Bacterial strains that release recombinant proteins in fermentation methods

Abstract

The present invention relates to a bacterial strain containing an open reading frame encoding a recombinant protein, thereby controlling a functional promoter. The present invention is characterized in that the bacterial strain contains an open reading frame encoding a mutant peptidoglycan-associated lipoprotein (PAL protein), thereby controlling a functional promoter, wherein the PAL protein is mutated so that the PAL protein does not contain a membrane anchor of the bacterial outer cell membrane. The present invention also relates to plasmids encoding recombinant proteins and mutant PAL proteins, and to a method for fermenting and producing recombinant proteins using the bacterial strain according to the present invention. In this way, compared with the prior art, an increased product yield in the culture residue can be achieved without causing a large number of bacterial cells to die.

Description

Technical Field

[0001] The present invention relates to 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-related lipoprotein (Pal protein) under the control of a functional promoter, and the Pal protein has been mutated so that it does not have a membrane anchor of the bacterial outer cell membrane. The present invention also relates to a plasmid encoding a recombinant protein and a mutated Pal protein, and to a method for fermenting and producing a recombinant protein using the bacterial strain according to the present invention. Background Art

[0002] Compared with mammalian cell culture, recombinant proteins can be produced economically and efficiently in bacteria due to the short production time and simple operation in bacteria. Due to its extensively studied genetics and physiology, the Gram-negative enterobacterium Escherichia coli is currently the most commonly used organism for the production of recombinant proteins. Particularly attractive are methods for producing recombinant proteins in E. coli, in which the target protein is released directly into the fermentation medium in high yield and correctly folded, because this avoids the complex cell disruption and protein folding processes. A series of E. coli strains and methods for using E. coli strains to achieve the release of recombinant proteins into the culture medium are disclosed in the literature.

[0003] i) Firstly, it is possible to use so-called "leaky" strains. These are understood to mean E. coli mutants that are defective in the outer membrane and therefore partially discharge periplasmic proteins into the culture medium. This is a non-specific mechanism. An example of such a "leaky" mutant is a strain with a defect in the Tol-Pal complex, such as a tol- and pal-deletion mutant. It is known that tol- and pal-deletion mutants release endogenous periplasmic proteins into the fermentation medium, such as, for example, alkaline phosphatase PhoA or RNase I. This strain is extremely sensitive to EDTA, various detergents and antibiotics, and exhibits 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 limitedly suitable for cultivation under high cell density fermentation conditions.

[0004] ii) Another method to achieve the release of recombinant proteins is to express proteins that can permeate the cell envelope of E. coli, thereby promoting the release of proteins into the culture medium. The expression of bacteriocin release protein (BRP), a small lipoprotein that causes outer membrane degradation and cell lysis, has been well described. By overexpressing ColE1 BRP (Kil protein) or enterocin DF13 BRP, interleukin-2, β-glucanase, alkaline phosphatase or β-lactamase can be discharged 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).

[0005] Iii) Wan and Baneyx describe another method for making cell envelope unstable (1998, Protein Expr. Purif. 14, pp. 13-22). This involves destroying membrane integrity by overexpressing the soluble C-terminal domain of TolA protein (TolAIII). This results in a phenotype similar to tolA-deletion mutants, an increase in the release of protein RNase I and alkaline phosphatase from the periplasm to the culture medium, and causes sensitivity to deoxycholate (Levengood-Freyermuth et al. 1993, J. Bacteriol. 175, pp. 222-228). By overexpression of TolAIII protein, the release of OmpA-TEM-β-lactamase protein significantly increases. However, compared with the control, the expression of β-lactamase has decreased by 1.5-2 times. In addition, as in the tolA mutant, the expression of TolAIII greatly impairs the viability of the cell. Three hours after the start of TolAIII expression, the number of colony forming units (CFU) in the shake flask, a measure of culture viability, decreased 1000-fold. In addition, the cells were sensitive to low concentrations of SDS (0.02%), indicating the presence of a clear membrane defect.

[0006] Such cells are not suitable for cultivation at high cell densities for the relatively long periods of time required for the production of complex eukaryotic proteins.Another disadvantage of TolAIII co-expression is that TolAIII itself can be found in large amounts in the culture supernatant, thus contaminating the outbound target protein. Summary of the invention

[0007] It is an object of the present invention to provide a bacterial strain which releases recombinant proteins into the culture medium with increased yields without strong cell lysis.

[0008] This object is achieved by 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 mutant peptidoglycan-associated lipoprotein (Pal protein) under the control of a functional promoter, the mutant Pal protein having been mutated so that it does not have a membrane anchor to the bacterial outer cell membrane.

[0009] The advantage of using this bacterial strain in a fermentation method is that an additional protein (i.e., a mutant form of the Pal protein) is expressed together with the recombinant protein. The expression of the mutant Pal protein results in limited permeabilization of the outer cell membrane of the bacteria, so that the recombinant protein can be released from the cell without causing cell death. Due to the improved release, the recombinant protein can be isolated with an increased yield. The present invention provides a corresponding bacterial strain, a method for producing a recombinant protein by fermentation using a bacterial strain, and a corresponding plasmid. In this way, compared with the prior art, it is possible to obtain an increased product yield in the culture supernatant without a large number of bacterial cells dying.

[0010] The bacterial strain is preferably characterized in that the bacterial strain is a Gram-negative bacterium, particularly preferably a bacterial strain of the family Enterobacteriaceae, particularly preferably a strain of the species Escherichia coli.

[0011] The peptidoglycan-associated lipoprotein (Pal, Pal protein) is a periplasmic protein of a bacterium, anchored in the outer membrane of the bacterial cell by its lipidated N-terminal, and interacts with the peptidoglycan layer by the C-terminal. In addition, Pal is a component of the Pal-Tol system (Godlewska et al. 2009, FEMS Microbiol. Lett. 298, pp. 1-11) and interacts with various other proteins of the periplasm, such as Lpp and ompA (Cascales et al., 2002, J. Bacteriol. 184, pp. 754-759; Godlewska, supra). In technical applications, the Pal protein is used, for example, to anchor antibody fragments on the cell surface of bacteria in the form of fusion proteins consisting of the Pal protein and the antibody fragment (Fuchs et al. 1991, Biotechnology (NY) 9, pp. 1369-1372; Dhillon et al. 1999, Lett. Appl. Microbiol. 28, pp. 350-354).

[0012] Wild-type Pal gene refers to the form of Pal gene naturally produced by evolution and present in wild-type bacterial genome. Wild-type Pal gene expresses wild-type Pal precursor protein, which includes the amino acid sequence (SEQ ID No.4) of signal peptide and wild-type Pal protein. After cutting off the signal sequence of 21 amino acids, the mature wild-type Pal protein from Escherichia coli (strain K-12) includes a sequence of 152 amino acids (see Genbank entry X05123, Uniprot P0A912) and carries amino acid cysteine ​​at position 1, which is highly conserved in bacterial lipoproteins and is membrane anchored (see, for example, Zückert et al. 2014, Biochimica et Biophysica Acta 1843, pages 1509-1516 or Konovalova and Silhavy 2015, Phil. Trans. R. Soc. B 370: 20150030). In contrast, the mutant Pal protein lacks a membrane anchor in the bacterial outer cell membrane.

[0013] The Pal precursor protein (the precursor form of the Pal protein) refers to the Pal protein that contains the amino acid sequence and signal sequence of the Pal protein and does not carry post-translational modifications.

[0014] Mature wild-type Pal protein refers to a Pal protein that contains the amino acid sequence of the wild-type Pal protein and no longer carries the signal sequence (because it has been removed), and in which there are possible post-translational modifications. Such post-translational modifications are, for example, acylation of the Pal protein with a membrane anchor.

[0015] An open reading frame (ORF) refers to a region between the start codon and the stop codon of DNA or RNA and encodes the amino acid sequence of a protein. ORF is also called a coding region.

[0016] ORF is surrounded by non-coding regions. A gene refers to a DNA fragment containing all the basic information for producing biologically active RNA. A gene comprises a DNA fragment that produces a single-stranded RNA copy by transcription and an expression signal that participates in regulating the copy process. The expression signal includes, for example, at least one promoter, a transcription start site, a translation start site, and a ribosome binding site. In addition, terminators and one or more operators can serve as expression signals.

[0017] In the case of a functional promoter, the ORF under the regulation of the promoter is transcribed into RNA.

[0018] A monocistronic messenger RNA (mRNA) contains only one open reading frame.

[0019] An operon is a functional unit of DNA that contains multiple ORFs, a promoter, and possibly other expression signals.

[0020] The bacterial strain may contain (1) one ORF encoding a recombinant protein, (2) multiple ORFs encoding the same recombinant protein or (3) multiple ORFs encoding different recombinant proteins. The second possibility is used, for example, to increase the expression and yield of the recombinant protein, while the third possibility is particularly useful for expressing proteins consisting of multiple polypeptides (subunits). An example is the expression of antibody fragments that are subsequently assembled after transport out of the cytoplasm.

[0021] Each of these ORFs can be located in a separate gene; alternatively, multiple ORFs can be organized into an operon, ie, regulated by a common expression signal.

[0022] Furthermore, each operon may contain an ORF encoding a mutant Pal protein. Even if one or more ORFs encoding one or more recombinant proteins and an ORF encoding a mutant Pal protein are located in one operon, the mutant Pal protein is always expressed as a separate protein rather than a fusion protein, because, by definition, the ORF encoding a mutant Pal protein is characterized by its own translation start and stop codons.

[0023] If the recombinant protein consists of multiple polypeptide chains (subunits), it is preferred that the ORFs of the individual polypeptide chains (subunits) are organized in one operon.

[0024] The ORF encoding the mutated Pal protein is preferably transcribed as a monocistronic messenger RNA. This means that the mutated Pal protein is expressed by its own separate gene. The gene is referred to as the mutated Pal gene.

[0025] The ORFs encoding the recombinant protein and the mutated Pal protein can be expressed by chromosomes or plasmids. In the case of separate genes, they can also be expressed by separate plasmids compatible with each other in terms of replication origin and selection markers. The ORF is preferably plasmid-encoded.

[0026] Compared to the wild-type Pal protein, the ORF used in the present invention and encoding the mutant Pal protein contains changes in the amino acid sequence, such as substitution, deletion and / or insertion.

[0027] According to the present invention, the sequence of the ORF encoding the mutated Pal protein is preferably a DNA sequence that leads to the expression of the mutated Pal protein, which DNA sequence is derived from the same organism that is also used for the production of the recombinant protein.

[0028] The source of the DNA sequence of the ORF according to the present invention encoding the mutated Pal protein is not limited to the bacterial strain used to produce the recombinant protein, as long as the corresponding Pal protein in non-mutated form is functional in the bacterial strain used to produce the recombinant protein. In this regard, functionality means that the above-mentioned Pal protein can assume all the biological functions of the Pal protein of the bacterial strain used to produce the recombinant protein. In particular, this means that the phenotypic characteristics of the bacterial strain with a chromosomal deletion of the Pal gene can be supplemented by additionally introducing a copy of the above-mentioned Pal gene.

[0029] The terms "one or the ORF encoding a recombinant protein" and "a / recombinant protein" used in the singular form in the context of the present invention may also refer to a plurality of ORFs and / or a plurality of different recombinant proteins. Preferably, it is 1 to 3 different recombinant proteins, particularly preferably 1 recombinant protein or 2 different recombinant proteins. Cloning and expression of the recombinant protein in bacteria is achieved as in the prior art. The recombinant protein has, for example, a signal sequence for transport to the periplasm.

[0030] A recombinant protein is a protein that is not naturally expressed at all or expressed in different amounts by the wild-type bacterial genome. The bacteria are used to produce the recombinant protein and according to the invention, the protein is released into the culture medium.

[0031] In order to achieve the release of the recombinant protein into the culture medium, after protein biosynthesis in the cytosol, both the recombinant protein and the mutated Pal protein need to be transported to the periplasm. In order to be transported to the periplasm, it is necessary to connect the 5' end of the coding DNA sequence of the protein to be produced in the frame with the 3' end of the signal sequence of the protein output. In principle, all signal sequences are suitable for this purpose, which allow the target protein to be translocated in the bacterial strain used by means of Sec or Tat devices. 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, Staphylococcus protein A, StII, etc. (Choi and Lee 2004, Appl. Microbiol. Biotechnol. 64, pp. 625-635). According to the present invention, the preferred recombinant protein is the signal sequence of the phoA gene or ompA gene of Escherichia coli, or the signal sequence of the cyclodextrin glycosyltransferase (α-CGTase) of Klebsiella pneumoniae M5a1 disclosed as SEQ ID No. 1 and 3 in US 2008 / 0076157, or a signal sequence derived therefrom. Preferably, the mutated Pal protein carries the natural signal sequence of the Pal protein in its precursor form, such as Chen and Henning (1987, Eur. J. Biochem. 163, pp. 73-77). Preferably, the recombinant protein and the mutated Pal protein carry different signal sequences in their precursor form, and these two signal sequences lead to protein export.

[0032] The expression of the ORF encoding the mutated Pal protein and the recombinant protein can be controlled by one promoter or multiple different promoters. Preferably, the ORF encoding the mutated Pal protein and the recombinant protein is controlled by different promoters.

[0033] The DNA fragments encoding the mutated Pal protein can first be amplified by PCR using oligonucleotides as primers and a DNA template encoding the Pal protein (e.g., genomic DNA isolated from E. coli), and then connected to a DNA molecule containing a signal peptide sequence and produced in a similar manner in each case using common molecular biology techniques, thereby forming an in-frame fusion. Alternatively, the entire DNA molecule can also be produced by gene synthesis. The DNA molecule consisting of the coding sequence of the signal sequence and the mutated Pal protein in question can then be introduced into a vector (e.g., a plasmid), or directly integrated into the chromosome of a bacterial strain by known methods. Preferably, the DNA molecule is introduced into a plasmid, such as, for example, a derivative of a known expression vector, such as pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3 or pET. The plasmid is introduced into bacterial cells (transformation) using methods known to those skilled in the art.

[0034] The plasmid used may carry a selection marker. Suitable for use as a selection marker are genes encoding resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, kanamycin, etc. Preferably, the plasmid comprises a gene whose expression mediates tetracycline resistance. In addition, suitable for use as a selection marker are auxotrophic markers encoding essential genes that have been deleted in the bacterial strain in question containing the plasmid. If two plasmids are transformed into a cell, then two different antibiotic resistances or two different auxotrophic markers are preferentially used to select the presence of the two plasmids.

[0035] Suitable as promoters are all promoters known to those skilled in the art, such as constitutive promoters, for example GAPDH promoter, or inducible promoters, for example Lac, tac, trc, λPL, ara, cumate or tet promoters or sequences derived therefrom. The ORF encoding the recombinant protein and the ORF encoding the mutated Pal protein can be controlled as an operon by the promoter, or can be controlled by different promoters. Preferably, the ORF 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 is expressed under the control of the ara (arabinose) promoter.

[0036] The amino acid sequence of the mutated Pal protein can be generated in a known manner, for example by removing conserved cysteine ​​residues or introducing other mutations that prevent modification of the membrane anchor. The skilled person is aware of methods in the literature for checking whether mutations introduced after expression in a bacterial strain result in a mutated Pal protein that no longer contains a membrane anchor (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, supra; Mizuno 1979, J. Biochem. 86, pp. 991-1000).

[0037] In a preferred embodiment, the bacterial strain is characterized in that the ORF encoding the mutant Pal protein has been mutated such that it encodes a mutant Pal protein which is mutated at one or more of amino acid positions 1 to 6, preferably 1 to 4 and particularly preferably 1 to 2. In this context, the amino acid position refers to the amino acid following the signal sequence, i.e. amino acid position 1 is the first amino acid after the signal sequence.

[0038] Preferably, the mutation is a deletion (lack of an amino acid) or a substitution (exchange of an amino acid), with deletion being particularly preferred.

[0039] Particularly preferably, the bacterial strain is characterized in that the ORF encoding the mutant Pal protein has been mutated such that it encodes a mutant Pal protein in which the N-terminal cysteine ​​residue has been substituted.

[0040] In another preferred embodiment, the bacterial strain is characterized in that the ORF encoding the mutant Pal protein has been mutated so that it encodes a mutant Pal protein in which the N-terminal cysteine ​​residue is absent. Preferably, the amino acid is deleted at position 1, particularly preferably the cysteine ​​residue is deleted.

[0041] The N-terminus or amino terminus refers to the end of the Pal protein with a free amino group (NH2). The N-terminal cysteine ​​residue is the cysteine ​​residue at amino acid position 1 after the signal sequence.

[0042] Particularly preferably, the amino acid sequence of the mutated Pal protein is the sequence specified in SEQ ID No. 7 (Pal22A) and wherein the N-terminal cysteine ​​has been exchanged for the amino acid alanine, with the result that alanine follows the first 21 amino acids of the signal sequence.

[0043] In a preferred embodiment, the bacterial strain is characterized in that the ORF encoding the mutant Pal protein has been mutated so that it encodes a mutant 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 mutant Pal protein is SEQ ID No. 5 (PalΔ 22-27 ) and wherein alanine follows the first 21 amino acids of the signal sequence. The sequence after the signal peptide of the mutated Pal protein is identical to amino acids 7-152 of the mature wild-type Pal protein of E. coli (the amino acid sequence of the wild-type protein is shown in SEQ ID No. 4).

[0044] Preferably, the recombinant protein is a heterologous protein. A heterologous protein is understood to mean a protein that does not belong to the proteome, i.e. the entire natural protein set of a bacterial strain, preferably an E. coli K12 strain. All proteins naturally present in the bacterial strain used, for example in the E. coli K12 strain, can be derived from a known genomic sequence, for example from the Genbank entry for E. coli K12 with the accession number NC_000913.

[0045] Particularly preferred as heterologous proteins are eukaryotic proteins containing one or more disulfide bonds. Particularly preferred are eukaryotic proteins in the functional form of dimers or multimers.

[0046] The most important classes of heterologous proteins include antibodies and fragments thereof, cytokines, growth factors, protein kinases, protein hormones, lipocalins, anticalins, enzymes, binding proteins and molecular scaffolds and proteins derived therefrom. Examples of protein classes are, in particular, heavy chain antibodies and fragments thereof (e.g. nanobodies), single chain antibodies, interferons, interleukins, interleukin receptors, interleukin receptor antagonists, G-CSF, GM-CSF, M-CSF, leukemia inhibitors, stem cell growth factor, tumor necrosis factor, growth hormone, insulin-like growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, hepatocyte growth factor, bone morphogenetic factor, nerve growth factor, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GNF), and glial cell-derived neurotrophic factor (GNF). factors, angiogenesis inhibitors, tissue plasminogen activator, coagulation factors, trypsin inhibitor, elastase inhibitor, complement components, hypoxia-induced stress proteins, oncoproducts, transcription factors, viral components, proinsulin, urokinase, erythropoietin, thrombopoietin, neurotrophins, protein C, glucocerebrosidase, superoxide dismutase, renin, lysozyme, P450, chymotrypsin, lipocortin, reptin, serum albumin, streptokinase, tenecteplase, CNTF and cyclodextrin glycosyltransferase.

[0047] Examples of proteins derived from molecular scaffolds are, inter alia, evibodies (derived from CTLA-4), affibodies (protein A of Staphylococcus aureus), avidin multimers (of the human A domain family), transferrin (of transferrin), DARPins (of ankyrin repeat proteins), adnectins (of fibronectin III), peptide aptamers (of thioredoxin), microbodies (of microproteins), affilins (of ubiquitin), α-crystallin, charybdo toxins, tetranectin, PDZ domains of the RAS binding protein AF-6, Kunitz-type domains of protein inhibitors.

[0048] Preferably, the bacterial strain is characterized in that it also contains a wild-type Pal gene encoding peptidoglycan-associated lipoprotein (Pal). Particularly preferably, the wild-type Pal gene is under the control of a functional promoter.

[0049] The present invention further provides 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, and the Pal protein has been mutated so that it has no membrane anchor of the bacterial outer cell membrane.

[0050] The preferred and particularly preferred features mentioned for the bacterial strain according to the invention also apply to the features mentioned for the plasmid according to the invention, wherein the preferred and particularly preferred embodiments mentioned for the bacterial strain according to the invention are also preferred or particularly preferred in the plasmid, respectively.

[0051] Similarly, the mentioned definitions and preferred embodiments apply to the ORF encoding the signal peptide and the mutated Pal protein as well as to the ORF encoding the recombinant protein.

[0052] For example, one or more ORFs encoding one or more recombinant proteins can be in one operon. Preferably, the ORF encoding the mutated Pal protein is located in a separate gene. This means that the mutated Pal protein and the recombinant protein can be expressed independently of each other. In the particularly preferred case where the promoter of the recombinant protein is different from the promoter of the mutated Pal protein and can be induced differently, the plasmid has a particular advantage, that is, after the bacteria are transformed with the plasmid, the optimal time point for expressing the protein can be selected independently of each other.

[0053] Introducing the plasmid according to the invention into a bacterial strain using methods known to a person skilled in the art and expressing the recombinant protein and the mutated Pal protein via the plasmid has the advantage that the release of the recombinant proteins from the bacterial strain cells is improved and they can be isolated in increased yields.

[0054] Compared with the chromosomal integration of the ORF encoding the recombinant protein and the mutated Pal protein, the advantage of using a plasmid is that the cells of the bacterial strain carrying the plasmid have a selection advantage and can be selected by standard methods. Since each plasmid contains at least one replication origin (ORI), another advantage is that the plasmid can replicate autonomously. In addition, a particular advantage is that the plasmid can be present in the cells of the bacterial strain in high copy number and be inherited.

[0055] The present invention further provides a method for producing a recombinant protein by fermentation, characterized in that the bacterial strain according to the present invention is cultured in a fermentation medium, the fermentation medium is removed from the cells after fermentation, and the recombinant protein is separated from the fermentation medium.

[0056] The cells of the bacterial strain transformed by chromosomal integration or with one or two expression plasmids are cultivated in shake flasks or bioreactors (fermenters) by conventional methods known to those skilled in the art.

[0057] In principle, all conventional media known to those skilled in the art for cultivating bacteria can be used as fermentation media (growth media, culture media). In this regard, complex media or minimal salt media with complex components (e.g., peptone, tryptone, yeast extract, molasses or corn steep liquor) added in a specific ratio can be used. In addition, components such as vitamins, salts, amino acids and trace elements can be added to the culture media, which can improve cell growth.

[0058] The fermentation is preferably carried out in a conventional bioreactor, such as a stirred tank, a bubble column fermenter or a gas lift fermenter. A stirred tank fermenter is particularly preferred.

[0059] Fermentation involves culturing cells of a protein-producing strain in a growth medium with constant monitoring and precise control of various parameters (e.g., nutrient feed, oxygen partial pressure, pH and temperature of the culture). The culturing time is preferably 24-65 h.

[0060] The main carbon source used in the fermentation can be in principle all sugars, sugar alcohols or organic acids or their salts that can be utilized by cells. In this regard, preferably glucose, lactose, arabinose or glycerol are used. Glucose and arabinose are particularly preferred. A variety of different carbon sources can also be fed together. In this case, the carbon source can be initially fully loaded into the fermentation medium at the beginning of the fermentation, or any carbon source is not loaded at the beginning or only a portion of the carbon source is loaded, and then the carbon source is fed during the fermentation. Particularly preferred in this respect is an embodiment, wherein a portion of the carbon source is initially loaded and a portion of the carbon source is fed. Particularly preferably, the carbon source glucose is initially loaded with a concentration of 10-30g / l, and when the concentration drops to less than 5g / l during the fermentation, the feeding is started, and it is configured to keep the concentration lower than 5g / l.

[0061] If the expression of the recombinant protein and / or the mutated Pal protein is controlled by an inducible promoter, the expression is induced by adding an inducer corresponding to the promoter to the fermentation batch. Suitable inducers are, for example, arabinose, lactose, IPTG, tetracycline or cumate. The inducer can be metered in a single dose or multiple doses at any desired time point during the fermentation process. 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 metered in as a single dose, and the induction of the expression of the mutated Pal protein is configured so that a mixture of glucose and arabinose is fed at a certain time point when the glucose concentration drops to less than 5 g / l. At this point, the proportion of arabinose in the mixture is preferably 33% by weight to 66% by weight; particularly preferably 33% by weight.

[0062] Preferably, expression of the mutant Pal protein is induced during a phase of the culture when cells are dividing little to no longer (i.e., before or at the beginning of the plateau phase of the growth curve). This time point is determined by a little to no increase in cell density, measured as OD 600 value or CDW value (see below).

[0063] Preferably, the medium in the fermenter is stirred before inoculation and bubbled with sterile compressed air; at this point, the oxygen content is calibrated to 100% saturation and a target value for O2 saturation during the fermentation is selected, which is between 10% and 70% of this value, preferably between 20% and 60%, and particularly preferably 30%. After the O2 saturation falls below the target value, the regulation cascade is started so that the O2 saturation returns to the target value, and the gas supply and stirring speed can be adjusted.

[0064] The pH of the culture is preferably pH 6 to pH 8. Preferably, a pH of 6.5 to 7.5 is set; particularly preferably, the pH of the culture is maintained at 6.8 to 7.2.

[0065] The temperature of the culture is preferably from 15° C. to 45° C. Preference is given to a temperature range of from 20° C. to 40° C., particular preference is given to a temperature range of from 25° C. to 35° C. and very particular preference is given to a temperature of 30° C.

[0066] According to the present invention, the fermentation medium is removed from the cells after fermentation, and the recombinant protein is isolated from the fermentation medium. As known in the prior art, this can be accomplished by conventional methods. Typically, in a first step, cells are removed from the recombinant protein released into the culture medium by a separation method (e.g., centrifugation or filtration). The recombinant protein can then be concentrated, for example, by ultrafiltration.

[0067] Expression of the mutant Pal protein improves the release of the recombinant protein into the culture supernatant. Due to the improved release, the recombinant protein can be isolated with increased yield.

[0068] Increased yield is understood to mean that the amount released into the culture medium is preferably at least 110%, particularly preferably at least 150%, and especially preferably at least 200%, compared to the amount of recombinant protein that can be produced according to the prior art using a wild-type bacterial strain containing the gene for the recombinant protein or a wild-type bacterial strain containing the gene for the recombinant protein and additionally expressing a protein that destabilizes the bacterial cell envelope. This means that the yield of the 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 the yield that can be achieved with the corresponding bacterial strains of the prior art.

[0069] For example, Example 1 shows that when expressing PalΔ22-27 When the expression of Pal22A or Pal22A was 2-3 times higher than that of the wild-type bacterial strain or the bacterial strain expressing TolAIII, the expression of PalΔ 22-27 The mutant of increased CGTase production by 2-3 times (see Table 2). Example 3 (see Table 3) similarly demonstrated a significant increase.

[0070] Another advantage of the Pal mutant is that, at the same time, cell lysis is only slightly increased over a 5-24 h incubation period compared to the corresponding wild-type cells. Wan and Baneyx (1998, supra) demonstrated that after a few hours, TolAIII overproduction resulted in a decrease in CFU counts of approximately 1400-3000-fold compared to non-induced cells and concluded that the long-term viability of the bacterial cells was significantly reduced due to the effects on the outer membrane. In contrast, in the induced mutant PalΔ 22-27 24 h after protein expression, the CFU count decreased only 2-fold compared to the corresponding wild-type bacterial strain (see Example 3).

[0071] By means of co-expression of mutated Pal proteins in bacterial strains (preferably in E. coli), Fab antibody fragments can also be produced extracellularly. In this case, the bacterial cells must simultaneously synthesize the corresponding fragments of the light chain (comprising domains VL and CL) and heavy chain (comprising domains VH and CH1) of the antibody, which are then secreted into the periplasm. Outside the cytoplasm, the two chains are assembled to form a functional Fab fragment. The corresponding ORFs can be located in different genes. The ORFs encoding the antibody fragments of the light chain and the heavy chain are preferably organized in an operon. As a result of the simultaneous production of the Pal proteins mutated according to the present invention, the heavy and light chains of the antibody are released into the fermentation medium at increased concentrations.

[0072] The main advantages of the method are that it is suitable for the production of recombinant proteins that require longer culture times and is suitable for high cell density fermentations. Thus, complex eukaryotic proteins can be produced as recombinant proteins.

[0073] Preferably, the method is characterized in that, after removing the fermentation medium, the recombinant protein is purified from the fermentation medium.

[0074] The recombinant protein can be further purified by standard methods (eg precipitation or chromatography). Methods such as affinity chromatography, which utilize the native conformation of the already correctly folded protein, are particularly preferred.

[0075] Preferably, the method is characterized in that the expression of the mutated Pal protein is induced. This means that the expression of the mutated Pal protein or the expression of the recombinant protein and the expression of the mutated Pal protein is induced. This means that, in this preferred embodiment, the expression of the mutated Pal protein is in any case controlled by an inducible promoter. In contrast, the expression of the recombinant protein can be controlled by a constitutive or inducible promoter. Particularly preferably, the ORF encoding the recombinant protein and the ORF encoding the mutated Pal protein are both controlled by an inducible promoter, and particularly preferably by different inducible promoters. Therefore, it is preferred that the expression of the mutated Pal protein and the recombinant protein can be induced.

[0076] Since the promoters of the genes encoding the recombinant protein and the mutated Pal protein can preferably be induced independently of each other, the optimal time points for expressing the recombinant protein and expressing the mutated Pal protein can be selected independently of each other.

[0077] By using an inducible promoter, the expression of the corresponding protein can be induced at any desired time point of the fermentation. Preferably, the expression of the mutated Pal protein is induced after the expression of the recombinant protein is induced. Particularly preferably, the expression of the mutated Pal protein is induced at least 1 hour, particularly preferably at least 2 hours, further particularly preferably 15 to 24 hours and particularly preferably 19 hours after the expression of the recombinant protein is induced.

[0078] Induction of expression of recombinant protein and mutated Pal protein is triggered by adding an inducer to the culture medium, and it is necessary to select the inducer according to the gene used. In a preferred embodiment in which 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 a preferred embodiment in which 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.

[0079] According to the present invention, even after inducing expression of a mutated Pal protein and further culturing 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, a strong drop in optical density due to cell lysis does not occur, and only a slight drop in viable cell count measured in colony forming units (CFU) occurs.

[0080] The higher the measured optical density (OD), the higher the cell density. Bacterial strains with unstable cell envelopes usually show relatively high cell lysis. In the case of relatively high cell lysis, the cell density decreases and thus the OD value also decreases.

[0081] The advantage of the invention is that the recombinant protein is released into the culture medium without strong lysis of the bacterial strain cells. Therefore, the method according to the invention is characterized in that it is suitable for both the production of complex eukaryotic proteins with correspondingly long culture times and for high cell density fermentations.

[0082] In the context of the present invention, fermentations that reach a cell dry weight of >50 g / l 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).

[0083] A long culture time means a culture time of at least 24 h, preferably at least 48 h, and particularly preferably at least 72 h.

[0084] In the context of the present invention, relatively low (or only slightly increased or not strong) cell lysis means that the OD of the bacterial strain culture measured after induction of the expression of the mutated Pal protein is less than 1.0% compared to the wild-type bacterial strain not carrying the Pal mutation, preferably for a culture time of 5-24 hours. 600 There is little or no decrease in the A, CDW or viable cell count values. Bacterial strains expressing mutated Pal proteins have a major advantage, particularly in the cultivation of bacterial strains containing modifications that disrupt the stability of their cell envelope, where cell lysis is significantly increased, as shown by Wan and Baneyx (1998, supra) for cells expressing the TolAIII protein.

[0085] Therefore, the cell culture according to the fermentation process of the present invention is preferably characterized by an OD of 0.05477% as compared to the bacterial culture at the same time point of the fermentation process. 600 The difference between the fermentation method and the method of the present invention is that the cultured bacterial strain does not contain the ORF encoding the mutated Pal protein contained in the bacterial strain of the present invention, and the optical density (OD) measured at 600 nm in the fermentation method of the present invention is 5 to 24 hours, particularly preferably 7 hours, after inducing the expression of the mutated Pal protein. 600 ) is reduced by no more than 20%, particularly preferably by no more than 10%, and particularly preferably by no more than 5%. The optical density of the cell culture at 600 nm is determined spectrophotometrically.

[0086] The optical density of a cell culture determined by means of 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 the cell division activity and of cell lysis, which leads to a decrease in the cell density.

[0087] Alternatively, a certain amount of the culture may be separated by filtration or centrifugation (preferably centrifugation), then dried and weighed, and the cell weight is determined as the cell dry weight (CDW).

[0088] Another way in which the viability of bacterial strains expressing the mutated Pal protein can be compared with the viability of bacterial strains not expressing the mutated Pal protein is to determine the viable cell count as "colony forming units (CFU)" based on a defined culture volume. The experimental procedure is described in Wan and Baneyx (1998, supra). The CFU unit specifies the cells that can reproduce in the culture. In the case of bacterial strains expressing the mutated Pal protein and the recombinant protein, the viable cell count is reduced by a maximum of 100 times, preferably by a maximum of 10 times, and particularly preferably by a maximum of 2 times, compared to bacterial strains not expressing the mutated Pal protein.

[0089] In contrast, Wan and Baneyx (1998, supra) indicated in Table 2 that 3 hours after induction of TolAIII expression with IPTG, the viable cell counts determined as CFU per ml increased further in the wild-type bacterial strain, whereas they decreased significantly in the bacterial strain expressing TolAIII. The authors concluded that, although TolAIII overexpression had relatively little effect on bacterial growth during the logarithmic growth phase of the bacterial strain, the viability of the bacterial cells decreased significantly in the long term due to permeabilization of the outer cell membrane.

[0090] The negligible effect of the expressed mutant Pal protein on cell integrity is another significant advantage of the present invention. In addition, since the ORF encoding the mutant Pal protein is preferably cloned under an inducible promoter, the expression of the mutant Pal protein can be controlled and the culture time for the desired effect to occur is narrowly limited, thereby limiting the potential impact on bacterial cell viability. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 A plasmid map of plasmid pCGT-Pal is shown.

[0092] Figure 2 A plasmid map of plasmid pCGT-TolAIII is shown.

[0093] Figure 3 A plasmid map of plasmid pJF118ut-CD154 is shown.

[0094] Figure 4 A plasmid map of plasmid pJF118ut-CD154-Pal is shown.

[0095] Figure 5 A plasmid map of plasmid pCGT-Pal22A is shown.

[0096] The abbreviations in the figures indicate the DNA regions encoding the following functions:

[0097] tac p / o: tac promoter / operator

[0098] pBAD p / o: arabinose promoter / operator

[0099] bla: β-lactamase gene (ampicillin resistance)

[0100] TcR: Tetracycline resistance

[0101] lacIq: repressor of the tac promoter

[0102] cgt-SP: signal peptide of CGTase

[0103] CGTase: Cyclodextrin glycosyltransferase

[0104] ColE1: Origin of replication

[0105] phoA-SP: phoA signal peptide

[0106] AFA-SP: A derivative of the CGTase signal peptide

[0107] rrnB terminator: The terminator region of the rrnB gene

[0108] trpA terminator: The terminator region of the trpA gene

[0109] Pal: peptidoglycan-associated lipoprotein

[0110] Pal-SP: signal peptide of Pal

[0111] TolAIII: Domain structure of TolA protein

[0112] OmpA-SP: signal peptide of OmpA

[0113] HisTag: Histidine tag

[0114] Light chain: Antibody fragment consisting of the domains VL and CL

[0115] Heavy chain: Antibody fragment containing domains VH and CH1

[0116] ScaI / MauBI / EcoRI: restriction sites of corresponding restriction endonucleases DETAILED DESCRIPTION

[0117] Example

[0118] The invention is described in more detail below with reference to exemplary embodiments but the invention is not limited thereto.

[0119] All molecular biology methods used, such as polymerase chain reaction (PCR), gene synthesis, isolation and purification of DNA, modification of DNA by restriction enzymes and ligases, transformation, etc., were performed in a manner known to the person skilled in the art, described in the literature or recommended by the respective manufacturers.

[0120] Plasmid Description:

[0121] pCGT:

[0122] The production of plasmid pCGT is described in Example 4 of US 2008 / 0254511 A1 and Figure 4 Plasmid maps are described in detail in .

[0123] Essentially, the plasmid contains not only a gene for tetracycline resistance but also, inter alia, a structural gene for cyclodextrin glycosyltransferase (CGTase) of Klebsiella pneumoniae M5a1 including a native CGTase signal sequence. The expression of the CGTase gene is controlled by the tac promoter.

[0124] pCGT-Pal:

[0125] To obtain pCGT-Pal (see Figure 1 The DNA fragment xI (described in detail in SEQ ID No. 1) was generated by gene synthesis by Eurofins Genomics. The DNA fragment xI (described in detail in SEQ ID No. 1) contains:

[0126] - nucleotides 1136-1304 from GenBank entry X81837.1, containing the arabinose promoter (pBAD promoter) and the operators O1 and I2+I1 and the CAP binding site (nucleotides 10-178 of SEQ ID No. 1),

[0127] - Shine-Dalgarno sequence (nucleotides 203-208 of SEQ ID No. 1) and

[0128] - a nucleotide fragment encoding a fusion of the following i, ii and iii:

[0129] i The signal sequence of the peptidoglycan-associated lipoprotein of Escherichia coli K12 (nucleotides 136-198 from GenBank entry X05123.1, encoding amino acids -21 to -1, nucleotides 216-278 of SEQ ID No. 1),

[0130] ii nucleotides 217-654 from GenBank entry X05123.1, encoding amino acids 7-152 of a peptidoglycan-associated lipoprotein from Escherichia coli K12 (nucleotides 279-716 of SEQ ID No. 1) and

[0131] iii. the terminator of the trpA gene of Escherichia coli (nucleotides 749-774 of SEQ ID No. 1).

[0132] The DNA fragment xI was cut with the restriction enzyme MauBI and ligated with the expression vector pCGT cut with the same restriction enzyme. Cloning was performed in a non-directional manner; however, plasmids were preferably used in which the insertion direction of the DNA fragment was opposite to the reading direction of the gene encoding CGTase and verified by restriction pattern and sequencing of the restriction enzyme ScaI. The plasmid was called pCGT-Pal and encodes the protein PalΔ 22-27 , which is also synonymously named PalD22-27 (detailed in SEQ ID No. 5).

[0133] pCGT-Pal22A:

[0134] To obtain pCGT-Pal22A (see Figure 5 The DNA fragment xIA (described in detail in SEQ ID No. 6) was generated by gene synthesis by Eurofins Genomics. The DNA fragment xIA (described in detail in SEQ ID No. 6) contains:

[0135] - nucleotides 1136-1304 from GenBank entry X81837.1, containing the arabinose promoter (pBAD promoter) and the operators O1 and I2+I1 and the CAP binding site (nucleotides 10-178 of SEQ ID No. 6),

[0136] - Shine-Dalgarno sequence (nucleotides 203-208 of SEQ ID No. 6) and

[0137] - a nucleotide fragment encoding a fusion of the following i, ii and iii:

[0138] i The signal sequence of the peptidoglycan-associated lipoprotein of Escherichia coli K12 (nucleotides 136-198 from GenBank entry X05123.1, encoding amino acids -21 to -1, nucleotides 216-278 of SEQ ID No. 6),

[0139] ii amino acids 1-152 of the peptidoglycan-associated lipoprotein of Escherichia coli K12 (referenced to GenBank: X05123.1, nucleotides 279-734 of SEQ ID No. 6), wherein the amino acid cysteine ​​at position 1 has been exchanged for alanine, and

[0140] iii. the terminator of the trpA gene of Escherichia coli (nucleotides 767-792 of SEQ ID No. 6).

[0141] The DNA fragment xIA was cut with the restriction enzyme MauBI and ligated with the expression vector pCGT that had been cut with the same restriction enzyme. Cloning was performed in a non-directional manner; however, a plasmid in which the insertion direction of the DNA fragment was opposite to the reading direction of the gene encoding CGTase was preferably used and verified by restriction pattern and sequencing of the restriction enzyme ScaI. The plasmid was called pCGT-Pal22A and encoded the protein Pal22A (detailed description in SEQ ID No. 7).

[0142] pCGT-TolAIII:

[0143] Overexpression of TolAIII corresponds to the prior art (Wan and Baneyx 1998, supra); therefore, pCGT-TolAIII was chosen as a comparative example representing the prior art, in which the outer cell envelope of bacteria containing this plasmid and expressing TolAIII was disrupted, thereby increasing the release of the recombinant protein.

[0144] To obtain pCGT-TolAIII (see Figure 2 The DNA fragment was generated by gene synthesis by Eurofins Genomics.

[0145] DNA fragment xII (described in detail in SEQ ID No. 2) contains:

[0146] - nucleotides 1136-1304 from GenBank entry X81837.1, containing the arabinose promoter (pBAD promoter) and the operators O1 and I2+I1 and the CAP binding site (nucleotides 10-178 of SEQ ID No. 2),

[0147] - Shine-Dalgarno sequence (nucleotides 203-208 of SEQ ID No. 2) and

[0148] - a nucleotide fragment encoding a fusion of the following i, ii and iii:

[0149] i The signal sequence of ompA of Escherichia coli K12 (nucleotides 216-278 of SEQ ID No. 2),

[0150] ii amino acids 291-421 of the TolA protein of Escherichia coli K12 (encoding TolAIII, nucleotides 279-671 of SEQ ID No. 2) and

[0151] iii. the terminator of the trpA gene of Escherichia coli (nucleotides 714-729 of SEQ ID No. 2).

[0152] DNA fragment xII was cut with restriction enzyme MauBI and ligated with expression vector pCGT (see above) cut with the same restriction enzyme. Cloning was performed in a non-directional manner; however, plasmids were preferably used in which the insertion direction of the DNA fragment was opposite to the reading direction of the gene encoding CGTase and verified by restriction pattern or sequencing of restriction enzymes ScaI and EcoRI. The plasmid was called pCGT-TolAIII.

[0153] pJF118ut-CD154:

[0154] The plasmid pJF118ut described in US 2008 / 076157 A is used as the starting vector for cloning and expressing the Fab fragment gene of humanized monoclonal anti-CD154 antibody 5c8, and its sequence is published in Karpusas et al.2001 (Structure 9, pp. 321-329). pJF118ut is a derivative of known expression vector pKK223-3 (Amersham Pharmacia Biotech), is deposited in the DSMZ-German Microorganism and Cell Culture Co., Ltd. Collection Center (DSMZ-German Microorganism Collection Center) (Braunschweig), and is numbered as DSM 18596.

[0155] To obtain pJF118ut-CD154 (see Figure 3 The DNA fragment xIII (described in detail in SEQ ID No. 3) contains a fusion consisting of the following i, ii, iii, iv and v:

[0156] i the signal sequence disclosed in US 2008 / 076157 as SEQ ID No. 2 and derived from the CGTase signal sequence of Klebsiella pneumoniae M5a1 (nucleotides 25-114 of SEQ ID No. 3), and

[0157] ii The reading frame of the heavy chain (VH-CH1 domain) of the Fab fragment of the humanized monoclonal anti-CD154 antibody 5c8, encoding the Figure 3 Amino acids 1-221 of the sequence published in (nucleotides 115-777 of SEQ ID No. 3),

[0158] iii phoA signal sequence (nucleotides 800-862 of SEQ ID No. 3),

[0159] iv light chain of the Fab fragment of humanized monoclonal anti-CD154 antibody 5c8 (V L -C L domain), such as Karpusias et al. 2001 Figure 3 (nucleotides 863-1516 of SEQ ID No. 3), and

[0160] v Nucleotides 1517-1546 of SEQ ID No. 3, encoding a linker of 4 amino acids in length and a hexahistidine tag.

[0161] DNA fragment xIII was cut with restriction enzymes EcoRI and PdmI and ligated with expression vector pJF118ut cut with EcoRI and SmaI. The resulting plasmid was named pJF118ut-CD154, in which the gene expression of the heavy and light chains of the Fab fragment was controlled by the tac promoter.

[0162] pJF118ut-CD154-Pal:

[0163] The DNA fragment xI encoding the Pal variant under the control of the arabinose promoter and flanked by the trpA terminator was inserted into the plasmid pJF118ut-CD154 via the MauBI restriction sites as described above for pCGT-Pal. Cloning was performed in a non-directional manner; however, it is preferred to use a plasmid in which the DNA fragment xI is inserted in the opposite reading direction to the DNA fragment xIII encoding CD154, verified by restriction pattern and sequencing of the restriction enzyme ScaI. The plasmid is called pJF118ut-CD154-Pal (see Figure 4 (plasmid map).

[0164] Example 1: Production of cyclodextrin glycosyltransferase (CGTase) in shake flasks

[0165] In order to produce CGTase from Klebsiella pneumoniae M5a1, E. coli strain W3110 (ATCC 27325) was transformed with plasmid pCGT, pCGT-Pal, pCGT-Pal22A or pCGT-TolAIII by a common method (e.g., TSS transformation). Plasmid-containing cells were selected by tetracycline (20 mg / l). The E. coli strains were named W3110 / pCGT, W3110 / pCGT-Pal, W3110 / pCGT-Pal22A and W3110 / pCGT-TolAIII, respectively.

[0166] The transformed strain was grown at 30°C in 10 ml LB medium (5 g / l yeast extract (Oxoid LP0021), 10 g / l tryptone (Oxoid LP0042), 5 g / l NaCl), supplemented with 1 ml / l trace element solution (0.15 g / l Na2MoO4×2H2O; 2.5 g / l Na3BO3; 0.7 g / l CoCl2×6H2O; 0.25 g / l CuSO4×5H2O; 1.6 g / l MnCl2×4H2O; 0.3 g / l ZnSO4×7H2O), 3 g / l glucose, 10 g / l lactose, 0.55 g / l CaCl2 and 20 mg / l tetracycline). The medium used was an auto-induction medium, i.e. no additional inducer was added for the tac promoter. The lactose present in the medium can be taken up by the cells after metabolism of the glucose present in the medium. This resulted in induction of protein expression from the tac promoter. CGTase was produced from all four plasmids.

[0167] Due to the addition of 0.2% (w / v) arabinose to the culture medium after 48 h, the plasmids pCGT-Pal and pCGT-Pal22A additionally induced the expression of the Pal variants, which were also present on the plasmid and were under the control of the arabinose promoter, in contrast to the plasmid pCGT-TolAIII additionally induced the expression of TolAIII. Control cultures containing the plasmid pCGT were also appropriately mixed with arabinose to achieve better comparability.

[0168] After 72 hours of cultivation, samples were collected, cells were removed from the culture medium by centrifugation, and the CGTase content in the culture supernatant was determined based on the amount of cyclodextrin (CD) produced by amylase by the following enzyme assay:

[0169] Assay buffer: 5 mM Tris HCl buffer, 5 mM CaCl2 × 2H2O, pH 6.5

[0170] Substrate solution: 10% starch solution (Merck No. 1.01252) in assay buffer, pH 6.5

[0171] Assay mixture: 0.2 ml substrate solution + 0.2 ml centrifuged (5 min, 12 000 rpm) culture supernatant

[0172] Reaction temperature: 40℃

[0173] Enzyme assay:

[0174] *Pre-adjust the temperature of the substrate solution and the centrifuged culture supernatant (at 40°C for about 5 minutes)

[0175] * The assay mixture was prepared by rapid mixing (vortex mixer) of the substrate solution and the centrifuged culture supernatant. If necessary, the centrifuged culture supernatant was diluted with assay buffer to obtain a CD value of 0.9-1.5 g / l in the subsequent HPLC analysis;

[0176] *Incubate at 40°C for 3 minutes

[0177] *Terminate the enzyme reaction by adding 0.6 ml methanol and mixing quickly (vortex mixer)

[0178] * Chill the mixture on ice (approximately 5 minutes)

[0179] * Centrifuge (5 min, 12 000 rpm) and remove the clear supernatant

[0180] *CD amount produced by HPLC analysis: Nucleodur100-3 NH2-RP column (150 mm×4.6 mm, Macherey-Nagel) was used on an Agilent HP 1100 HPLC system and 64% acetonitrile in water (v / v) was used as the mobile phase at a flow rate of 2.1 ml / min. The analysis was performed by RI detector (1260 Infinity RI, Agilent) and quantified based on the peak area and α-CD standards (Cavamax W6-8 Pharma, Wacker Chemie AG).

[0181] Calculation of enzyme activity: A = G*V1*V2 / (t*MG)[U / ml]

[0182] A = activity,

[0183] G = CD content, unit: mg / l

[0184] V1 = dilution factor in the assay mixture

[0185] V2 = dilution factor of the culture supernatant used before the assay;

[0186] If not diluted, then: V2 = 1

[0187] t = reaction time, unit: minutes

[0188] MG = molecular weight, unit: g / mol (MGCD = 973 g / mol)

[0189] 1 unit (U) 1μmol / l product (CD) / min

[0190] Table 1 shows the CGTase yields achieved respectively.

[0191] Table 1: CGTase production in the shake flask supernatant after 72 h of culture.

[0192] strain Shake flask: CGTase (U / ml) W3110 / pCGT 39 W3110 / pCGT-Pal 134 W3110 / pCGT-Pal22A 95 W3110 / pCGT-TolAIII 42

[0193] Example 2: Fermentation production of cyclodextrin glycosyltransferase (CGTase) in a stirred tank fermenter

[0194] The strains W3110 / pCGT, W3110 / pCGT-Pal and W3110 / pCGT-TolAIII described in Example 1 were used to produce cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae M5a1. The production of CGTase was carried out in stirred tank fermenters.

[0195] Using a preliminary culture grown in LB medium of Example 1 for 7 hours in a shake flask, 1.2 l of fermentation medium (1.5 g / l KH2PO4; 5 g / l (NH4)2SO4; 0.5 g / l MgSO4×7H2O; 0.225 g / l CaCl2×2H2O, 0.075 g / l FeSO4×7H2O; 1 g / l Na3citrate×2H2O; 0.5 g / l NaCl; 1 ml / l trace element solution (0.15 g / l Na2MoO4×2H2O; 2.5 g / l Na3BO3; 0.7 g / l CoCl2×6H2O; 0.25 g / l CuSO4×5H2O; 1.6 g / l MnCl2×4H2O; 0.3 g / l ZnSO4×7H2O); 5 mg / l vitamin B1; 3 g / l plant peptone (BD211906); 1.5 g / l yeast extract (Oxoid LP0021); 10 g / l glucose; 20 mg / l tetracycline) in a fermenter inoculated to 0.1 OD 600. Fermentation thus started (time point 0, start of fermentation). During the fermentation, the temperature was set to 30°C and the pH was kept constant at 7.0 by metering in NH4OH or H3PO4. Glucose was metered in during the fermentation, striving for a glucose concentration of <5 g / l. After 22 hours (at the end of the logarithmic growth phase), the expression of CGTase was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to 0.15 mM. By switching the pure glucose feed to a glucose / arabinose mixture with a constant feed of 3 g / l*h glucose:arabinose in a ratio of 2:1, the expression of the Pal variant was induced 24 h or 41 h after the start of the fermentation, and the expression of TolAIII was induced 41 h after the start of the fermentation.

[0196] Before inoculation, the culture medium in the fermenter was stirred at a speed of 400 rpm and bubbled with 1.67 vvm (volume of air / volume of culture medium / minute) of compressed air through a sterile filter. Under these starting conditions, an optical oxygen sensor (VisiFerm DO225, Hamilton) was calibrated to 100% saturation before inoculation. The target value of O2 saturation during the fermentation process was set to 30% of this value. O2 saturation was measured by an oxygen sensor during the fermentation process and captured by the fermenter control 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 1500 rpm under software control to return the O2 saturation to the target value.

[0197] After 48 h of fermentation, samples were collected, cells were removed from the fermentation medium by centrifugation, and the CGTase content in the fermentation supernatant was determined by the activity assay method as in Example 1.

[0198] Table 2 shows the yields of cyclodextrin glycosyltransferase obtained respectively.

[0199] Table 2: CGTase production in the fermentation supernatant after 48 h of cultivation.

[0200] strain CGTase (relative yield, unit: %) W3110 / pCGT 100 W3110 / pCGT-Pal, induced after 24h 430 W3110 / pCGT-Pal, induced after 41h 690 W3110 / pCGT-TolAIII 260

[0201] During the fermentation of W3110 / pCGT and W3110 / pCGT-Pal (41 h post-induction), the optical density (OD) at 600 nm was measured on a spectrophotometer (Beckman Coulter DU 730). 600 ) were used to determine cell growth and to study the effect of Pal variant expression on cell growth.

[0202] In addition, the cell dry weight of the culture was determined. To determine the cell dry weight, a sample containing 1 ml of the fermenter culture was transferred to a reaction vessel with a predetermined empty weight. After centrifugation (5 minutes, 12 000 rpm), the supernatant was removed and the cell pellet was dried in an incubator (at 60° C., ≥48 h). Afterwards, the weight of the container containing the dried cell pellet was weighed, and the cell dry weight (CDW) was calculated from the difference between the weight of the container containing the dried cell pellet and the weight of the empty container. The results are summarized in Table 3.

[0203] Table 3: Optical density (OD) of CGTase-producing bacteria in fermenters 600 ) and cell dry weight (CDW).

[0204]

[0205] Example 3: Production of Fab antibody fragments in shake flasks

[0206] The production of CD154 Fab fragments was performed in shake flasks using E. coli strains W3110 / pJF118ut-CD154 and W3110 / pJF118ut-CD154-Pal using a method similar to that described in Example 1. After 72 h, samples were collected, cells were removed from the culture medium by centrifugation, and the supernatant was analyzed to determine the CD154 Fab fragments released into the culture medium. The cell pellets were collected in PBS buffer and the cells were disrupted in a FastPrep homogenizer (MP Biomedicals). The CD154 Fab fragments present in the cells were determined from the cell lysates thus obtained.

[0207] CD154 Fab fragments were quantified by sandwich ELISA assays known to those skilled in the art. This involved the use of an immobilized anti-human IgG (Fd) antibody (The Binding Site, product No. PC075) as a trap and a peroxidase-conjugated goat anti-human κ light chain antibody (Sigma, product no. A 7164) as a detection antibody. Quantification was achieved by peroxidase conversion of the chromogenic substrate Dako TMB+ (Dako #S1599) and the associated absorbance change at 450 nm. The ELISA was calibrated using the Fab fragment "Human Fab / κ" (Bethyl Laboratories, product number: P80-115).

[0208] Table 3: Yields of CD154 antibody fragments in culture supernatants.

[0209] strain CD154 antibody fragment in supernatant (mg / l) W3110 / pJF118ut-CD154 59 W3110 / pJF118ut-CD154-Pal 87

[0210] In addition to the product yield, the viable cell count of the culture was determined after 72 h. For this purpose, the culture samples were diluted 10% with LB medium in a final volume of 1 ml. 5 100 μl of the dilution was then plated on LB agar plates containing 20 mg / l tetracycline. The colonies that grew were counted and the viable cell count of the starting culture was calculated taking into account the dilution factor. For strain W3110 / pJF118ut-CD154, it was 5.5·10 8 cells / ml, and for strain W3110 / pJF118ut-CD154-Pal it was 2.7·10 8 cells / ml.

[0211] PCT

[0212] Output (original electronic form)

[0213] (This page is not part of the international application and is not counted as part of the international application)

[0214] SEQUENCE LISTING <110> Wacker Chemie AG Johanna Koch Markus Brenner <120> Bacterial strains that release recombinant proteins in fermentation methods <130> CO11724 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 816 <212> DNA <213> Artificial sequence <220> <223> DNS-fragment xI <220> <221> misc_feature <222> (1)..(816) <400> 1 caattcgcgc gcgtaacaaa agtgtctata atcacggcag aaaagtccac attgattatt 60 tgcacggcgt cacactttgc tatgccatag cattttatc cataagatta gcggatccta 120 cctgacgctt tttatcgcaa ctctctactg tttctcata cccgtttttt tgggctagaa 180 atattttgt ttagaattc taggaggaa attaatgca actgacaaa gtgctgaaag 240 ggctgatgat tgctctgcct gttatggcaa tgcggcagc cagcaatgac ggcagcgaag 300 gcatgctggg tgccggcact ggtatggatg cgaacggcgg caacggcaac atgtcttccg 360 aagagcaggc tcgtctgcaa atgcacagc tgcagcagaa cacatcgtt tacttcgatc 420 tggacaagta cgatatccgt tctgacttcg ctcaatgct ggatgcacat gcaaacttcc 480 tgcgtagcaa cccgtcttac aaagtcaccg tagaggtca cgcggacgaa cgtggtactc 540 cggaatacaa catctccctg ggtgaacgtc gtgcgaacgc cgttaagatg tacctgcagg 600 gtaaaggcgt ttctgcagac cagatctcca tcgtttctta cggtaaagaa aaacctgcag 660 tactgggtca tgacgaagcg gcatactcca aaaaccgtcg tgcggtactg gtttactaag 720 aattgcaagc tggccgacgc gtcccacagc cgccagttcc gctggcggca tttaacttt 780 ctttaatgaa gccggaaaaa tcccgcgcgc gaaggc 816 <210> 2 <211> 771 <212> DNA <213> Artificial Sequence <220> <223> DNS - fragment xII <220> <221> misc_feature <222> (1)..(771) <400> 2 caattcgcgc gcgtaacaaa agtgtctata atcacggcag aaaagtccac attgattatt 60 tgcacggcgt cacactttgc tatgccatag catttttatc cataagatta gcggatccta 120 cctgacgctt tttatcgcaa ctctctactg tttctccata cccgtttttt tgggctagaa 180 ataattttgt ttaagaattc taaggaggaa attatatgaa aaagacagct atcgcgattg 240 cagtggcact ggctggtttc gctaccgtag cgcaggctgc cgcagaggca gatgatattt 300 tcggtgagct aagctctggt aagaatgcac cgaaaacggg gggaggggcg aaagggaaca 360 atgcttcgcc tgccgggagt ggtaatacta aaaacaatgg cgcatcaggg gccgatatca 420 ataactatgc cgggcagatt aaatctgcta tcgaaagtaa gttctatgac gcatcgtcct 480 atgcaggcaa aacctgtacg ctgcgcataa aactggcacc cgatggtatg ttactggata 540 tcaaacctga aggtggcgat cccgcacttt gtcaggctgc gttggcagca gctaaacttg 600 cgaagatccc gaaaccacca agccaggcag tatatgaagt gttcaaaaac gcaccattgg 660 acttcaaacc gtaagaattg caagctggcc gacgcgtccc acagccgcca gttccgctgg 720 cggcatttta actttcttta atgaagccgg aaaaatcccg cgcgcgaagg c 771 <210> 3 <211> 1599 <212> DNA <213> Artificial sequence <220> <223> DNS - fragment xIII <220> <221> misc_feature <222> (1)..(1599) <400> 3 acagaattct aaggaggaaa ttatatgaaa agaaaccgtt tttttaatac ctcggctgct 60 attgccattt cgattgcatt acagatcttt tttccgtccg cttccgcttt cgctcaggtt 120 cagctggtgc agagcggtgc cgaagttgtt aaaccgggtg caagcgttaa actgagctgt 180 aaagcaagcg gctatatttt taccagctat tatatgtatt gggtgaaaca ggcaccgggt 240 cagggtctgg aatggattgg tgaaattaat ccgagcaatg gcgataccaa tttaatgaa 300 aaatttaaa gcaaagccac cctgaccgtt gataaaagcg caagcaccgc atatatggaa 360 ctgagcagcc tgcgtagcga agataccgca gtttattatt gtacccgtag tgatggtcgc 420 aatgatatgg atagctgggg tcagggcacc ctggtcaccg ttagcagcgc aagcaccaaa 480 ggtccgagcg tttttccgct ggcaccgagc agcaaaagca ccagcggtgg caccgcagca 540 ctgggttgtc tggttaaaga ttattttccg gaaccggtta cagttagctg gaatagcggt 600 gcactgacca gtggtgttca tacctttccg gcagttctgc aaagcagcgg tctgtatagc 660 ctgagcagcg ttgttaccgt tccgagcagt agcctgggca cccagaccta tatttgtaat 720 gttaatcata aaccgagcaa caccaaagtg gataaaaaag ttgaaccgaa aagctgctaa 780 taaccatgga gaaaataaag tgaaacaaag cactattgca ctggcactct taccgttact 840 cttcacccct gttaccaaag ccgatattgt gctcacccag agtccggcaa ccctgagcgt 900 tagtccgggt gaacgtgcaa ccattagctg tcgtgcaagc cagcgtgtta gcagcagcac 960 ctatagctat atgcattggt atcagcagaa accgggtcag cctccgaaac tgctgattaa 1020 atatgcaagc aatctggaaa gcggtgttcc ggcacgtttt agcggtagcg gtagtggcac 1080 cgattttacc ctgaccatta gcagcgttga accggaagat tttgccacct attattgtca 1140 gcatagctgg gaaattcctc cgacctttgg tggtggcacc aaactcgaga ttaaacgtac 1200 cgttgcagca ccgagcgtgt ttatctttcc gcctagtgat gaacagctga aaagcggcac 1260 cgcaagcgtt gtttgtctgc tgaataactt ttatccgcgt gaagcaaaag ttcagtggaa 1320 agttgataat gcactgcaaa gcggtaatag ccaagaaagc gttaccgaac aggatagcaa 1380 agatagcacc tatagcctgt caagcaccct gaccctgagc aaagcagatt atgaaaaaca 1440 caaagtgtat gcctgcgaag ttacccatca gggtctgagc agtccggtta caaaaagttt 1500 taatcgtggt gaatgtagct cttctgccca tcaccaccat caccattaat aagcttctag 1560 aagcttggct gttttggcgg atgagagaag attttcgac 1599 <210> 4 <211> 173 <212> PRT <213> Escherichia coli <220> <221> Peptide <222> (1)..(173) <223> Wild-type - Pal - protein <400> 4 Met Gln Leu Asn Lys Val Leu Lys Gly Leu Met Ile Ala Leu Pro Val 1 5 10 15 Met Ala Ile Ala Ala Cys Ser Ser Asn Lys Asn Ala Ser Asn Asp Gly 20 25 30 Ser Glu Gly Met Leu Gly Ala Gly Thr Gly Met Asp Ala Asn Gly Gly 35 40 45 Asn Gly Asn Met Ser Ser Glu Glu Gln Ala Arg Leu Gln Met Gln Gln 50 55 60 Leu Gln Gln Asn Asn Ile Val Tyr Phe Asp Leu Asp Lys Tyr Asp Ile 65 70 75 80 Arg Ser Asp Phe Ala Gln Met Leu Asp Ala His Ala Asn Phe Leu Arg 85 90 95 Ser Asn Pro Ser Tyr Lys Val Thr Val Glu Gly His Ala Asp Glu Arg 100 105 110 Gly Thr Pro Glu Tyr Asn Ile Ser Leu Gly Glu Arg Arg Ala Asn Ala 115 120 125 Val Lys Met Tyr Leu Gln Gly Lys Gly Val Ser Ala Asp Gln Ile Ser 130 135 140 Ile Val Ser Tyr Gly Lys Glu Lys Pro Ala Val Leu Gly His Asp Glu 145 150 155 160 Ala Ala Tyr Ser Lys Asn Arg Arg Ala Val Leu Val Tyr 165 170 <210> 5 <211> 167 <212> PRT <213> Artificial Sequence <220> <223> PalD22-27 <220> <221> Peptide <222> (1)..(167) <400> 5 Met Gln Leu Asn Lys Val Leu Lys Gly Leu Met Ile Ala Leu Pro Val 1 5 10 15 Met Ala Ile Ala Ala Ala Ser Asn Asp Gly Ser Glu Gly Met Leu Gly 20 25 30 Ala Gly Thr Gly Met Asp Ala Asn Gly Gly Asn Gly Asn Met Ser Ser 35 40 45 Glu Glu Gln Ala Arg Leu Gln Met Gln Gln Leu Gln Gln Asn Asn Ile 50 55 60 Val Tyr Phe Asp Leu Asp Lys Tyr Asp Ile Arg Ser Asp Phe Ala Gln 65 70 75 80 Met Leu Asp Ala His Ala Asn Phe Leu Arg Ser Asn Pro Ser Tyr Lys 85 90 95 Val Thr Val Glu Gly His Ala Asp Glu Arg Gly Thr Pro Glu Tyr Asn 100 105 110 Ile Ser Leu Gly Glu Arg Arg Ala Asn Ala Val Lys Met Tyr Leu Gln 115 120 125 Gly Lys Gly Val Ser Ala Asp Gln Ile Ser Ile Val Ser Tyr Gly Lys 130 135 140 Glu Lys Pro Ala Val Leu Gly His Asp Glu Ala Ala Tyr Ser Lys Asn 145 150 155 160 Arg Arg Ala Val Leu Val Tyr 165 <210> 6 <211> 834 <212> DNA <213> Artificial Sequence <220> <223> DNS - fragment xIA <220> <221> misc_feature <222> (1)..(834) <400> 6 caattcgcgc gcgtaacaaa agtgtctata atcacggcag aaaagtccac attgattatt 60 tgcacggcgt cacactttgc tatgccatag catttttatc cataagatta gcggatccta 120 cctgacgctt tttatcgcaa ctctctactg tttctccata cccgtttttt tgggctagaa 180 ataattttgt ttaagaattc taaggaggaa attatatgca actgaacaaa gtgctgaaag 240 ggctgatgat tgctctgcct gttatggcaa ttgcggcagc ttcttccaac aagaacgcca 300 gcaatgacgg cagcgaaggc atgctgggtg ccggcactgg tatggatgcg aacggcggca 360 acggcaacat gtcttccgaa gagcaggctc gtctgcaaat gcaacagctg cagcagaaca 420 acatcgttta cttcgatctg gacaagtacg atatccgttc tgacttcgct caaatgctgg 480 atgcacatgc aaacttcctg cgtagcaacc cgtcttacaa agtcaccgta gaaggtcacg 540 cggacgaacg tggtactccg gaatacaaca tctccctggg tgaacgtcgt gcgaacgccg 600 ttaagatgta cctgcagggt aaaggcgttt ctgcagacca gatctccatc gtttcttacg 660 gtaaagaaaa acctgcagta ctgggtcatg acgaagcggc atactccaaa aaccgtcgtg 720 cggtactggt ttactaagaa ttgcaagctg gccgacgcgt cccacagccg ccagttccgc 780 tggcggcatt ttaactttct ttaatgaagc cggaaaaatc ccgcgcgcga aggc 834 <210> 7 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> Pal22A <220> <221> Peptide <222> (1)..(173) <400> 7 Met Gln Leu Asn Lys Val Leu Lys Gly Leu Met Ile Ala Leu Pro Val 1 5 10 15 Met Ala Ile Ala Ala Ala Ser Ser Asn Lys Asn Ala Ser Asn Asp Gly 20 25 30 Ser Glu Gly Met Leu Gly Ala Gly Thr Gly Met Asp Ala Asn Gly Gly 35 40 45 Asn Gly Asn Met Ser Ser Glu Glu Gln Ala Arg Leu Gln Met Gln Gln 50 55 60 Leu Gln Gln Asn Asn Ile Val Tyr Phe Asp Leu Asp Lys Tyr Asp Ile 65 70 75 80 Arg Ser Asp Phe Ala Gln Met Leu Asp Ala His Ala Asn Phe Leu Arg 85 90 95 Ser Asn Pro Ser Tyr Lys Val Thr Val Glu Gly His Ala Asp Glu Arg 100 105 110 Gly Thr Pro Glu Tyr Asn Ile Ser Leu Gly Glu Arg Arg Ala Asn Ala 115 120 125 Val Lys Met Tyr Leu Gln Gly Lys Gly Val Ser Ala Asp Gln Ile Ser 130 135 140 Ile Val Ser Tyr Gly Lys Glu Lys Pro Ala Val Leu Gly His Asp Glu 145 150 155 160 Ala Ala Tyr Ser Lys Asn Arg Arg Ala Val Leu Val Tyr 165 170

Claims

1. A bacterial strain for preparing and releasing a recombinant protein, the bacterial strain containing an open reading frame encoding the recombinant protein under the control of a functional promoter, characterized in that The bacterial strain contains an open reading frame encoding a mutant peptidoglycan-associated lipoprotein (Pal protein) under the control of a functional promoter, the mutant peptidoglycan-associated lipoprotein having been mutated such that it does not have a membrane anchor for the outer cell membrane of the bacterium, The 5' end of the DNA sequence encoding the protein to be produced is linked in frame to the 3' end of a signal sequence for protein export, so that after protein biosynthesis in the cytosol, both the recombinant protein and the mutant peptidoglycan-associated lipoprotein are transported to the periplasm, and The bacterial strain additionally contains a wild-type peptidoglycan-associated lipoprotein gene, and The bacterial strain is a strain of the species Escherichia coli, wherein the open reading frame encoding the mutant peptidoglycan-associated lipoprotein has been mutated such that the open reading frame encodes a mutant peptidoglycan-associated lipoprotein in which amino acids 1 to 6 are deleted.

2. The bacterial strain according to claim 1, characterized in that The recombinant protein is a heterologous protein.

3. A method for producing recombinant protein by fermentation, characterized in that The bacterial strain according to any one of claims 1 to 2 is cultured in a fermentation medium, the fermentation medium is separated from the cells after fermentation, and the recombinant protein is isolated from the fermentation medium.

4. The method according to claim 3, characterized in that After separating the fermentation medium from the cells, the recombinant protein is purified from the fermentation medium.

5. The method according to claim 3 or 4, characterized in that Inducing the expression of the mutant peptidoglycan-associated lipoprotein.

6. The method according to claim 3 or 4, characterized in that After inducing the expression of the recombinant protein, the expression of the mutant peptidoglycan-associated lipoprotein is induced.

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