Improved cysteine-producing strains

Abstract

The present invention relates to an improved cysteine-producing strain, in particular to a microbial strain suitable for the fermentative production of L-cysteine, characterized in that the relative enzymatic activity of the enzymes identified by the number EC 2.7. 9.2 in the KEGG database is inactivated or the specific activity is reduced based on the wild-type enzymes, and the enzyme represented by the number EC 2.7. 9.2 in the KEGG database forms an increased amount of L-cysteine as compared to a microbial strain having a wild-type enzymatic activity in which the gene encoding the enzymatic activity is represented by ppsA. The invention also provides methods of producing L-cysteine using these microbial cells.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080102380.3, filed on June 26, 2020, entitled "Improved Cysteine-Producing Strains". Technical Field

[0002] This invention relates to microbial strains suitable for the fermentation production of L-cysteine, characterized in that the relative enzyme activity of the enzyme class identified by KEGG database number EC2.7.9.2 is inactivated or reduced relative to wild-type enzyme activity, and that the strain produces an increased amount of L-cysteine ​​compared to microbial strains having wild-type enzyme activity of the enzyme class identified by KEGG database number EC2.7.9.2, wherein the gene encoding said enzyme activity is identified by ppsA. Furthermore, this invention provides a method for producing L-cysteine ​​using these microbial cells. Background Technology

[0003] Cysteine ​​(abbreviated as Cys or C) is an α-amino acid with a side chain -CH2-SH. Because the naturally occurring enantiomer is L-cysteine, and because only L-cysteine ​​is a proteogenic amino acid, in the context of this invention, the term cysteine ​​without a descriptive symbol refers to L-cysteine. Oxidation of the thiol group can lead to the formation of a disulfide bond between two cysteine ​​residues, subsequently forming cystine. The same statement applies to cystine, i.e., in the absence of a descriptive symbol, it refers to the L-enantiomer (or L-cysteine, or (R,R)-3,3′-dithiobis(2-aminopropionic acid)) in this invention. L-cysteine ​​is a semi-essential amino acid in humans because it can be formed from the amino acid methionine.

[0004] In all organisms, cysteine ​​plays a crucial role in sulfur metabolism and is used in the synthesis of proteins, glutathione, biotin, lipoic acid, thiamine, taurine, methionine, and other sulfur-containing metabolites. Furthermore, L-cysteine ​​serves as a precursor in the biosynthesis of coenzyme A.

[0005] The biosynthesis of cysteine ​​has been studied in detail in bacteria, especially in gut bacteria. An overview of cysteine ​​biosynthesis can be found in Wada and Takagi, Appl. Microbiol. Biotechnol. (2006) 73:48-54.

[0006] The amino acid L-cysteine ​​is of economic importance.

[0007] For example, it is used as a food additive (especially in the baking industry), as a raw material in cosmetics, and as a starting material for the production of active pharmaceutical ingredients (especially N-acetylcysteine ​​and S-carboxymethylcysteine).

[0008] In addition to the classic preparation method of cysteine ​​by extraction from keratin-containing materials (such as hair, mane, horns, hooves, and feathers) or biotransformation via enzymatic conversion of precursors, methods for producing cysteine ​​by fermentation also exist. Existing techniques for producing cysteine ​​using microbial fermentation are disclosed, for example, in EP 0858510B1, EP 0885962B1, EP1382684B1, EP 1220940B2, EP 1769080B1, and EP 2138585B1. The bacterial host organisms used include strains of the genus Corynebacterium and members of the family Enterobacteriaceae, such as Escherichia coli (…). Escherichia coli ) or Pantothecin pineapple ( Pantoea ananatis ).

[0009] Various methods can be used to improve cysteine ​​production in microbial strains. In addition to the classic approach of obtaining improved cysteine-producing strains through mutation and selection, specific genetic modifications of strains are also employed to achieve efficient overproduction of cysteine.

[0010] For example, the introduction of the cysE allele leads to increased cysteine ​​production. The cysE allele encodes a serine O-acetyltransferase with reduced cysteine ​​feedback inhibition (EP 0858510B1; Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611). The feedback-resistant CysE enzyme largely decouples the formation of O-acetyl-L-serine (a direct precursor of cysteine) from cellular cysteine ​​levels.

[0011] O-acetyl-L-serine is formed from L-serine and acetyl-CoA. Therefore, it is crucial to provide sufficient L-serine for cysteine ​​production. This can be achieved by introducing the serA allele, which encodes a 3-phosphoglycerate dehydrogenase with reduced L-serine feedback inhibition. Thus, the formation of the L-serine biosynthetic precursor 3-hydroxypyruvate is largely decoupled from cellular L-serine levels. Examples of such SerA enzymes are described in EP 0620853B1 and EP 1496111B1. Alternatively, Bell et al., Eur. J. Biochem. (2002) 269: 4176-4184 discloses modifications to the serA gene to deregulate enzyme activity.

[0012] In addition, it is known that cysteine ​​yield in fermentation can be increased by weakening or disrupting genes encoding cysteine-degrading enzymes such as tryptophanase TnaA or cystathionine β-lyase MalY or MetC (EP 1571223B1).

[0013] Increasing cysteine ​​transport out of cells is another way to increase product yield in culture media. This can be achieved by overexpressing the so-called efflux gene, which encodes a membrane-bound protein that mediates cysteine ​​export from cells.

[0014] Several efflux genes (EP 0885962B1, EP 1382684B1) for cysteine ​​export have been described. Exporting cysteine ​​from cells to the fermentation medium has several advantages: 1) Continuous withdrawal of L-cysteine ​​from intracellular equilibrium results in a low level of this amino acid in the cell, and thus the feedback inhibition of the sensitive enzyme by L-cysteine ​​ceases: (1) L-cysteine ​​(intracellular) <-> L-cysteine ​​(culture medium) 2) L-cysteine ​​secreted into the culture medium is oxidized in the presence of oxygen to form L-cysteine ​​disulfide, which is introduced into the culture medium during culture (EP 0885962B1): (2) 2L-cysteine ​​+ 1 / 2O2 -> L-cysteine ​​+ h2O Because L-cysteine ​​has a much lower solubility in aqueous solution at neutral pH compared to cysteine, the disulfide has precipitated at a low concentration, forming a white precipitate. (3) L-cystine (dissolved) -> L-cystine (precipitate) The precipitation of L-cystine reduces the level of the product dissolved in the medium, thereby also causing the reaction equilibrium of (1) and (2) to be directed to the product side.

[0015] 3) If the amino acid can be obtained directly from the fermentation medium, purifying the product is significantly less complicated than if the product accumulates in the cell and requires cell destruction first.

[0016] Besides genetic modification of cysteine-producing strains, optimization of the fermentation process, i.e., how to culture cells, also plays an important role in the development of efficient production methods. Various culture parameters, such as the nature and stoichiometry of carbon and energy sources, temperature, oxygen supply (EP 2707492B1), pH and composition of the culture medium, can affect the product yield and / or product profile in cysteine ​​fermentation production.

[0017] Due to rising raw material and energy costs, there is a ongoing need to increase the product yield in cysteine ​​production in order to improve the economic viability of this method. Summary of the Invention

[0018] The purpose of this invention is to provide a microbial strain for the fermentation production of cysteine, which, compared with known strains from the prior art, enables the production of higher yields of L-cysteine ​​or L-cystine during fermentation.

[0019] This objective is achieved by a microbial strain suitable for L-cysteine ​​fermentation production, characterized in that the relative enzyme activity of the enzyme class identified by number EC2.7.9.2 in the KEGG database is inactivated or the specific activity relative to the wild-type enzyme is reduced, and it produces an increased amount of L-cysteine ​​compared to a microbial strain having wild-type enzyme activity of the enzyme class identified by number EC2.7.9.2 in the KEGG database, wherein the gene encoding said enzyme activity is identified by ppsA.

[0020] In the KEGG database, the enzyme activity of enzymes identified by the number EC 2.7.9.2 is defined as their ability to reversibly produce pyruvate from phosphoenolpyruvate according to the following formula: (4) Phosphoenolpyruvate + Phosphate + AMP <-> Pyruvate + H2O + ATP (AMP: adenosine monophosphate; ATP: adenosine triphosphate) Therefore, this enzyme activity is also known as phosphoenolpyruvate synthase (PEP synthase, EC 2.7.9.2) or synonymously as pyruvate-H2O dual kinase. The gene encoding this protein is abbreviated as ppsA in the context of this invention.

[0021] Enzyme activity detection (enzyme assay, PEP synthase assay): The PEP synthase activity of microbial strains can be determined by granulating cells from the culture in a liquid medium, washing the cells, and preparing a cell extract, for example using FastPrep-24. TM 5G cell homogenizer (MP Biomedicals). The protein content of the extract can be measured, for example, via "Qubit". ® The protein assay kit (ThermoFisher Scientific) was used to determine this.

[0022] According to Equation (4), for example, using the Malachite Green Phosphate Assay Kit (Sigma Aldrich), PEP synthase activity can be measured by the stoichiometric production of phosphate from the reaction of pyruvate with ATP. Alternatively, the stoichiometric production of AMP or phosphoenolpyruvate or the stoichiometric consumption of pyruvate or ATP can also be determined (see Equation 4). For example, an assay for determining PEP synthase activity via ATP-dependent consumption of pyruvate is described in Berman and Cohn, J. Biol. Chem. (1970) 245: 5309-5318. Berman and Cohn, J. Biol. Chem. (1970) 245: 5309-5318 also describes an assay for the ATP-dependent formation of phosphoenolpyruvate.

[0023] Specific enzyme activity was calculated based on 1 mg of total protein from the cell extract (without any further purification or treatment) (U / mg protein). It should be noted that comparisons of different PEP synthases require that the cell extract be prepared in the same manner. As already described, the cell extract can be prepared, for example, using FastPrep-24. TM The cells were prepared using a 5G cell homogenizer (MPBiomedicals).

[0024] Alternatively, different enzymes can be compared by making their specific activities based on 1 mg of the enzyme (U / mg of purified protein) purified in the same manner. A method for purifying PEP synthase and for determining the specific activity of purified protein is described, for example, in Berman and Cohn, J. Biol. Chem. (1970) 245: 5309-5318.

[0025] Relative enzyme activity can be determined by setting the specific enzyme activity (as determined in a PEP synthase assay of a microbial strain carrying the Wt allele relative to the gene encoding the PEP synthase) to 100%. The specific enzyme activity measured for a sample in a PEP synthase assay is labeled as a percentage relative to such a strain possessing the Wt enzyme.

[0026] An open reading frame (ORF, synonym of cds or coding sequence) is a region of DNA or RNA that begins with a start codon and ends with a stop codon and encodes the amino acid sequence of a protein. ORFs are also called coding regions or structural genes.

[0027] A gene is a portion of DNA containing all the essential information for producing biologically active RNA. A gene contains the DNA portion that produces single-stranded RNA copies through transcription, as well as expression signals involved in regulating this copying process. Expression signals include, for example, at least one promoter, transcription initiation, translation initiation, and ribosome binding site. Additionally, terminators and one or more operons may serve as expression signals.

[0028] In the context of this invention, proteins, such as PpsA, begin with an uppercase letter, while sequences encoding said proteins (cds) are identified by lowercase letters (e.g., ppsA).

[0029] Therefore, *E. coli* ppsA refers to the cds of the ppsA gene from *E. coli*, as identified by nucleotides 333-2711 in SEQ ID NO: 1. *E. coli* PpsA refers to the protein encoded by the aforementioned cds (*E. coli* ppsA*) and identified in SEQ ID NO: 2. This protein is phosphoenolpyruvate synthase.

[0030] Pantotheca ananatis ppsA refers to the cds of the ppsA gene from Pantotheca ananatis, as identified by nucleotides 417-2801 in SEQ ID NO: 3. Pantotheca ananatis PpsA refers to the protein encoded by the cds (Pantotheca ananatis ppsA) and identified in SEQ ID NO: 4.

[0031] The abbreviation WT (Wt) refers to the wild type. Wild-type genes are those that are naturally occurring through evolution and exist in the wild-type genome. The DNA sequence of the Wt gene is publicly available in databases such as NCBI.

[0032] Alleles define the state of genes that can be transformed into each other through mutation (i.e., by altering the nucleotide sequence of DNA). Genes that are naturally present in microorganisms are called wild-type alleles, and variants derived from them are called mutant alleles of the gene.

[0033] Homologous genes or homologous sequences should be understood to mean that the DNA sequence of the gene or DNA segment is at least 80% identical, preferably at least 90% identical, and particularly preferably at least 95% identical.

[0034] DNA identity is determined using the "Nucleotide Blast" procedure, which can be found at http: / / blast.ncbi.nlm.nih.gov / and is based on the blastn algorithm. Default parameters are used as algorithm parameters for aligning two or more nucleotide sequences. The default general parameters are: Maximum Target Sequence = 100; Short Queries = "Auto-adjust parameters for short input sequences"; Expected Threshold = 10; Word Size = 28; Auto-adjust Short Input Sequence Parameters = 0. The corresponding default scoring parameters are: Match / Mismatch Score = 1, -2; Gap Costs = Linear.

[0035] Use the "Protein Blast" program at http: / / blast.ncbi.nlm.nih.gov / to compare protein sequences. This program uses the BLASTP algorithm. Default parameters are used as algorithm parameters for aligning two or more protein sequences. The default general parameters are: Maximum Target Sequence = 100; Short Query = "Auto-adjust parameters for short input sequences"; Expected Threshold = 10; Word Size = 3; Auto-adjust parameters for short input sequences = 0. Default scoring parameters are: Matrix = BLOSUM62; Gap Cost = Existence: 11; Extension: 1; Composition Adjustment = Conditional Composition Score Matrix Adjustment.

[0036] In the microorganisms according to the invention, the relative enzyme activity of the enzymes identified by the number EC 2.7.9.2 in the KEGG database is inactivated or reduced by at least 10%, particularly preferably at least 25%, especially preferably at least 60%, and particularly preferably at least 70% relative to the wild-type enzyme. The enzyme activity 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%).

[0037] In a preferred embodiment, the microbial strain is characterized in that it no longer has any enzyme activity of the enzyme class identified by EC2.7.9.2 in the KEGG database, that is, the relative enzyme activity of the enzyme class identified by EC2.7.9.2 in the KEGG database is reduced by 100% relative to the specific activity of the wild-type enzyme.

[0038] In the context of this invention, "compared to (corresponding) wild-type enzymes / compared to (corresponding) wild-type enzymes / related to (corresponding) wild-type enzymes" means compared to the activity of a protein encoded by a non-mutant form of a gene from a microorganism (i.e., a gene that is naturally produced through evolution and is present in the wild-type genome of the microorganism).

[0039] Microbial strains suitable for the fermentation production of L-cysteine ​​include all microorganisms containing dysregulated biosynthetic metabolic pathways (homologous or heterologous) that result in the synthesis of cysteine, cystine, or derivatives thereof. Such strains are disclosed, for example, in EP 0885962B1, EP 1382684B1, EP 1220940B2, EP 1769080B1, and EP 2138585B1.

[0040] Microorganisms suitable for L-cysteine ​​fermentation production are preferably characterized by having one of the following modifications: a) The distinguishing feature of the microbial strain is the modified 3-phosphoglycerate dehydrogenase (serA), which has L-serine feedback inhibition that is reduced by at least two times compared to the corresponding wild-type enzyme (as described in EP 1950287B1).

[0041] Compared to the corresponding wild-type enzyme, the L-serine feedback inhibition of a particularly preferred variant of 3-phosphoglycerate dehydrogenase (serA) is reduced by at least 5-fold, particularly preferably by at least 10-fold, and in a more preferred embodiment by at least 50-fold.

[0042] b) The microbial strain contains serine O-acetyltransferase (cysE) which, compared to the corresponding wild-type enzyme, has cysteine ​​feedback inhibition that is reduced by at least 2-fold (e.g., EP 0858510B1 or Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611).

[0043] Compared to the corresponding wild-type enzyme, a particularly preferred variant of serine O-acetyltransferase (cysE) has cysteine ​​feedback inhibition that is reduced by at least 5-fold, particularly preferably by at least 10-fold, and in a more preferred embodiment by at least 50-fold.

[0044] c) Compared to the corresponding wild-type cells, the microbial strain exhibited cysteine ​​efflux from the cells, with the cysteine ​​content increasing by at least 2-fold due to the overexpression of efflux genes.

[0045] Compared to wild-type cells, overexpression of efflux genes preferably leads to an increase of at least 5-fold, particularly preferably at least 10-fold, and especially preferably at least 20-fold increase in cysteine ​​efflux from the cell.

[0046] The efflux gene preferably originates from the group consisting of: ydeD (see EP0885962B1), yfiK (see EP 1382684B1), cydDC (see WO 2004 / 113373A1), bcr (see US2005-221453AA), and emrAB (see US 2005-221453AA) from *E. coli*, or corresponding homologous genes from different microorganisms.

[0047] Such strains are known, for example, EP 0858510B1 and EP 0885962B1.

[0048] Furthermore, the microbial strains suitable for the fermentation production of L-cysteine ​​are preferably characterized in that at least one cysteine-degrading enzyme is reduced to a level where the cell contains only a maximum of 50% of the activity of this enzyme compared to wild-type cells. The cysteine-degrading enzyme is preferably derived from the group consisting of tryptophanase (TnaA) and cystathionine β-lyase (MalY, MetC).

[0049] The microbial strains suitable for fermenting L-cysteine ​​production described in the preceding paragraph are dysregulated in terms of their cysteine ​​content, resulting in an increased amount of L-cysteine ​​compared to microbial strains that are not dysregulated in cysteine ​​metabolism and possess wild-type enzyme activity of the enzyme class identified by KEGG database number EC2.7.9.2. This is because, in cells of microbial strains with undysregulated cysteine ​​metabolism and 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 (cf. Table 2). An increased amount refers to any amount exceeding 0.05 g / L L-cysteine ​​measured in the culture after 24 hours of culture.

[0050] Preferably, the microbial strain is characterized in that it is a strain from the Enterobacteriaceae or Corynebacteriaceae families, particularly preferably a strain from the Enterobacteriaceae family. Such strains are commercially available, for example, from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig).

[0051] Preferably, the microbial strain is selected from the group consisting of: *Escherichia coli*, *Panthera pineapple*, and *Corynebacterium glutamicum*, and particularly preferably from the group consisting of: *Escherichia coli* and *Panthera pineapple*. Particularly preferably, the microbial strain is a strain of the *Escherichia coli* species.

[0052] Particularly preferred are the *Escherichia coli* strains selected from *Escherichia coli* K12, and more particularly preferred are *Escherichia coli* K12 W3110. These strains are commercially available from DSMZ-German Collection of Microorganisms and CellCultures GmbH (Braunschweig), including, for example, *Escherichia coli* K12 W3110 DSM 5911 (id. ATCC 27325) and *Panthera pineapple* DSM 30070 (id. atcc 11530). The PpsA is preferably a PpsA from *Escherichia coli* with SEQ ID NO: 2 or a PpsA from *Panthera pineapple* with SEQ ID NO: 4.

[0053] Preferably, the microbial strain is characterized by containing at least one mutation in the ppsA gene. Additionally, in this preferred embodiment, the strain also produces an increased amount of L-cysteine ​​compared to wild-type cells. Preferably, the genetic modification in the ppsA gene causes the protein expressed by the gene to have a reduced relative enzyme activity, or no activity, compared to the specific activity of the wild-type enzyme, for an enzyme class identified by the KEGG database number EC 2.7.9.2.

[0054] Furthermore, the production strain according to the present invention can be further optimized to further improve cysteine ​​production.

[0055] For example, optimization can be genetically achieved by additionally expressing one or more genes suitable for improving production characteristics. These genes can be expressed in the production strain as individual gene constructs or in combination as expression units (so-called operons) in a manner known per se.

[0056] In addition, the production strain can be optimized by inactivating genes other than ppsA, whose gene products have an adverse effect on cysteine ​​production.

[0057] However, optimization is also possible in ways known per se, through mutagenesis and selection of strains with improved cysteine ​​production.

[0058] In the context of this invention, genetic modifications in the ppsA gene are defined as meaning...

[0059] a) The coding sequence of the ppsA gene is partially or completely deleted. b) The coding sequence of the ppsA gene is modified by one or more insertions or 5' and / or 3' extensions. c) The ppsA structural gene contains one or more mutations, especially point mutations, which result in reduced or complete inactivity of the expressed phosphoenolpyruvate synthase. d) The ppsA structural gene contains one or more mutations, especially point mutations, that lead to strong attenuation or complete repression of ppsA expression or reduced mRNA stability, or e) Attenuation or complete suppression of ppsA gene expression or ppsA mRNA translation due to genetic modification of the 5' and / or 3' non-coding ppsA sequences (promoter, 5'-UTR, Shine-Dalgarno sequence, and / or terminator). Furthermore, the protein expressed by this sequence exhibits reduced relative PEP synthase activity compared to the wild-type enzyme.

[0060] In the context of this invention, any combination of genetic modifications in the ppsA gene listed in a) through e) is also possible. In summary, therefore, in the context of this invention, it is possible to form less PpsA protein or no protein at all and / or to express a less active or inactive PpsA protein.

[0061] Alternative methods include attenuating or completely inactivating ppsA enzyme activity at the gene transcription level using a so-called "antisense RNA" strategy known to those skilled in the art. It is also conceivable to reduce or completely inactivate ppsA enzyme activity by adding inhibitors (which are chemical or protein inhibitors).

[0062] Preferably, modifying the ppsA gene in the strain according to the invention involves completely or partially deleting the ppsA structural gene, mutating the ppsA structural gene in a manner that causes reduced enzyme activity or enzyme inactivation, or mutating the ppsA structural gene and / or its untranscribed or untranslated gene regions in a manner that causes reduced or complete inhibition of ppsA gene expression or translation or additionally reduces the stability of ppsA mRNA, these gene regions regulating expression and located on the 5' and 3' flanking positions.

[0063] Particularly preferably, the inactivation of the ppsA gene in the strain according to the invention is caused by the complete or partial deletion of ppsA cds (i.e., in the case of nucleotides 333–2711 of ppsA cds in Escherichia coli, SEQ ID NO: 1, or in the case of nucleotides 417–2801 of ppsA cds in Pantotheca pineapple, SEQ ID NO: 3) or by mutation of the ppsA structural gene in a manner that causes reduced enzyme activity or enzyme inactivation or decreased mRNA stability.

[0064] In a preferred embodiment, the microbial strain is characterized in that the mutated gene is selected from the group consisting of: the ppsA gene from *Escherichia coli*, the ppsA gene from *Panthera pineapple*, and genes homologous to these genes. The ppsA gene from *E. coli* is disclosed in an entry in the NCBI gene database with gene ID 946209, and the ppsA gene from *Panthera pineapple* is disclosed in an entry in the NCBI gene database with gene ID 11796889. The above-given definition applies to the term "homologous gene". Particularly preferred is the mutated ppsA gene from *E. coli*. In another preferred embodiment, cds is the cds of the ppsA gene from *E. coli*, disclosed in SEQ ID NO: 1 nucleotides 333-2711 (encoding the protein having SEQ ID NO: 2) or the cds of the ppsA gene from *Panthera pineapple*, disclosed in SEQ ID NO: 3 nucleotides 417-2801 (encoding the protein having SEQ ID NO: 4).

[0065] In a preferred embodiment, the microbial strain is characterized in that the DNA sequence encoding the ppsA gene is SEQ ID NO: 5 or a sequence homologous to it, particularly preferably SEQ ID NO: 5. The above-defined term "homologous sequence" applies.

[0066] In this case, based on the ppsA-MHI gene having the DNA sequence in SEQ ID NO: 5, a mutation in the DNA sequence identified in SEQ ID NO: 1 results in a three-amino acid mutation in the WT protein sequence identified in SEQ ID NO: 2, namely, 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), encoding a ppsA-MHI protein having the amino acid sequence disclosed in SEQ ID NO: 6.

[0067] Different methods for inactivating and mutating the ppsA gene are known to those skilled in the art. In the simplest case, the parental strain can be mutagenized in a known manner (e.g., by chemical methods using mutagenic chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine or by physical methods using UV radiation), wherein mutations are randomly generated in the genomic DNA, and the desired ppsA mutant is then selected from the resulting plurality of mutants, for example, after these mutants have been individually isolated, by the absence of a color reaction based on enzyme activity or by genetic methods by detecting a defective ppsA gene.

[0068] In contrast to complex random mutagenesis and the selection of sought ppsA mutants, the ppsA gene can be targeted for inactivation in a relatively simple manner, for example, through known homologous recombination mechanisms. Cloning systems for targeted gene inactivation via homologous recombination are known to those skilled in the art and are commercially available, for example in "Quick and Easy..." E. coli The user manual for the "Gene Deletion Kit" discloses that it is based on Red from Gene Bridges GmbH. ® / 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 the references cited therein).

[0069] According to existing technology, the ppsA gene or a portion thereof can be isolated, and exogenous DNA can be cloned into the ppsA gene, thereby disrupting the protein-defined open reading frame of the ppsA gene. Therefore, a DNA construct suitable for targeted inactivation of the ppsA gene can consist of a 5' portion of DNA homologous to the genomic ppsA gene, followed by a gene segment containing exogenous DNA, and then a 3' portion of DNA that is again homologous to the genomic ppsA gene.

[0070] Therefore, the possible regions in the ppsA gene used for homologous recombination may include not only the region encoding phosphoenolpyruvate synthase. These possible regions may also contain DNA sequences side-attached to the ppsA gene, specifically in the 5' region before the start of the coding region (gene transcription promoter, e.g., nucleotides 1-332 in SEQ ID NO: 1, or nucleotides 1-416 in SEQ ID NO: 3) and the 3' region after the end of the coding region (gene transcription terminator, e.g., nucleotides 2712-3000 in SEQ ID NO: 1, or nucleotides 2802-3062 in SEQ ID NO: 3), which, when modified by homologous recombination, can lead to the inactivation of the ppsA gene, just as modifications to the coding region would.

[0071] The exogenous DNA is preferably a selectable marker expression cassette. It consists of a gene transcription promoter functionally linked to the actual selectable marker gene, optionally followed by a gene transcription terminator. In this case, the selectable marker also contains 5' and 3' flanking homologous sequences of the ppsA gene.

[0072] Preferably, the markers are selected from the 5' and 3' flanking homologous sequences containing the ppsA gene, each having a length of at least 30 nucleotides, and particularly preferably at least 50 nucleotides.

[0073] Therefore, the DNA construct for inactivating the ppsA gene can start from the 5' end and consist of the following: a sequence homologous to the ppsA gene, followed by an expression cassette of a selection marker, for example, selected from the class of antibiotic resistance genes, and then another sequence homologous to the ppsA gene.

[0074] In a preferred embodiment, the DNA construct for inactivating the ppsA gene consists of, 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 of a selectable marker selected from antibiotic resistance gene classes, and subsequently, another sequence homologous to the ppsA gene of at least 30 nucleotides in length, particularly preferably at least 50 nucleotides in length.

[0075] Selective marker genes are typically genes whose gene products enable the parent strain to grow under selective conditions where the original parent strain cannot grow.

[0076] Preferred selectable marker genes are selected from the group consisting of antibiotic resistance genes, such as ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, chloramphenicol resistance genes, or neomycin resistance genes. Other preferred selectable marker genes allow parental strains with metabolic defects (e.g., amino acid auxotrophs) to grow under selective conditions by correcting these defects through expression of the selectable marker gene. Finally, another possibility is a selectable marker gene whose gene product chemically alters and thus inactivates the inherently toxic compounds of the parental strain (e.g., acetamase genes, which cleave acetamide, a compound toxic to many microorganisms, into non-toxic products acetic acid and ammonia).

[0077] 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 especially preferred.

[0078] There are also systems based on homologous recombination that, in addition to targeting gene inactivation, offer the option to remove selectable markers from the genome, thereby enabling the generation of dual and multiple mutants. One such system is the so-called "LambdaRed" technology, touted as "Quick and Easy." E. coli The "Gene Deletion Kit" is commercially available and is based on Red from Gene Bridges GmbH. ® / 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 the references cited therein.

[0079] Examples of strains according to the invention with inactivated ppsA genes are *Escherichia coli* W3110-ppsA and *Panthera pineapple* ppsA disclosed in the embodiments. Both strains are characterized in that their ppsA genes have been inactivated through homologous recombination.

[0080] Another such system for targeted gene inactivation based on homologous recombination is a method of gene inactivation or genetic modification known to those skilled in the art and described in Example 3, and is based on a combination of Lambda Red recombination and anti-selection. This system is described, for example, in Sun et al., Appl. Env. Microbiol. (2008) 74: 4241-4245. A DNA construct is used to inactivate, for example, the ppsA gene, starting from the 5' end, consisting of sequences homologous to the ppsA gene, followed by two expression cassettes in any order, each consisting of: a) an expression cassette for a selection marker selected from the class of antibiotic resistance genes and b) an expression cassette for the sacB gene encoding fructan sucrase, and finally, another sequence homologous to the ppsA gene.

[0081] In the first step, the DNA construct is transformed into the production strain, and antibiotic-resistant clones are isolated. The obtained clones are characterized by the fact that they cannot grow on sucrose due to the co-binding sacB gene. These two marker genes can be removed by the principle of anti-selection, as in the second step, a suitable DNA fragment replaces these two marker genes through homologous recombination. The clones obtained in this step then regain their ability to grow on sucrose, and then also their sensitivity to antibiotics. This method was used in Example 3 with the ppsA WT gene exchanged for E. For the triple mutant ppsA-MHI (SEQ ID NO: 5) of *E. coli* (SEQ ID NO: 1) described below.

[0082] The examples disclose an *E. coli* strain W3110-ppsA-MHI, which exhibits weakened PpsA enzyme activity due to a mutation in the coding sequence of the ppsA gene. W3110-ppsA-MHI contains the cds of a PpsA triple mutant, PpsA-V126M-R427H-V434I (ppsA-MHI). The cds of the mutant gene in ppsA-MHI correspond to the DNA sequence SEQ ID NO: 5 and encode a PpsA protein having the sequence SEQ ID NO: 6. PpsA-MHI is characterized in that, compared to the WT sequence identified in SEQ ID NO: 2, the protein having the sequence SEQ ID NO: 6 contains the following changes in its amino acid sequence: 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).

[0083] Due to these mutations, the PpsA-MHI protein has only 26.8% of the relative enzyme activity compared to the specific wild-type enzyme activity (see Example 5, Table 1).

[0084] In the case of the Escherichia coli ppsA gene, preferably, at least one mutation in cds results in at least one of the following changes in the amino acid sequence of SEQ ID NO: 2: valine at position 126, arginine at position 427 and / or valine at position 434, wherein any one of these three amino acids may be exchanged for any other amino acid.

[0085] A particularly preferred mutation is one that causes simultaneous mutations in three amino acids in the amino acid sequence of the WT protein identified in SEQ ID NO: 2.

[0086] The mutation in the ppsA-MHI gene according to the invention can be introduced into the ppsA WT gene in a manner known per se, for example, by so-called “site-directed” mutagenesis using a commercially available cloning kit, as disclosed, for example, in the user manual for the “QuickChange II Site-Directed Mutagenesis Kit” from Agilent. Alternatively, the ppsA-MHI gene according to the invention can also be generated by DNA synthesis in a known manner.

[0087] According to the strains of the present invention, the ppsA structural gene is characterized by mutation in a manner that weakens enzyme activity. For example, the Escherichia coli ppsA-MHI triple mutant can be generated by the above-described combination of Lambda Red recombination and anti-selective screening of genetic modifications (see, for example, Sun). et al.(Appl. Env. Microbiol. (2008) 74: 4241-4245), as disclosed in the embodiments.

[0088] Particularly preferred strains are Escherichia coli W3110 ΔppsA (described in Example 1) and Escherichia coli W3110ppsA-MHI (described in Example 3).

[0089] The present invention further provides a fermentation method for producing L-cysteine, characterized by the use of microbial cells according to the present invention.

[0090] The main product of the method of this invention is L-cysteine, from which compounds L-cysteine ​​and thiazolidinediones can be formed. L-cysteine ​​and thiazolidinediones are formed during fermentation and accumulate in the culture supernatant and precipitate. Thiazolidine is a 2-methyl-2,4-thiazolidinedionic acid, which can be formed as a byproduct of cysteine ​​production as an adduct of cysteine ​​and pyruvate (EP 0885962B1).

[0091] In the context of this invention, the yield of total cysteine ​​is defined as the sum of cysteine, cystine, and thiazolidinedone produced. As described in Example 7, this is determined from the whole culture. For example, it can be quantified by colorimetric determination using Gaitonde (Gaitonde, MK (1967) Biochem. J. 104, 627-633).

[0092] No existing technology discloses any method or production strain that can improve the production of amino acids, particularly cysteine, by weakening or inactivating phosphoenolpyruvate synthase activity.

[0093] As illustrated in the embodiments of this application, weakening or inactivating ppsA enzyme activity in microbial strains suitable for cysteine ​​production significantly increased the yield of total cysteine ​​(i.e., the sum of cysteine, cystine, and thiazolidinediones produced) during fermentation. This is entirely unexpected from the prior art.

[0094] As outlined in Table 4 of Example 7, surprisingly, a significantly higher cysteine ​​yield was achieved in the fermentation of the ppsA mutant of *E. coli* W3110 compared to the corresponding wild-type strain. Contrary to the prior art and unexpectedly to those skilled in the art, the reduction or inactivation of phosphoenolpyruvate synthase activity resulted in an improved cysteine-producing strain.

[0095] The results summarized in Tables 2 and 3 of Example 6 confirm this novel and inventive method for improving cysteine-producing strains, wherein in Escherichia coli, the ppsA gene or a mutated ppsA gene is inactivated to produce a PpsA enzyme with reduced enzyme activity, and in Pantothenica pineapple has resulted in increased cysteine ​​yield in shake-flask culture.

[0096] Therefore, for those skilled in the art, the reduction or inactivation of phosphoenolpyruvate synthase activity is also a new and useful measure for improving cysteine ​​production in other cysteine-producing strains.

[0097] Therefore, in the microbial strains according to the invention and suitable for cysteine ​​production, the enzymatic activity of the protein encoded by the ppsA gene in the producing strain is weakened or completely inhibited, and simultaneously, cysteine ​​production increases. Example 7 shows that, compared with strains containing the ppsA WT gene, strains that can produce cysteine ​​and encode the ppsA mutant ppsA-MHI (instead of the Wt enzyme) with reduced PpsA enzyme activity achieve significantly higher cysteine ​​yields during fermentation.

[0098] In the fermentation method discussed, not only the biomass of the production strain according to the invention is formed, but also cysteine ​​and its oxidation product cystine. The formation of biomass and cysteine ​​can be time-dependent, or the biomass and cysteine ​​can form in a manner that separates them from each other over time. Cultivation is carried out in a manner well known to those skilled in the art. For this purpose, cultivation can be carried out in shake flasks (laboratory scale) or alternatively by fermentation (production scale).

[0099] The preferred fermentation production scale method is one with a fermentation volume greater than 1L, the preferred production scale is greater than 10L, the preferred production scale is greater than 1000L, and the preferred fermentation volume is greater than 10000L.

[0100] Culture media are familiar to those skilled in the art from the practice of microbial culture. They typically consist of carbon sources, nitrogen sources, and additives such as vitamins, salts, and trace elements, as well as sulfur sources to optimize cell growth and cysteine ​​production.

[0101] Carbon sources are those that the producing strains can use to form cysteine ​​products. These 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.

[0102] However, the production method according to the invention also covers all carbon sources in the form of disaccharides, particularly sucrose, lactose, maltose or cellobiose.

[0103] Furthermore, the production method according to the invention also includes all carbon sources in the form of higher sugars, glycosides, or carbohydrates having more than two sugar units, such as maltodextrin, starch, cellulose, hemicellulose, pectin, or monomers or oligomers (enzymatically or chemically) released therefrom by hydrolysis. Hydrolysis of the higher carbon sources can occur upstream of the production method according to the invention, or in situ during the production method according to the invention.

[0104] Other available carbon sources besides sugars or carbohydrates include acetic acid (or its acetates), 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 conceivable.

[0105] Carbon sources associated with the production method according to the invention include isolated pure substances or mixtures of individual carbon sources that have not been further purified for economic efficiency, such as those obtained by chemically or enzymatically digesting plant materials as hydrolysates. These include, for example, hydrolysates of starch (glucose monosaccharides), beet (glucose, fructose, and arabinose monosaccharides), sugarcane (sucrose disaccharides), pectin (galacturonic acid monosaccharides), or lignocellulose (glucose monosaccharides from cellulose, xylose, arabinose, and mannose, and galactose monosaccharides from hemicellulose, as well as non-carbohydrate lignin). Furthermore, waste products from the digestion of plant materials can also be used as carbon sources, such as molasses (beet) or bagasse (sugarcane).

[0106] Preferred carbon sources for culturing production strains are glucose, fructose, sucrose, mannose, xylose, arabinose, and plant hydrolysates that can be obtained from starch, lignocellulose, sugarcane, or sugar beets.

[0107] The preferred carbon source is glucose and sucrose in isolated form or as a component of plant hydrolysis products.

[0108] The preferred carbon source is glucose.

[0109] Nitrogen sources are those that can be used by the producing strains to form biomass. These include ammonia, or its salts, in gaseous or aqueous NH4OH form, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, or ammonium nitrate. Additionally, 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 extracts, peptones, malt extracts, soybean peptones, casein amino acids, corn steep liquor (liquid or dried as so-called CSD), and NZ amines and yeast nitrogen bases.

[0110] To efficiently produce cysteine ​​and cysteine ​​derivatives, a sulfur source needs to be added in a metered manner, either as a batch addition or as a continuous feed. Continuous metering can be done as a pure feed solution or as a mixture with other feed components, such as glucose.

[0111] Suitable sulfur sources are salts of sulfates, sulfites, dithionites, thiosulfates, or sulfides, and the corresponding acids can also be used under given stability conditions.

[0112] Preferred sulfur sources are salts of sulfates, sulfites, thiosulfates, and sulfides.

[0113] The preferred sulfur sources are sulfates and thiosulfates.

[0114] Thiosulfates, such as sodium thiosulfate and ammonium thiosulfate, are particularly preferred.

[0115] Culture can be carried out in a so-called batch mode, which involves inoculating the culture medium with a starter culture of the production strain and then growing the cells without further supplying nutrient sources.

[0116] Culture can also be carried out in a so-called fed-batch mode, which involves supplementing the diet with additional nutrient sources (feeds) after the initial stage of growth in the batch mode to compensate for its consumption. 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 thereof. These feed components can be metered together as a mixture or metered separately in separate feeding stages. Furthermore, other culture medium components can be added to the feed, as well as additives that specifically increase cysteine ​​production. Feed can be provided continuously or partially (discontinuously), or a combination of continuous and discontinuous feeding. Fertilized batch culture is preferred.

[0117] Preferred carbon sources in the feed are glucose, sucrose, and plant hydrolysates containing glucose or sucrose, as well as mixtures of preferred carbon sources in any mixing ratio.

[0118] Glucose is the preferred carbon source in the feed.

[0119] Preferably, the carbon source of the culture is added in metered amounts so that the carbon source content in the fermenter does not exceed 10 g / L during the production stage. A maximum concentration of 2 g / L is preferred, 0.5 g / L is particularly preferred, and 0.1 g / L is especially preferred.

[0120] The preferred nitrogen source in the feed is ammonia, either in gaseous form or in an aqueous solution of NH4OH, and its salts ammonium sulfate, ammonium phosphate, ammonium acetate and ammonium chloride, as well as urea, KNO3, NaNO3 and ammonium nitrate, yeast extract, peptone, malt extract, soybean peptone, casein amino acids, corn extract, and NZ amines and yeast nitrogen bases.

[0121] The preferred nitrogen sources in feed are ammonia or ammonium salts, urea, yeast extract, soybean peptone, malt extract or corn steep liquor (in liquid or dry form).

[0122] The preferred sulfur sources in the feed are sulfates, sulfites, thiosulfates, and sulfide salts.

[0123] Sulfates and thiosulfates are particularly preferred sulfur sources in the feed.

[0124] Thiosulfates, such as sodium thiosulfate and ammonium thiosulfate, are particularly preferred as sulfur sources in feed.

[0125] As additional medium additives, salts of elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, and iron, as well as salts of elements molybdenum, boron, cobalt, manganese, zinc, copper, and nickel, can be added in trace amounts (i.e., at µM concentration). Furthermore, organic acids (e.g., acetates, citrates), amino acids (e.g., isoleucine), and vitamins (e.g., vitamin B1, vitamin B6) can be added to the culture medium.

[0126] Culture is carried out under pH and temperature conditions that promote the growth of the producing strain and cysteine ​​production. A useful pH range is from pH 5 to pH 9. A preferred pH range is from pH 5.5 to pH 8. Particularly preferred is a pH range from pH 6.0 to pH 7.5.

[0127] The preferred temperature range for the growth of the production strain is 20°C to 40°C. A particularly preferred temperature range is 25°C to 37°C, and even more preferred is 28°C to 34°C.

[0128] The growth of the production strain can occur optionally under oxygen-free conditions (anaerobic culture) or alternatively under oxygen-supply conditions (aerobic culture). Preferably, aerobic culture is performed with oxygen.

[0129] When aerobic culture of the strain according to the invention is used for cysteine ​​production, an oxygen saturation of at least 10% (v / v), preferably at least 20% (v / v), and particularly preferably at least 30% (v / v) is set. According to the prior art, the oxygen saturation in the culture is automatically regulated by a combination of gas supply and stirring speed.

[0130] Oxygen supply is ensured by introducing compressed air or pure oxygen. Aerobic culture with compressed air is preferred. A useful range for compressed air supply in aerobic culture is 0.05 vvm to 10 vvm (vvm: the amount of compressed air introduced into a fermentation batch, indicated by liters of compressed air per liter of fermentation volume per minute). It is preferred to introduce compressed air at 0.2 vvm to 8 vvm, particularly preferred at 0.4 to 6 vvm, and especially preferred at 0.8 to 5 vvm.

[0131] The maximum stirring speed is 2500 rpm, preferably 2000 rpm, and especially preferably 1800 rpm.

[0132] The incubation time is from 10 hours to 200 hours. A preferred incubation time is 20 hours to 120 hours. A particularly preferred incubation time is 30 hours to 100 hours.

[0133] The culture batches obtained by the above method contain cysteine, which is oxidized to cystine in the culture supernatant in either dissolved or precipitated form. The cysteine ​​or cystine contained in the culture batch can be used directly without further post-processing or separated separately from the culture batch.

[0134] Preferably, the method is characterized in that the cysteine ​​formed is isolated. Known method steps per se can be used to separate cysteine ​​and cystine, including centrifugation, decantation, dissolution of the crude product with an inorganic acid, filtration, extraction, chromatography or crystallization, or precipitation. These process steps can be combined in any form to separate cysteine ​​to a desired purity. The desired purity depends on the further application.

[0135] The cystine obtained after post-processing can be reduced to cysteine ​​for further use. A method for reducing L-cystine to L-cysteine ​​in an electrochemical manner is disclosed in EP 0235908.

[0136] Various analytical methods are available for identifying, quantifying, and determining the purity of cysteine ​​or cystine products, including spectrophotometry, NMR, gas chromatography, HPLC, mass spectrometry, gravimetric analysis, or combinations of these methods.

[0137] This invention can also be used to produce improved microbial strains for the fermentation production of compounds whose biosynthesis begins with 3-phosphoglyceric acid and proceeds via L-serine to L-cysteine ​​and L-cysteine. This invention also includes microbial strains for the fermentation production of L-serine and L-cysteine ​​derivatives, including condensation products of phosphoserine, O-acetylserine, N-acetylserine, and thiazolidinyl, L-cysteine, and pyruvate. Attached Figure Description

[0138] The accompanying figure shows the plasmids used in the examples.

[0139] Figure 1 The 3.4kb carrier pKD13 used in Examples 1 and 2 is shown.

[0140] Figure 2 The 6.3kb vector pKD46 used in Examples 1 and 3 is shown.

[0141] Figure 3 The 5kb vector pKa-SacB used in Example 3 is shown.

[0142] Figure 4 The 4.2kb carrier pACYC184 used in Example 4 is shown. Detailed Implementation

[0143] The present invention will be further illustrated by the following embodiments, but is not limited thereto: Example 1: Production of ppsA deletion mutant in E. coli The parental strain used for gene isolation and strain development was Escherichia coli K12 W3110 (commercially purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM5911).

[0144] The target of gene inactivation is the coding sequence of the ppsA gene from *E. coli*. The DNA sequence of the ppsA gene from *E. coli* K12 (Genbank GeneID: 946209) is disclosed in SEQ ID NO: 1. Nucleotides 333–2711 (identified by *E. coli* ppsA) encode a phosphoenolpyruvate synthase protein having the amino acid sequence disclosed in SEQ ID NO: 2 (*E. coli* PpsA).

[0145] Using Red from Gene Bridges GmbH ® / ET ® The technology inactivates the ppsA gene in E. coli, as detailed below (described in "Quick and Easy"). E. coli See the "Technical Protocol, Quick & Easy" section in the user manual for the "Gene Deletion Kit". E. coli Gene Deletion Kit, by Red ® / ET ®Recombination, Cat. No. K006, Version 2.3, June 2012” and references cited therein, such as Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645). For this purpose, plasmids pKD13, pKD46 and pCP20 were used: 3.4kb plasmid pKD13 ( Figure 1 It is publicly available in the GenBank gene database under accession number AY048744.1.

[0146] 6.3kb plasmid pKD46 ( Figure 2 It is publicly available in the GenBank gene database under accession number AY048746.1.

[0147] The 9.4kb plasmid pCP20 was published in Cherepanov and Wackernagel, Gene 158 (1995): 9–14.

[0148] To inactivate the ppsA gene in E. coli W3110 via homologous recombination using the Lambda Red system, the following steps were performed: 1. Using plasmid pKD46 (the so-called "red recombinase" plasmid), Figure 2 Transform Escherichia coli W3110 and isolate an ampicillin-resistant clone (named W3110 x pKD46).

[0149] 2. In the PCR reaction (“Phusion”) TM High-fidelity DNA polymerase, Thermo Scientific TM In ), plasmid pKD13 ( Figure 1 DNA from primers pps-5f (SEQ ID NO: 7) and pps-6r (SEQ ID NO: 8) produces ppsA-specific DNA fragments suitable for their inactivation.

[0150] Primer pps-5f contains 30 nucleotides (nt) from the 5' region of the ppsA gene (nt 333-362 in SEQ ID NO: 1) and is linked to a 20 nt specific to plasmid pKD13. Figure 1 It is referred to as "pr-1" in China.

[0151] Primer pps-6r contains a 30nt region from the 3' region of the ppsA gene (nt 2682-2711 in SEQ ID NO: 1, in reverse complementary form) and a 20nt region specifically for plasmid pKD13 linked thereto (in... Figure 1 It is referred to as "pr-2" in China.

[0152] Using DNA from plasmid pKD13 and primers pps-5f and pps-6r, a 1.4 kb PCR product was generated. This product contains 30 nt portions of DNA specific to the ppsA gene from *E. coli* W3110 at both the 5' and 3' ends. Furthermore, the PCR product contains the expression cassette of the kanamycin resistance gene contained in pKD13, as well as so-called "FRT repetitives" flanking the 5' and 3' ends of the kanamycin expression cassette. Figure 1 The short segments of DNA (referred to as "FRT1" and "FRT2") are used as identifier sequences for the "FLP recombinase" (contained on plasmid pCP20) in a later working step used to remove the antibiotic marker kanamycin.

[0153] 3. The 1.4 kb PCR product was isolated and treated with Dpn I, a restriction endonuclease familiar to those skilled in the art, which cleaves only methylated DNA to remove residual pKD13 plasmid DNA. Unmethylated DNA from the PCR reaction was not degraded.

[0154] 4. A 1.4 kb PCR product specific to the ppsA gene and containing a kanamycin resistance gene expression cassette was transformed into *E. coli* W3110 x pKD46, and the kanamycin resistance clone was isolated on LB Kan plates at 30°C. LB Kan 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.

[0155] 5. Ten of the obtained kanamycin resistance clones were purified on LBkan plates (i.e., clones were isolated by single-cell PCR) and examined in a PCR reaction to determine whether the kanamycin resistance cassette had been correctly integrated into the ppsA gene.

[0156] Kanamycin-resistant clones of *E. coli* W3110 cultured in Lbkan medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L kanamycin) were isolated from cells for PCR reaction using a DNA isolation kit (Qiagen). TM High-fidelity DNA polymerase, Thermo Scientific TMGenomic DNA of *Escherichia coli* strain W3110 was used as a control. Primers used for the PCR reaction were pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10). Primer pps-7f in reverse complementary form contained nt167-188 from SEQ ID NO: 1, and primer pps-8r contained nt2779-2800 from SEQ ID NO: 1.

[0157] Wild-type E. coli W3110 DNA produced a 2630 bp DNA fragment in the PCR reaction, as expected for the intact gene. In contrast, the kanamycin-resistant clone in the study produced an approximately 1660 bp DNA fragment in the PCR reaction, as expected if this 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 indicates that the kanamycin resistance gene has been successfully integrated into the ppsA gene locus, and therefore the ppsA gene has been inactivated. Clones containing the inactivated ppsA gene were selected and labeled W3110-ΔppsA::kan.

[0158] 6. To eliminate the kanamycin selection marker, W3110-ΔppsA::kan was transformed with plasmid pCP20 and the transformants were selected at 30°C. The 9.4kb vector pCP20 is disclosed in Cherepanov and Wackernagel (1995), Gene 158:9-14. The gene for the FLP recombinase is present on the pCP20 vector. The FLP recombinase marks the FRT sequence flanking the kanamycin resistance gene expression cassette and causes the removal of the kanamycin expression cassette. For this purpose, 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 inhibited.

[0159] The result of this step is a clone in which the ppsA gene has been inactivated and sensitivity to kanamycin has been restored (a so-called "cure" of antibiotic selection markers). Removing the kanamycin box from the genome of the ΔppsA mutant allows the introduction of additional mutations to produce double or multiple mutants.

[0160] W3110-ΔppsA::kan regained kanamycin sensitivity after treatment with pCP20 plasmid, as shown in the following tests: By laying boards on LB and LBkan boards: Growth was positive on LB plates, but no further growth was observed on LBkan plates, indicating that the kanamycin box was successfully removed from the genome.

[0161] Through PCR reaction: Therefore, genomic DNA was isolated from kanamycin-sensitive clones (Qiagen DNA Isolation Kit) and primers pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10) were used for PCR reactions (“Phusion”). TM High-fidelity DNA polymerase, Thermo Scientific TM Wild-type E. coli W3110 DNA produced a DNA fragment of approximately 2630 bp in the PCR reaction, as expected for the complete gene. In contrast, kanamycin-sensitive clones produced a DNA fragment of approximately 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.

[0162] The strain isolated from this step was identified as *Escherichia coli* W3110-ΔppsA. This strain is notable for containing an inactivated ppsA gene and for regaining sensitivity to the antibiotic kanamycin.

[0163] Example 2: ppsA deletion mutant produced in Pantotheca pineapple

[0164] The parent strain used for gene isolation and strain development is Pantotheca pineapple (available commercially from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM 30070).

[0165] The target of gene inactivation is the ppsA gene from *U. pineapple pantotheca*. The DNA sequence of the ppsA gene from *U. pineapple pantotheca* (Genbank GeneID: 31510655) is disclosed in SEQ ID NO: 3. Nucleotides 417–2801 (identified by *U. pineapple pantotheca* ppsA) encode a phosphoenolpyruvate synthase protein (*U. pineapple pantotheca* PpsA) having the amino acid sequence disclosed in SEQ ID NO: 4 (P).

[0166] Using Red from Gene Bridges GmbH, as detailed below ® / ET ® Technology (described in "Quick and Easy") E. coli See the "Technical Protocol, Quick & Easy" section in the user manual for the "Gene Deletion Kit". E. coli Gene Deletion Kit, by Red ® / ET ® The ppsA gene in *Plasmodium pineappleense* was inactivated using plasmids pKD13 and pRedET (Recombination, Cat. No. K006, Version 2.3, June 2012) and references cited therein, such as Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645).

[0167] 3.4kb plasmid pKD13 ( Figure 1 It is publicly available in the GenBank gene database under accession number AY048744.1.

[0168] In "Quick and Easy" E. coli The user manual for the "Gene Deletion Kit" discloses the commercially available 9.3kb plasmid pRedET; see "Technical Protocol, Quick & Easy". E. coli GeneDeletion Kit, by Red ® / ET ® Recombination, Cat. No. K006, Version 2.3, June 2012. "

[0169] To inactivate the ppsA gene in *Ureaplasma pineapple* via homologous recombination using the Lambda Red system, the following steps were performed: 1. Transform Pantotheca pineapple using plasmid pRedET (the so-called "red recombinase e" plasmid) and isolate tetracycline-resistant clones (called Pantotheca pineapple x pRedET).

[0170] 2. In the PCR reaction (“Phusion”) TM High-fidelity DNA polymerase, Thermo Scientific TM In ), plasmid pKD13 ( Figure 1 DNA from primers ppsapa-3f (SEQ ID NO: 11) and ppsapa-4r (SEQ ID NO: 12) produces ppsA-specific DNA fragments suitable for their inactivation.

[0171] Primer ppsapa-3f contains 49 nt from the 5' region of the ppsA gene (nt 417-465 in SEQ ID NO: 3) and a 20 nt specific to plasmid pKD13 linked thereto. Figure 1 It is referred to as "pr-1" in China.

[0172] Primer ppsapa-4r contains a 49nt region from the 3' region of the ppsA gene (nt 2753-2801 in reverse complementary form of SEQ ID NO: 3) and a 20nt region specifically for plasmid pKD13 linked thereto. Figure 1 It is referred to as "pr-2" in China.

[0173] Using DNA from plasmid pKD13 and primers ppsapa-3f and ppsapa-4r, a 1.4kb PCR product was generated. This product contains 49nt portions of DNA specific to the ppsA gene from *Ureaplasma pineapple* at both the 5' and 3' ends. Furthermore, the PCR product contains the expression cassette of the kanamycin resistance gene contained in pKD13, as well as so-called "FRT repetitives" (FRTs) flanking the 5' and 3' ends of the kanamycin expression cassette. Figure 1 These are referred to as "FRT1" and "FRT2" in the text, short segments of DNA that allow for the removal of the antibiotic marker kanamycin from ppsA deletion mutants as needed.

[0174] 3. The 1.4 kb PCR product was isolated and treated with Dpn I, a restriction endonuclease familiar to those skilled in the art, which cleaves only methylated DNA to remove residual pKD13 plasmid DNA. Unmethylated DNA from the PCR reaction was not degraded.

[0175] 4. A 1.4 kb PCR product specific to the ppsA gene and containing a kanamycin resistance gene was transformed into P. *Plasmodium pineapple* x 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.

[0176] 5. Purify the kanamycin resistance clone on LBkan plates (i.e., isolate the clone by single-cell PCR) and check in a PCR reaction to determine whether the kanamycin resistance cassette has been correctly integrated into the ppsA gene.

[0177] Genomic DNA for PCR was isolated from cells cultured with kanamycin-resistant clones of *Pueraria pineapple* from 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). TM High-fidelity DNA polymerase, Thermo Scientific TM Genomic DNA from wild-type *Panthera pineapple* strains was used as a control. Primers used for the PCR reaction were ppsapa-1f (SEQ ID NO: 13) and ppsapa-2r (SEQ ID NO: 14). In reverse complementary form, primer ppsapa-1f contains nt 281-302 from SEQ ID NO: 3, and primer ppsapa-2r contains nt 2901-2922 from SEQ ID NO: 3.

[0178] Wild-type *Umbrella pineapple* DNA produced a 2640 bp DNA fragment in the PCR reaction, as expected for the intact gene. In contrast, the kanamycin-resistant clone in this study produced an approximately 1670 bp DNA fragment in the PCR reaction, as expected if this 1.4 kb PCR product had 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 indicates that the kanamycin resistance gene has been successfully integrated into the ppsA gene locus, and therefore the ppsA gene has been inactivated. Clones containing the inactivated ppsA gene were selected and labeled as *Umbrella pineapple*-ΔppsA::kan.

[0179] Example 3: Production of Escherichia coli W3110-ppsA-MHI

[0180] Escherichia coli W3110-ppsA-MHI was produced by using a combination of Lambda Red recombination and anti-selective screening of gene modifications, as known to those skilled in the art. The mutation is characterized by a mutation in the ppsA structural gene that causes a reduction in enzyme activity (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), which encodes a protein having the sequence identified as SEQ ID NO: 6 (PpsA-MHI).

[0181] The procedure is as follows: 1. By PCR, using primers pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10), a 2.6 kb DNA fragment containing the ppsA WT gene (nt 167 to nt 2800 in SEQ ID NO: 1), i.e. cds and portions of the 5' and 3' flanking sequences, was isolated from the genomic DNA of Escherichia coli W3110.

[0182] 2. ppsA-MHI was obtained from the ppsA WT gene by sequentially introducing mutations into it via site-directed mutagenesis. This was performed using the commercially available cloning kit "QuickChange II Site-Directed Mutagenesis Kit" from Agilent, following the instructions in the user manual.

[0183] 3. To exchange the ppsA WT gene of Escherichia coli W3110 for ppsA-MHI, the first step was to use primers pps-9f (SEQ ID NO: 15) and pps-10r (SEQ ID NO: 16) to PCR from plasmid pKan-SacB ( Figure 3 A 3.2kb Kan-sacB box was separated from the sample.

[0184] The plasmid pKan-sacB contains an expression cassette of the kanamycin (Kan) resistance gene and the sacB gene encoding fructan sucrase.

[0185] Primer pps-9f contains a 30 nt starting from the initiation ATG of the ppsA gene (nt 333-362 in SEQ ID NO: 1) and a 20 nt linked thereto that is specific to plasmid pKa-SacB (in Figure 3 In China, it is referred to as "pr-f".

[0186] Primer pps-10r contains a 30nt stop codon from the ppsA gene (nt 2682-2711 in reverse complementary form of SEQ ID NO: 1) and a 21nt specific to plasmid pKa-SacB linked thereto (in Figure 3 In China, it is referred to as "pr-r".

[0187] 4. Transform Escherichia coli W3110 x pKD46 (for its production, see Example 1) with the ppsA-specific 3.2kb PCR product and isolate kanamycin-resistant clones.

[0188] 5. Inoculate these clones onto LBSC plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar and 15 mg / L kanamycin).

[0189] Clones containing the integrated sacB gene produce toxic fructans from sucrose, leading to growth inhibition. Such clones are selected and examined in PCR reactions to determine if the Kan-sacB box has been correctly integrated into the ppsA gene. Genomic DNA for PCR reactions has previously been obtained using a DNA isolation kit (Qiagen) from cells cultured with a kanamycin-resistant clone of *E. coli* W3110 in Ibkan medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L kanamycin). TM High-fidelity DNA polymerase, Thermo Scientific TM Genomic DNA from wild-type Escherichia coli strain W3110 was used as a control. Primers used for the PCR reaction were pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10).

[0190] Wild-type E. coli W3110 DNA produced a 2630 nt DNA fragment in the PCR reaction, as expected for the intact gene. In contrast, the kanamycin-resistant clone produced a DNA fragment of approximately 3400 nt in the PCR reaction, as expected if the 3.2 kb PCR product had 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 indicates that the Kan-sacB box has been successfully integrated into the ppsA gene locus, and therefore the ppsA gene has been inactivated. Clones containing the integrated Kan-sacB box were selected and labeled as W3110-ΔppsA::kan-sacB x pKD46.

[0191] 6. In the next step, the ppsA-MHI gene is exchanged using a Kan-sacB box. For this purpose, in the PCR reaction (“Phusion”... TM High-fidelity DNA polymerase, Thermo Scientific TM In this study, primers pps-11f (SEQ ID NO: 17) and pps-12r (SEQ ID NO: 18) were used to amplify a 2.5 kb DNA fragment from the ppsA-MHI DNA fragment obtained in step 2. In reverse complementary form, primer pps-11f contained nt300-319 from SEQ ID NO: 1, and primer pps-12r contained nt2743-2763 from SEQ ID NO: 1.

[0192] 7. The 2.5 kb ppsA-MHI gene was transformed into *E. coli* W3110-ΔppsA::kan-sacB x pKD46, and clones were selected on LBS plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar) without kanamycin. Only clones that no longer contained the active sacB gene were allowed to grow on LBS plates.

[0193] These clones were inoculated onto LBkan plates to select those clones that no longer contained the active Kan gene and whose growth was inhibited in the presence of kanamycin.

[0194] Clones that showed positive growth in the presence of sucrose and negative growth in the presence of kanamycin were selected and examined in PCR to determine whether the Kan-sacB box had been correctly replaced by the ppsA-MHI gene.

[0195] Genomic DNA was obtained from cells cultured 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 wild-type *E. coli* strain W3110 was used as a control. Primers used for PCR were pps-7f (SEQ ID NO: 9) and pps-8r (SEQ ID NO: 10). The PCR product, expected to be 2630 nt in size, was analyzed by DNA sequencing (Eurofins Genomics). Clones containing the correctly integrated ppsA-MHI gene produced DNA sequences as disclosed in SEQ ID NO: 5, encoding a protein corresponding to the sequence from SEQ ID NO: 6. Clones containing the correct ppsA-MHI gene with mutants V126M, R427H, and V434I were selected and identified as *E. coli* W3110-ppsA-MHI.

[0196] Example 4: Production of cysteine-producing strains

[0197] The cysteine-specific production plasmid used is pACYC184-cysEX-GAPDH-ORF306-serA317, derived from the parent vector pACYC184. Figure 4pACYC184-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 tetracycline resistance genes (parental vector pACYC184), but also the cysEX allele, which encodes a serine O-acetyltransferase with reduced cysteine ​​feedback inhibition, and the efflux gene ydeD (ORF306), whose expression is controlled by the constitutive GAPDH promoter.

[0198] In addition, pACYC184-cysEX-GAPDH-ORF306-serA317 contains the serA317 gene fragment, which was cloned after the ydeD (ORF306) efflux gene and encodes the N-terminal 317 amino acids (total length: 410 amino acids) of the SerA protein. The *E. coli* serA gene is disclosed in the GenBank gene database with gene ID 945258. serA317 is disclosed in Bell Labs. et al. , Eur. J. Biochem. (2002) 269: 4176-4184, referred to herein as “NSD:317”, and encodes a serine feedback resistance variant of 3-phosphoglycerate dehydrogenase. Expression of serA317 is controlled by the serA promoter.

[0199] Strains of *Escherichia coli* W3110, *Escherichia coli* W3110-ΔppsA, *Escherichia coli* W3110-ppsA-MHI, *Pantotheca pineappleica*, and *Pantotheca pineappleica*-ΔppsA::kan were each transformed with plasmid pACYC184-cysEX-GAPDH-ORF306-serA317 (referred to as pCYS in the following examples). Transformation was performed by electroporation according to prior art, as described in EP 0885962B1.

[0200] Transformants carrying 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 examined against the transformed pCYS plasmid using plasmid isolation and restriction analysis with the QIAprep Spin Plasmid Kit (Qiagen). Transformants containing the correctly incorporated pCYS plasmid were cultured to examine ppsA enzyme activity (Example 5) and determine cysteine ​​production (Examples 6 and 7).

[0201] Example 5: Assay of ppsA enzyme activity

[0202] The ppsA enzyme activities of *E. coli* strains W3110, W3110-ΔppsA, and W3110-ppsA-MHI, transformed with the production plasmid pCYS, were determined (Example 4). Cells from the three strains cultured in shake flasks in 50 ml SM1 medium (composition as described in 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 absorbed into 10 ml of assay buffer (100 mM Tris-HCl, pH 8.0; 10 mM MgCl2) and cell extracts were prepared.

[0203] Using the FastPrep-24 cell homogenizer from MP Biomedicals TM 5G. For this purpose, two 1ml cell suspensions were broken up in 1.5ml tubes pre-made by the manufacturer and containing glass beads (“Lysing Matrix B”) (3 x 20 seconds, shaking at a frequency of 6000 rpm, with a 30-second pause between each interval). The resulting homogenate was centrifuged, and the supernatant was used as a cell extract for determining activity.

[0204] The protein content of the extract was determined using a Qubit 3.0 fluorometer from Thermo Fisher Scientific. ® The protein assay kit should be selected according to the manufacturer's instructions.

[0205] To determine ppsA enzyme activity, the phosphate assay kit “Malachite Green Phosphate Assay Kit” (catalog number MAK307) from Sigma Aldrich was used according to the manufacturer’s instructions. The basis is the conversion of pyruvate to phosphoenolpyruvate via ATP in equilibrium reaction (4) to determine ppsA enzyme activity. This produces a stoichiometric amount of phosphate, which is used to determine the activity.

[0206] - These assays contain 1 ml of assay buffer (100 mM Tris-HCl, pH 8.0; 10 mM MgCl2).

[0207] - Incubate all measurements at 30°C.

[0208] - At 0, 10, 20, 30 and 60 minutes after the start of incubation, remove 50 µl of the corresponding assay, add it to 750 µl of H2O, and finally mix with 200 µl of reagent from the "Malachite Green Phosphate Assay Kit".

[0209] - After incubation for 30 min, the amount of phosphate formed was determined by measuring absorbance at 620 nm using a phosphate standard curve and according to the manufacturer's instructions. Finally, the ppsA enzyme activity in U / ml extract (1 U = µmol substrate turnover / min) was determined from the amount of phosphate measured based on the sampling time from the corresponding assay. Specific ppsA enzyme activity was calculated by basing ppsA enzyme activity on total protein (U / mg protein) in 1 mg of cell extract.

[0210] Table 1: Determination of ppsA enzyme activity

[0211] Example 6: Cysteine ​​is produced in the shake flask.

[0212] As a pre-culture for culturing in shake flasks, 3 ml of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) containing 15 mg / L tetracycline was inoculated with the corresponding strain and incubated in a shaker at 30°C and 135 rpm for 16 h. The strains studied were *Escherichia coli* W3110, W3110-ΔppsA, and W3110-ppsA-MHI, and in the second experiment, *Pantotheca pineappleensis* and *Pantotheca pineappleensis-ΔppsA::kan*, each transformed with the production plasmid pCYS (Example 4).

[0213] Master culture: Subsequently, a portion of the corresponding pre-culture was transferred to a 300 ml Erlenmeyer flask (with baffle) containing 30 ml of SM1 medium containing 15 g / L glucose, 5 mg / L vitamin B1 and 15 mg / L tetracycline.

[0214] The composition of SM1 medium is as follows: 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 citrate2H2O, 0.1 g / L NaCl; 1 ml / L trace element solution.

[0215] The composition of the trace element solution is as follows: 0.15 g / L Na2MoO4x 2H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2x6H2O, 0.25 g / L CuSO4x 5H2O, 1.6 g / L MnCl2x 4H2O, and 0.3 g / L ZnSO4x 7H2O.

[0216] Inoculate the master culture with sufficient pre-culture to establish an initial cell density OD of 0.025 / ml. 600 / ml (optical density of the master culture, measured at 600 nm). From here, incubate the entire 30 ml batch at 30°C and 135 rpm for 24 h.

[0217] After 24 hours, samples were taken and cell density (OD) was measured. 600 The total cysteine ​​content in the culture supernatant and the concentration of cysteine ​​in the culture supernatant was determined by colorimetric assay by Gaitonde (Gaitonde, MK (1967), Biochem. J. 104, 627-633) for the quantitative determination of cysteine. It should be noted that under highly acidic reaction conditions, this assay cannot distinguish cysteine ​​from the condensation product of cysteine ​​and pyruvate, 2-methylthiazolidin-2,4-dicarboxylic acid (thiazolidin), which is described in EP 0885962B1. L-cysteine ​​(formed by the oxidation of two cysteine ​​molecules according to equation (2)) was also detected as cysteine ​​in the assay by reduction with dithiothreitol in a dilute solution at pH 8.0. Results for the mentioned Escherichia coli strains are reported in Table 2, and results for Pantotheca pineapple strains are reported in Table 3.

[0218] Table 2: Cell density and total homocysteine ​​content after 24 hours of culture in shake flasks

[0219] Table 3: Cell density and total homocysteine ​​content after 24 hours of culture in shake flasks

[0220] Example 7: Cysteine ​​is produced in the fermenter: In a production-scale fed-batch fermentation, comparisons were made between Escherichia coli W3110 x pCYS, W3110-ppsA-MHI xpCYS, and W3110-ΔppsA x pCYS.

[0221] Pre-culture 1: Inoculate the corresponding strain into a 100ml Erlenmeyer flask with 20ml LB medium containing 15mg / L tetracycline and incubate on a shaker (150rpm, 30°C) for 7 hours.

[0222] Pre-culture 2: Subsequently, the entire preculture 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).

[0223] The culture was incubated at 30°C in a conical flask (1 L volume) with shaking at 150 rpm for 17 h (Infors incubator shaker). After this incubation, the cell density OD was measured. 600 / ml is 3 to 5.

[0224] Main training: Fermentation in DASGIP from Eppendorf ® The fermentation was conducted in a parallel bioreactor microbial system 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 MgSO4 x 7 H2O, 0.015 g / L CaCl2 x 2 H2O, 0.075 g / L FeSO4 x 7 H2O, 1 g / L sodium citrate x 2 H2O, and 1 ml of trace element solution (see Example 6), 0.005 g / L vitamin B1, and 15 mg / L tetracycline.

[0225] The pH in the fermenter was initially adjusted to 6.5 by pumping in a 25% NH4OH solution. During fermentation, the pH was maintained at 6.5 via automatic correction with 25% NH4OH. For inoculation, 100 ml of pre-culture 2 was pumped into the fermenter vessel. 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 a rate of 2 vvm (air volume / volume of culture medium / min). Under these initial conditions, the oxygen probe was calibrated to 100% saturation prior to inoculation.

[0226] The target value for O2 saturation during fermentation is set at 30%. Once the O2 saturation has dropped below the target value, the regulation cascade is activated to restore the O2 saturation to the target value. This involves first continuously increasing the gas supply (up to a maximum of 5 vvm) and then continuously increasing the stirring speed (up to a maximum of 1500 rpm).

[0227] 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 x 5H2O was fed at a rate of 1.5 ml / h.

[0228] Once the glucose concentration in the fermenter decreased from an initial 15 g / L to approximately 2 g / L, a 56% (w / w) glucose solution was continuously metered in. The feed rate was adjusted so that the glucose concentration in the fermenter no longer exceeded 2 g / L. Glucose levels were determined using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0229] Fermentation time was 48 hours. Afterward, samples were taken from the fermentation batches, and the content of L-cysteine ​​and its derivatives in the culture supernatant (mainly L-cysteine ​​and thiazolidinone) and the precipitate (L-cysteine) was determined separately. For this purpose, the Gaitonde colorimetric assay (Gaitonde, MK (1967), Biochem. J. 104, 627-633) was used in each case. L-cysteine ​​present in the precipitate must first be dissolved in 8% (v / v) hydrochloric acid and then quantified in the same manner. Finally, the total amount of cysteine ​​was determined as the sum of cysteine ​​in the pellets and the supernatant.

[0230] As summarized in Table 4, the cell density OD of the studied strains... 600 The g / ml ratio was comparable, although slightly higher for the control strain W3110xpCYS. In contrast, the volumetric production of cysteine ​​(in g / L) was significantly higher in W3110-ppsA-MHIxpCYS and in W3110-ΔppsAxpCYS (approximately 3-fold higher) than in the control strain W3110xpCYS containing the wild-type ppsA gene.

[0231] Therefore, under controlled fermentation conditions, the result achieved at production scale is a significant improvement in cysteine ​​production due to reduced or inactivated ppsA enzyme activity, which is a suitable measure for improving the strain. This result has not been described previously and is also unexpected to those skilled in the art due to the prior art.

[0232] Table 4: Cell density and total cysteine ​​content after 24 hours of culture in the fermenter

[0233] Abbreviations used in the attached diagram: bla: Gene that confers resistance to ampicillin (β-lactamase) rrnB term: rrnB terminator used for transcription. kanR: Gene that confers kanamycin resistance ORI: Origin of Replication pr-1: Primer binding site 1 pr-2: Primer binding site 2 FRT1: Marker sequence of FLP recombinase 1 FRT2: Marker sequence 2 of FLP recombinase araC: araC gene (repressor gene) ParaC: The promoter of the araC gene ParaB: The promoter of the araB gene Gam: λ phage Gam recombinant gene Bet: λ phage Bet recombinant gene Exons: λ phage in vitro recombinant genes ORI101: Temperature-sensitive origin of replication RepA: Gene for plasmid replication protein A sacB: L-sucrase gene pr-f: Primer binding site f (forward) pr-r: The binding site r of the primer (reverse) OriC: Replication origin C IHF: The binding site of the DNA-binding protein IHF (“integrative host factor”). CamR: Gene that confers resistance to chloramphenicol TetR: Gene that confers tetracycline resistance P15A ORI: Origin of replication.

Claims

1. A microbial strain suitable for the fermentation production of L-cysteine, characterized in that, - Number EC from the KEGG database 2.7.9.2 The relative enzyme activity of the identified enzyme is inactive or reduced relative to the wild-type enzyme, and - With EC numbers from the KEGG database Compared to microbial strains with wild-type enzyme activity of the enzymes identified in 2.7.9.2, the microbial strains suitable for fermenting and producing L-cysteine ​​produce an increased amount of L-cysteine. The gene encoding the enzyme activity is identified by ppsA.

2. The microbial strain according to claim 1, characterized in that, The microbial strain is a strain from the Enterobacteriaceae or Corynebacterialae families.

3. The microbial strain according to claim 1 or 2, characterized in that, The microbial strain was selected from Escherichia coli (E. coli) Escherichia coli ), Pantothecinus pineapple Pantoeaananatis ) and Corynebacterium glutamicum ( Corynebacterium glutamicum A group consisting of ).

4. The microbial strain according to claim 1 or 2, characterized in that, The microbial strains were selected from the group consisting of Escherichia coli and Pantotheca pineapple.

5. The microbial strain according to claim 1 or 2, characterized in that, The microbial strain is a strain of Escherichia coli.

6. The microbial strain according to claim 1 or 2, characterized in that, The microbial strain contains at least one mutation in the ppsA gene.

7. The microbial strain according to claim 6, characterized in that, The mutated genes were selected from a group consisting of the ppsA gene from Escherichia coli, the ppsA gene from Pantotheca pineapple, and genes homologous to these genes, wherein the genes homologous to these genes are DNA sequences that are at least 80% identical to these genes.

8. The microbial strain according to claim 6, characterized in that, The encoding DNA sequence of the ppsA gene is SEQ ID NO:

5.

9. The microbial strain according to claim 1 or 2, characterized in that, Among the strains described, the one identified by the KEGG database number EC... 2.7.9.2 The relative enzyme activity of the identified enzyme is reduced by at least 25% relative to the specific activity of the wild-type enzyme.

10. The microbial strain according to claim 1 or 2, characterized in that, Among the strains described, the one identified by the KEGG database number EC... 2.7.9.2 The relative enzyme activity of the identified enzyme is reduced by at least 70% relative to the specific activity of the wild-type enzyme.

11. The microbial strain according to claim 1 or 2, characterized in that, The microbial strain does not have the EC number from the KEGG database. 2.7.9.2 Enzyme activity of the enzyme class identified.

12. A fermentation method for producing L-cysteine, characterized in that, Use the microbial strain according to any one of claims 1 to 11.

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