Improved cysteine-producing strain

Abstract

To provide a microorganism strain suitable for fermentative production of L-cysteine.SOLUTION: A microorganism strain suitable for fermentative production of L-cysteine is provided which has: relative enzyme activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database that is inactivated or reduced relative to the specific activity of the wild-type enzyme; and formation of an increased amount of L-cysteine relative to a microorganism strain having wild-type enzyme activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database, where the gene encoding the enzyme activity is identified by ppsA. Also provided is a method of producing L-cysteine using those microorganism cells.SELECTED DRAWING: Figure 4

Description

[Technical field]

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

[0002] Cysteine, abbreviated 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 protein-forming amino acid, and therefore, when the term cysteine ​​is used in the context of the present invention without a descriptor, L-cysteine ​​is meant. The same description applies to the two cysteine ​​residues that join together to form a disulfide bond by oxidation of the sulfhydryl group, resulting in the formation of cystine, i.e., in the absence of a descriptor, the L-enantiomer (or L-cystine, or (R,R)-3,3'-dithiobis(2-aminopropionic acid)) is meant in the present invention. L-cysteine ​​is a semi-essential amino acid for humans, since it is formed from the amino acid methionine.

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

[0004] The biosynthesis of cysteine ​​has been extensively studied in bacteria, particularly in enterobacteria. 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 bakery industry), as a raw material for cosmetics and as a starting material for the production 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, horn, hooves and feathers, or by biotransformation by enzymatic conversion of precursors, there are also processes for the fermentative production of cysteine. Prior art on the fermentative production of cysteine ​​using microorganisms is disclosed, for example, in EP0858510B1, EP0885962B1, EP1382684B1, EP1220940B2, EP1769080B1 and EP2138585B1. Bacterial host organisms used include strains of the genus Corynebacterium and members of the family Enterobacteriaceae, such as, for example, Escherichia coli or Pantoea ananatis.

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

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

[0009] O-acetyl-L-serine is formed from L-serine and acetyl-CoA. It is therefore very important to provide sufficient amounts of L-serine for the production of cysteine. This can be achieved by introducing a serA allele that encodes a 3-phosphoglycerate dehydrogenase with reduced feedback inhibition by L-serine. As a result, the formation of 3-hydroxypyruvate, the biosynthetic precursor of L-serine, is largely uncoupled from the L-serine level in the cell. Examples of such SerA enzymes are described in EP0620853B1 and EP1496111B1. Alternatively, Bell et al., Eur. J. Biochem. (2002) 269: 4176-4184, disclose modifications to the serA gene to deregulate the enzyme activity.

[0010] It is known that cysteine ​​yield in fermentation can be increased 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 product yield in the medium. This can be achieved by overexpressing so-called efflux genes, which code for membrane-bound proteins that mediate the transport of cysteine ​​out of the cell.

[0012] Various efflux genes for cysteine ​​export have been described (EP0885962B1, EP1382684B1). Export of cysteine ​​from the cell into the fermentation medium has several advantages: 1) L-cysteine ​​is continually withdrawn from the intracellular reaction equilibrium, so that the intracellular level of this amino acid is kept low and feedback inhibition of sensitive enzymes by L-cysteine ​​is stopped: (1) L-cysteine ​​(intracellular) <-> L-cysteine ​​(culture medium).

[0013] 2) L-cysteine ​​secreted into the medium is oxidized to form the disulfide L-cystine in the presence of oxygen introduced into the medium during cultivation (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, the disulfide precipitates already at low concentrations, forming a white precipitate: (3) L-cystine (dissolved) -> L-cystine (precipitated)

[0015] Precipitation of L-cystine reduces the level of product dissolved in the medium, thereby shifting the equilibrium of reactions (1) and (2) toward the product.

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

[0017] In addition to the genetic modification of the cysteine-producing strain, the optimization of the fermentation process, i.e. the method of culturing the cells, also plays an important role in the development of an efficient production process. For example, various culture parameters such as the nature and measurement of the carbon and energy sources, the temperature, the oxygen supply (EP2707492B1), the pH and the composition of the culture medium can affect the product yield and / or the product spectrum in the fermentative production of cysteine.

[0018] Due to ever-increasing raw material and energy costs, there is a constant need to increase product yields in cysteine ​​production to improve the economic viability of the process.

[0019] The object of the present invention is to provide a microbial strain for the fermentative production of cysteine, which makes it possible to achieve a higher yield of L-cysteine ​​or L-cystine in fermentation compared to strains known from the prior art.

[0020] This object is achieved by a microbial strain suitable for the fermentative production of L-cysteine, characterized in that the relative enzymatic activity of the enzyme class identified 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 that it forms increased amounts of L-cysteine ​​compared to a microbial strain having the wild-type enzymatic activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database, the gene encoding said enzymatic activity being identified by ppsA.

[0021] The enzymatic activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database has the formula: (4) Phosphoenolpyruvate + phosphate + AMP <-> Pyruvate + H2O + ATP (AMP: adenosine monophosphate, ATP: adenosine triphosphate) It is defined as the reaction that can produce pyruvate from phosphoenolpyruvate by the reversible reaction:

[0022] This enzymatic activity is therefore also called phosphoenolpyruvate synthase (PEP synthase, EC 2.7.9.2) or equivalently pyruvate-HO dikinase. The gene encoding this protein is abbreviated as ppsA in the context of the present 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 liquid medium, washing the cells, and preparing a cell extract, for example, using a FastPrep-24™ 5G cell homogenizer (MP Biomedicals). The protein content of the extract can be determined by the “Qubit® Protein Assay Kit” (Thermo Fisher Scientific).

[0024] PEP synthase enzyme activity can be measured by the stoichiometric production of phosphate from the reaction of pyruvate with ATP according to formula (4), for example, using the "Malachite Green Phosphate Assay Kit" (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). An assay for determining PEP synthase enzyme activity via the ATP-dependent consumption of pyruvate is 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 the assay of the ATP-dependent formation of phosphoenolpyruvate.

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

[0026] Alternatively, different enzymes can be compared based on the specific activity of 1 mg of enzyme each purified by the same method (U / mg purified protein). Methods for purifying PEP synthase and determining 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 in relation to the gene encoding PEP synthase by setting the specific enzyme activity determined in the PEP synthase assay of a microbial strain carrying the Wt allele 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 that strain containing the Wt enzyme.

[0028] Open reading frame (ORF, synonymous with cds or coding sequence) refers to a region of DNA or RNA that begins with a start codon, ends with a stop codon, and codes for the amino acid sequence of a protein. ORFs are also called coding regions or structural genes.

[0029] A gene refers to a section of DNA that contains all the basic information to produce biologically active RNA. A gene includes a section of DNA that is transcribed to produce a single-stranded RNA copy, and expression signals involved in regulating the copying process. Expression signals include, for example, at least one promoter, a transcription start site, a translation start site, and a ribosome binding site. In addition, a terminator and one or more operators can be expression signals.

[0030] In the context of the present invention, proteins, such as PpsA, begin with a capital letter, whereas the sequences encoding said proteins (cds) are specified in lower case (eg ppsA).

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

[0032] P. ananatis ppsA refers to the cds of the ppsA gene from P. ananatis, as identified in SEQ ID NO: 3 from nucleotides 417 to 2801. P. ananatis PpsA is the protein encoded by said cds (P. ananatis ppsA) and is identified in SEQ ID NO: 4.

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

[0034] Alleles define the states of a gene that can be converted into one another by mutation, i.e. by a change in the nucleotide sequence of DNA. A gene that occurs naturally in a microorganism is called a wild-type allele, and variants derived from it are called mutant alleles of the gene.

[0035] A homologous gene or sequence is to be understood as meaning that the DNA sequences or DNA portions of said genes are at least 80% identical, preferably at least 90% identical, particularly preferably at least 95% identical.

[0036] The degree of DNA identity is determined by the "nucleotide blast" program, which is based on the blastn algorithm found at http: / / blast.ncbi.nlm.nih.gov / . Default parameters are used as algorithm parameters for alignment of two or more nucleotide sequences. 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. 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 / , which uses the blastp algorithm. Default parameters are used as algorithm parameters for alignment of two or more protein sequences. 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. Default scoring parameters are: Matrix=BLOSUM62; Gap Costs=Existence:11 Extention:1; Compositional adjustments=Conditional compositional score matrix adjustment.

[0038] In the microorganism of the invention, the relative enzymatic activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database is inactivated or reduced by preferably at least 10%, particularly preferably at least 25%, particularly preferably at least 60%, particularly preferably at least 70% compared to the specific activity of the wild-type enzyme. An 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 in that it no longer has the enzymatic activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database, i.e. the relative enzymatic activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database is reduced by 100% compared to the specific activity of the wild-type enzyme.

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

[0041] Microbial strains suitable for the fermentative production of L-cysteine ​​include all microorganisms that contain deregulated biosynthetic metabolic pathways (homologous or heterologous) leading to the synthesis of cysteine, cystine or derivatives derived therefrom. Such strains are described, for example, in EP0885962B1, EP1382684B1, EP1220940B2, EP1769080B1 and EP2138585B1.

[0042] Microorganisms suitable for the fermentative production of L-cysteine ​​are preferably characterized by having one of the following modifications: a) The microbial strain is distinguished by a modified 3-phosphoglycerate dehydrogenase (serA) that exhibits at least a two-fold reduced feedback inhibition by L-serine compared to the corresponding wild-type enzyme (e.g. as described in EP1950287B1).

[0043] Compared to the corresponding wild-type enzyme, particularly preferred mutants of 3-phosphoglycerate dehydrogenase (serA) have at least a 5-fold, particularly preferred at least a 10-fold and in a more preferred embodiment at least a 50-fold reduction in feedback inhibition by L-serine.

[0044] b) The microbial strain comprises a serine O-acetyltransferase (cysE) that has at least two-fold reduced feedback inhibition by cysteine ​​compared to the corresponding wild-type enzyme (e.g., as described in EP0858510B1 or 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) have at least a 5-fold, particularly preferred at least a 10-fold and in a more preferred embodiment at least a 50-fold reduction in feedback inhibition by cysteine.

[0046] c) the microbial strain exhibits at least a two-fold increase in the transport of cysteine ​​out of the cell due to overexpression of the efflux gene, as compared to the corresponding wild-type cell.

[0047] Compared to wild-type cells, overexpression of the efflux gene preferably increases the transport of cysteine ​​out of the cell by at least 5-fold, particularly preferably by at least 10-fold, particularly preferably by at least 20-fold.

[0048] The efflux 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 a different microorganism.

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

[0050] Furthermore, the microbial strain suitable for the fermentative production of L-cysteine ​​is preferably characterized in that at least one cysteine ​​degrading enzyme is attenuated in comparison with the wild-type cell to such an extent that the cell contains only up to 50% of its enzymatic activity, the cysteine ​​degrading enzyme preferably being from the group consisting of tryptophanase (TnaA) and cystathionine β-lyase (MalY, MetC).

[0051] The microbial strains suitable for the fermentative production of L-cysteine ​​described in the previous paragraph are not deregulated in terms of cysteine ​​metabolism, and are deregulated in terms of their cysteine ​​metabolism so as to form an increased amount of L-cysteine ​​compared to a microbial strain having a wild-type enzyme activity of the enzyme class identified by the number EC2.7.9.2 in the KEGG database. In the cells of a microbial strain that is not deregulated in terms of cysteine ​​metabolism and has a wild-type enzyme activity of the enzyme class identified by the number EC2.7.9.2 in the KEGG database, the amount of L-cysteine ​​in the culture is about 0 g / l (see Table 2), and an increased amount means that the amount of L-cysteine ​​measured in the culture after 24 hours of cultivation is more than 0.05 g / l.

[0052] The microbial strain is characterized in that it is a strain of the family Enterobacteriaceae or Corynebacteriaceae, particularly preferably a strain of the family Enterobacteriaceae. Such strains are commercially available, inter alia, 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, particularly preferably from the group consisting of Escherichia coli and Pantoea ananatis. Particularly preferably, the microbial strain is a strain of the species Escherichia coli.

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

[0055] Preferably, the microbial strain is characterized by comprising at least one mutation in the ppsA gene.At the same time, the strain also forms an increased amount of L-cysteine ​​in this preferred embodiment compared to wild-type cells.Preferably, the genetic modification in the ppsA gene causes the protein expressed by said gene to have a reduced or no relative enzymatic activity of the enzyme class identified by the number EC2.7.9.2 in the KEGG database compared to the specific activity of the wild-type enzyme in the protein expressed by said gene.

[0056] Additionally, the production strains 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, said genes being expressed in the production strain by known methods either as separate genetic constructs or combined as expression units (so-called operons).

[0058] Furthermore, the production strain can be optimized by inactivating, in addition to the ppsA gene, further genes whose gene products adversely affect cysteine ​​production.

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

[0060] In the context of the present invention, a genetic 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 modified by one or more insertions or 5' and / or 3' extensions; c) the ppsA structural gene contains one or more mutations, in particular point mutations, such that the enzymatic activity of the expressed phosphoenolpyruvate synthase is attenuated or completely inactivated; d) the ppsA structural gene contains one or more mutations, in particular point mutations, that result in a strong attenuation or complete inactivation of ppsA expression or a decrease in mRNA stability, or e) expression of the ppsA gene or translation of the ppsA mRNA is attenuated or completely inactivated due to genetic modification of the 5' and / or 3' non-coding ppsA sequences (promoter, 5'-UTR, Shine-Dalgarno sequence and / or terminator), meaning that the protein expressed by said sequences has 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 under a) to e) is also possible. Thus, in summary, in the context of the present invention, it is possible that less PpsA protein is formed or not formed at all and / or that the expressed PpsA protein is less active or inactive.

[0062] In another approach, the ppsA enzyme activity can be attenuated or completely inactivated at the level of gene transcription by the so-called "antisense RNA" strategy known to those skilled in the art. The ppsA enzyme activity can also be attenuated or completely inactivated by adding inhibitors, either chemical or protein inhibitors.

[0063] Preferably, modification of the ppsA gene in the strain of the invention comprises complete or partial deletion of the ppsA structural gene, mutation of the ppsA structural gene in a manner that leads to attenuation of the enzymatic activity or inactivation of the enzyme, or attenuation or complete inhibition of expression or translation of the ppsA structural gene and / or the ppsA gene, or a decrease in the stability of the ppsA mRNA, regulating expression and mutation of its non-transcribed or non-translated gene regions located 5' and 3' to it.

[0064] Particularly preferably, the inactivation of the ppsA gene in the strain of the invention is caused either by a complete or partial deletion of the ppsA cds (i.e. nucleotides 333 to 2711 of SEQ ID NO: 1 for the E. coli ppsA cds or nucleotides 417 to 2801 of SEQ ID NO: 3 for the P. ananatis ppsA cds) or by a mutation of the ppsA structural gene in a manner that leads to an attenuation of the enzyme activity or inactivation of the enzyme or a decrease in the mRNA stability.

[0065] In a preferred embodiment, the microbial strain is characterized in that the above-mentioned mutated gene is selected from the group consisting of the ppsA gene from Escherichia coli, the ppsA gene from Pantoea ananatis and genes homologous thereto. The ppsA gene from E. coli is disclosed in the NCBI gene database entry under gene ID 946209 and the ppsA gene from P. ananatis is disclosed in the NCBI gene database entry under gene ID 11796889. The term "homologous gene" is as defined above. Particularly preferably, the mutated ppsA gene is the ppsA gene from E. coli. In a further preferred embodiment, the cds is the cds of the ppsA gene from E. coli disclosed in nucleotides 333 to 2711 of SEQ ID NO: 1 (encoding the protein having SEQ ID NO: 2) or the cds of the ppsA gene from Pantoea ananatis disclosed in nucleotides 417 to 2801 of SEQ ID NO: 3 (encoding the 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 SEQ ID NO: 5 or a homologous sequence thereof, particularly preferably SEQ ID NO: 5. The term "homologous sequence" is as defined above.

[0067] In this case, the mutations in the DNA sequence identified in SEQ ID NO:1 result in three amino acid mutations in the WT protein sequence identified in SEQ ID NO:2, namely 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), in accordance with a ppsA-MHI gene having a DHA sequence shown in SEQ ID NO:5 encoding a ppsA-MHI protein having the amino acid sequence as 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 by mutagenic chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine, or physically by UV irradiation), and mutations are randomly generated in the genomic DNA, and then the desired ppsA mutant is selected from the large number of mutants generated, for example, by the absence of a color reaction based on the enzyme activity after each mutant is isolated, or by genetic means through the detection of a malfunctioning ppsA gene.

[0069] In contrast to complex mutagenesis and selection of ppsA mutants, the ppsA gene can be subjected to targeted inactivation in a relatively simple manner, for example by the known mechanism of homologous recombination. Cloning systems for targeted gene inactivation by homologous recombination are known to the skilled artisan and commercially available, for example as disclosed in the user manual "Quick and Easy E. coli Gene Deletion Kit" by Red® / ET® technology from Gene Bridges GmbH (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 references therein).

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

[0071] Thus, the potential regions for homologous recombination in the ppsA gene can include not only the region encoding phosphoenolpyruvate synthase. The potential regions can also include DNA sequences flanking the ppsA gene, i.e. the 5' region before the start of the coding region (gene transcription promoter, e.g. nucleotides 1 to 332 of SEQ ID NO: 1, or nucleotides 1 to 416 of SEQ ID NO: 3) and the 3' region after the end of the coding region (gene transcription terminator, e.g. nucleotides 2712 to 3000 of SEQ ID NO: 1, or nucleotides 2802 to 3062 of SEQ ID NO: 3), the modification of which by homologous recombination can result in the inactivation of the ppsA gene, as can the modification of the coding region.

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

[0073] Preferably, the selection marker comprises homologous sequences flanking the 5' and 3' of the ppsA gene, each having a length of at least 30 nucleotides, particularly preferably at least 50 nucleotides.

[0074] A DNA construct for inactivating the ppsA gene consists, starting from the 5' end, of a sequence homologous to the ppsA gene, followed by an expression cassette for a selection marker, e.g. chosen from the class of antibiotic resistance genes, followed by further sequences homologous to the ppsA gene.

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

[0076] A selectable marker gene is typically a gene, the product of which allows a parent strain to grow under selective conditions in which the original parent strain cannot grow.

[0077] Preferred selection marker genes are selected from the group of antibiotic resistance genes, such as, for example, ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, chloramphenicol resistance genes or neomycin resistance genes. Other preferred selection marker genes allow parent strains with metabolic defects (e.g., amino acid auxotrophy) to grow under selection conditions as a result of correction of the metabolic defect by expression of the selection marker genes described above. Finally, another possibility is a selection marker gene, the gene product of which chemically alters a compound that is essentially toxic to the parent strain, inactivating said compound (e.g., the gene for the enzyme acetamidase, which breaks down the compound acetamide, which is toxic to many microorganisms, into the non-toxic products acetate and ammonia).

[0078] Among the selection marker genes, the ampicillin resistance gene, the tetracycline resistance gene, the kanamycin resistance gene, and the chloramphenicol resistance gene are particularly preferred, with the tetracycline resistance gene and the kanamycin resistance gene being particularly preferred.

[0079] In addition to the inactivation of targeted genes, there are also systems based on homologous recombination that offer the option of removing selection markers from the genome, thereby making it possible to generate double and multiple mutants. Such a system is, for example, the so-called Lambda Red technology, which is commercially available as the "Quick and Easy E. coli Gene Deletion Kit" by Red® / ET® technology from Gene Bridges GmbH (see "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012" and references therein).

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

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

[0082] In the first step, the DNA construct is transformed into a production strain and antibiotic-resistant clones are isolated. The clones obtained are distinguished by the fact that they cannot grow on sucrose due to the co-integrated sacB gene. The two marker genes can be removed by the principle of counterselection and in a second step, the appropriate DNA fragment is replaced by the two marker genes by homologous recombination. The clones obtained in this step then regain the ability to grow on sucrose and then also regain sensitivity to antibiotics. This method is used in Example 3 to replace the ppsA WT gene of E. coli (SEQ ID NO: 1) 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 according to the invention in which the 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 a PpsA protein having the sequence of SEQ ID NO:6. PpsA-MHI is characterized in that the protein having the sequence of SEQ ID NO:6 contains the following changes in the amino acid sequence compared to the WT sequence specified in SEQ ID NO:2: 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 had only 26.8% relative enzyme 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 preferred that at least one of the mutations in the cds results in at least one of the following changes in the amino acid sequence of SEQ ID NO:2: a change to a valine at position 126, an arginine at position 427 and / or a valine at position 434, where these three amino acids can be exchanged for any other amino acid.

[0086] Mutations which result in the simultaneous mutation of three amino acids in the amino acid sequence of the WT protein defined in SEQ ID NO:2 are particularly preferred.

[0087] The mutations of the ppsA-MHI gene according to the present invention are 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 the QuickChange II Site-Directed Mutagenesis Kit from Agilent, Inc. Alternatively, the ppsA-MHI gene according to the present invention can also be produced by DNA synthesis in known manner.

[0088] For example, strains of the invention characterized by mutations in the ppsA structural gene in a manner that results in attenuated enzymatic activity, such as the ppsA-MHI triple mutation in E. coli, can be generated by using the above-mentioned Lambda Red recombination in combination with counterselection screens for genetic modification, as disclosed in the Examples (see, e.g., 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 in that the microbial cell of the present invention is used.

[0091] The primary product formed in the process according to the invention is L-cysteine, from which the compounds L-cystine and thiazolidine can be formed. L-cystine and thiazolidine are formed during fermentation and accumulate in both the culture supernatant and the sediment. Thiazolidine is 2-methyl-2,4-thiazolidinedicarboxylic acid, an adduct of cysteine ​​with pyruvic acid that can be formed as a by-product of cysteine ​​production (EP0885962B1).

[0092] In the context of the present invention, the total cysteine ​​yield is defined as the sum of cysteine, cystine and thiazolidine produced. It is determined from the whole culture as described in Example 7. It can be quantified, for example, using the colorimetric assay according to Gaitonde (Gaitonde, MK (1967) Biochem. J. 104, 627-633).

[0093] The prior art does not disclose processes or production strains that can improve the production of amino acids, especially cysteine, by attenuating or inactivating the enzymatic activity of phosphoenolpyruvate synthase.

[0094] As shown in the examples of the present application, attenuation or inactivation of the enzymatic activity of ppsA in a microbial strain suitable for cysteine ​​production significantly increases the total cysteine ​​yield in the fermentation process, i.e. the sum of cysteine, cystine and thiazolidine produced, which was completely unexpected from the prior art.

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

[0096] This novel and original means for improving cysteine ​​producing strains is confirmed by the results summarized in Tables 2 and 3 of Example 6, where inactivation of the ppsA gene in E. coli or a mutated ppsA gene resulting in a PpsA enzyme with attenuated enzymatic activity, as well as inactivation of the ppsA gene in Pantoea ananatis, already resulted in improved cysteine ​​yields in shake flask cultivations.

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

[0098] Thus, in the microbial strains of the 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, with a concomitant increase in cysteine ​​production. Example 7 demonstrates that a strain capable of cysteine ​​production and encoding the ppsA mutant ppsA-MHI, instead of the Wt enzyme, with reduced enzymatic activity of PpsA, achieves a significantly higher cysteine ​​yield in fermentation than a strain containing the WT gene for ppsA.

[0099] In the fermentation process in question, it is not only the biomass of the production strain of the invention that is formed, but also cysteine ​​and its oxidation product cystine. The formation of biomass and cysteine ​​is either temporally correlated or biomass and cysteine ​​are formed over time in a mutually separated manner. The cultivation is carried out in a manner well known to those skilled in the art. For this purpose, the cultivation can be carried out in shake flasks (laboratory scale) or in fermentation (production scale).

[0100] Production scale processes with fermentation in fermentation volumes of more than 1 L are preferred, with production scales of more than 10 L being preferred, with production scales of more than 1000 L being particularly preferred, and fermentation volumes of more than 10000 L being especially preferred.

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

[0102] Carbon sources are those that the producing strain can use to form the cysteine ​​product and 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 process of the invention also encompasses all carbon sources in the form of disaccharides, in particular sucrose, lactose, maltose or cellobiose.

[0104] Furthermore, the process of the invention also encompasses all carbon sources in the form of higher sugars, glycosides or carbohydrates with more than two sugar units, such as, for example, maltodextrins, starches, cellulose, hemicelluloses, pectins, or monomers or oligomers released therefrom by hydrolysis (enzymatically or chemically). Hydrolysis of higher carbon sources can be carried out upstream of the process of the invention or in situ during the process of the invention.

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

[0106] The carbon sources relevant to the production method of the present invention include both isolated pure substances or mixtures of individual carbon sources without further purification, which can be obtained by 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), sugar cane hydrolysates (sucrose disaccharides), pectin hydrolysates (galacturonic acid monosaccharides) or lignocellulose hydrolysates (glucose monosaccharides from cellulose, xylose, arabinose, mannose, and galactose monosaccharides from hemicellulose, and non-carbohydrate lignin). In addition, waste products from the digestion of vegetable raw materials, such as molasses (sugar beet) and bagasse (sugar cane), 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 which can be obtained from starch, lignocellulose, sugar cane or sugar beet.

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

[0109] A particularly preferred carbon source is glucose.

[0110] Nitrogen sources are those that the production strain can use to form biomass. These include ammonia in gaseous or aqueous solutions as NH4OH or its salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate or ammonium nitrate. Further suitable nitrogen sources are 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 complex mixtures of amino acids, such as yeast extract, proteose peptone, malt extract, soybean peptone, casamino acids, corn steep liquor (liquid or dried as the so-called CSD), and NZ amines and yeast nitrogen base.

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

[0112] Suitable sulfur sources are sulfates, sulfites, dithionites, thiosulfates or sulfide salts, and it is contemplated to use the respective acids with the given stability.

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

[0114] Particularly preferred sulfur sources are sulfate and thiosulfate.

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

[0116] Cultivation can be carried out in the so-called batch mode, which involves inoculating the medium with a starter culture of the production strain and then allowing the cells to grow without further supply of nutrients.

[0117] The cultivation can also be carried out in the so-called fed-batch mode, which involves the additional supply of nutrient sources (feed) after the initial stage of growth in batch mode to compensate for the consumption of the nutrient sources (feed). The feed can consist of a carbon source, a nitrogen source, a sulfur source, one or more vitamins or trace elements important for production, or a combination of these. The feed components can be metered together as a mixture or metered separately in individual feed sections. Furthermore, other medium components can be added to the feed, as well as additives that specifically increase cysteine ​​production. The feed can be supplied continuously or partially (discontinuously) or with a combination of continuous and discontinuous feeds. Cultivation in fed-batch mode is preferred.

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

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

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

[0121] Preferred sources of nitrogen in the feed are ammonia in gaseous or aqueous form as NH4OH and 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 amines and yeast nitrogen base.

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

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

[0124] Particularly preferred sources of sulfur in the feed are sulfate and thiosulfate.

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

[0126] Further medium additives include salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, and iron, and 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) can be added to the medium.

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

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

[0129] The growth of the producing strain can optionally be carried out without oxygen supply (anaerobic culture) or with oxygen supply (aerobic culture), aerobic culture with oxygen being preferred.

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

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

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

[0133] The culture time is 10 to 200 hours. A culture time of 20 to 120 hours is preferable. A culture time of 30 to 100 hours is particularly preferable.

[0134] The culture batch obtained by the above-mentioned method contains cysteine ​​in dissolved form in the culture supernatant or in oxidized form as precipitated cystine. The cysteine ​​or cystine contained in the culture batch can be used further as it is without further work-up or can be separated from the culture batch.

[0135] Preferably, the method is characterized in that the cysteine ​​formed is isolated. The isolation of cysteine ​​and cystine can utilize known process steps including centrifugation, decantation, dissolution of the crude product with mineral acid, filtration, extraction, chromatography or crystallization, or precipitation. Said process steps can be combined in any manner to isolate cysteine ​​at the desired purity. The desired purity depends on the subsequent use.

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

[0137] A variety of analytical methods are available for identifying, quantifying and determining the purity of the cysteine ​​or cystine product, including spectrophotometry, NMR, gas chromatography, HPLC, mass spectrometry, gravimetry or a combination of these analytical methods.

[0138] The present invention can also be used to produce improved microbial strains for the fermentative production of compounds whose biosynthesis begins with 3-phosphoglycerate, through L-serine, to L-cysteine ​​and L-cystine. This also includes microbial strains for the fermentative production of L-serine and L-cysteine ​​derivatives, including phosphoserine, O-acetylserine, N-acetylserine and thiazolidine, the condensation product of L-cysteine ​​and pyruvic acid. [Brief description of the drawings]

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

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

[0141] Example 1: Construction of a ppsA deletion mutant 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 from E. coli. The DNA sequence of the ppsA gene from E. coli K12 (Genbank Gene ID: 946209) is disclosed in SEQ ID NO: 1. Nucleotides 333 to 2711 (identified as E. coli ppsA) encode a phosphoenolpyruvate synthase protein having the amino acid sequence disclosed in SEQ ID NO: 2 (E. coli PpsA).

[0143] E. coli ppsA was inactivated using Red® / ET® from Gene Bridges GmbH as detailed below (described in the user manual of the "Quick & Easy E. coli Gene Deletion Kit" and see "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012" and references therein, 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 -3.4 kb plasmid pKD13 (Figure 1) is disclosed in the "GenBank" gene database under the accession number AY048744.1. The -6.3 kb plasmid pKD46 (Figure 2) is disclosed in the gene database of "GenBank" under the accession number AY048746.1. The -9.4 kb plasmid pCP20 is disclosed in Cherepanov and Wackernagel, Gene 158 (1995): 9-14.

[0144] To inactivate the ppsA gene of E. coli W3110 using the Lambda Red system, the following procedure was performed:

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

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

[0147] Primer pps-5f contains 30 nucleotides (nt) from the 5′ region of the ppsA gene (nt 333 to 362 in SEQ ID NO:1) joined to a 20 nt fragment specific for plasmid pKD13 (referred to as “pr-1” in FIG. 1 ).

[0148] Primer pps-6r contains 30 nt from the 3′ region of the ppsA gene (nt 2682 to 2711 of SEQ ID NO:1, reverse complement) bound to a 20 nt fragment specific to plasmid pKD13 (referred to as “pr-2” in FIG. 1 ).

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

[0150] 3. The 1.4 kb PCR product was isolated and treated with the restriction endonuclease DpnI, which is well known to those skilled in the art and which cuts only methylated DNA, to remove residual pKD13 plasmid DNA. Unmethylated DNA from the PCR reaction was not degraded.

[0151] 4. The 1.4-kb PCR product specific for 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 resulting kanamycin-resistant clones were purified on LBkan plates (i.e., isolation of clones by differentiation) and checked by PCR reaction to confirm whether the kanamycin resistance cassette had been correctly integrated 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 a kanamycin-resistant clone 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 of the E. coli W3110 wild-type strain 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 nt 167-188 from SEQ ID NO: 1, and primer pps-8r contained nt 2779-2800 from SEQ ID NO: 1 in reverse complement form.

[0154] E. coli W3110 wild-type DNA generated a 2630 bp DNA fragment in the PCR reaction, as expected for an intact gene. In contrast, the kanamycin-resistant clone under study generated a DNA fragment of approximately 1660 bp in the PCR reaction, as expected if the 1.4 kb PCR product had been integrated into the ppsA gene at the site defined by primers pps-5f and pps-6r. This result indicated that the kanamycin resistance gene had been successfully integrated into the ppsA locus, and the ppsA gene had been inactivated. A clone containing the inactivated ppsA gene was selected and designated W3110-ΔppsA::kan.

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

[0156] This step resulted in inactivation of the ppsA gene and in clones in which sensitivity to kanamycin was restored (so-called "curing" of the antibiotic selection marker). By removing the kanamycin cassette from the genome of the ΔppsA mutant, further mutations can be introduced to generate double or multiple mutants.

[0157] W3110-ΔppsA::kan regained kanamycin sensitivity after treatment with the pCP20 plasmid, as confirmed by the following: - By plating on LB and LBkan plates: Growth on LB plates was positive, whereas no growth was observed on LBkan plates, suggesting successful removal of the kanamycin cassette from the genome.

[0158] PCR reaction: For this purpose, genomic DNA was isolated from kanamycin-sensitive clones (Qiagen DNA isolation kit) and used in a PCR reaction ("Phusion™ High-Fidelity" DNA Polymerase, Thermo Scientific™) with primers pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10). E. coli W3110 wild-type DNA generated a DNA fragment of about 2630 bp in the PCR reaction, as expected for the intact gene. In contrast, kanamycin-sensitive clones gave rise to a DNA fragment of about 300 bp in the PCR reaction, corresponding to the expected size of the 5' and 3' fragments of the inactivated ppsA gene remaining after homologous recombination.

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

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

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

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

[0163] To inactivate the ppsA gene of P. ananatis using the Lambda Red system, the following procedure was performed:

[0164] 1. P. ananatis was transformed with the plasmid pRedET (the so-called "Red recombinase" plasmid) and a tetracycline resistant clone was isolated (designated P. ananatis x pRedET).

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

[0166] Primer ppsapa-3f contains 49 nt from the 5' region of the ppsA gene (nt 417 to 465 in SEQ ID NO:3) bound to a 20 nt fragment specific to plasmid pKD13 (referred to as "pr-1" in Figure 1).

[0167] Primer ppsapa-4r contains 49 nt from the 3′ region of the ppsA gene (nt 2753 to 2801 of SEQ ID NO:3, reverse complement) bound to a 20 nt fragment specific to plasmid pKD13 (referred to as “pr-2” in FIG. 1 ).

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

[0169] 3. The 1.4 kb PCR product was isolated and treated with the restriction endonuclease DpnI, which is well known to those skilled in the art and which cuts only methylated DNA, to remove residual pKD13 plasmid DNA. Unmethylated DNA from the PCR reaction was not degraded.

[0170] 4. A 1.4-kb PCR product specific for the ppsA gene and containing an expression cassette for the kanamycin resistance gene was transformed into P. ananatis × pRedET, and kanamycin-resistant clones were isolated on LBkan plates at 30°C. 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 on LBkan plates (i.e., isolation of clones by differentiation) and checked by PCR reaction to confirm whether the kanamycin resistance cassette was correctly integrated 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 a kanamycin-resistant clone 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 a P. ananatis wild-type strain 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 nt 281-302 of SEQ ID NO: 3, and primer ppsapa-2r contained nt 2901-2922 of SEQ ID NO: 3 in reverse complement form.

[0173] P. ananatis wild-type DNA generated a 2640 bp DNA fragment in the PCR reaction, as expected for an intact gene. In contrast, the kanamycin-resistant clone under study generated a 1670 bp DNA fragment in the PCR reaction, as expected for a PCR product of approximately 1.4 kb integrated 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 indicated that the kanamycin resistance gene had been successfully integrated into the ppsA locus, inactivating the ppsA gene. A clone containing the inactivated ppsA gene was selected and designated P. ananatis-ΔppsA::kan.

[0174] Example 3 Preparation of E. coli W3110-ppsA-MHI E. coli W3110-ppsA-MHI is characterized by a mutation in the ppsA structural gene that causes attenuation of the enzyme activity, and was generated using a combination of Lambda Red recombination and counterselection screening of genetic modifications known to those skilled in the art (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 specified in SEQ ID NO: 6 (PpsA-MHI).

[0175] The steps are as follows: 1. A 2.6 kb DNA fragment containing a portion of the ppsA WT gene (nt 167 to nt 2800 of SEQ ID NO: 1), i.e., the cds and 5' and 3' flanking sequences, was isolated from the genomic DNA of E. coli W3110 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 successively introducing mutations into the ppsAWT gene by “site-directed” mutagenesis, which was performed using a commercial cloning kit from Agilent, “QuickChange II Site-Directed Mutagenesis Kit,” following the instructions in the user manual.

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

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

[0179] Primer pps-9f contains 30 nt starting from the initiation ATG of the ppsA gene (nt 333 to 362 in SEQ ID NO: 1) to which is linked 20 nt specific for plasmid pKan-SacB (referred to as "pr-f" in Figure 3).

[0180] Primer pps-10r contains 30 nt from the termination codon of the ppsA gene (nt 2682 to 2711 in SEQ ID NO: 1, reverse complement) bound to 21 nt specific for plasmid pKan-SacB (referred to as “pr-r” in Figure 3 ).

[0181] 4. E. coli W3110xpKD46 (for its generation see Example 1) was transformed with the ppsA specific 3.2 kb PCR product and kanamycin resistant clones were isolated.

[0182] 5. Clones were plated 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 led to growth inhibition. Such clones were selected and checked by PCR reaction 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 a kanamycin-resistant clone 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 of the E. coli W3110 wild-type strain 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 generated a 2630 nt DNA fragment in the PCR reaction, as expected for the intact gene. In contrast, the kanamycin-resistant clone generated a ∼3400 nt DNA fragment in the PCR reaction, as expected if the 3.2 kb PCR product had been integrated 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 indicated that the Kan-sacB cassette had been successfully integrated into the ppsA locus, inactivating the ppsA gene. A clone containing the integrated Kan-sacB cassette was selected and designated 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 2.5 kb DNA fragment was amplified from the ppsA-MHI DNA fragment from step 2 in a PCR reaction ("Phusion™ High-Fidelity" DNA Polymerase, Thermo Scientific™) with primers pps-11f (SEQ ID NO: 17) and pps-12r (SEQ ID NO: 18). Primer pps-11f contained nt 300 to 319 of SEQ ID NO: 1, and primer pps-12r contained nt 2743 to 2763 of SEQ ID NO: 1 in reverse complement form.

[0186] 7. The 2.5 kb ppsA-MHI gene was transformed into E. coli W3110-ΔppsA::kan-sacB×pKD46, and clones were selected on LBS plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar) lacking kanamycin. Only clones that did not contain an active sacB gene grew on the LBS plates.

[0187] These clones were plated on LB kan plates to select clones that did not contain an active Kan gene and whose growth was inhibited in the presence of kanamycin.

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

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

[0190] Example 4: Generation of cysteine-producing strains The cysteine-specific production plasmid used was the plasmid pACYC184-cysEX-GAPDH-ORF306-serA317, derived from the parent vector pACYC184 (Figure 4). pACYC184-cysEX-GAPDH-ORF306-serA317 is a derivative of the 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 feedback inhibition by cysteine, and the efflux gene ydeD (ORF306), whose expression is controlled by the constitutive GAPDH promoter.

[0191] Furthermore, pACYC184-cysEX-GAPDH-ORF306-serA317 further contains a serA317 gene fragment cloned after the ydeD (ORF306) efflux gene and encoding the N-terminal 317 amino acids of the SerA protein (full length: 410 amino acids). The serA gene of E. coli 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 mutant of 3-phosphoglycerate dehydrogenase. Expression of serA317 is controlled by the serA promoter.

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

[0193] Transformants carrying the plasmid 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 plasmid by plasmid isolation with QIAprep Spin Plasmid Kit (Qiagen) and restriction analysis. Transformants containing correctly integrated plasmid pCYS were cultured to check ppsA enzyme activity (Example 5) and to determine cysteine ​​production (Examples 6 and 7).

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

[0195] A cell homogenizer FastPrep-24™ 5G was used. For this purpose, 2×1 ml of cell suspension was disrupted in a 1.5 ml tube previously prepared by the manufacturer and containing glass beads ("Lysing Matrix B") (3×20 s, with a shaking frequency of 6000 rpm and a 30 s pause each time between intervals). The resulting homogenate was centrifuged and the supernatant was used as cell extract for activity determination.

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

[0197] To measure ppsA enzyme activity, a phosphate detection kit "Malachite Green Phosphate Assay Kit" (catalog number MAK307) from Sigma Aldrichi was used according to the manufacturer's instructions. Its principle is the conversion of pyruvate by ATP in an equilibrium reaction (4) by ppsA enzyme activity to form phosphoenolpyruvate, which generates the stoichiometric amount of phosphate used to determine the activity. -The assay contained 100 μg of cell extract in 1 ml of assay buffer (100 mM Tris-HCl, pH 8.0; 10 mM MgCl2), 4 mM sodium pyruvate, and 4 mM ATP. - The various assays were incubated at 30°C. - After 0, 10, 20, 30 and 60 minutes of incubation, 50 μl was removed from each assay and added to 750 μl of H2O and finally mixed with 200 μl of the reagent from the "Malachite Green Phosphate Assay Kit". After 30 min of incubation, the amount of phosphate formed was measured photometrically 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 extract in U / ml (1 U = μmol substrate turnover / min) was determined from the measured amount of phosphate based on the sampling time from each assay. The specific ppsA enzyme activity was calculated based on 1 mg of total protein in the cell extract (U / mg protein).

[0198] [Table 1]

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

[0200] Main culture: A portion of each preculture was then transferred to a 300 ml Erlenmeyer flask (with baffles) containing 30 ml of SM1 medium containing 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 Nacitrate3x2H2O, 0.1 g / L NaCl; 1 ml / L trace element solution.

[0202] Composition of trace element solution: 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] The culture was grown to an initial cell density (OD) of 0.025 / ml. 600 The culture was inoculated with enough preculture to establish a 30 ml / ml (optical density of the main culture measured at 600 nm). Starting with this, the entire 30 ml batch was incubated at 30° C., 135 rpm for 24 hours.

[0204] After 24 hours, samples were taken and the cell density OD 600The total cysteine ​​content in / ml and in the culture supernatant was determined and cysteine ​​quantification was carried out by colorimetric assay according to Gaitonde (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-methylthiazolidine-2,4-dicarboxylic acid (thiazolidine), the condensation product of cysteine ​​and pyruvic acid, which is described in EP 0 885 962 B1. L-cystine, which is formed by oxidation of two cysteine ​​molecules according to formula (2), is likewise detected as cysteine ​​in the assay by reduction with dithiothreitol in a dilute solution at pH 8.0. The results are shown in Table 2 for the mentioned E. coli strains and in Table 3 for the P. ananatis strains.

[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 production-scale fed-batch fermentation.

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

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

[0210] The culture was incubated in an Erlenmeyer flask (volume 1 L) at 30° C. for 17 h with shaking at 150 rpm (Infors incubator shaker). After this incubation, the cell density OD 600 / ml ranged from 3 to 5.

[0211] Main medium: Fermentation was carried out in an Eppendorf DASGIP® Parallel Bioreactor System for Microbiology fermenter. A culture vessel with a total volume 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 Na3x2H2O citrate and 1 ml of 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 in a 25% NH4OH solution. During the fermentation, the pH was maintained at 6.5 by automatic correction with 25% NH4OH. For inoculation, 100 ml of preculture 2 was pumped into the fermentation vessel. The initial volume was therefore 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 starting conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0213] The target value of O2 saturation during fermentation was set to 30%. After O2 saturation fell below the target value, a regulating cascade was started to bring O2 saturation back to the target value. For this, first the gas supply was continuously increased (up to a maximum of 5 vvm) and then the stirring speed was continuously increased (up to a maximum of 1500 rpm).

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

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

[0216] The fermentation time was 48 h. Afterwards, samples were taken from the fermentation batches and the content of L-cysteine ​​and its derivatives was determined separately in the culture supernatant (mainly L-cysteine ​​and thiazolidine) and in the precipitate (L-cystine). For this purpose, in both cases the colorimetric assay according to Gaitonde (Gaitonde, MK (1967), Biochem. J. 104, 627-633) was performed. The L-cystine present in the precipitate had to be first dissolved in 8% (v / v) hydrochloric acid before it could be quantified by the same method. Finally, the total amount of cysteine ​​was determined as the sum of the cysteine ​​in the pellet and in the supernatant.

[0217] The cell density OD of the studied strains as summarized in Table 4 600 / ml were comparable but somewhat higher for the control strain W3110×pCYS. In contrast, cysteine ​​production (g / L) was significantly higher (approximately threefold) in both W3110-ppsA-MHI×pCYS and W3110-ΔppsA×pCYS than in the control strain W3110×pCYS containing the wild-type ppsA gene.

[0218] It has been shown that under controlled fermentation conditions attenuation or inactivation of the ppsA enzyme activity leads to a significant improvement in cysteine ​​production and is therefore a suitable tool for improving the strain at production scale, a result which has not been previously described and which is unexpected for the person skilled in the art in view of the prior art.

[0219] [Table 4]

[0220] Abbreviations used in the figure: bla: a gene that confers resistance to ampicillin (β-lactamase) rrnB term: rrnB terminator for transcription kanR: a gene conferring resistance to kanamycin ORI: origin of replication pr-1: primer binding site 1 pr-2: primer binding site 2 FRT1: FLP recombinase recognition sequence 1 FRT2: FLP recombinase recognition sequence 2 araC: araC gene (repressor gene) P araC: promoter of the araC gene P araB: promoter of the araB gene Gam: the Gam recombination gene of phage λ Bet: the Bet recombination gene of phage λ Exo: Exo recombination gene of phage λ ORI101: a temperature-sensitive replication origin RepA: gene for plasmid replication protein A sacB: levansucrase gene pr-f: primer binding site f (forward) pr-r: primer binding site r (reverse) OriC: origin of replication C IHF: Binding site for the DNA-binding protein IHF ("integration 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 fermentative production of L-cysteine, comprising: - the relative enzymatic activity of the enzyme class identified 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 - forming an increased amount of L-cysteine ​​compared to a microbial strain having a wild-type enzyme activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database, The microbial strain, wherein the gene encoding the enzymatic activity is identified by ppsA.

2. The microbial strain of claim 1 , wherein the microbial strain is an Enterobacteriaceae or Corynebacteriaceae strain.

3. 3. The microbial strain according to claim 1 or 2, wherein the microbial strain is selected from the group consisting of Escherichia coli, Pantoea ananatis and Corynebacterium glutamicum.

4. The microbial strain according to any one of claims 1 to 3, wherein the microbial strain is selected from the group consisting of Escherichia coli and Pantoea ananatis.

5. The microbial strain according to any one of claims 1 to 4, wherein the microbial strain is a strain of the Escherichia coli species.

6. The microbial strain according to any one of claims 1 to 5, comprising at least one mutation in the ppsA gene.

7. The microbial strain according to claim 6 , wherein the mutated gene is selected from the group consisting of the ppsA gene from Escherichia coli, the ppsA gene from Pantoea ananatis, and genes homologous to these genes.

8. The microbial strain according to claim 6 or 7, wherein the coding DNA sequence of the ppsA gene is SEQ ID NO:

5.

9. The microbial strain according to any one of claims 1 to 8, wherein in said strain the relative enzymatic activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database is reduced by at least 25% compared to the specific activity of the wild-type enzyme.

10. The microbial strain according to any one of claims 1 to 8, wherein in said strain the relative enzymatic activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database is reduced by at least 70% compared to the specific activity of the wild-type enzyme.

11. The microbial strain according to any one of claims 1 to 8, which does not have an enzyme activity of the enzyme class identified by the number EC 2.7.9.2 in the KEGG database.

12. A fermentation process for producing cysteine, in which a microbial strain according to any one of claims 1 to 11 is used.

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