plasmid chart see. Fig. 1 To obtain this, a DNA fragment was produced by Eurofins Genomics using gene synthesis. This DNA fragment xI (specified in SEQ ID No. 1) contained: the nucleotides 1136-1304 from GenBank entry X81837.1, containing the arabinose promoter (pBAD promoter), as well as the operators O1 and I2+I1 and the CAP binding site (nucleotides 10-178 of SEQ ID No. 1), the Shine-Dalgarno sequence (nucleotides 203-208 of SEQ ID No. 1) and a nucleotide fragment encoding a fusion of the Spr protein from E. coli K12 (nucleotides 216-782 of SEQ ID No. 1) and ii the terminator of the trpA gene from E. coli (Nucleotides 812-837 of SEQ ID NO:1).

[0097] This DNA fragment xI was cut with the restriction enzyme MauBI and ligated to the expression vector pCGT, which had been cut with the same restriction enzyme. Cloning was performed without directionality; however, the plasmid in which the DNA fragment was inserted in the same reading direction as the gene encoding CGTase was used. Detection was achieved via the restriction pattern of the restriction enzyme BamHI and sequencing. This plasmid was designated pCGT-Spr and encodes the Spr protein. pCGT-YdhO:

[0098] To pCGT-YdhO (plasmid chart see. Fig. 2 To obtain this, a DNA fragment was produced by Eurofins Genomics using gene synthesis. This DNA fragment xII (specified in SEQ ID No. 2) contained: the nucleotides 1136-1304 from GenBank entry X81837.1, containing the arabinose promoter (pBAD promoter), as well as the operators O1 and I2+I1 and the CAP binding site (nucleotides 10-178 of SEQ ID No. 2), the Shine-Dalgarno sequence (nucleotides 203-208 of SEQ ID No. 2) and a nucleotide fragment encoding a fusion of the YdhO protein from E. coli K12 (nucleotides 216-1028 of SEQ ID No. 2) and ii the terminator of the trpA gene from E. coli (Nucleotides 1064-1089 of SEQ ID NO:2).

[0099] This DNA fragment xII was cut with the restriction enzyme MauBI and ligated to the expression vector pCGT, which had been cut with the same restriction enzyme. Cloning was performed without directionality; however, the plasmid in which the DNA fragment was inserted in the same reading direction as the gene encoding CGTase was used. Detection was achieved via the restriction pattern of the restriction enzyme BamHI and sequencing. This plasmid was designated pCGT-YdhO and encodes the YdhO protein. pJF118ut-CD154:

[0100] The plasmid pJF118ut, described in US 2008 / 076157 A, served as the starting vector for cloning and expressing the genes of the Fab fragment of the humanized monoclonal anti-CD154 antibody 5c8, whose sequence is published in Karpusas et al. 2001 (Structure 9, pp. 321-329). pJF118ut is a derivative of the known expression vector pKK223-3 (Amersham Pharmacia Biotech) and is registered with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig) under number DSM 18596.

[0101] To pJF118ut-CD154 (plasmid map see. Fig. 3 To obtain this, a DNA fragment was produced by gene synthesis from the company eurofins Genomics. This DNA fragment xIII (specified in SEQ ID No. 3) comprised a fusion consisting of: i the signal sequence disclosed in US 2008 / 076157 under SEQ ID No. 2, derived from the signal sequence of a CGTase from Klebsiella pneumoniaeM5a1 (nucleotides 25-114 of SEQ ID No. 3) and ii the heavy chain reading frame (VH-CH1 domains) of the Fab fragment of the humanized monoclonal anti-CD154 antibody 5c8, encoding amino acids 1-221 of the in Karpusas et al. 2001 in Fig. 3 published sequence (nucleotides 115-777 of SEQ ID No. 3), iii the phoA signal sequence (nucleotides 800-862 of SEQ ID No. 3), iv the reading frame for the light chain (VL-CL domains) of the Fab fragment of the humanized monoclonal anti-CD154 antibody 5c8, as published by Karpusas et al. 2001 in Fig. 3 published (nucleotides 863-1516 of SEQ ID No. 3) and v the nucleotides 1517-1546 of SEQ ID No. 3, encoding a 4-amino acid long linker and a hexa-histidine tag.

[0102] This DNA fragment xIII was cut with the restriction enzymes EcoRI and PdmI and ligated with the expression vector pJF118ut, which had been cut with EcoRI and SmaI. The resulting plasmid, in which the expression of the genes for the heavy and light chains of the Fab fragment was under the control of the tac promoter, was designated pJF118ut-CD154. pJF118ut-CD154-Spr:

[0103] The DNA fragment xI encoding Spr was inserted into the plasmid pJF118ut-CD154 via the MauBI cleavage site under the control of the arabinose promoter and flanked by the trpA terminator, as described above for pCGT-Spr. Cloning was performed in an undirected manner; however, the plasmid in which the DNA fragment xI was inserted in the opposite reading direction to the DNA fragment xIII encoding CD154 was used for analysis. Detection was achieved via the restriction pattern of the restriction enzyme BamHI and sequencing. This plasmid was designated pJF118ut-CD154-Spr (see plasmid chart). Fig. 4 ). Example 1: Fermentative production of cyclodextrin glycosyltransferase (CGTase) in a stirred tank fermenter

[0104] For the production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae M5a1, the E. coli-Strain W3110 (ATCC 27325) with the plasmids pCGT, pCGT-Spr, and pCGT-YdhO was transformed using standard methods (e.g., TSS transformation). Selection for plasmid-containing cells was performed using tetracycline (20 mg / L). E . E. coli The strains were designated W3110 / pCGT, W3110 / pCGT-Spr and W3110 / pCGT-YdhO. a) CGTase production took place in stirred tank fermenters.

[0105] Der mit 1,2 l des Fermentationsmediums (1,5 g / l KH 2 PO 4 ; 5 g / l (NH 4 ) 2 SO 4 ; 0,5 g / l MgSO 4 × 7 H 2 O; 0,225 g / l CaCl 2 × 2 H 2 O, 0,075 g / l FeSO 4 × 7 H 2 O; 1 g / l Na 3 Citrat × 2 H 2 O; 0.5 g / l NaCl; 1 ml / l Spurenelementlösung (0.15 g / l Na 2 MoO 4 × 2 H 2 O; 2.5 g / l Na 3 BO 3 ; 0.7 g / l CoCl 2 × 6 H 2 O; OR; 1.6 g / l MnCl 2 × 4 H 2 O; 0.3 g / l ZnSO 4 × 7 H 2 O); 5 mg / l Vitamin B1; 3 g / l Phyton-Pepton (BD 211906); 1.5 g / l Hefeextrakt (Oxoid LP0021); 10 g / l Glukose; 20 mg / l Tetracyclin) befüllte Fermenter wurde ad 0,1 OD 600 mit einer Vorkultur von W3110 / pCGT bzw.W3110 / pCGT-Spr, which had been cultured for 7 h in a shake flask in LB medium (5 g / l yeast extract (Oxoid LP0021), 10 g / l tryptone (Oxoid LP0042), 5 g / l NaCl), additionally containing 1 ml / l trace element solution (0.15 g / l Na2MoO4 × 2 H2O; 2.5 g / l Na3BO3; 0.7 g / l CoCl2 × 6 H2O; 0.25 g / l CuSO4 × 5 H2O; 1.6 g / l MnCl2 × 4 H2O; 0.3 g / l ZnSO4 × 7 H2O), 3 g / l glucose, 0.55 g / l CaCl2 and 20 mg / l tetracycline, were inoculated. This started the fermentation (time point 0, start of fermentation). During fermentation, a temperature of 30 °C was maintained, and the pH was kept constant at 7.0 by adding NH₄OH or H₃PO₄. Glucose was added throughout the fermentation, with a target glucose concentration of < 5 g / L. CGTase expression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to 0.15 mM after 21 h (at the end of the logarithmic growth phase).For Spr production, basal expression of the arabinose promoter was used, which leads to the formation of small amounts of the Spr protein during fermentation. The fact that the arabinose promoter also enables small production of the target protein in the absence of an inducer is described in the prior art (Malachowska and Olszewski, Microb Cell Fact (2018) 17:40).

[0106] The medium in the fermenter was stirred at 400 rpm before inoculation and aerated with compressed air purified via a sterile filter at a rate of 1.67 Vvm (volume of air per volume of culture medium per minute). Under these initial conditions, the optical oxygen sensor (VisiFerm DO225, Hamilton) was calibrated to 100% saturation before inoculation. The target value for the O₂ saturation during fermentation was set to 30% of this value. The O₂ saturation was measured via the oxygen sensor during fermentation and recorded by the fermenter's DCU (Digital Control Unit, Sartorius Stedim). After the O₂ saturation dropped below the target value, the stirring speed was continuously increased to a maximum of 1,500 rpm under software control to bring the O₂ saturation back up to the target value.

[0107] After 65 hours of fermentation, samples were taken, the cells were separated from the fermentation medium by centrifugation, and the CGTase content in the fermentation supernatant was determined using the following enzyme test: Test buffer: 5 mM Tris-HCl buffer, 5 mM CaCl 2 x 2 H 2 0, pH 6.5 Substrate solution: 10% starch solution (Merck No. 1.01252) in test buffer, pH 6.5 Test approach: 0.2 ml substrate solution + 0.2 ml centrifuged (5 min, 12,000 rpm) culture supernatant Reaction temperature: 40°C

[0108] Enzyme test: * Pre-tempering of substrate solution and centrifuged culture supernatant (approx. 5 min at 40°C) * Preparation of the test mixture by rapidly mixing (whirl mixer) of substrate solution and centrifuged culture supernatant, whereby the centrifuged culture supernatant is optionally diluted with test buffer, so that a value of 0.9-1.5 g / l CD is determined in the subsequent HPLC analysis; * Incubation for 3 min at 40°C * Stopping the enzyme reaction by adding 0.6 ml of methanol, rapid mixing (whirl mixer) * Cooling the mixture on ice (approx. 5 min) * Centrifuging (5 min, 12,000 rpm) and pipetting off the clear supernatant * Analysis of the amount of CD produced by HPLC: The analysis was performed on an Agilent HP 1100 HPLC system with a Nucleodur 100-3 NH2-RP column (150 mm x 4.6 mm, Macherey nail) and 64% acetonitrile in water (v / v) as the mobile phase, at a flow rate of 2.1 ml / min.Detection was performed using an RI detector (1260 Infinity RI, Agilent) and quantification was carried out using peak area and an α-CD standard (Cavamax W6-8 Pharma, Wacker Chemie AG). A=G*V1*V2 / t*MGU / ml A = Activity, G = CD content in mg / L, V1 = Dilution factor in the test setup, V2 = Dilution factor of the culture supernatant before use in the test; if undiluted: V2 = 1, t = Reaction time in min, MW = Molecular weight in g / mol (MW CD = 973 g / mol), 1 Unit (U) ≙ 1 µmol / L product (CD) / min

[0109] Table 1 shows the respective cyclodextrin glycosyltransferase yields achieved. Table 1: CGTase yields in the fermentation supernatant after 65 h cultivation. tribe CGTase (U / ml) W3110 / pCGT 31 W3110 / pCGT-Spr 295

[0110] To investigate whether the release of CGTase through Spr production is due to increased cell lysis, the dry cell mass and viable cell count (VCC) of the cultures were determined at the end of fermentation. To determine the VCC, samples of the cultures were diluted 10⁸ times with LB medium in a final volume of 1 ml, and 100 µl of the dilution were plated onto LB agar plates containing 20 mg / l tetracycline. The colonies grown were counted, and, taking the dilution factor into account, the VCC of the starting cultures was calculated to be 50 × 10⁹ for W3110 / pCGT and 39 × 10⁹ for W3110 / pCGT-Spr. To determine the dry cell mass, samples containing 1 ml of fermentation culture were transferred to reaction vessels whose tare weight had been previously determined. After centrifugation (5 min, 12,000 rpm), the supernatants were removed and the cell pellets were dried in an incubator (≥ 48 h at 60°C).The containers with the dried cell pellets were then weighed, and the cell dry weight (CDW) was calculated from the difference between the mass of the containers with dried cell pellets and the mass of the empty containers. The cell dry weights were 45 g / l for W3110 / pCGT and 55 g / l for W3110 / pCGT-Spr.

[0111] Strong cell lysis leads to the release of host cell protein. This was ruled out by analyzing the protein content of the culture supernatants via SDS-PAGE ( Fig. 5Samples 1 and 2). After 65 h of fermentation, samples were taken and the cells were separated from the fermentation medium by centrifugation. The cell-free supernatant was diluted 1:10 with water, 4 µl of which was mixed with 4 µl of 2×Tris-Glycine SDS Sample Buffer (Invitrogen, Carlsbad, USA, Cat. No. LC2676) containing 1 mL / l NuPAGE Antioxidant (Invitrogen, Carlsbad, USA, Cat. No. NP0005) and 5% dithioerythritol, and boiled for 5 min at 99 °C in a thermoblock. The sample was centrifuged for 20 s at 10,000 rpm and 2 µl at a time were loaded onto a 12% NuPAGE Bis-Tris gel (1.0 mm × 15 wells, Invitrogen, Carlsbad, USA, Cat. No. NP0343). The proteins were separated by electrophoresis in 1×MOPS buffer (NuPAGE MOPS SDS Running Buffer (20×), Invitrogen, Carlsbad, USA, Cat. No. NP0001; diluted to 1× with water) for 3 h at 95 V. See Blue Prestained (Invitrogen, Carlsbad, USA; Cat. No. 100006636) was used as a standard.The proteins were stained with Instant Blue (Coomassie-based staining solution for protein gels, Expedeon Ltd., Cambridge, UK, Cat. No. ISB1L) for 1 h at room temperature and then destained overnight in water at room temperature. Apart from the CGTase protein band, only minor impurities with other proteins were visible in the gel. In particular, no increased release of the overproduced Spr protein (18 kDa) was observed.

[0112] b) In addition, CGTase production was carried out as described above using strains W3110 / pCGT-Spr and W3110 / pCGT-YdhO. YdhO production was induced 48 h after the start of fermentation by switching from pure glucose feeding to a constant glucose-arabinose mixture of 3 g / l*h in a 10:1 glucose:arabinose ratio. Spr production was induced 59 h after the start of fermentation by a single addition of arabinose to the culture, resulting in a final concentration of 1 g / l. Harvesting took place 65 h after the start of fermentation.

[0113] Table 2 shows the CGTase yields obtained in this way. Table 2: CGTase yields in the fermentation supernatant after 65 h cultivation. tribe CGTase (U / ml) W3110 / pCGT-Spr 1130 W3110 / pCGT-YdhO 1187

[0114] As previously described, the cell dry mass was determined after 65 h of cultivation. Values ​​of 50 g / l for W3110 / pCGT-Spr and 41 g / l for W3110 / pCGT-YdhO were obtained.

[0115] The culture supernatant of W3110 / pCGT-Spr was analyzed via SDS-PAGE and showed that, apart from the release of CGTase, no significant release occurred. E . E. coli -own proteins were produced ( Fig. 5 , Sample 3). Example 2: Fermentative production of a Fab antibody fragment

[0116] The CD154 fab fragment was produced using the E. coli- Strain W3110 was transformed with the plasmids pJF118ut-CD154 and pJF118ut-CD154-Spr using standard methods (e.g., TSS transformation). Selection for plasmid-containing cells was performed using tetracycline (20 mg / L). E . coli- Strains were designated W3110 / pJF118ut-CD154 and W3110 / pJF118ut-CD154-Spr.

[0117] The fermentation of this E . E. coli -Strains were fermented in stirred tank fermenters, as is the case for the fermentation of E. colipCGT strains are described in Example 1. In contrast to Example 1, CD154-Fab production was induced by 0.15 mM IPTG 26 h after the start of fermentation, and Spr production was induced by switching from pure glucose feeding to a constant glucose-arabinose mixture of 3 g / l*h in a 2:1 glucose:arabinose ratio after 41 h.

[0118] After 48 hours, samples were taken, the cells were separated from the culture medium by centrifugation, and the supernatant was analyzed to determine the CD154 Fab fragment released into the culture medium.

[0119] The CD154 Fab fragment was quantified using a sandwich ELISA assay known to those skilled in the art. An immobilized anti-human IgG (Fd) antibody (The Binding Site, Product No. PC075) served as the capture antibody, and a peroxidase-conjugated goat anti-human kappa light chain antibody (Sigma, Product No. A 7164) served as the detection antibody. Quantification was achieved by the conversion of the chromogenic substrate Dako TMB+ (Dako # S1599) by the peroxidase and the resulting change in absorbance at 450 nm. The Fab fragment "Human Fab / Kappa" (Bethyl Laboratories, Product No.: P80-115) was used to calibrate the ELISA. Table 3: CD154 antibody fragment yields in the culture supernatant after 48 h. tribe CD154 antibody fragment (mg / l) in supernatant W3110 / pJF118ut-CD154 5 W3110 / pJF118ut-CD154-Spr 1045

[0120] To investigate the influence of Spr expression on cell growth, the dry cell mass of the cultures was determined at the end of fermentation after 48 h, as described in Example 1. The results are summarized in Table 4. Table 4: Cell dry mass (CDW) of CD154-producing strains in the fermenter after 48 h. tribe CDW (g / l) W3110 / pJF118ut-CD154 48 W3110 / pJF118ut-CD154-Spr 43

Claims

1. Method for fermentative production of recombinant proteins, characterized in that a bacterial strain containing an open reading frame encoding a signal peptide and a recombinant protein under the control of a functional promoter, wherein the bacterial strain contains an additional open reading frame encoding a signal peptide and a peptidoglycan peptidase under the control of a functional promoter, wherein the peptidoglycan peptidase is (i) Spr, (ii) YdhO or (iii) Spr and YdhO, and wherein the protein chosen as peptidoglycan peptidase has murein DD-endopeptidase activity, is cultured in a fermentation medium, the fermentation medium is removed from the cells after the fermentation and the recombinant protein is isolated from the fermentation medium, wherein the recombinant protein is released from the periplasm of the cell and the yield of recombinant protein which is released into the culture medium is at least 1.1 times as high as the yield which can be achieved with corresponding bacterial strains without overproduction of the peptidoglycan peptidase, and wherein the bacterial strain is a strain of the species Escherichia coli.

2. Method for fermentative production of recombinant proteins according to Claim 1, characterized in that Spr is the sequence specified by amino acids 27-188 in SEQ ID No. 5.

3. Method for fermentative production of recombinant proteins according to one or more of Claims 1 to 2, characterized in that YdhO is the sequence specified by amino acids 28-271 in SEQ ID No. 7.

4. Method for fermentative production of recombinant proteins according to one or more of Claims 1 to 3, characterized in that the functional promoter which controls the expression of the open reading frame encoding the peptidoglycan peptidase is an inducible promoter.

5. Method for fermentative production of recombinant proteins according to one or more of Claims 1 to 4, characterized in that the recombinant protein is a heterologous protein.

6. Method according to one or more of Claims 1 to 5, characterized in that the expression of the peptidoglycan peptidase is induced.

7. Method according to one or more of Claims 1 to 6, characterized in that the expression of the peptidoglycan peptidase is induced after the induction of the expression of the recombinant protein.

8. Method according to one or more of Claims 1 to 7, characterized in that, 5 to 24 hours after induction of the expression of the additional peptidoglycan peptidase, the cell dry weight after the removal of the fermentation medium differs by not more than 20% from the cell dry weight of a cell culture at the same time point from a fermentation method for a bacterial strain which does not express the additional peptidoglycan peptidase.