Improved cysteine-producing strain

Abstract

The present invention relates to a microbial strain suitable for the fermentative production of L-cysteine, wherein the relative enzymatic activity of an enzyme class identified by EC 2.7.9.2 in the KEGG database is inactivated or reduced compared to the specific activity of the wild-type enzyme, and the enzyme class identified by EC 2.7.9.2 in the KEGG database forms an increased amount of L-cysteine ​​compared to a microbial strain having the wild-type enzymatic activity, and the gene encoding said enzymatic activity is identified by ppsA. The present invention also relates to a method for producing L-cysteine ​​using these microbial cells.

Description

Technical Field

[0001] The present invention relates to a microbial strain suitable for the fermentative production of L-cysteine, wherein the relative enzymatic activity of the enzyme class specified by the number EC 2.7.9.2 in the KEGG database is inactivated or reduced compared to the specific activity of the wild-type enzyme, and said microbial strain forms an increased amount of L-cysteine compared to a microbial strain having the wild-type enzymatic activity of the enzyme class specified by the number EC 2.7.9.2 in the KEGG database, wherein the gene encoding said enzymatic activity is identified by ppsA. Furthermore, the present invention provides a method for producing L-cysteine using these microbial cells.

[0002] Cysteine, abbreviated as Cys or C, is an α-amino acid with the side chain -CH₂-SH. The naturally occurring enantiomeric form is L-cysteine, which is the only amino acid that forms proteins. Therefore, when the term cysteine is used without a descriptor in the context of the present invention, it means L-cysteine. By oxidation of the sulfhydryl group, two cysteine residues combine to form a disulfide bond, resulting in the formation of cystine, to which the same description applies, i.e., in the absence of a descriptor, the present invention refers to the L-enantiomer (or L-cystine, or (R,R)-3,3'-dithiobis(2-aminopropionic acid)). L-cysteine is a semi-essential amino acid for humans, as it is formed from the amino acid methionine.

[0003] In all organisms, cysteine occupies an important position in sulfur metabolism and is used for the synthesis of proteins, glutathione, biotin, lipoic acid, thiamine, taurine, methionine and other sulfur-containing metabolites. In addition, L-cysteine functions as a precursor in the biosynthesis of coenzyme A.

[0004] Cysteine ​​biosynthesis has been studied in detail in bacteria, particularly intestinal bacteria. An overview of cysteine ​​biosynthesis can be found in Wada and Takagi, Appl. Microbiol. Biotechnol. (2006) 73: 48-54.

[0005] The amino acid L-cysteine ​​is economically important. It is used, for example, as a food additive (especially in the baking industry), as a raw material for cosmetics, and as a starting material for the manufacture of active pharmaceutical ingredients (especially N-acetylcysteine ​​and S-carboxymethylcysteine).

[0006] In addition to the classical preparation of cysteine ​​by extraction from keratin-containing materials such as hair, bristles, keratin, hooves, and feathers, or by in vivo transformation through enzymatic conversion of precursors, there are also processes of fermentation production of cysteine. Prior art relating to fermentation production of cysteine ​​using microorganisms is disclosed, for example, in EP0858510B1, EP0885962B1, EP1382684B1, EP1220940B2, EP1769080B1, and EP2138585B1. Examples of bacterial host organisms used include strains of the genus Corynebacterium and members of the family Enterobacteriaceae, such as Escherichia coli or Pantoea ananatis.

[0007] Various methods are available to improve cysteine ​​production in microbial strains. In addition to the classical approach of obtaining improved cysteine ​​producers through mutation and selection, strains have also been genetically modified to achieve effective overproduction of cysteine.

[0008] For example, the introduction of the cysE allele encoding a serine O-acetyltransferase with reduced cysteine-mediated feedback inhibition resulted in increased cysteine ​​production (EP0858510B1; Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611). The feedback-resistant CysE enzyme significantly cleaved the formation of O-acetyl-L-serine, a direct precursor of cysteine, from intracellular cysteine ​​levels.

[0009] O-acetyl-L-serine is formed from L-serine and acetyl-CoA. Therefore, it is crucial to provide a sufficient amount of L-serine for cysteine ​​production. This can be achieved by introducing the serA allele, which encodes a 3-phosphoglycerate dehydrogenase with reduced feedback inhibition ability by L-serine. As a result, the formation of 3-hydroxypyruvate, a biosynthetic precursor of L-serine, is significantly cleaved from intracellular L-serine levels. Examples of such SerA enzymes are described in EP0620853B1 and EP1496111B1. Alternatively, Bell et al., Eur. J. Biochem. (2002) 269: 4176-4184 discloses modifications to the serA gene to de-regulate enzyme activity.

[0010] It is known that cysteine ​​yield in fermentation can be increased, for example, by attenuating or disrupting genes encoding cysteine-degrading enzymes such as tryptophanase TnaA or cystathionine β-lyase MalY or MetC (EP1571223B1).

[0011] Increasing the transport of cysteine ​​out of the cell is another way to increase the yield of products in the culture medium. This can be achieved by overexpressing so-called efflux genes, which encode membrane-bound proteins that mediate the transport of cysteine ​​out of the cell.

[0012] Various leachate genes for cysteine ​​export have been described (EP0885962B1, EP1382684B1). Exporting cysteine ​​from cells to fermentation media offers several advantages: 1) L-cysteine ​​is continuously drawn out of intracellular reaction equilibrium, resulting in a low level of the amino acid in the cell and the cessation of L-cysteine-mediated feedback inhibition of sensitive enzymes: (1) L-cysteine ​​(intracellular) <-> L-cysteine ​​(culture medium).

[0013] 2) L-cysteine ​​secreted into the culture medium is oxidized in the presence of oxygen introduced into the culture medium during cultivation to form disulfide L-cystine (EP0885962B1): (2) 2L-cysteine ​​+ 1 / 2O2 -> L-cystine + H2O.

[0014] Since the solubility of L-cystine in aqueous solution at neutral pH is very low compared to cysteine, disulfides already precipitate at low concentrations, forming a white precipitate. (3) L-cystine (dissolves) -> L-cystine (precipitates)

[0015] The precipitation of L-cystine reduces the level of product dissolved in the culture medium, thereby shifting the reaction equilibrium of (1) and (2) towards the product side.

[0016] 3) When the amino acids are obtained directly from the fermentation medium, the purification of the product is significantly less complicated than when the product accumulates in cells and cell disruption is required first.

[0017] In addition to genetic modification of cysteine-producing strains, optimizing the fermentation process, i.e., cell culture methods, also plays a crucial role in developing efficient production processes. For example, various culture parameters such as the properties and measurement of carbon and energy sources, temperature, oxygen supply (EP2707492B1), pH, and culture medium composition can affect the product yield and / or product spectrum in cysteine ​​fermentation production.

[0018] As raw material and energy costs are constantly increasing, there is always a need to increase the product yield in cysteine ​​production in order to improve the economic viability of the process.

[0019] The object of the present invention is to provide a microbial strain for cysteine ​​fermentation production, thereby enabling higher yields of L-cysteine ​​or L-cystine in fermentation compared to strains known from the prior art.

[0020] This objective is achieved by a microbial strain suitable for the fermentation production of L-cysteine, characterized by the relative enzymatic activity of the enzyme class identified by KEGG database number EC2.7.9.2 being inactivated or reduced compared to the specific activity of the wild-type enzyme, and by the formation of increased amounts of L-cysteine ​​compared to a microbial strain having the wild-type enzymatic activity of the enzyme class identified by KEGG database number EC2.7.9.2, with the gene encoding the said enzymatic activity identified by ppsA.

[0021] The enzyme activity of the enzyme class identified by the number EC2.7.9.2 in the KEGG database is given by the formula: (4) Phosphoenolpyruvate + Phosphate + AMP <-> Pyruvate + H2O + ATP (AMP: adenosine monophosphate, ATP: adenosine triphosphate) It is defined as a compound that can produce pyruvate from phosphoenolpyruvate through a reversible reaction.

[0022] Therefore, this enzyme activity is also called phosphoenolpyruvate synthase (PEP synthase, EC2.7.9.2) or synonymously pyruvate-H2O dikinase. The gene encoding this protein is abbreviated as ppsA in the context of this invention.

[0023] Detection of enzyme activity (enzyme assay, PEP synthase assay): The PEP synthase activity of a microbial strain can be determined by pelleting cells from a culture in a liquid medium, washing the cells, and preparing a cell extract using, for example, FastPrep-24™ 5G Cell Homogenizer (manufactured by MP Biomedicals). The protein content of the extract can be determined using the "Qubit® Protein Assay Kit" (manufactured by Thermo Fisher Scientific).

[0024] PEP synthase enzyme activity can be measured by the stoichiometric production of phosphate from the reaction of pyruvate and ATP according to formula (4), using, for example, the "Malachite Green Phosphate Assay Kit" (manufactured by Sigma Aldrich). Alternatively, the stoichiometric production of AMP or phosphoenolpyruvate, or the stoichiometric consumption of pyruvate or ATP can also be determined (see Formula 4). Assays for determining PEP synthase enzyme activity via the ATP-dependent consumption of pyruvate are described, for example, in Berman and Cohn, J. Biol. Chem. (1970) 245: 5309-5318. Similarly, Berman and Cohn, J. Biol. Chem. (1970) 245: 5309-5318 describes assays for the ATP-dependent formation of phosphoenolpyruvate.

[0025] The specific enzyme activity is calculated based on the enzyme activity relative to 1 mg of total protein in the cell extract without further purification or treatment (U / mg protein). It should be noted that to compare different PEP synthase enzymes, cell extracts must be prepared in the same manner. As already mentioned, cell extracts can be prepared using, for example, FastPrep-24™ 5G Cell Homogenizer (manufactured by MP Biomedicals).

[0026] Alternatively, different enzymes can also be compared based on the specific activity of 1 mg of each enzyme purified in the same manner (U / mg purified protein). Methods for purifying PEP synthase and measuring the specific activity of the purified protein are described, for example, in Berman and Cohn, J. Biol. Chem. (1970) 245: 5309-5318.

[0027] Relative enzyme activity can be determined by setting the specific enzyme activity determined in the PEP synthase assay of the microbial strain having the Wt allele in relation to the gene encoding PEP synthase to 100%. The value measured in the PEP synthase assay for the specific enzyme activity of a sample is specified as a percentage relative to the said strain containing the Wt enzyme.

[0028] An open reading frame (ORF, synonymous with cds or coding sequence) refers to a region of DNA or RNA that starts with a start codon, ends with a stop codon, and encodes the amino acid sequence of a protein. An ORF is also referred to as a coding region or a structural gene.

[0029] A gene refers to a section of DNA that contains all the essential information for producing a biologically active RNA. A gene includes a section of DNA from which a single-stranded RNA copy is generated by transcription, as well as expression signals involved in the regulation of the copying process. Expression signals include, for example, at least one promoter, transcription start site, translation start site, and ribosome binding site. Furthermore, terminators and one or more operators may be expression signals.

[0030] In the context of the present invention, a protein, for example PpsA, starts with a capital letter, whereas the sequence (cds) encoding said protein is identified in lowercase (for example ppsA).

[0031] Therefore, E. coli ppsA refers to the cds of the E. coli-derived ppsA gene, as identified by SEQ ID NO: 1, starting from nucleotides 333-2711. E. coli PpsA is the protein encoded by the aforementioned cds (E. coli ppsA), identified by SEQ ID NO: 2. The protein is phosphoenolpyruvate synthase.

[0032] P. ananatis ppsA refers to the cds of the ppsA gene derived from P. ananatis, as identified by Sequence ID No. 3, starting from nucleotides 417-2801. P. ananatis PpsA is the protein encoded by the aforementioned cds (P. ananatis ppsA), as identified by Sequence ID No. 4.

[0033] The abbreviation WT (Wt) refers to the wild type. A wild-type gene is a gene that arises naturally through evolution and is present in the wild-type genome. The DNA sequences of Wt genes are publicly available in databases such as NCBI.

[0034] Alleles define the states of genes that can be interconverted through mutation, that is, through changes in the nucleotide sequence of DNA. Genes that exist naturally in microorganisms are called wild-type alleles, and variants derived from them are called mutant alleles of the gene.

[0035] Homologous genes or homologous sequences should be understood to mean that the DNA sequences or portions of the DNA of the genes are at least 80% identical, preferably at least 90% identical, and particularly preferably at least 95% identical.

[0036] The degree of DNA identity is determined by the "nucleotide blast" program, based on the blastn algorithm found at http: / / blast.ncbi.nlm.nih.gov / . Default parameters are used as algorithm parameters for the alignment of two or more nucleotide sequences. The default general parameters are: Max target sequences=100; Short queries="Automatically adjust parameters for short input sequences"; Expect Threshold=10; Word size=28; Automatically adjust parameters for short input sequences=0. The corresponding default scoring parameters are: Match / Mismatch Scores=1, -2; Gap Costs=Linear.

[0037] Protein sequences are compared using the "protein blast" program at http: / / blast.ncbi.nlm.nih.gov / . This program uses the blastp algorithm. Default parameters are used as algorithm parameters for the alignment of two or more protein sequences. The default general parameters are: Max target sequences=100; Short queries="Automatically adjust parameters for short input sequences"; Expect Threshold=10; Word size=3; Automatically adjust parameters for short input sequences=0. The default scoring parameters are: Matrix=BLOSUM62; Gap Costs=Existence:11 Extension:1; Compositional adjustments=Conditional compositional score matrix adjustment.

[0038] In the microorganisms of the present invention, the relative enzymatic activity of the enzyme class identified by the KEGG database number EC2.7.9.2 is inactivated or reduced by at least 10%, most preferably at least 25%, most preferably at least 60%, and most preferably at least 70% compared to the specific activity of the wild-type enzyme. The enzymatic activity of the enzyme encoded by the ppsA gene, reduced by at least 10% (or 25% / 60% / 70%), is also referred to as a residual activity of up to 90% (or 75% / 40% / 30%).

[0039] In a preferred embodiment, the microbial strain is characterized by no longer possessing the enzyme activity of the enzyme class identified by the KEGG database number EC2.7.9.2, i.e., the relative enzyme activity of the enzyme class identified by the KEGG database number EC2.7.9.2 is 100% lower compared to the specific activity of the wild-type enzyme.

[0040] In the context of the present invention, "compared to / in comparison with / in comparison with (the corresponding) wild-type enzyme" means a comparison with the activity of a protein encoded by a non-mutant form of a gene of microbial origin, i.e., a protein that arises naturally through evolution and is present in the wild-type genome of the microbial organism.

[0041] Microbial strains suitable for L-cysteine ​​fermentation production include all microorganisms that possess deregulated biosynthetic metabolic pathways (homogeneous or heterogeneous) that result in the synthesis of cysteine, cystine, or derivatives thereof. Such strains are described, for example, in EP0885962B1, EP1382684B1, EP1220940B2, EP1769080B1, and EP2138585B1.

[0042] Microorganisms suitable for the fermentation production of L-cysteine ​​are preferably characterized by having one of the following modifications. a) Microbial strains are distinguished by a modified 3-phosphoglycerate dehydrogenase (serA) in which feedback inhibition by L-serine is reduced by at least twofold compared to the corresponding wild-type enzyme (e.g., described in EP1950287B1).

[0043] Compared to the corresponding wild-type enzyme, particularly preferred mutants of 3-phosphoglycerate dehydrogenase (serA) exhibit at least 5-fold, particularly preferably at least 10-fold, and in more preferred embodiments at least 50-fold reductions in L-serine-mediated feedback inhibition.

[0044] b) The microbial strain contains serine O-acetyltransferase (cysE) with at least twofold reduced cysteine-mediated feedback inhibition compared to the corresponding wild-type enzyme (e.g., EP0858510B1 or described in Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611).

[0045] Compared to the corresponding wild-type enzyme, particularly preferred mutants of serine O-acetyltransferase (cysE) exhibit at least 5-fold, particularly preferably at least 10-fold, and in more preferred embodiments at least 50-fold reductions in cysteine-mediated feedback inhibition.

[0046] c) The microbial strain exhibits at least a twofold increase in extracellular cysteine ​​transport compared to the corresponding wild-type cell, due to the overexpression of eluting genes.

[0047] Compared to wild-type cells, overexpression of leachate genes preferably increases the transport of cysteine ​​to the extracellular space by at least 5 times, particularly preferably at least 10 times, and especially preferably at least 20 times.

[0048] Leaked genes are preferably derived from the group consisting of ydeD (see EP0885962B1), yfiK (see EP1382684B1), cydDC (see WO2004 / 113373A1), bcr (see US2005-221453AA), and emrAB (see US2005-221453AA) from E. coli, or from corresponding homologous genes from different microorganisms.

[0049] Such strains are known, for example, in EP0858510B1 and EP0885962B1.

[0050] Furthermore, microbial strains suitable for L-cysteine ​​fermentation production are preferably characterized in that, compared to wild-type cells, at least one cysteine-degrading enzyme is attenuated to such an extent that the cells contain only up to 50% of its enzyme activity. The cysteine-degrading enzyme is preferably derived from the group consisting of tryptophanase (TnaA) and cystathionine β-lyase (MalY, MetC).

[0051] The microbial strains suitable for L-cysteine ​​fermentation production described in the previous paragraph are not deregulated with respect to cysteine ​​metabolism and are deregulated with respect to their cysteine ​​metabolism to form increased amounts of L-cysteine ​​compared to microbial strains with wild-type enzyme activity of the enzyme class identified by KEGG database number EC2.7.9.2. In cells of microbial strains that are not deregulated with respect to cysteine ​​metabolism and have wild-type enzyme activity of the enzyme class identified by KEGG database number EC2.7.9.2, the amount of L-cysteine ​​in the culture is approximately 0 g / l (see Table 2), and the increased amount means that the amount of L-cysteine ​​measured in the culture after 24 hours of incubation is greater than 0.05 g / l.

[0052] The microbial strain is characterized by being a strain of the Enterobacteriaceae or Corynebacteriaceae family, particularly preferably a strain of the Enterobacteriaceae family. Such strains are commercially available, in particular, from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig).

[0053] Preferably, the microbial strain is selected from the group consisting of Escherichia coli, Pantoea ananatis, and Corynebacterium glutamicum, and particularly preferably from the group consisting of Escherichia coli and Pantoea ananatis. Particularly preferably, the microbial strain is a strain of the Escherichia coli species.

[0054] Particularly preferred, the E. coli strain is selected from E. coli K12, and especially preferably from E. coli K12 W3110. Such strains are commercially available, in particular from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig), and include, for example, E. coli K12 W3110 DSM 5911 (id. ATCC27325) and Pantoea ananatis DSM30070 (id. ATCC11530). The PpsA is preferably a PpsA derived from E. coli having SEQ ID NO: 2, or a PpsA derived from P. ananatis having SEQ ID NO: 4.

[0055] Preferably, the microbial strain is characterized by containing at least one mutation in the ppsA gene. Simultaneously, the strain also forms an increased amount of L-cysteine ​​compared to wild-type cells in this preferred embodiment. Preferably, the genetic modification in the ppsA gene causes a decrease in the relative enzyme activity of the enzyme class identified by KEGG database number EC2.7.9.2 in the protein expressed by the gene, or causes it to lack such enzyme activity, compared to the specific activity of the wild-type enzyme.

[0056] Furthermore, the production strain of the present invention can be further optimized to further improve cysteine ​​production.

[0057] Optimization can be achieved genetically, for example, by additionally expressing one or more genes suitable for improving production characteristics. These genes can be expressed in the production strain by known methods, either as separate gene constructs or combined as expression units (so-called operons).

[0058] Furthermore, the production strain can be optimized by inactivating not only the ppsA gene, but also another gene whose gene product negatively affects cysteine ​​production.

[0059] However, optimization is also possible by known methods, specifically through mutagenesis and selection of strains with improved cysteine ​​production.

[0060] In the context of the present invention, gene modification in the ppsA gene is a) The coding sequence of the ppsA gene is partially or completely deleted. b) The coding sequence of the ppsA gene has been altered by one or more insertions or 5' and / or 3' extensions. c) The ppsA structural gene contains one or more mutations, particularly point mutations, resulting in reduced or complete inactivation of the expressed phosphoenolpyruvate synthase enzyme activity. d) The ppsA structural gene contains one or more mutations, particularly point mutations, that result in a strong reduction or complete inactivation of ppsA expression, or a decrease in mRNA stability, or e) Expression of the ppsA gene or translation of ppsA mRNA is attenuated or completely inactivated due to genetic alterations of the 5' and / or 3' non-coding ppsA sequence (promoter, 5'-UTR, Shine-Dalgarno sequence and / or terminator), The protein expressed by this sequence is defined as having reduced relative PEP synthase enzyme activity compared to the specific activity of the wild-type enzyme.

[0061] In the context of the present invention, any combination of the genetic modifications in the ppsA gene listed in a) to e) is also possible. Thus, in summary, in the context of the present invention, it is possible for fewer PpsA proteins to be formed or not formed at all, and / or for the expressed PpsA proteins to be less active or inactive.

[0062] Another approach involves attenuating or completely inactivating ppsA enzyme activity at the gene transcription level using the so-called "antisense RNA" strategy known to those skilled in the art. ppsA enzyme activity may also be attenuated or completely inactivated by adding inhibitors, which are either chemical or protein inhibitors.

[0063] Preferably, modifications to the ppsA gene in the strain of the present invention include complete or partial deletion of the ppsA structural gene, mutation of the ppsA structural gene by a method that causes attenuation of enzyme activity or inactivation of the enzyme, or regulation of expression by a method that causes attenuation or complete repression of the expression or translation of the ppsA structural gene and / or the ppsA gene, or a decrease in the stability of the ppsA mRNA, and include mutations in its untranscribed or untranslated gene region located at the 5' and 3' ends.

[0064] Particularly preferably, inactivation of the ppsA gene in the strains of the present invention is caused by either complete or partial deletion of ppsA cds (i.e., nucleotides 333-2711 of SEQ ID NO: 1 in the case of ppsA cds of E. coli, or nucleotides 417-2801 of SEQ ID NO: 3 in the case of ppsA cds of P. ananatis), or by mutation of the ppsA structural gene in a manner that causes reduced enzyme activity, enzyme inactivation, or decreased mRNA stability.

[0065] In a preferred embodiment, the microbial strain is characterized in that the mutant gene described above is selected from the group consisting of the ppsA gene from Escherichia coli, the ppsA gene from Pantoea ananatis, and genes homologous to those genes. The ppsA gene from E. coli is disclosed in the NCBI gene database entry as gene ID 946209, and the ppsA gene from P. ananatis is disclosed in the NCBI gene database entry as gene ID 11796889. The definition described above applies to the term "homologous gene." Particularly preferably, the mutant ppsA gene is the ppsA gene from E. coli. In a further preferred embodiment, the cds are either the cds of the ppsA gene from E. coli disclosed in nucleotides 333-2711 of SEQ ID NO: 1 (encoding a protein having SEQ ID NO: 2), or the cds of the ppsA gene from Pantoea ananatis disclosed in nucleotides 417-2801 of SEQ ID NO: 3 (encoding a protein having SEQ ID NO: 4).

[0066] In a preferred embodiment, the microbial strain is characterized in that the coding DNA sequence of the ppsA gene is sequence number 5 or its homologous sequence, particularly preferably sequence number 5. The term "homologous sequence" is defined as described above.

[0067] In this case, the mutation in the DNA sequence identified in SEQ ID NO: 1 results in three amino acid mutations in the WT protein sequence identified in SEQ ID NO: a mutation of valine at position 126 to methionine (V126M), a mutation of arginine at position 427 to histidine (R427H), and a mutation of valine at position 434 to isoleucine (V434I), according to the ppsA-MHI gene having the DHA sequence shown in SEQ ID NO: 5, which encodes the ppsA-MHI protein having the amino acid sequence disclosed in SEQ ID NO: 6.

[0068] Various methods for inactivating and mutating the ppsA gene are known to those skilled in the art. In the simplest case, the parent strain can be subjected to mutagenesis by known methods (e.g., chemically with mutagenic chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine, or physically by UV irradiation), and mutations are randomly generated in the genomic DNA. Then, after each mutant is unified, the desired ppsA mutant can be selected from the numerous mutants generated, for example, by genetic means, either by the absence of a color reaction based on enzyme activity or by the detection of a non-functional ppsA gene.

[0069] In contrast to complex mutagenesis and selection of ppsA mutants, the ppsA gene can be targeted and inactivated relatively simply by known mechanisms, such as homologous recombination. Cloning systems for target gene inactivation by homologous recombination are known to those skilled in the art and are commercially available, for example, disclosed in the user manual "Quick and Easy E. coli Gene Deletion Kit" by Gene Bridges GmbH using Red® / ET® technology (see "Red® / ET® Recombination, Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012" and its references).

[0070] According to prior art, the ppsA gene or a portion thereof can be isolated, and foreign DNA can be cloned into the ppsA gene, thereby interrupting the protein-defining open reading frame of the ppsA gene. Therefore, a DNA construct suitable for targeted inactivation of the ppsA gene may consist of a 5' section of DNA homologous to the genomic ppsA gene, followed by a gene segment containing foreign DNA, and then a 3' section of DNA homologous to the genomic ppsA gene.

[0071] Therefore, the regions in the ppsA gene that are susceptible to homologous recombination may include not only the region encoding phosphoenolpyruvate synthase. Possible regions may also include DNA sequences adjacent to the ppsA gene, namely the 5' region before the start of the coding region (the gene transcription promoter, e.g., nucleotides 1-332 of SEQ ID NO: 1, or nucleotides 1-416 of SEQ ID NO: 3) and the 3' region after the end of the coding region (the gene transcription terminator, e.g., nucleotides 2712-3000 of SEQ ID NO: 1, or nucleotides 2802-3062 of SEQ ID NO: 3), and modifications to these regions by homologous recombination may result in inactivation of the ppsA gene, as may modifications to the coding region.

[0072] The exogenous DNA is preferably a selection marker expression cassette. It consists of a gene transcription promoter functionally linked to the actual selection marker gene, optionally followed by a gene transcription terminator. In this case, the selection marker also includes homologous sequences adjacent to the 5' and 3' ends of the ppsA gene.

[0073] Preferably, the selection markers include homologous sequences adjacent to the 5' and 3' ends of the ppsA gene, each having a length of at least 30 nucleotides, and particularly preferably at least 50 nucleotides.

[0074] The DNA construct for inactivating the ppsA gene begins at the 5' end and consists of a sequence homologous to the ppsA gene, followed by an expression cassette of a selection marker, for example, selected from a class of antibiotic resistance genes, and then further sequences homologous to the ppsA gene.

[0075] In a preferred embodiment, the DNA construct for inactivating the ppsA gene comprises a sequence homologous to the ppsA gene, starting at the 5' end and having a length of at least 30 nucleotides, particularly preferably at least 50 nucleotides, followed by an expression cassette of a selection marker selected from a class of antibiotic resistance genes, and further sequences homologous to the ppsA gene, also having a length of at least 30 nucleotides, particularly preferably at least 50 nucleotides.

[0076] A selection marker gene is generally a gene whose product enables the parent plant to grow under selective conditions under which the original parent plant cannot grow.

[0077] Preferred selection marker genes are selected from a group of antibiotic resistance genes, such as ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, chloramphenicol resistance genes, or neomycin resistance genes. Other preferred selection marker genes allow parental strains with metabolic defects (e.g., amino acid nutritional requirements) to grow under selective conditions as a result of correcting metabolic defects through the expression of the aforementioned selection marker genes. Finally, another possibility is a selection marker gene whose gene product chemically alters and inactivates compounds that are essentially toxic to the parental strain (e.g., the gene for acetamidase, an enzyme that breaks down acetamide, a compound toxic to many microorganisms, into its non-toxic products, acetate and ammonia).

[0078] Among the selected marker genes, ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, and chloramphenicol resistance genes are particularly preferred. Tetracycline resistance genes and kanamycin resistance genes are particularly preferred.

[0079] In addition to inactivating target genes, homologous recombination-based systems also exist that offer the option of removing select markers from the genome, thereby enabling the generation of dual and multiple mutants. One such system is the so-called Lambda Red technology, commercially available as the "Quick and Easy E. coli Gene Deletion Kit" by Gene Bridges GmbH using Red® / ET® technology (see "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012" and its cited references).

[0080] Examples of strains of the present invention possessing an inactivated ppsA gene are the E. coli W3110-ΔppsA strain and the P. ananatis-ΔppsA::kan strain disclosed in the examples. Both strains are characterized by the inactivation of the ppsA gene by homologous recombination.

[0081] Another such system for target gene inactivation based on homologous recombination is known to those skilled in the art and is described in Example 3, and is a method for gene inactivation or gene modification based on a combination of Lambda Red recombination and counterselection screening. The system described above is, for example, described in Sun et al., Appl. Env. Microbiol. (2008) 74: 4241-4245. The DNA construct is used for inactivation of the ppsA gene, for example, consisting of two expression cassettes in any order, starting from the 5' end and consisting of a sequence homologous to the ppsA gene, followed by a) an expression cassette of a selection marker selected from a class of antibiotic resistance genes and b) an expression cassette of the sacB gene encoding the enzyme levansculas, and finally a further sequence homologous to the ppsA gene.

[0082] In the first step, the DNA construct is transformed into a producing strain, and antibiotic-resistant clones are isolated. The resulting clones are distinguished by the fact that they cannot grow on sucrose due to the sacB gene they both incorporate. The two marker genes can be removed by the principle of counterselection, and in the second step, appropriate DNA fragments are replaced by homologous recombination for the two marker genes. The clones obtained in this step then regain the ability to grow on sucrose and subsequently regain sensitivity to antibiotics. This method is used in Example 3 to replace the ppsA WT gene (SEQ ID NO: 1) of E. coli with the triple mutant ppsA-MHI (SEQ ID NO: 5) described below.

[0083] The E. coli strain W3110-ppsA-MHI is disclosed in the examples as an example of a strain of the present invention in which PpsA enzyme activity is attenuated due to a mutation in the coding sequence of the ppsA gene. W3110-ppsA-MHI contains the cds of the PpsA triple mutant PpsA-V126M-R427H-V434I (ppsA-MHI). The cds of the mutant gene of ppsA-MHI correspond to the DNA sequence of SEQ ID NO: 5 and encode the PpsA protein having the sequence of SEQ ID NO: 6. Compared to the WT sequence identified by SEQ ID NO: 2, PpsA-MHI is characterized in that the protein having the sequence of SEQ ID NO: 6 contains the following amino acid sequence changes: valine at position 126 is mutated to methionine (V126M), arginine at position 427 is mutated to histidine (R427H), and valine at position 434 is mutated to isoleucine (V434I).

[0084] Due to these mutations, the PpsA-MHI protein exhibited only 26.8% of the relative enzymatic activity compared to the specific wild-type enzyme activity (see Table 1 in Example 5).

[0085] In the case of the E. coli ppsA gene, it is preferable that at least one mutation in cds results in at least one of the following changes in the amino acid sequence of SEQ ID NO: a change in valine at position 126, arginine at position 427, and / or valine at position 434, where these three amino acids can be replaced with any other amino acid.

[0086] Mutations that result in simultaneous mutations of three amino acids in the amino acid sequence of the WT protein identified by SEQ ID NO: 2 are particularly preferred.

[0087] The mutation in the ppsA-MHI gene according to the present invention can be introduced into the ppsA WT gene by known methods, for example, by so-called "site-directed" mutagenesis using a commercially available cloning kit, as disclosed in the user manual for Agilent's QuickChange II Site-Directed Mutagenesis Kit. Alternatively, the ppsA-MHI gene according to the present invention can also be generated by known methods through DNA synthesis.

[0088] For example, strains of the present invention characterized by mutations in the ppsA structural gene, such as the ppsA-MHI triple mutation in E. coli, which induce a reduction in enzyme activity, can be produced by combining the Lambda Red recombination described above with allele selection screening for gene modification, as disclosed in the examples (see, for example, Sun et al., Appl. Env. Microbiol. (2008) 74: 4241-4245).

[0089] Particularly preferred strains are E. coli W3110ΔppsA (described in Example 1) and E. coli W3110ppsA-MHI (described in Example 3).

[0090] The present invention further provides a fermentation process for producing L-cysteine, characterized by using the microbial cells of the present invention.

[0091] The primary product formed by the method according to the present invention is L-cysteine, from which the compounds L-cystine and thiazolidinedione can be formed. L-cystine and thiazolidinedione are formed during fermentation and accumulate in both the culture supernatant and the precipitate. Thiazolidinedione is 2-methyl-2,4-thiazolidinedicarboxylic acid, an adduct of cysteine ​​and pyruvate, which may be formed as a byproduct of cysteine ​​production (EP0885962B1).

[0092] In the context of the present invention, the total cysteine ​​yield is defined as the sum of the cysteine, cystine, and thiazolidinedions produced. This is determined from the total culture, as described in Example 7. For example, it can be quantified using a colorimetric assay by Gaitonde (Gaitonde, MK (1967) Biochem. J. 104, 627-633).

[0093] No prior art discloses a process or strain capable of improving the production of amino acids, particularly cysteine, by attenuating or inactivating the enzymatic activity of phosphoenolpyruvate synthase.

[0094] As shown in the embodiments of this application, attenuation or inactivation of the enzymatic activity of ppsA in microbial strains suitable for cysteine ​​production significantly increases the total cysteine ​​yield in the fermentation process, i.e., the sum of cysteine, cystine, and thiazolidinediones produced. This was entirely unexpected from the prior art.

[0095] It was surprising that significantly higher cysteine ​​yields were achieved in the fermentation of the E. coli W3110 ppsA mutant compared to the corresponding wild-type strain. Contrary to prior art and unexpected to those skilled in the art, the reduction or inactivation of phosphoenolpyruvate synthase activity resulted in improved cysteine-producing strains.

[0096] This novel and original method for improving cysteine-producing strains has been confirmed by the results summarized in Tables 2 and 3 of Example 6, showing that inactivation of the ppsA gene or a mutant ppsA gene resulting in reduced PpsA enzyme activity in Escherichia coli, as well as inactivation of the ppsA gene in Pantoea ananatis, have already resulted in improved cysteine ​​yield in shaking flask cultures.

[0097] Therefore, to those skilled in the art, attenuation or inactivation of phosphoenolpyruvate synthase activity is also a novel and useful means of improving cysteine ​​production in other cysteine-producing strains.

[0098] Therefore, in the microbial strain of the present invention suitable for cysteine ​​production, the enzymatic activity of the protein encoded by the ppsA gene of the producing strain is attenuated or completely inhibited, while cysteine ​​production is increased. Example 7 demonstrates that a strain encoding ppsA-MHI, a ppsA mutant with reduced PpsA enzymatic activity and capable of cysteine ​​production instead of the Wt enzyme, achieves a significantly higher cysteine ​​yield in fermentation than a strain containing the WT gene for ppsA.

[0099] In the fermentation process in question, what is formed is not only the biomass of the production strain of the present invention, but also cysteine ​​and its oxidation product, cystine. The formation of biomass and cysteine ​​may be transiently correlated, or they may be formed over time in a manner that separates them from each other. Cultivation is carried out by methods well known to those skilled in the art. For this purpose, cultivation can be carried out in a shaking flask (laboratory scale) or by fermentation (production scale).

[0100] A production scale of fermentation with a fermentation volume of more than 1 L is preferred, a production scale of more than 10 L is preferred, a production scale of more than 1000 L is particularly preferred, and a fermentation capacity of more than 10000 L is particularly preferred.

[0101] Culture media are well known to those skilled in the art from the practice of microbial culture. They typically consist of a carbon source, a nitrogen source, and additives such as vitamins, salts, trace elements, and a sulfur source that optimizes cell growth and cysteine ​​production.

[0102] A carbon source is anything that the producing strain can use to form cysteine ​​products. Carbon sources include all forms of monosaccharides, including C6 sugars (hexoses) such as glucose, mannose, fructose, or galactose, and C5 sugars (pentoses) such as xylose, arabinose, or ribose.

[0103] However, the manufacturing method of the present invention also encompasses all carbon sources in the form of disaccharides, particularly sucrose, lactose, maltose, or cellobiose.

[0104] Furthermore, the manufacturing method of the present invention also encompasses all carbon sources in the form of higher sugars such as maltodextrin, starch, cellulose, hemicellulose, and pectin, glycosides, or carbohydrates having two or more sugar units, or monomers or oligomers released from them (enzymatically or chemically) by hydrolysis. Hydrolysis of higher carbon sources can be carried out upstream of the manufacturing method of the present invention, or in situ during the manufacturing method of the present invention.

[0105] Other usable carbon sources besides sugars or carbohydrates include acetic acid (or acetates derived therefrom), ethanol, glycerol, citric acid (and its salts), or pyruvate (and its salts). However, gaseous carbon sources such as carbon dioxide or carbon monoxide are also possible.

[0106] The carbon sources related to the manufacturing method of the present invention include both isolated pure substances or mixtures of individual carbon sources without further purification, which can be obtained by the chemical or enzymatic digestion of plant raw materials as hydrolysates to increase economic efficiency. These include, for example, starch hydrolysates (glucose monosaccharides), sugar beet hydrolysates (glucose, fructose, and arabinose monosaccharides), sugarcane hydrolysates (sucrose disaccharides), pectin hydrolysates (galacturonic acid monosaccharides), or lignocellulose hydrolysates (glucose monosaccharides from cellulose, xylose, arabinose, and mannose, and galactose monosaccharides from hemicellulose, and non-carbohydrate lignin). Furthermore, waste products from the digestion of vegetable raw materials, such as molasses (sugar beet) and bagasse (sugarcane), can also be used as carbon sources.

[0107] Preferred carbon sources for culturing the production strain are glucose, fructose, sucrose, mannose, xylose, arabinose, and plant hydrolysates that can be obtained from starch, lignocellulose, sugarcane, or sugar beet.

[0108] Particularly preferred carbon sources are glucose and sucrose, either in isolated forms or as components of plant hydrolysates.

[0109] A particularly preferred carbon source is glucose.

[0110] Nitrogen sources are those that can be used by the production strain to form biomass. These include ammonia in gaseous or aqueous form as NH4OH, or its salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, or ammonium nitrate. Furthermore, suitable nitrogen sources include known nitrates such as KNO3, NaNO3, ammonium nitrate, Ca(NO3)2, Mg(NO3)2, and other nitrogen sources such as urea. Nitrogen sources also include, for example, yeast extract, proteose peptone, malt extract, soy peptone, casamino acids, corn steep liquor (liquid or dried as so-called CSD), and complex mixtures of amino acids such as NZ amines and yeast nitrogen bases.

[0111] Efficient production of cysteine ​​and cysteine ​​derivatives requires the metered addition of a sulfur source, either in a single batch or through continuous feeding. Continuous metered addition can be carried out as a pure feed solution or as a mixture with further feed components, such as glucose.

[0112] Suitable sulfur sources are sulfates, sulfites, dithionites, thiosulfates, or sulfide salts, and it is also possible to use each of these acids with the required stability.

[0113] Preferred sources of sulfur are sulfates, sulfites, thiosulfates, and sulfide salts.

[0114] Particularly preferred sources of sulfur are sulfates and thiosulfates.

[0115] Particularly preferred are thiosulfates such as sodium thiosulfate and ammonium thiosulfate.

[0116] Culturing can be carried out in a so-called batch mode, which involves inoculating a starter culture of the production strain into a culture medium and then growing the cells without further supplying nutrients.

[0117] Culturing can also be carried out in a so-called fed-batch mode, which involves supplying additional nutrients (supplies) after the initial stages of growth in batch mode to compensate for the consumption of nutrients (supplies). The supplies may consist of a carbon source, a nitrogen source, a sulfur source, one or more vitamins or trace elements important for production, or a combination thereof. The supplies may be weighed together as a mixture or separately into individual supplies. Furthermore, other culture medium components may be added to the supplies, as well as additives that specifically increase cysteine ​​production. Supplies may be supplied continuously, partially (discontinuously), or in combinations of continuous and discontinuous supplies. Culturing in fed-batch mode is preferred.

[0118] Preferred carbon sources in the feed are glucose, sucrose, glucose-containing or sucrose-containing plant hydrolysates, and mixtures of preferred carbon sources in any mixed ratio.

[0119] A particularly preferred carbon source in the supply is glucose.

[0120] Preferably, the carbon source of the culture is measured so that the amount of carbon source in the fermenter during the production stage does not exceed 10 g / L. A maximum concentration of 2 g / L is preferred, a maximum concentration of 0.5 g / L is particularly preferred, and a maximum concentration of 0.1 g / L is especially preferred.

[0121] Preferred nitrogen sources in the feed are ammonia in gaseous or aqueous form as NH4OH, as well as its salts ammonium sulfate, ammonium phosphate, ammonium acetate and ammonium chloride, as well as urea, KNO3, NaNO3 and ammonium nitrate, yeast extract, proteose peptone, malt extract, soy peptone, casamino acids, corn steep liquor, NZ amine and yeast nitrogen bases.

[0122] Particularly preferred nitrogen sources in the feed are ammonia or ammonium salts, urea, yeast extract, soy peptone, malt extract, or corn steep liquor (in liquid or dry form).

[0123] Preferred sulfur sources in the feed are sulfates, sulfites, thiosulfates, and sulfide salts.

[0124] Particularly preferred sulfur sources in the feed are sulfates and thiosulfates.

[0125] Particularly preferred sulfur sources in the feed are thiosulfates such as sodium thiosulfate and ammonium thiosulfate.

[0126] Further culture medium additives may include salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, and iron, as well as trace amounts (i.e., μM concentrations) of salts of the elements molybdenum, boron, cobalt, manganese, zinc, copper, and nickel. In addition, organic acids (e.g., acetic acid, citric acid), amino acids (e.g., isoleucine), and vitamins (e.g., vitamin B1, vitamin B6) may be added to the culture medium.

[0127] Culturing is carried out under pH and temperature conditions that promote the growth of the producing strain and cysteine ​​production. The useful pH range is from pH 5 to pH 9. A pH range of pH 5.5 to pH 8 is preferred. A pH range of pH 6.0 to pH 7.5 is particularly preferred.

[0128] The preferred temperature range for propagation of the producing strain is 20°C to 40°C. The temperature range is particularly preferred to be 25°C to 37°C, and especially preferred to be 28°C to 34°C.

[0129] The resulting strain can be propagated either without supplying oxygen (anaerobic culture) or with supplying oxygen (aerobic culture). Aerobic culture using oxygen is preferred.

[0130] In the case of aerobic culture of the strain of the present invention for cysteine ​​production, the oxygen content is set to a saturation of at least 10% (v / v), preferably at least 20% (v / v), and particularly preferably at least 30% (v / v). According to the prior art, the oxygen saturation during culture is automatically adjusted by a combination of gas supply and stirring speed.

[0131] Oxygen supply is ensured by introducing compressed air or pure oxygen. Aerobic culture using compressed air is preferred. The useful range for compressed air supply in aerobic culture is 0.05vvm to 10vvm (vvm: the introduction of compressed air into a fermentation batch, specified as liters of compressed air per liter of fermentation volume per minute). The introduction of compressed air at 0.2vvm to 8vvm is preferred, the introduction of compressed air at 0.4 to 6vvm is particularly preferred, and the introduction of compressed air at 0.8 to 5vvm is especially preferred.

[0132] The maximum stirring speed is 2500 rpm, preferably 2000 rpm, and particularly preferably 1800 rpm.

[0133] The incubation period is 10 to 200 hours. An incubation period of 20 to 120 hours is preferred. An incubation period of 30 to 100 hours is particularly preferred.

[0134] The culture batch obtained by the method described above contains cysteine ​​either dissolved in the culture supernatant or oxidized as precipitated cystine. The cysteine ​​or cystine contained in the culture batch can be used further without further purification (work-up) or separated from the culture batch.

[0135] Preferably, this method is characterized by the isolation of the formed cysteine. Known process steps, including centrifugation, decantation, dissolution of the crude product with mineral acid, filtration, extraction, chromatography or crystallization, or precipitation, can be used for the isolation of cysteine ​​and cystine. These process steps can be combined in any form to isolate cysteine ​​to a desired purity. The desired purity depends on subsequent use.

[0136] The cystine obtained by purification can be reduced to cysteine ​​for further use. A process for reducing L-cystine to L-cysteine ​​by an electrochemical process is disclosed in EP0235908.

[0137] A variety of analytical methods can be used to identify, quantify, and determine the purity of cysteine ​​or cystine products, including spectrophotometric methods, NMR, gas chromatography, HPLC, mass spectrometry, gravimetric methods, or combinations thereof.

[0138] The present invention can also be used to produce improved microbial strains for the fermentation production of compounds, the biosynthesis of which begins with 3-phosphoglycerate and proceeds via L-serine to L-cysteine ​​and L-cystine. This also includes microbial strains for the fermentation production of L-serine and L-cysteine ​​derivatives, including phosphoserine, O-acetylserine, N-acetylserine and thiazolidinediones, and condensation products of L-cysteine ​​and pyruvate. [Brief explanation of the drawing]

[0139] The figure shows the plasmid used in the example. [Figure 1] Figure 1 shows the 3.4kb vector pKD13 used in Example 1 and Example 2. [Figure 2] Figure 2 shows the 6.3kb vector pKD46 used in Examples 1 and 3. [Figure 3]Figure 3 shows the 5kb vector pKan-SacB used in Example 3. [Figure 4] Figure 4 shows the 4.2kb vector pACYC184 used in Example 4. [Examples]

[0140] The present invention will be further illustrated by the following examples, but the present invention is not limited thereto.

[0141] Example 1: Generation of ppsA deletion mutants in Escherichia coli The parent strain used for gene isolation and strain development was Escherichia coli K12 W3110 (commercially available from DSMZ-German Collection of Microorganism and Cell Cultures GmbH under strain number DSM5911).

[0142] The target of gene inactivation was the coding sequence of the ppsA gene derived from E. coli. The DNA sequence of the ppsA gene from E. coli K12 (Genbank Gene ID: 946209) is disclosed in Sequence ID No. 1. Nucleotides 333-2711 (identified as E. coli ppsA) encode a phosphoenolpyruvate synthase protein having the amino acid sequence disclosed in Sequence ID No. 2 (E. coli PpsA).

[0143] E. coli ppsA was inactivated using Red® / ET® from Gene Bridges GmbH, as detailed below (see the user manual for the "Quick & Easy E. coli Gene Deletion Kit," "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012," and its cited references, e.g., Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645). For this purpose, plasmids pKD13, pKD46, and pCP20 were used: The -3.4kb plasmid pKD13 (Figure 1) is disclosed in the GenBank gene database under accession number AY048744.1. The -6.3kb plasmid pKD46 (Figure 2) is disclosed in the GenBank gene database under accession number AY048746.1. The plasmid pCP20, measuring -9.4kb, is disclosed in Cherepanov and Wackernagel, Gene 158 (1995): 9-14.

[0144] To inactivate the ppsA gene in E. coli W3110 using the Lambda Red system, the following steps were performed:

[0145] 1. E. coli W3110 was transformed with plasmid pKD46 (the so-called "Red recombinase" plasmid, Figure 2), and an ampicillin-resistant clone was isolated (referred to as W3110×pKD46).

[0146] 2. A ppsA-specific DNA fragment suitable for inactivation was produced using plasmid pKD13 DNA (Figure 1) and a PCR reaction using primers pps-5f (SEQ ID NO: 7) and pps-6r (SEQ ID NO: 8) with "Phusion® High-Fidelity" DNA polymerase, Thermo Scientific®.

[0147] The primer pps-5f contains 30 nucleotides (nt) from the 5' region of the ppsA gene (nt333-362 in SEQ ID NO: 1), to which 20 nt specific to plasmid pKD13 (referred to as "pr-1" in Figure 1) are bound.

[0148] The primer pps-6r contains 30 nt (nt2682~2711 in SEQ ID NO: 1, reverse complement) from the 3' region of the ppsA gene, to which 20 nt (referred to as "pr-2" in Figure 1) specific to plasmid pKD13 are bound.

[0149] Using primers pps-5f and pps-6r, a 1.4kb PCR product was produced from the DNA of plasmid pKD13, containing 30nt sections of DNA specific to the ppsA gene derived from E. coli W3110 at both the 5' and 3' ends. Furthermore, the PCR product contained the expression cassette of the kanamycin resistance gene contained in pKD13, so-called "FRT direct repeats" (referred to as "FRT1" and "FRT2" in Figure 1) adjacent to the 5' and 3' ends of the kanamycin expression cassette, and short sections of DNA used as recognition sequences for "FLP recombinase" (contained in plasmid pCP20) in a later step to remove the antibiotic marker kanamycin.

[0150] 3.1.4kb PCR products were isolated and treated with DpnI, a restriction endonuclease well known to those skilled in the art that cleaves only methylated DNA, to remove residual pKD13 plasmid DNA. Unmethylated DNA from the PCR reaction was not degraded.

[0151] 4. A 1.4kb PCR product specific to the ppsA gene and containing an expression cassette for the kanamycin resistance gene was transformed into E. coli W3110×pKD46, and kanamycin-resistant clones were isolated on LBkan plates at 30°C. The LBkan plates contained LB medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl), 1.5% agar, and 15 mg / L kanamycin.

[0152] 5. Ten of the obtained kanamycin-resistant clones were purified using an LBkan plate (i.e., clone isolation by specificization), and checked by PCR to confirm whether the kanamycin-resistant cassette was correctly incorporated into the ppsA gene.

[0153] Genomic DNA used in the PCR reaction ("Phusion® High-Fidelity" DNA polymerase, Thermo Scientific®) was isolated from cells in a culture of kanamycin-resistant clones of E. coli W3110 in Lbkan medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L kanamycin) using a DNA isolation kit (Qiagen). Genomic DNA from wild-type E. coli W3110 was used as a control. The primers used in the PCR reaction were pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10). Primer pps-7f contained nt167-188 from SEQ ID NO: 1, and primer pps-8r contained nt2779-2800 from SEQ ID NO: 1 in reverse complementation.

[0154] E. coli W3110 wild-type DNA produced a 2630 bp DNA fragment in the PCR reaction, as expected from a complete gene. In contrast, the kanamycin-resistant clone under study produced a DNA fragment of approximately 1660 bp in the PCR reaction, as expected when a 1.4 kb PCR product is incorporated into the ppsA gene at the site defined by primers pps-5f and pps-6r. This result indicates that the kanamycin-resistant gene was successfully incorporated into the ppsA locus, inactivating the ppsA gene. Clones containing the inactivated ppsA gene were selected and named W3110-ΔppsA::kan.

[0155] 6. To remove the kanamycin selection marker, W3110-ΔppsA::kan was transformed with plasmid pCP20, and transformants were selected at 30°C. The 9.4kb vector pCP20 is disclosed in Cherepanov and Wackernagel (1995), Gene 158: 9-14. The FLP recombinase gene is present on vector pCP20. FLP recombinase recognizes the FRT sequence adjacent to the kanamycin resistance gene expression cassette, resulting in the removal of the kanamycin expression cassette. For this purpose, clones obtained at 30°C were incubated at 37°C. Under these conditions, FLP recombinase expression was induced, and replication of the pCP20 vector was suppressed.

[0156] As a result of this process, the ppsA gene is inactivated, and clones with restored sensitivity to kanamycin are obtained (so-called "curing" of the antibiotic selection marker). Further mutations can be introduced to generate double or multiple mutants by removing the kanamycin cassette from the genome of the ΔppsA mutant.

[0157] W3110-ΔppsA::kan regained kanamycin sensitivity after treatment with the pCP20 plasmid. This was confirmed as follows: -By plating onto LB plates and LBkan plates: Growth was positive on LB plates, but no growth was observed on LBkan plates, suggesting that the kanamycin cassette was successfully removed from the genome.

[0158] By PCR reaction: For this purpose, genomic DNA was isolated from kanamycin-sensitive clones (Qiagen DNA isolation kit) and used in PCR reactions with primers pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10) using "Phusion® High-Fidelity" DNA Polymerase, Thermo Scientific®. E. coli W3110 wild-type DNA produced approximately 2630 bp of DNA fragments in the PCR reaction, which was as expected for the complete gene. In contrast, kanamycin-sensitive clones produced approximately 300 bp of DNA fragments in the PCR reaction, which corresponded to the expected sizes of the 5' and 3' fragments of the inactivated ppsA gene remaining after homologous recombination.

[0159] The strain isolated from this process was named E. coli W3110-ΔppsA. This strain is identified by the fact that it contains an inactivated ppsA gene and has regained sensitivity to the antibiotic kanamycin.

[0160] Example 2: Creation of ppsA deletion mutants in Pantoea ananatis The parent strain used for gene isolation and strain development was Pantoea ananatis (commercially available as strain number DSM30070 from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH).

[0161] The target of gene inactivation was the ppsA gene derived from Pantoea ananatis. The DNA sequence of the P. ananatis ppsA gene (Genbank Gene ID: 31510655) is disclosed in Sequence ID No. 3. Nucleotides 417-2801 (identified as P. ananatis ppsA) encode a phosphoenolpyruvate synthase protein having the amino acid sequence disclosed in Sequence ID No. 4 (P. ananatis PpsA).

[0162] The ppsA gene of P. ananatis was inactivated using Red(registered trademark) / ET(registered trademark) from Gene Bridges GmbH, as detailed below (see the user manual for the "Quick & Easy E. coli Gene Deletion Kit," "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red(registered trademark) / ET(registered trademark) Recombination, Cat. No. K006, Version 2.3, June 2012," and its cited references, e.g., Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645). For this purpose, plasmids pKD13 and pRedET were used: The -3.4kb plasmid pKD13 (Figure 1) is disclosed in the GenBank gene database under accession number AY048744.1. -The commercially available 9.3kb plasmid pRedET is described in the user manual for the "Quick & Easy E. coli Gene Deletion Kit." Please refer to "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red(registered trademark) / ET(registered trademark) Recombination, Cat. No. K006, Version 2.3, June 2012."

[0163] To inactivate the ppsA gene in P. ananatis using the Lambda Red system, the following steps were taken:

[0164] 1. P. ananatis was transformed with plasmid pRedET (the so-called "Red recombinase" plasmid), and tetracycline-resistant clones were isolated (referred to as P. ananatis × pRedET).

[0165] 2. A ppsA-specific DNA fragment suitable for inactivation was prepared using plasmid pKD13 DNA (Figure 1) and a PCR reaction using primers ppsapa-3f (SEQ ID NO: 11) and ppsapa-4r (SEQ ID NO: 12) with "Phusion® High-Fidelity" DNA polymerase (Thermo Scientific®).

[0166] The primer ppsapa-3f contains 49 nt (nt417~465 in SEQ ID NO: 3) from the 5' region of the ppsA gene, to which 20 nt (referred to as "pr-1" in Figure 1) specific to plasmid pKD13 are bound.

[0167] The primer ppsapa-4r contains 49 nt (nt2753~2801 in SEQ ID NO: 3, reverse complement) from the 3' region of the ppsA gene, to which 20 nt (referred to as "pr-2" in Figure 1) specific to plasmid pKD13 are bound.

[0168] Using primers ppsapa-3f and ppsapa-4r, a 1.4kb PCR product was produced from the DNA of plasmid pKD13, containing 49nt DNA sections specific to the ppsA gene derived from P. ananatis at both the 5' and 3' ends. Furthermore, the PCR product contained an expression cassette of the kanamycin resistance gene contained in pKD13, so-called "FRT direct repeats" (referred to as "FRT1" and "FRT1" in Figure 1) adjacent to the 5' and 3' ends of the kanamycin expression cassette, and, if necessary, short sections of DNA that enable the removal of kanamycin, an antibiotic marker, in ppsA deletion mutants.

[0169] 3.1.4kb PCR products were isolated and treated with DpnI, a restriction endonuclease well known to those skilled in the art that cleaves only methylated DNA, to remove residual pKD13 plasmid DNA. Unmethylated DNA from the PCR reaction was not degraded.

[0170] 4. A 1.4kb PCR product containing an expression cassette for the kanamycin resistance gene, specific to the ppsA gene, was transformed into P. ananatis × pRedET, and kanamycin-resistant clones were isolated on LBkan plates at 30°C. The LBkan plates contained LB medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl), 1.5% agar, and 15 mg / L kanamycin.

[0171] 5. Kanamycin-resistant clones were purified using LBkan plates (i.e., isolated by specificization), and checked by PCR to confirm whether the kanamycin-resistant cassette was correctly incorporated into the ppsA gene.

[0172] Genomic DNA used in the PCR reaction ("Phusion® High-Fidelity" DNA polymerase, Thermo Scientific®) was isolated from cells in a culture of kanamycin-resistant clones of P. ananatis in Lbkan medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L kanamycin) using a DNA isolation kit (Qiagen). Genomic DNA of wild-type P. ananatis was used as a control. The primers used in the PCR reaction were ppsapa-1f (SEQ ID NO: 13) and ppsapa-2r (SEQ ID NO: 14). Primer ppsapa-1f contained nt281-302 of SEQ ID NO: 3, and primer ppsapa-2r contained nt2901-2922 of SEQ ID NO: 3 in reverse complementation.

[0173] Wild-type P. ananatis DNA produced a 2640 bp DNA fragment in the PCR reaction, as expected from a complete gene. In contrast, the kanamycin-resistant clone under study produced a 1670 bp DNA fragment in the PCR reaction, as expected when a PCR product of approximately 1.4 kb is incorporated into the ppsA gene at the site defined by primers ppsapa-3f (SEQ ID NO: 11) and ppsapa-4r (SEQ ID NO: 12). This result indicates that the kanamycin-resistant gene was successfully incorporated into the ppsA locus, inactivating the ppsA gene. Clones containing the inactivated ppsA gene were selected and named P. ananatis-ΔppsA::kan.

[0174] Example 3 Fabrication of E. coli W3110-ppsA-MHI E. coli W3110-ppsA-MHI was constructed using a combination of Lambda Red recombination, known to those skilled in the art, and counter-selection screening of genetic modifications, characterized by reduced enzyme activity due to mutations in the ppsA structural gene (see, for example, Sun et al., Appl. Env. Microbiol. (2008) 74: 4241-4245). The DNA sequence of the gene ppsA-MHI is disclosed in SEQ ID NO: 5 (ppsA-MHI) and encodes a protein having the sequence identified in SEQ ID NO: 6 (PpsA-MHI).

[0175] The procedure is as follows: 1. A 2.6kb DNA fragment containing a portion of the ppsA WT gene (nt167~nt2800 of SEQ ID NO: 1), i.e., cds and 5' and 3' flanking sequences, was isolated from E. coli W3110 genomic DNA by PCR using pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10).

[0176] 2. ppsA-MHI was obtained from the ppsAWT gene by sequentially introducing mutations into the ppsAWT gene through "site-directed" mutagenesis. This was performed using Agilent's commercially available cloning kit, "QuickChange II Site-Directed Mutagenesis Kit," following the instructions in the user manual.

[0177] 3. To replace the ppsA WT gene of E. coli W3110 with ppsA-MHI, a 3.2kb Kan-sacB cassette was first isolated from the plasmid pKan-SacB (Figure 3) by PCR using primers pps-9f (SEQ ID NO: 15) and pps-10r (SEQ ID NO: 16).

[0178] The plasmid pKan-sacB contained expression cassettes for both the kanamycin (Kan) resistance gene and the sacB gene, which encodes the enzyme levansculasase.

[0179] The primer pps-9f contains 30 nt starting from the ppsA gene's start ATG (nt333-362 in SEQ ID NO: 1), to which 20 nt specific to plasmid pKan-SacB (referred to as "pr-f" in Figure 3) is bound.

[0180] The primer pps-10r contains 30 nt (nt2682-2711 in SEQ ID NO: 1, reverse complement) from the stop codon of the ppsA gene, to which 21 nt (referred to as "pr-r" in Figure 3) specific to the plasmid pKan-SacB are bound.

[0181] 4. E. coli W3110×pKD46 (see Example 1 for its production) was transformed with a ppsA-specific 3.2kb PCR product, and kanamycin-resistant clones were isolated.

[0182] 5. Clones were seeded onto LBSC plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar, and 15 mg / L kanamycin).

[0183] Clones containing the integrated sacB gene produced toxic levan from sucrose, which inhibited growth. Such clones were selected and checked by PCR to determine whether the Kan-sacB cassette was correctly integrated into the ppsA gene. Genomic DNA used in the PCR reaction ("Phusion® High-Fidelity" DNA polymerase, Thermo Scientific®) was previously isolated from cells in a culture of kanamycin-resistant clones of E. coli W3110 in Lbkan medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L kanamycin) using a DNA isolation kit (Qiagen). Genomic DNA from wild-type E. coli W3110 was used as a control. The primers used in the PCR reaction were pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10).

[0184] E. coli W3110 wild-type DNA produced a 2630 nt DNA fragment in the PCR reaction, as expected for a complete gene. In contrast, the kanamycin-resistant clone produced a DNA fragment of approximately 3400 nt in the PCR reaction, as expected when a 3.2 kb PCR product is incorporated into the ppsA gene at the site defined by primers pps-9f (SEQ ID NO: 15) and pps-10r (SEQ ID NO: 16). This result indicates that the Kan-sacB cassette was successfully incorporated into the ppsA locus, and the ppsA gene was inactivated. Clones containing the incorporated Kan-sacB cassette were selected and named W3110-ΔppsA::kan-sacB×pKD46.

[0185] 6. In the next step, the Kan-sacB cassette was replaced with the ppsA-MHI gene. For this purpose, a 2.5kb DNA fragment was amplified from the ppsA-MHI DNA fragment from step 2 in a PCR reaction using primers pps-11f (SEQ ID NO: 17) and pps-12r (SEQ ID NO: 18) ("Phusion® High-Fidelity" DNA Polymerase, Thermo Scientific®). Primer pps-11f contained nt300~319 of SEQ ID NO: 1, and primer pps-12r contained nt2743~2763 of SEQ ID NO: 1 in reverse complementation.

[0186] The 7.2.5kb ppsA-MHI gene was transformed into E. coli W3110-ΔppsA::kan-sacB×pKD46, and clones were selected using kanamycin-free LBS plates (10g / L tryptone, 5g / L yeast extract, 7% sucrose, 1.5% agar). Only clones lacking the active sacB gene were grown on the LBS plates.

[0187] These clones were seeded onto LBkan plates, and clones that did not contain the active Kan gene and whose proliferation was inhibited in the presence of kanamycin were selected.

[0188] Clones that showed positive growth in the presence of sucrose and negative growth in the presence of kanamycin were selected and checked by PCR to determine whether the KansacB cassette was correctly replaced by the ppsA-MHI gene.

[0189] Genomic DNA was obtained from cells in LB medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl) using a DNA isolation kit (Qiagen). Genomic DNA from the wild-type E. coli W3110 strain was used as a control. The primers used for the PCR reaction were pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10). PCR products with a predicted size of 2630 nt were analyzed by DNA sequencing (Eurofins Genomics). Clones containing the correctly integrated ppsA-MHI gene generated the DNA sequence disclosed in SEQ ID NO: 5, encoding the protein corresponding to the sequence from SEQ ID NO: 6. Clones containing the correct ppsA-MHI gene, including mutants V126M, R427H, and V434I, were selected and named E. coli W3110-ppsA-MHI.

[0190] Example 4: Generation of cysteine-producing strains The cysteine-specific production plasmid used was plasmid pACYC184-cysEX-GAPDH-ORF306-serA317, derived from the parent vector pACYC184 (Figure 4). pACYC184-cysEX-GAPDH-ORF306-serA317 is a derivative of plasmid pACYC184-cysEX-GAPDH-ORF306 disclosed in EP0885962B1. Plasmid pACYC184-cysEX-GAPDH-ORF306 contains not only the origin of replication and the tetracycline resistance gene (parent vector pACYC184), but also the cysEX allele encoding a serine O-acetyltransferase with reduced cysteine-mediated feedback inhibition, and the efflux gene ydeD (ORF306), whose expression is controlled by the constitutive GAPDH promoter.

[0191] Furthermore, pACYC184-cysEX-GAPDH-ORF306-serA317 was cloned after the ydeD(ORF306) leachate and further contains the serA317 gene fragment encoding the N-terminal 317 amino acids of the SerA protein (full length: 410 amino acids). The E. coli serA gene is disclosed in the GenBank gene database as gene ID 945258. serA317 is disclosed in Bell et al., Eur. J. Biochem. (2002) 269: 4176-418, where it is referred to as "NSD:317" and encodes a serine feedback-resistant variant of 3-phosphoglycerate dehydrogenase. serA317 expression is regulated by the serA promoter.

[0192] E. coli W3110 strain, E. coli W3110-ΔppsA strain, E. coli W3110-ppsA-MHI strain, P. ananatis, and P. ananatis-ΔppsA::kan strain were each transformed with plasmid pACYC184-cysEX-GAPDH-ORF306-serA317 (referred to as pCYS in the following examples). Transformation was carried out by electroporation according to the prior art as described in EP0885962B1.

[0193] Transformants containing plasmids were selected on LBtet agar plates (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 1.5% agar, 15 mg / L tetracycline). The selected transformants were checked for transformed pCYS plasmids by plasmid isolation and restriction analysis using the QIAprep Spin Plasmid Kit (Qiagen). Transformants containing correctly incorporated plasmid pCYS were cultured, and ppsA enzyme activity was checked (Example 5), and cysteine ​​production was determined (Examples 6 and 7).

[0194] Example 5: Measurement of ppsA enzyme activity The ppsA enzyme activity of E. coli strains W3110, W3110-ΔppsA, and W3110-ppsA-MHI, each transformed with the pCYS plasmid (Example 4), was determined. Cells from shaking flask cultures of the three strains in 50 ml of SM1 medium (see Example 6 for its composition) were pelletized by centrifugation for 10 minutes and washed once with 10 ml of 0.9% (w / v) NaCl. Cell extracts were prepared by transferring the cell pellet to 10 ml of assay buffer (100 mM Tris-HCl, pH 8.0; 10 mM MgCl2).

[0195] A cell homogenizer, FastPrep-24™ 5G, was used. For this purpose, a 2 × 1 ml cell suspension was lysed in a 1.5 ml tube containing glass beads ("Lysing Matrix B"), pre-prepared by the manufacturer (3 × 20 seconds, shaking frequency of 6000 rpm with 30-second pauses between each interval). The resulting homogenate was centrifuged, and the supernatant was used as the cell extract to determine activity.

[0196] The protein content of the extract was measured using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions, with a Thermo Fisher Scientific Qubit 3.0 Fluorometer.

[0197] To measure ppsA enzyme activity, we used the Sigma Aldrichi Malachite Green Phosphate Assay Kit (catalog number MAK307) according to the manufacturer's instructions. The principle involves the formation of phosphoenolpyruvate through the conversion of pyruvate by ATP in the equilibrium reaction (4) mediated by ppsA enzyme activity. This generates a stoichiometric amount of phosphate used to determine the activity. -This assay included 100 μg of cell extract, 4 mM sodium pyruvate, and 4 mM ATP in 1 ml of assay buffer (100 mM Tris-HCl, pH 8.0; 10 mM MgCl2). - Various assays were incubated at 30°C. - At 0, 10, 20, 30, and 60 minutes after the start of incubation, 50 μl was taken from each assay, added to 750 μl of H2O, and finally mixed with 200 μl of reagent from the "Malachite Green Phosphate Assay Kit". - After a 30-minute incubation, the amount of phosphate formed was photometrically measured by measuring the absorbance at 620 nm using a phosphate standard curve, according to the manufacturer's instructions. Finally, the ppsA enzyme activity in the U / ml (1U = μmol substrate turnover / min) extract was determined from the amount of phosphate measured, based on the sampling time from each assay. Specific ppsA enzyme activity was calculated based on the total protein per 1 mg of the cell extract (U / mg protein).

[0198] [Table 1]

[0199] Example 6: Cysteine ​​production in a shaking flask As a pre-culture for culturing in a shaking flask, each strain was inoculated into 3 ml of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) further containing 15 mg / L tetracycline, and incubated for 16 hours at 30°C in a 135 rpm shaker. The strains studied were E. coli W3110, W3110-ΔppsA, and W3110-ppsA-MHI, and in the second experiment, P. ananatis and P. ananatis-ΔppsA::kan, each transformed with the productive plasmid pCYS (Example 4).

[0200] Main culture: Subsequently, a portion of each pre-culture was transferred to a 300 ml Erlenmeyer flask (with baffles) containing 30 ml of SM1 medium with 15 g / L glucose, 5 mg / L vitamin B1, and 15 mg / L tetracycline.

[0201] Composition of SM1 medium: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L (NH4)2SO4, 0.3 g / L MgSO4x7H2O, 0.015 g / L CaCl2x2H2O, 0.002 g / L FeSO4x7H2O, 1 g / L Sodium citrate3x2H2O, 0.1 g / L NaCl; 1 ml / L trace element solution.

[0202] Composition of trace element solutions: 0.15 g / L Na2MoO4x2H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2x6H2O, 0.25 g / L CuSO4x5H2O, 1.6 g / L MnCl12x4H2O, 0.3 g / L ZnSO4x7H2O.

[0203] This culture was prepared with an initial cell density (OD) of 0.025 / ml. 600 The culture was inoculated with a sufficient amount of pre-culture to establish the optical density of the culture (measured at 600 nm) per ml. Starting from there, the entire 30 ml batch was incubated at 30°C and 135 rpm for 24 hours.

[0204] After 24 hours, a sample was taken, and the cell density (OD) was measured. 600The total cysteine ​​content in the culture supernatant and per ml was determined, and cysteine ​​was quantified by Gaitonde's colorimetric assay (Gaitonde, MK (1967), Biochem. J. 104, 627-633). It should be noted that under very acidic reaction conditions, this assay does not distinguish between cysteine ​​and 2-methylthiazolidinedion-2,4-dicarboxylic acid (thiazolidinedion), which is a condensation product of cysteine ​​and pyruvate, as described in EP0885962B1. L-cystine, formed by the oxidation of two cysteine ​​molecules according to formula (2), is similarly detected as cysteine ​​in an assay by reduction with dithiothreitol in a diluted solution at pH 8.0. The results are shown in Table 2 for the E. coli strain and in Table 3 for the P. ananatis strain.

[0205] [Table 2]

[0206] [Table 3]

[0207] Example 7: Cysteine ​​production in fermenters E. coli W3110×pCYS, W3110-ppsA-MHI×pCYS, and W3110-ΔppsA×pCYS were compared in a production-scale fed-batch fermentation.

[0208] Preculture 1: Each strain from a 100 ml Erlenmeyer flask was inoculated into 20 ml of LB medium containing 15 mg / L of tetracycline, and incubated in a shaker (150 rpm, 30°C) for 7 hours.

[0209] Preculture 2: Subsequently, the entire pre-culture 1 was transferred to 100 ml of SM1 medium supplemented with 5 g / L glucose, 5 mg / L vitamin B1, and 15 mg / L tetracycline (see Example 6 for the composition of SM1 medium).

[0210] The culture was placed in an Erlenmeyer flask (1 L capacity) and shaken at 150 rpm (Infors incubator shaker) at 30°C for 17 hours. After this incubation, the cell density OD was measured. 600 The value per ml was between 3 and 5.

[0211] Main culture medium: Fermentation was carried out in an Eppendorf "DASGIP® Parallel Bioreactor System for Microbiology" fermenter. A culture vessel with a total capacity of 1.8 L was used. The fermentation medium (900 ml) contained 15 g / L glucose, 10 g / L tryptone (Difco), 5 g / L yeast extract (Difco), 5 g / L (NH4)2SO4, 1.5 g / L KH2PO4, 0.5 g / L NaCl, 0.3 g / L MgSO4x7H2O, 0.015 g / L CaCl2x2H2O, 0.075 g / L FeSO4x7H2O, 1 g / L sodium citrate3x2H2O, and 1 ml trace element solution (see Example 6), 0.005 g / L vitamin B1, and 15 mg / L tetracycline.

[0212] The pH in the fermenter was initially adjusted to 6.5 by pumping a 25% NH4OH solution. During fermentation, the pH was maintained at 6.5 by automatic correction with 25% NH4OH. For inoculation, 100 ml of pre-culture 2 was pumped into the fermenter. Thus, the initial volume was approximately 1 L. The culture was initially stirred at 400 rpm and aerated with sterile compressed air through a sterile filter at an aeration rate of 2 vvm (volume of air per volume of medium per minute). Under these initial conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0213] The target O2 saturation level during fermentation was set at 30%. After the O2 saturation level fell below the target, an adjustment cascade was initiated to return the O2 saturation level to the target. To this end, the gas supply was first continuously increased (up to a maximum of 5 vvm), followed by a continuous increase in the stirring speed (up to a maximum of 1500 rpm).

[0214] Fermentation was carried out at 30°C. After 2 hours of fermentation, a sulfur source in the form of a sterile 60% (w / v) stock solution of sodium thiosulfate × 5H2O was supplied at a rate of 1.5 ml per hour.

[0215] When the glucose content in the fermenter decreased from the initial 15 g / L to approximately 2 g / L, a 56% (w / w) glucose solution was continuously metered and supplied. Thereafter, the supply rate was adjusted to ensure that the glucose concentration in the fermenter did not exceed 2 g / L. Glucose was measured using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0216] The fermentation period was set to 48 hours. Afterward, samples were removed from the fermentation batch, and the content of L-cysteine ​​and its derivatives in the culture supernatant (mainly L-cysteine ​​and thiazolidinediones) and precipitate (L-cystine) was measured separately. For this purpose, a colorimetric assay by Gaitonde (Gaitonde, MK (1967), Biochem. J. 104, 627-633) was performed in both cases. L-cystine present in the precipitate needed to be dissolved in 8% (v / v) hydrochloric acid before being quantified by the same method. Finally, the total amount of cysteine ​​was determined as the sum of cysteine ​​in the pellet and supernatant.

[0217] As summarized in Table 4, the cell density OD of the studied strains 600While the g / ml values ​​were comparable, they were somewhat higher for the control strain W3110×pCYS. In contrast, cysteine ​​production (g / L) was significantly higher (approximately 3 times) in both W3110-ppsA-MHI×pCYS and W3110-ΔppsA×pCYS compared to the control strain W3110×pCYS containing the wild-type ppsA gene.

[0218] Under controlled fermentation conditions, the reduction or inactivation of ppsA enzyme activity led to a significant improvement in cysteine ​​production, and thus, at a production scale, it was shown to be an appropriate means of improving the strain. This result is not previously described and is unexpected to those skilled in the art, even considering the prior art.

[0219] [Table 4]

[0220] Abbreviations used in the diagram: bla: A gene that confers resistance to ampicillin (β-lactamase). rrnB term: rrnB terminator for transcription kanR: A gene that confers resistance to kanamycin. ORI: origin of replication pr-1: Primer binding site 1 pr-2: Primer binding site 2 FRT1: Recognition sequence 1 of FLP recombinase FRT2: Recognition sequence 2 of FLP recombinase araC: araC gene (repressor gene) P araC: Promoter of the araC gene P araB: Promoter of the araB gene Gam:λ phage Gam recombinant gene Bet:λ phage Bet recombinant gene Exo:λ phage Exo recombinant gene ORI101: Temperature-sensitive replication origin RepA: The gene for plasmid replication protein A sacB: Levans sucrase gene pr-f: Primer binding site f (forward) pr-r: Primer binding site r (reverse) OriC: origin of replication C IHF: Binding site of DNA-binding protein IHF ("integrated host factor") CamR: A gene that confers resistance to chloramphenicol. TetR: A gene that confers resistance to tetracycline. P15A ORI: Origin of replication

Claims

1. A microbial strain suitable for the fermentation production of L-cysteine, - The relative enzyme activity of the enzyme class identified by number EC2.7.9.2 in the KEGG database is inactivated or reduced compared to the specific activity of the wild-type enzyme, and - Compared to microbial strains with wild-type enzyme activity of the enzyme class identified by number EC2.7.9.2 in the KEGG database, it forms an increased amount of L-cysteine. The gene encoding the enzyme activity is identified by ppsA, and the ppsA gene contains at least one mutation, and the mutant gene is selected from the group consisting of the ppsA gene of Escherichia coli defined by nucleotides 333 to 2711 of SEQ ID NO: 1, the ppsA gene of Pantoea ananatis defined by nucleotides 417 to 2801 of SEQ ID NO: 3, and homologous genes of these genes. The homologous gene is at least 90% identical to the DNA sequence or portion of the DNA of the ppsA gene. The microbial strain is selected from the group consisting of Echerichia coli and Pantoea ananatis, and contains a mutant serine O-acetyltransferase (cysE) in which cysteine-mediated feedback inhibition is reduced by at least twofold compared to the corresponding wild-type enzyme.

2. The microbial strain according to claim 1, wherein the microbial strain is a strain of the Escherichia coli species.

3. The microbial strain according to claim 1 or 2, wherein the coding DNA sequence of the ppsA gene is sequence number 5.

4. The microbial strain according to any one of claims 1 to 3, wherein the relative enzyme activity of the enzyme class identified by the number EC2.7.9.2 in the KEGG database is at least 25% lower than the specific activity of the wild-type enzyme.

5. The microbial strain according to any one of claims 1 to 3, wherein the relative enzyme activity of the enzyme class identified by the number EC2.7.9.2 in the KEGG database is at least 70% lower than the specific activity of the wild-type enzyme.

6. A microbial strain according to any one of claims 1 to 3, which does not have the enzymatic activity of the enzyme class identified by the number EC2.7.9.2 in the KEGG database.

7. A fermentation process for producing cysteine, using a microbial strain according to any one of claims 1 to 6.

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