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

Description

[0001] This document describes a bacterial strain containing an open reading frame encoding a recombinant protein under the control of a functional promoter, characterized in that the bacterial strain contains an open reading frame encoding a mutated peptidoglycan-associated lipoprotein (Pal protein) under the control of a functional promoter, wherein the Pal protein is mutated such that it lacks a membrane anchor for the outer cell membrane of the bacterium. Also described herein are a plasmid encoding a recombinant protein and the mutated Pal protein, as well as a method for the fermentative production of recombinant proteins using the bacterial strain.

[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 correct folding, as this avoids complex cell disruption and protein folding processes. A number of such methods are described in the literature. E. coli -strains and processes with E. coli -strains revealed through which a release of recombinant proteins into the culture medium is achieved. i) One possibility is the use of so-called "leaky" strains. These include mutants of E. coliTo understand why some mutants have a defect in their 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, such as alkaline phosphatase PhoA or RNase I, into the fermentation medium. Such strains are extremely sensitive to EDTA, various detergents, and antibiotics, and exhibit growth defects (Lazzaroni and Portalier 1981, J. Bact. 145, pp. 1351–1358; Lazzaroni and Portalier 1992, Mol. Microbiol. 6, pp. 735–742; Chen et al. 2014, Microb. Biotechnol. 7, pp. 360–370; Bernstein et al. 1972, J. Bacteriol. 112, pp. 74–83). Therefore, they are only of limited use for cultivation under high-cell-density fermentation conditions.ii) Another approach to achieve the release of recombinant proteins is the expression of a protein that encodes the cell envelope of . E. coliThis permeabilization promotes the release of proteins into the culture medium. The expression of bacteriocin release proteins (BRPs), small lipoproteins that lead to the degradation of the outer membrane and cell lysis, is well-documented. Overexpression of ColE1-BRP (Kil protein) or cloacin DF13 BRP has been shown to release interleukin-2, β-glucanase, alkaline phosphatase, or β-lactamase into the culture medium (Beshay et al. 2007, Biotechnol. Lett. 29, pp. 1893-1901; Robbens et al. 1995, Protein Expr. Purif. 6, pp. 481-486; Sommer et al. 2010, J. Biotechnol. 145, pp. 350-358). iii) Another approach to destabilizing the cell envelope is described in Wan and Baneyx (1998, Protein Expr. Purif. 14, pp. 13-22). Here, membrane integrity was disrupted by overexpressing the soluble, C-terminal domain of the TolA protein (TolAIII).This resulted in a similar phenotype to that seen in tolA deletion mutants, with increased release of the proteins RNaseI and alkaline phosphatase from the periplasm into the medium, as well as sensitivity to deoxycholate (Levengood-Freyermuth et al. 1993, J. Bacteriol. 175, pp. 222-228). Overexpression of the TolAIII protein significantly increased the release of an OmpA-TEM β-lactamase protein. However, β-lactamase expression decreased by a factor of 1.5-2 compared to the control. Furthermore, cell viability was severely impaired by TolAIII expression, just as in tolA mutants. The number of colony-forming units (CFU), a measure of culture viability, decreased by a factor of 1000 as early as 3 hours after the start of TolAIII expression in the shake flask. Furthermore, the cells were sensitive to low concentrations of SDS (0.02%), indicating significant membrane defects.

[0003] Such cells are not suitable for cultivation at high cell densities over the extended period necessary for the production of complex eukaryotic proteins. A further disadvantage of TolAIII co-expression is that TolAIII itself is found in large quantities in the culture supernatant, thus contaminating the extracted target protein.

[0004] The object of the present invention is to provide a bacterial strain which releases a recombinant protein into the culture medium in increased yield without causing severe cell lysis.

[0005] This problem is solved by a bacterial strain containing an open reading frame encoding a recombinant protein under the control of a functional promoter, characterized in that it contains an open reading frame encoding a mutated peptidoglycan-associated lipoprotein (Pal protein) under the control of a functional promoter, wherein the mutated Pal protein is mutated such that it lacks a membrane anchor for the outer cell membrane of the bacterium.

[0006] The invention is defined in the attached claims.

[0007] The use of the bacterial strain defined above in a fermentation process has the advantage that, along with the recombinant protein, an additional protein, namely a mutated form of the Pal protein, is expressed. The expression of the mutated Pal protein leads to restricted permeabilization of the bacterium's outer cell membrane, allowing recombinant proteins to be released from the cell without causing cell death. This improved release enables the isolation of recombinant proteins in increased yields. The disclosure provides a corresponding bacterial strain, a process for the fermentative production of recombinant proteins using this bacterial strain, and a corresponding plasmid. In this way, higher product yields in the culture supernatant can be achieved compared to the prior art, without significant bacterial cell death.

[0008] The bacterial strain is preferably characterized by the fact that the bacterial strain is a gram-negative bacterium, particularly preferably a bacterial strain of the genus Enterobacteriaceae, particularly preferably a strain of the species Escherichia It concerns E. coli.

[0009] Peptidoglycan-associated lipoprotein (Pal, Pal protein) is a bacterial periplasmic protein whose lipidated N-terminus is anchored in the outer membrane of the bacterial cell and whose C-terminus interacts with the peptidoglycan layer. Pal is also a component of the Pal-Tol system (Godlewska et al. 2009, FEMS Microbiol. Lett. 298, pp. 1-11) and interacts with various other periplasmic proteins, such as lpp and ompA (Cascales et al., 2002, J. Bacteriol. 184, pp. 754-759; Godlewska et al.). In technical applications, the Pal protein is used, for example, to anchor antibody fragments, in the form of a fusion protein consisting of Pal protein and antibody fragment, to the cell surface of bacteria (Fuchs et al. 1991, Biotechnology (NY) 9, pp. 1369-1372; Dhillon et al. 1999, Lett. Appl. Microbiol. 28, pp. 350-354).

[0010] The wild-type Pal gene refers to the form of the Pal gene that arose naturally through evolution and is present in the wild-type bacterial genome. The wild-type Pal gene expresses the wild-type Pal precursor protein, which comprises a signal peptide and the amino acid sequence of the wild-type Pal protein (SEQ ID No. 4). After cleavage of the 21-amino-acid signal sequence, the mature wild-type Pal protein consists of... E. coli (Strain K-12) has a sequence of 152 amino acids (see gene bank entry X05123, Uniprot P0A912) and carries the highly conserved amino acid cysteine ​​at amino acid position 1, which is acylated with the membrane anchor in bacterial lipoproteins (see Zückert et al. 2014, Biochimica et Biophysica Acta 1843, pp. 1509-1516 or Konovalova and Silhavy 2015, Phil. Trans. R. Soc. B 370: 20150030). In contrast, the mutated Pal protein lacks a membrane anchor for the outer cell membrane of the bacterium.

[0011] The term Pal precursor protein (precursor form of the Pal protein) refers to the Pal protein that includes the amino acid sequence of the Pal protein and the signal sequence and does not carry any post-translational modifications.

[0012] Mature wild-type Pal protein refers to the Pal protein that contains the amino acid sequence of the wild-type Pal protein but no longer carries a signal sequence, as this has been cleaved off, and which may contain post-translational modifications. One such post-translational modification is, for example, the acylation of the Pal protein with the membrane anchor.

[0013] 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.

[0014] 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.

[0015] For a functional promoter, the ORF under the regulation of this promoter is transcribed into an RNS.

[0016] A messenger RNA (mRNA) that contains only one open reading frame is called monocistronic.

[0017] An operon is a functional unit of DNA that includes multiple ORFs, a promoter, and possibly other expression signals.

[0018] The bacterial strain can contain (1) one ORF encoding a recombinant protein, (2) multiple ORFs encoding the same recombinant protein, or (3) multiple ORFs encoding different 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 relevant for the expression of proteins composed of multiple polypeptides (subunits). An example is the expression of antibody fragments, which are then assembled after transport from the cytoplasm. Each of these ORFs can be located in a separate gene, or multiple ORFs can be organized within a single operon, meaning they are regulated by common expression signals.

[0019] Each operon can also contain an ORF encoding the mutated Pal protein. Even if one or more ORFs encoding one or more recombinant proteins and the ORF encoding the mutated Pal protein are located in an operon, the mutated Pal protein is always expressed as a separate protein and not as a fusion protein, since the ORF encoding the mutated Pal protein is, by definition, characterized by its own translation start and stop codons.

[0020] 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) are organized in one operon.

[0021] It is preferred that the ORF encoding the mutated Pal protein is transcribed as monocistronic messenger RNA. This means that the mutated Pal protein is expressed by a separate, distinct gene. This gene is referred to as the mutated Pal gene.

[0022] The ORFs encoding the recombinant protein and the mutated Pal protein can be expressed chromosomally or from a plasmid. If they are separate genes, they can also be expressed by separate plasmids that are compatible with each other in terms of origin of replication and selection markers. Plasmid-encoded ORFs are preferred.

[0023] The ORF used in this invention encoding the mutated Pal protein contains changes in the amino acid sequence such as substitutions, deletions and / or insertions compared to the wild-type Pal protein.

[0024] According to the present invention, the ORF sequence encoding the mutated Pal protein is preferably a DNA sequence that leads to the expression of a mutated Pal protein originating from the same organism used to produce the recombinant proteins.

[0025] The origin of the DNA sequence of the ORF according to the invention encoding the mutated Pal protein is not limited to the bacterial strain used for the production of the recombinant protein, as long as the corresponding Pal protein in its non-mutated form is functional in the bacterial strain used for the production of the recombinant protein. In this context, "functional" means that all biological functions of the Pal protein in the bacterial strain used for the production of the recombinant protein can be assumed by the aforementioned Pal protein. In particular, this means that the phenotypic expressions of a bacterial strain with a chromosomal deletion of the Pal gene can be complemented by an additionally introduced copy of the aforementioned Pal gene.

[0026] The terms "an ORF encoding 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. 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.

[0027] 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.

[0028] To achieve the release of the recombinant protein into the culture medium, it is necessary that both the recombinant protein and the mutated Pal protein 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 protein to be produced must be articulated. into link a frame 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 mutated Pal protein in its precursor form carries the native signal sequence of the Pal protein, as described in Chen and Henning (1987, Eur. J. Biochem. 163, pp. 73-77). Preferably, the recombinant protein and the mutated Pal protein in their precursor form carry different signal sequences, both of which induced protein export.

[0029] The expression of the ORFs encoding the mutated Pal protein and the recombinant protein can be controlled by one or more promoters. It is preferred that the ORFs for the mutated Pal protein and for the recombinant protein are controlled by different promoters.

[0030] The DNA segment encoding the mutated Pal protein can first be isolated by PCR using oligonucleotides as primers and a DNA template encoding the Pal protein, 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 of the mutated Pal 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).

[0031] 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 either two different antibiotic resistances or two different auxotrophy markers.

[0032] 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 mutated Pal protein can be controlled as operons by a single promoter or by different promoters. Preferably, the ORFs encoding the recombinant protein and the ORF encoding the mutated Pal protein are controlled by different inducible promoters. Particularly preferably, the recombinant protein is expressed under the control of the tac promoter and the mutated Pal protein under the control of the ara (arabinose) promoter.

[0033] The amino acid sequence of the mutated Pal protein can be produced in a known manner, for example by removing the conserved cysteine ​​residue or by introducing other mutations that prevent modification with a membrane anchor. Methods are known to those skilled in the art to verify whether an introduced mutation results in the mutated Pal protein no longer containing a membrane anchor after expression in a bacterial strain (Hayashi and Wu 1990, J. Bioenerg. Biomembr. 22, pp. 451-471; Giam et al. 1984, Eur. J. Biochem. 141, pp. 331-337; Lazzaroni and Portalier 1992; Mizuno 1979, J. Biochem. 86, pp. 991-1000).

[0034] In a preferred embodiment, the bacterial strain is characterized in that the ORF encoding the mutated Pal protein is mutated such that it codes for a mutated Pal protein that is mutated at one or more of the amino acid positions 1 to 6, preferably 1 to 4, and particularly preferably 1 to 2. The amino acid position refers to the amino acids that follow the signal sequence; i.e., amino acid position 1 is the first amino acid after the signal sequence.

[0035] Preferably, the mutation is a deletion (missing amino acids) or substitution (exchange of amino acids), in particular preferably a deletion.

[0036] The bacterial strain is particularly favored in that the ORF encoding the mutated Pal protein is mutated in such a way that it codes for a mutated Pal protein in which the N-terminal cysteine ​​residue is substituted.

[0037] In a further preferred embodiment, the bacterial strain is characterized in that the ORF encoding the mutated Pal protein is mutated such that it codes for a mutated Pal protein lacking the N-terminal cysteine ​​residue. Preferably, the amino acid at position 1, and in particular preferably the cysteine ​​residue, is deleted.

[0038] The N-terminus, or amino-terminus, is the end of the Pal protein that possesses a free amino group (NH₂). The N-terminal cysteine ​​residue is the cysteine ​​residue at amino acid position 1 after the signal sequence.

[0039] In particular, the preferred amino acid sequence of the mutated Pal protein is the sequence specified in SEQ ID No. 7 (Pal22A), in which the N-terminal cysteine ​​has been replaced by the amino acid alanine, so that alanine follows the first 21 amino acids of the signal sequence.

[0040] In a preferred embodiment, the bacterial strain is characterized in that the ORF encoding the mutated Pal protein is mutated such that it codes for a mutated Pal protein in which amino acids 1 to 6, preferably 1 to 4, and particularly preferably 1 to 2 are missing. Particularly preferably, the amino acid sequence of the mutated Pal protein is the sequence specified in SEQ ID No. 5 (PalΔ 22-27), in which an alanine follows the first 21 amino acids of the signal sequence. The sequence following the signal peptide of the mutated Pal protein is identical to amino acids 7-152 of the mature wild-type Pal protein. E. coli (Amino acid sequence of the wild-type protein see SEQ ID No. 4).

[0041] 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. coliK12 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).

[0042] 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.

[0043] 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.

[0044] Examples of proteins derived from molecular scaffolds include evibodies (derived from CTLA-4) and affibodies (protein A of CTLA-4). S. aureus ) ,Avimers (of the human A-domain family), transbodies (of transferrin), DARPins (of the 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.

[0045] Preferably, the bacterial strain is characterized by the additional presence of the wild-type Pal gene encoding peptidoglycan-associated lipoprotein (Pal). Most preferably, the wild-type Pal gene is under the control of a functional promoter.

[0046] Another object of disclosure is a plasmid containing an open reading frame encoding a recombinant protein under the control of a functional promoter, characterized in that it contains an open reading frame encoding a signal peptide and a mutated Pal protein under the control of a functional promoter, wherein the Pal protein is mutated such that it does not have a membrane anchor for the outer cell membrane of the bacterium.

[0047] The preferred and particularly preferred features mentioned for the bacterial strain also apply to the features mentioned in the plasmid, wherein the preferred and particularly preferred embodiments mentioned for the bacterial strain are also preferred or particularly preferred in the plasmid.

[0048] Likewise, the aforementioned definitions and preferred embodiments apply to the ORF encoding a signal peptide and a mutated Pal protein, and to the ORF encoding the recombinant protein.

[0049] For example, one or more ORFs can encode one or more recombinant proteins within a single operon. Preferably, the ORF encoding the mutated Pal protein resides in a separate gene. This means that the mutated Pal protein and the recombinant proteins can be expressed independently. In the particularly preferred case where the promoter(s) of the recombinant protein(s) differ from the promoter of the mutated Pal protein and are differently inducible, the plasmid offers the distinct advantage that, during the transformation of bacteria with this plasmid, the optimal time for protein expression can be selected independently.

[0050] Introducing the plasmid into the bacterial strain using methods known to those skilled in the art and expressing the recombinant protein and the mutated Pal protein from this plasmid has the advantage that the release of the recombinant proteins from the cells of the bacterial strain is improved and these can be isolated in increased yield.

[0051] In contrast to the chromosomal integration of the ORFs encoding the recombinant protein and the mutated Pal protein, the advantage of using plasmids is that the plasmid-bearing cells of the bacterial strain have a selective advantage and can be selected by standard methods.

[0052] Since each plasmid contains at least one origin of replication (ORI), a further advantage is that plasmids replicate autonomously. Furthermore, it is particularly advantageous that plasmids can be present in high copy numbers in the cells of the bacterial strain and are inherited. The invention relates to a method for the fermentative production of recombinant proteins, characterized in that a bacterial strain as described herein 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.

[0053] 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 methods known to those skilled in the art in a shake flask or in a bioreactor (fermenter).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] If the expression of the recombinant protein and / or the mutated Pal protein 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 as a single or multiple dose. Alternatively, the inducer can be added continuously. Preferably, the expression of the recombinant protein is induced by adding IPTG, and the expression of the mutated Pal protein is induced by adding arabinose. Particularly preferably, IPTG is added as a single dose, and the induction of the expression of the mutated Pal protein is managed by adding a mixture of glucose and arabinose after the glucose concentration has fallen below 5 g / L.The proportion of arabinose in the mixture is preferably between 33 wt.% and 66 wt.%, particularly preferably 33 wt.%.

[0059] The induction of expression of the mutated Pal protein 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.

[0060] 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 most 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Expression of the mutated Pal protein improves the release of recombinant proteins into the culture supernatant. This improved release allows for the isolation of recombinant proteins in increased yield.

[0065] Increased yield means that preferably at least 110%, particularly preferably at least 150%, and especially preferably at least 200% of the amount of recombinant protein that can be produced according to the prior art with a wild-type bacterial strain containing a gene for the recombinant protein or with a wild-type bacterial strain containing a gene for the recombinant protein and additionally expressing a protein for destabilizing the bacterial cell wall is released into the culture medium. That is, the yield of recombinant protein released into the culture medium is preferably 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 obtained with corresponding prior art bacterial strains.

[0066] Example 1 shows that, compared to the corresponding wild-type or TolAIII-expressing bacterial strain, more than 2-3 times as much CGTase can be produced in the cell supernatant in the same culture volume when PalΔ 22-27 or Pal22A is expressed (see Table 1). In Example 2, CGTase production in the PalΔ 22-27-expressing mutant is also increased by a factor of 2-3 compared to the corresponding wild-type or TolAIII-expressing bacterial strain (see Table 2). Example 3 (see Table 3) also confirms a significant increase.

[0067] Another advantage of the described Pal mutants is that cell lysis is only slightly increased compared to corresponding wild-type cells during a culture period of 5–24 h. Wan and Baneyx (1998, so) show that TolAIII overproduction leads to a CFU count approximately 1400–3000 times lower than in uninduced cells after just a few hours and conclude that the long-term viability of the bacterial cells decreases significantly by affecting the outer membrane. In contrast, 24 h after induction of expression of the mutated PalΔ 22-27 protein, the CFU count decreases by only a factor of 2 compared to the corresponding wild-type bacterial strain (see Example 3).

[0068] Using the co-expression of the mutated Pal protein 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 Pal protein mutated according to the invention, the heavy and light chains of the antibody are released into the fermentation medium in increased concentrations.

[0069] This method has the major advantage of being suitable for the production of recombinant proteins that require long culture times, as well as for high-cell-density fermentation. This allows for the production of complex, eukaryotic proteins as recombinant proteins.

[0070] Preferably, the process is characterized in that, after the fermentation medium has been separated, the recombinant proteins are purified from the fermentation medium.

[0071] 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.

[0072] The method is preferably characterized in that the expression of the mutated Pal protein is induced. This means that either the expression of the mutated Pal protein or the expression of both the recombinant protein and the mutated Pal protein is induced. In this preferred embodiment, the expression of the mutated Pal protein is in any case 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. It is particularly preferred that both the ORF encoding the recombinant protein and the ORF encoding the mutated Pal protein are under the control of an inducible promoter, and especially preferably, of promoters with different inducibility levels. Therefore, the expression of both the mutated Pal protein and the recombinant protein is preferably inducible.

[0073] Since the promoters of the genes encoding the recombinant protein and the mutated Pal protein 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 mutated Pal protein.

[0074] By using inducible promoters, the expression of the corresponding proteins can be induced at any desired time point during fermentation. Preferably, the expression of the mutated Pal protein is induced after the induction of the expression of the recombinant protein. Particularly preferably, the expression of the mutated Pal protein is induced at least 1 hour, more preferably at least 2 hours, and furthermore particularly preferably 15 to 24 hours, and especially preferably 19 hours, after the induction of the expression of the recombinant protein.

[0075] The induction of expression of the recombinant protein and the mutated Pal protein 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 mutated Pal gene contains an arabinose (ara) promoter, expression of the mutated Pal protein 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.

[0076] According to the invention, even after induction of expression of the mutated Pal protein and further culture of the cells, the cell density of the strain is comparable to that of a bacterial strain that does not express a mutated Pal protein. In particular, there is no significant decrease in optical density due to cell lysis and only a slight decrease in the number of viable cells measured as colony-forming units (CFUs).

[0077] The measured optical density (OD) is higher the higher the cell density. Bacterial strains with destabilized cell walls generally show higher cell lysis. With higher cell lysis, the cell density decreases, and consequently, so does the OD value.

[0078] 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 for both the production of complex eukaryotic proteins requiring a correspondingly long culture period and for high-cell-density fermentation.

[0079] Within the scope of this invention, fermentations in which cell dry weights >50 g / l are achieved are considered high-cell-density fermentations. This is also reflected in the literature (Bruschi et al. 2014, Microb. Cell Fact. 13:99; Shokri and Larsson 2004, Microb. Cell Fact. 3:9; Knabben et al. 2010, J. Biotechnol. 150, pp. 73-79).

[0080] A long cultivation period means that the cultivation time is at least 24 h, preferably at least 48 h and most preferably at least 72 h.

[0081] Within the scope of the invention, reduced (or only slightly increased or no significant) cell lysis means that the OD 600, CDW, or viable cell count of the bacterial strain culture, determined after a culture period of preferably 5-24 hours following induction of expression of the mutated Pal protein, is only slightly or not at all reduced compared to the wild-type bacterial strain not carrying this Pal mutation. In particular, for the culture of bacterial strains containing modifications to destabilize their cell envelope, whose cell lysis is significantly increased, as shown by Wan and Baneyx (1998, ed.) for cells expressing the TolAIII protein, the bacterial strains expressing the mutated Pal protein offer a considerable advantage.

[0082] A cell culture produced according to the fermentation process according to the invention is therefore preferably characterized in that, 5 to 24 hours, particularly preferably 7 hours, after induction of expression of the mutated Pal protein, it exhibits an optical density determined at 600 nm (OD 600) that is at most 20%, particularly preferably at most 10%, and particularly preferably at most 5% lower than the OD 600 value 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 the mutated Pal protein encoding the ORF. The optical density of the cell culture at 600 nm is determined spectrophotometrically.

[0083] The optical density of the cell culture, determined using a spectrophotometer at 600 nm, depends on 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.

[0084] Alternatively, the cell mass can also be determined as cell dry weight (CDW) by isolating a defined amount of culture by filtration or centrifugation, preferably centrifugation, then drying and weighing it.

[0085] Another alternative that allows a comparison of the viability of a bacterial strain expressing the mutated Pal protein with that of a bacterial strain not expressing the mutated Pal protein is the determination of the viable cell count as colony forming units (CFU) per defined culture volume. The experimental procedure is described in Wan and Baneyx (1998, so). The CFU unit indicates the number of viable cells in the culture. The viable cell count of a bacterial strain expressing the mutated Pal protein and a recombinant protein is reduced by at most a factor of 100, preferably by at most a factor of 10, and most preferably by at most a factor of two compared to a bacterial strain not expressing the mutated Pal protein.

[0086] In contrast, Wan and Baneyx (1998, so) describe in Table 2 that 3 hours after induction of TolAIII expression with IPTG, the viable cell count, determined as the number of CFU / ml, continues to increase in the wild-type bacterial strain, while it is significantly reduced in the TolAIII-expressing bacterial strain. The authors conclude that while TolAIII overexpression has a lesser impact on bacterial growth during the logarithmic growth phase of the bacterial strain, it significantly reduces bacterial cell viability over the long term by permeabilizing the outer cell membrane.

[0087] The minimal impact on cell integrity caused by the expression of the mutated Pal protein is another significant advantage of the invention. Furthermore, by preferentially cloning the ORF encoding the mutated Pal protein under an inducible promoter, the expression of the mutated Pal protein can be controlled, thus narrowly limiting the culture time in which the desired effect occurs, which in turn limits any potential impact on the viability of the bacterial cells. Fig. 1 shows the plasmid map of the plasmid pCGT-Pal. Fig. 2 shows the plasmid map of the plasmid pCGT-TolAIII. Fig. 3 shows the plasmid chart of the plasmid pJF118ut-CD154. Fig. 4 shows the plasmid chart of the plasmid pJF118ut-CD154-Pal. Fig. 5 shows the plasmid map of the plasmid pCGT-Pal22A.

[0088] 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 CGTase signal peptide rrnB terminator: terminator region of the rrnB gene trpA terminator: terminator region of the trpA gene Pal: peptidoglycan-associated lipoprotein Pal-SP: signal peptide of Pal TolAIII: domain of the TolA protein OmpA-SP: signal peptide of OmpA HisTag: histidine tag light chain: Antibody fragment encompassing the VL and CL domains heavy chain: Antibody fragment encompassing the VH and CH1 domains ScaI / MauBI / EcoRI: Interfaces of the corresponding restriction endonucleases Examples

[0089] The invention will be described in more detail below using exemplary embodiments, without being limited by this.

[0090] 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:

[0091] 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.

[0092] 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-Pal:

[0093] To pCGT-Pal (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 i of the signal sequence of the peptidoglycan-associated lipoprotein from E. coli K12 (nucleotides 136-198 from GenBank entry X05123.1 encoding amino acids -21 to -1, nucleotides 216-278 from SEQ ID No. 1), ii the nucleotides 217-654 from GenBank entry X05123.1 encoding amino acids 7-152 of the peptidoglycan-associated lipoprotein from E. coli K12 (nucleotides 279-716 of SEQ ID No. 1) and iii the terminator of the trpA gene fromE . E. coli (Nucleotides 749-774 of SEQ ID NO:1).

[0094] 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 in an undirected manner; however, preference was given to the plasmid in which the DNA fragment was inserted in the opposite reading direction to the gene encoding CGTase. Detection was achieved via the restriction pattern of the restriction enzyme ScaI and sequencing. This plasmid was designated pCGT-Pal and encodes the protein PalΔ 22-27, also known as PalD22-27 (as reported in SEQ ID No. 5). pCGT-Pal22A :

[0095] To pCGT-Pal22A (plasmid map see. Fig. 5 To obtain this, a DNA fragment was produced by Eurofins Genomics using gene synthesis. This DNA fragment xIA (specified in SEQ ID No. 6) 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. 6), the Shine-Dalgarno sequence (nucleotides 203-208 of SEQ ID No. 6) and a nucleotide fragment encoding a fusion of i of the signal sequence of the peptidoglycan-associated lipoprotein from E. coli K12 (nucleotides 136-198 from GenBank entry X05123.1 encoding amino acids -21 to -1, nucleotides 216-278 from SEQ ID No. 6), ii the amino acids 1-152 of the peptidoglycan-associated lipoprotein from E. coli K12 (cf. GenBank: X05123.1, nucleotides 279-734 of SEQ ID No. 6), wherein the amino acid cysteine ​​at position 1 was replaced by an alanine and iii the terminator of the trpA gene from E. coli (Nucleotides 767-792 of SEQ ID NO:6).

[0096] This DNA fragment xIA was cut with the restriction enzyme MauBI and ligated to the expression vector pCGT, which had also been cut with the same restriction enzyme. Cloning was performed in an undirected manner; however, preference was given to the plasmid in which the DNA fragment was inserted in the opposite reading direction to the gene encoding CGTase. Detection was achieved via the restriction pattern of the restriction enzyme ScaI and sequencing. This plasmid was designated pCGT-Pal22A and encodes the protein Pal22A (specified in SEQ ID No. 7). pCGT-TolAIII:

[0097] The overexpression of TolAIII is state of the art (Wan and Baneyx 1998, so), therefore pCGT-TolAIII was chosen as a comparative example representing the state of the art, in which the outer cell envelope of a bacterium containing this plasmid and expressing TolAIII is destabilized and the release of recombinant proteins is increased.

[0098] To pCGT-TolAIII (plasmid map see. Fig. 2 To obtain this, a DNA fragment was produced by gene synthesis at the company eurofins Genomics. 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 i of the signal sequence of ompA from E. coliK12 (nucleotides 216-278 of SEQ ID No. 2), ii the amino acids 291-421 of the TolA protein from E. coli K12 (coding for TolAIII, nucleotides 279-671 of SEQ ID No. 2) and iii the terminator of the trpA gene from E. coli (Nucleotides 714-729 of SEQ ID NO:2).

[0099] This DNA fragment xII was cut with the restriction enzyme MauBI and ligated to the expression vector pCGT (so), which had been cut with the same restriction enzyme. Cloning was performed in an undirected manner; however, preference was given to the plasmid in which the DNA fragment was inserted in the opposite reading direction to the gene encoding CGTase, with detection via the restriction pattern of the restriction enzymes ScaI and EcoRI or by sequencing. This plasmid was designated pCGT-TolAIII. 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 Karpusias 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 Karpusias 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-Pal:

[0103] The DNA fragment xI encoding the Pal variant 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-Pal. Cloning was performed in an undirected manner; however, preference was given to the plasmid in which the DNA fragment xI was inserted in the opposite reading direction to the DNA fragment xIII encoding CD154. Detection was achieved via the restriction pattern of the restriction enzyme ScaI and sequencing. This plasmid was designated pJF118ut-CD154-Pal (see plasmid chart). Fig. 4 ). Example 1: Production of cyclodextrin glycosyltransferase (CGTase) in a shake flask

[0104] For the production of CGTase from Klebsiella pneumoniae M5a1 was the E . E. coli strainW3110 (ATCC 27325) was transformed with the plasmids pCGT, pCGT-Pal, pCGT-Pal22A, or pCGT-TolAIII 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 / pCGT, W3110 / pCGT-Pal, W3110 / pCGT-Pal22A and W3110 / pCGT-TolAIII.

[0105] The transformed strains were grown in 10 ml of 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 x 2 H₂O; 2.5 g / l Na₃BO₃; 0.7 g / l CoCl₂ x 6 H₂O; 0.25 g / l CuSO₄ x 5 H₂O; 1.6 g / l MnCl₂ x 4 H₂O; 0.3 g / l ZnSO₄ x 7 H₂O), 3 g / l glucose, 10 g / l lactose, 0.55 g / l CaCl₂ and 20 mg / l tetracycline, at 30 °C. The medium used was an autoinduction medium, meaning no additional inducer for the tac promoter was required. After the glucose in the medium was metabolized, the lactose could be taken up by the cells. This led to the induction of protein expression originating from the tac promoter. CGTase was produced by all four plasmids.The addition of 0.2% (w / v) arabinose to the culture medium after 48 h induced the expression of the Pal variants, which are also contained on the plasmid and under the control of the arabinose promoter, from the plasmids pCGT-Pal and pCGT-Pal22A, and the expression of TolAIII from the plasmid pCGT-TolAIII. The control cultures containing the plasmid pCGT were also treated with arabinose for better comparability.

[0106] After 72 hours of culture, samples were taken, the cells were separated from the culture medium by centrifugation, and the CGTase content in the culture supernatant was determined based on the amount of cyclodextrin (CD) enzymatically produced from starch 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 Enzyme test:

[0107] * 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).

[0108] Calculation of enzyme activity: A = G*V1*V2 / (t*MG) [U / 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 CGTase yields achieved. Table 1: CGTase yields in shake flask supernatant after 72 h cultivation. tribe Shaker flask: CGTase (U / ml) W3110 / pCGT 39 W3110 / pCGT-Pal 134 W3110 / pCGT-Pal22A 95 W3110 / pCGT-TolAIII 42 Example 2: Fermentative production of cyclodextrin glycosyltransferase (CGTase) in a stirred tank fermenter

[0110] The strains W3110 / pCGT, W3110 / pCGT-Pal and W3110 / pCGT-TolAIII described in Example 1 were used for the production of cyclodextrin glycosyl transferase (CGTase) from Klebsiella pneumoniae M5a1.

[0111] CGTase production took place in stirred tank fermenters.

[0112] The one with 1.2 1 of the fermentation medium (1.5 g / l KH 2 PO 4 ; 5 g / l (NH 4 ) 2 SO 4 ; 0.5 g / l MgSO 4 x 7 H 2 O; 0.225 g / l CaCl 2 x 2 H 2 O, 0.075 g / l FeSO 4 x 7 H 2 O; 1 g / l Na 3 Citrate x 2 H 2 O; 0.5 g / l NaCl; 1 ml / l trace element solution (0.15 g / l Na 2 MoO 4 x 2 H 2 O; 2.5 g / l Na 3 BO 3 ; 0.7 g / l CoCl 2 x 6 H 2 O; 0.25 g / l CuSO 4 x 5 H 2 O; 1.6 g / l MnCl 2 x 4H2O; The fermenter, filled to 0.1 OD 600 (0.3 g / l ZnSO₄ x 7 H₂O), 5 mg / l Vitamin B1, 3 g / l Phyton-Peptone (BD 211906), 1.5 g / l Yeast Extract (Oxoid LP0021), 10 g / l Glucose, and 20 mg / l Tetracycline), was inoculated with a pre-culture that had been cultivated for 7 h in a shake flask using the LB medium described in Example 1. This started the fermentation (time 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 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 22 h (at the end of the logarithmic growth phase). Expression of the Pal variant was induced 24 h and 41 h after the start of fermentation, and TolAIII expression was induced 41 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 2:1 glucose:arabinose ratio.

[0113] 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.

[0114] After 48 h 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 by the activity assay as described in Example 1. Table 2 shows the respective cyclodextrin glycosyltransferase yields obtained. Table 2: CGTase yields in the fermentation supernatant after 48 h cultivation. tribe CGTase (relative yield in %) W3110 / pCGT 100 W3110 / pCGT-Pal, induction after 24 h 430 W3110 / pCGT-Pal, induction after 41 h 690 W3110 / pCGT-TolAIII 260

[0115] To investigate the influence of the expression of the Pal variant on cell growth, the growth of the cells was determined during the fermentation process for W3110 / pCGT and W3110 / pCGT-Pal (induction after 41 h) by measuring the optical density at 600 nm (OD 600 ) on a spectrophotometer (Beckman Coulter DU 730).

[0116] Additionally, the cell dry weight of the cultures was determined. To determine the cell dry weight, samples containing 1 ml of fermenter 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). Subsequently, the vessels containing the dried cell pellets were weighed, and the cell dry weight (CDW) was calculated from the difference between the mass of the vessels with the dried cell pellets and the mass of the empty vessels. The results are summarized in Table 3. Table 3: Optical density (OD 600 ) and cell dry mass (CDW) of the CGTase-producing strains in the fermenter. tribe OD 600 CDW (g / l) Cultural duration Cultural duration 41 h 48 h 41 h 48 h W3110 / pCGT 142 153 51 55 W3110 / pCGT-Pal, induction after 41 h 146 150 52 52 Example 3: Production of a Fab antibody fragment in a shake flask

[0117] The CD154-Fab fragment was produced in a shake flask analogously to the procedure described in Example 1 using the E. coli strains W3110 / pJF118ut-CD154 and W3110 / pJF118ut-CD154-Pal. After 72 h, 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. The cell pellet was resuspended in PBS buffer, and the cells were lysed using a FastPrep homogenizer (MP Biomedicals). The intracellular CD154-Fab fragment was then determined from the resulting cell lysate.

[0118] 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 absorption at 450 nm. The Fab fragment "Human Fab / Kappa" (Bethyl Laboratories, Product No.: P80-115) was used to calibrate the ELISA. Table 3: Yields of CD154 antibody fragment in culture supernatant. tribe CD154 antibody fragment (mg / l) in supernatant W3110 / pJF118ut-CD154 59 W3110 / pJF118ut-CD154-Pal 87

[0119] In addition to the product yield, the viable cell count of the cultures was also determined after 72 h. For this purpose, samples of the cultures were diluted 10⁵ 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 that grew were counted, and the viable cell count of the original cultures was calculated taking the dilution factor into account. This was 5.5 × 10⁸ cells / ml for strain W3110 / pJF118ut-CD154 and 2.7 × 10⁸ cells / ml for strain W3110 / pJF118ut-CD154-Pal.

Claims

1. Method for fermentative production of recombinant proteins, characterized in that a bacterial strain of the species Escherichia coli containing an open reading frame encoding a recombinant protein under the control of a functional promoter and an open reading frame encoding a mutated peptidoglycan-associated lipoprotein (Pal protein) under the control of a functional promoter is cultured in a fermentation medium, the fermentation medium is removed from the cells after the fermentation, and recombinant proteins are isolated from the fermentation medium, the mutated Pal protein having been mutated such that it has no membrane anchor for the outer cell membrane of the bacterium and the bacterial strain additionally containing the wild-type Pal gene.

2. Method according to Claim 1, characterized in that the open reading frame encoding the mutated Pal protein has been mutated such that it encodes a mutated Pal protein which has been mutated at one or more of amino acid positions 1 to 6.

3. Method according to Claim 2, characterized in that the open reading frame encoding the mutated Pal protein has been mutated such that it encodes a mutated Pal protein in which the N-terminal cysteine residue has been substituted.

4. Method according to Claim 2, characterized in that the open reading frame encoding the mutated Pal protein has been mutated such that it encodes a mutated Pal protein in which the N-terminal cysteine residue is absent.

5. Method according to Claim 2, characterized in that the open reading frame encoding the mutated Pal protein has been mutated such that it encodes a mutated Pal protein in which amino acids 1 to 6 are absent.

6. Method according to one or more of Claims 1 to 5, characterized in that the recombinant protein is a heterologous protein.

7. Method according to one or more of Claims 1 to 6, characterized in that the recombinant proteins are purified from the fermentation medium after the removal of the fermentation medium.

8. Method according to one or more of Claims 1 to 7, characterized in that that the expression of the mutated Pal protein is induced.

9. Method according to one or more of Claims 1 to 8, characterized in that the expression of the mutated Pal protein is induced after the induction of the expression of the recombinant protein.