Improved cysteine-producing strain

Abstract

The present invention relates to a microbial strain having a deregulated cysteine ​​biosynthetic pathway, which is therefore suitable for the fermentative production of at least one substance selected from L-cysteine, L-cystine and thiazolidine. The invention is characterized in that the relative expression of the CRP gene by mutating the CRP promoter sequence is reduced compared to the expression of the CRP gene using a wild-type promoter sequence. The invention is particularly advantageous in that the above-mentioned microbial strain forms an increased amount of a substance selected from L-cysteine, L-cystine and thiazolidine compared to the corresponding microbial strain with the expression of the CRP gene using a wild-type promoter. Therefore, the present invention also relates to a method for producing at least one compound selected from L-cysteine ​​and its L-cystine and thiazolidine derivatives using said microbial strain.

Description

[Technical Field]

[0001] The present invention relates to a microbial strain comprising a deregulated cysteine ​​biosynthetic pathway, thereby suitable for the fermentation production of at least one substance selected from L-cysteine, L-cystine, and thiazolidinediones, characterized in that, as a result of a mutation in the crp promoter sequence, the relative expression of the crp gene is reduced compared to the expression of the crp gene having a wild-type promoter sequence. A particular advantage is that the microbial strain forms an increased amount of the substance selected from L-cysteine, L-cystine, and thiazolidinediones compared to a corresponding microbial strain having expression of the crp gene having a wild-type promoter. Accordingly, the present invention also provides a method for producing L-cysteine ​​and at least one compound selected from its derivatives, L-cystine and thiazolidinediones, using the microbial strain. [Background technology]

[0002] Cysteine, abbreviated as Cys or C, is an α-amino acid having a side chain -CH2-SH. The naturally occurring enantiomer is L-cysteine, and since L-cysteine ​​is the only proteinogenic amino acid, in the context of this invention, when the term cysteine ​​is used without a descriptive term, it means L-cysteine. Oxidation of the sulfhydryl group can result in two cysteine ​​residues that together form a disulfide bond, resulting in the formation of cystine, to which the same description applies; that is, when no descriptive term is present, in this invention it means L-enantiomer (or L-cystine, i.e., (R,R)-3,3'-dithiobis(2-aminopropionic acid)). Since L-cysteine ​​can be formed from the amino acid methionine, L-cysteine ​​is a semi-essential amino acid for humans. Thiazolidine refers to the compound 2-methyl-2,4-thiazolidinedicarboxylic acid, which is an adduct of cysteine ​​and pyruvate (European Patent No. 0885962).

[0003] In all living 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 for the biosynthesis of coenzyme A.

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

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

[0006] In addition to the classical preparation of cysteine ​​by extraction from keratin-containing materials such as hair, bristles, horns, hooves, and feathers, or by biotransformation through enzymatic conversion of precursors, methods for fermentation production of cysteine ​​also exist. Prior art relating to the fermentation preparation of cysteine ​​using microorganisms is disclosed, for example, in European Patent Nos. 0858510, 0885962, 1382684, 1220940, 1769080, 2138585, and International Publication No. 2021 / 259491. Bacterial host organisms used include strains of the genus Corynebacterium and members of the family Enterobacteriaceae, such as Escherichia coli or Pantoea ananatis.

[0007] Wild-type host organisms without further modifications contain the cysteine ​​biosynthesis pathway (see, for example, the KEGG pathway database: "Cysteine ​​and Methionine Metabolism"), but this pathway is regulated so that the amount of cysteine ​​produced is only what is necessary for cell proliferation. For example, as described in the review by Wada and Takagi 2006 (see above), cysteine ​​biosynthesis in WT strains is regulated by so-called key enzyme feedback inhibition. For example, L-serine inhibits the SerA enzyme 3-phosphoglycerate dehydrogenase, and L-cysteine ​​inhibits the CysE enzyme serine O-acetyltransferase. Both SerA and CysE are enzymes in the cysteine ​​biosynthesis pathway, and their feedback inhibition by L-serine and L-cysteine, respectively, prevents the formation of more cysteine ​​than is needed by the cell. Such wild-type strains, as disclosed for example for the Escherichia coli (E. coli) strain K12 W3110 in Table 2 of this invention, do not produce detectable cysteine ​​and are therefore unsuitable for cysteine ​​production despite the presence of the cysteine ​​biosynthesis pathway. Wild-type microbial strains become suitable for cysteine ​​production by deregulating the cysteine ​​biosynthesis pathway. Various methods are available to produce microbial strains that have deregulated cysteine ​​biosynthesis and are characterized by improved cysteine ​​production. In addition to conventional methods that use mutation and selection to arrive at improved cysteine-producing strains, specific gene modifications have also been performed on strains to achieve effective overproduction of cysteine.

[0008] For example, the introduction of a cysE allele encoding a serine O-acetyltransferase with reduced cysteine-mediated feedback inhibition resulted in increased cysteine ​​production (European Patent No. 0858510; Nakamori et al., Appl. Env. Microbiol. (1998) 64:1607-1611). Feedback-resistant CysE enzymes largely cleave the formation of O-acetyl-L-serine, a direct precursor of cysteine, from intracellular cysteine ​​levels.

[0009] O-acetyl-L-serine is formed from L-serine and acetyl-CoA. Therefore, it is crucial to provide a sufficient amount of L-serine for cysteine ​​production. This can be achieved by introducing the serA allele, which encodes a 3-phosphoglycerate dehydrogenase with reduced feedback inhibition by L-serine. As a result, the formation of 3-phosphohydroxypirubate, a biosynthetic precursor of L-serine, is largely cleaved from intracellular L-serine levels. Examples of such SerA enzymes are described in European Patent No. 0620853 and European Patent No. 1496111. Alternatively, Bell et al., Eur.J.Biochem.(2002)269:4176-4184 disclose modifications to the serA gene for deregulating enzyme activity.

[0010] Increasing the transport of cysteine ​​outside the cell is another method for increasing the yield of products in the culture medium. This can be achieved by overexpressing so-called efflux genes, which encode membrane-bound proteins that mediate the export of cysteine ​​outside the cell. Various efflux genes for cysteine ​​export have been described (European Patent No. 0 885 962, European Patent No. 1 382 684). Exporting cysteine ​​from cells into fermentation media has several advantages.

[0011] 1) L-cysteine ​​is constantly removed from intracellular reaction equilibrium, resulting in low levels of this amino acid in the cell and cessation of L-cysteine-mediated feedback inhibition of sensitive enzymes. (1) L-cysteine ​​(intracellular) <-> L-cysteine ​​(culture medium)

[0012] 2) L-cysteine ​​secreted into the culture medium is oxidized in the presence of oxygen to form disulfide L-cystine, which is then introduced into the culture medium during cultivation (European Patent No. 0885962): (2) 2 L-cysteine ​​+ 1 / 2O2 -> L-cystine + H2O The solubility of L-cystine in an aqueous solution at neutral pH is extremely low compared to cysteine, so this disulfide already precipitates at low concentrations, forming a white precipitate. (3)L-cystine (dissolved) -> L-cystine (precipitated) The precipitation of L-cystine reduces the level of the product dissolved in the medium, thereby also shifting the reaction equilibria of (1) and (2) towards the product side.

[0013] 3) If amino acids can be obtained directly from the fermentation broth, the product accumulates intracellularly, and it is much less technically complex to purify the product than when cell disruption is first required.

[0014] A cysteine-producing strain / a microorganism strain capable of producing cysteine / a microorganism strain having a deregulated cysteine biosynthetic pathway / a microorganism strain having deregulated cysteine biosynthesis is characterized by at least one modification / feature selected from overexpression of a feedback-resistant SerA enzyme, a feedback-resistant CysE enzyme, and a cysteine export protein.

[0015] Furthermore, it is known that the 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 (European Patent No. 1 571 223).

[0016] In addition to the genetic modification of cysteine-producing strains, optimization of the fermentation process, i.e., how the cells are cultured, also plays an important role in the development of an efficient production process. Various culture parameters such as the nature and measurement of the carbon and energy sources, temperature, oxygen supply (European Patent No. 2 707 492), pH and composition of the culture medium can affect the product yield and / or product spectrum in the fermentative production of cysteine.

[0017] As raw material and energy costs are constantly rising, there is always a need to increase the product yield in cysteine ​​production in order to improve the economic viability of this process. [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] European Patent No. 0885962 [Patent Document 2] European Patent No. 0858510 [Patent Document 3] European Patent No. 1382684 [Patent Document 4] European Patent No. 1220940 [Patent Document 5] European Patent No. 1769080 [Patent Document 6] European Patent No. 2138585 [Patent Document 7] International Publication No. 2021 / 259491 [Patent Document 8] European Patent No. 0620853 [Patent Document 9] European Patent No. 1496111 [Patent Document 10] European Patent No. 1571223 [Patent Document 11] European Patent No. 2707492 [Non-patent literature]

[0019] [Non-Patent Document 1] Wada and Takagi, Appl. Microbiol. Biotechnol. (2006) 73:48-54 [Non-Patent Document 2] Nakamori et al.,Appl.Env.Microbiol.(1998)64:1607-1611 [Non-Patent Document 3] Bell et al.,Eur.J.Biochem.(2002)269:4176-4184 [Overview of the project]

[0020] The object of the present invention is to provide a microbial strain for the fermentation production of cysteine, L-cystine, and / or thiazolidinediones, thereby enabling higher yields of L-cysteine, L-cystine, and / or thiazolidinediones in fermentation compared to known strains from the prior art. [Means for solving the problem]

[0021] This objective is achieved by a microbial strain that includes a deregulated cysteine ​​biosynthetic pathway and is thereby suitable for the fermentation production of at least one substance selected from L-cysteine, L-cystine, and thiazolidinediones, characterized in that, as a result of a mutation in the crp promoter sequence, the relative expression of the crp gene is reduced compared to the expression of the crp gene having a wild-type promoter sequence. [Modes for carrying out the invention]

[0022] Crp, encoded by the crp gene, is an abbreviation for cyclic AMP (cAMP) receptor protein (or "catabolite repressor protein"), also known as CAP (catabolite activator protein), and is an important transcription factor particularly known for mediating so-called catabolite repression, i.e., regulating gene expression according to the carbon (C) source. For example, glucose is a preferred carbon source in Escherichia coli (E. coli), and as long as glucose is available, the expression of genes for the metabolism of alternative carbon sources is suppressed. Crp is activated by the binding of the signaling molecule cAMP (cyclic AMP) (called Crp-cAMP). Crp-cAMP has an effect on the expression of target genes, thereby regulating not only carbon source utilization but also other cellular functions such as nitrogen fixation, biofilm formation, transport of the trace element iron, or osmotic equilibrium. Hanamura and Aiba, Nucleic Acids Res. (1991) 19:4413-4419, further reported that Crp-cAMP can suppress its own expression (negative autoregulation).

[0023] Transcriptome analyses (e.g., Gosset et al., J. Bacteriol. (2004) 186:3516-3524) have shown that the expression of numerous genes (over 400 genes) is influenced by Crp or Crp-cAMP. Furthermore, Crp is part of a branched regulatory network of global transcription factors that influence each other (as outlined in Figure 1 of Frendorf et al., Comput. Structural Biotechnol. J. (2019) 17:730-736) and affect the expression of target genes depending on the cellular metabolic state.

[0024] Therefore, when the expression of the crp gene is altered, it is impossible to predict in advance how increased or decreased crp expression generally affects cellular metabolism, or how increased or decreased crp expression affects biosynthetic metabolic pathways, such as the biosynthetic metabolic pathway for L-cysteine, due to the numerous genes regulated by Crp-cAMP and the mutually influencing global transcription factors.

[0025] Frendorf et al., 2019 (see above), provide an overview of numerous previously studied variants of the Crp protein. These previously studied variants are exclusively modifications of the Crp amino acid sequence caused by modifications of crp cds. European Patent Nos. 3686214, 3686215, and 3725800 (all from CJ Corp., South Korea) similarly disclose variants of the Crp amino acid sequence, as well as their use for producing L-amino acids, particularly L-threonine and L-tryptophan.

[0026] What has not been studied in the prior art is the effect on metabolism, particularly cysteine ​​biosynthesis, of altered expression of the crp WT gene resulting from mutations in the crp promoter in microbial strains with a dysregulated cysteine ​​biosynthesis pathway. The present invention differs from the prior art in that it preferably modifies the nucleotide sequence of the crp promoter rather than the amino acid sequence of Crp. In other words, the prior art has modified the amino acid sequence, and therefore the activity characteristics of the Crp protein as a transcription factor. In contrast, in the present invention, the amino acid sequence, and therefore the activity characteristics of the Crp protein (wild-type Crp), preferably remain unchanged. However, what is modified is the expression due to modification of the crp promoter, i.e., the amount of protein. From the prior art, it was not possible to predict how this treatment would affect cellular metabolism in general, and specifically cysteine ​​biosynthesis, in microbial strains with a dysregulated cysteine ​​biosynthesis pathway. For example, Liu et al., J.Agric.Food Chem 2020, 68:14928-14937, describes the Escherichia coli (E. coli) strain BW25113-pLH03 in Figure 2 of the publication. This strain showed both increased crp gene expression and improved L-cysteine ​​production compared to the JM109-pLH03 strain. Therefore, it was entirely unexpected that reduced expression of the crp gene in microbial strains with a deregulated cysteine ​​biosynthesis pathway, as disclosed in the present invention, would result in improved cysteine ​​production.

[0027] Mutations in the CRP promoter sequence, preferably as a result of shortening of the CRP promoter sequence, or as a result of a combination of insertion and shortening of the CRP promoter sequence, result in attenuated CRP expression, with CRP CDS particularly preferably remaining unchanged. Attenuated CRP expression results in improved L-cysteine ​​production in microbial strains having a deregulated cysteine ​​biosynthesis pathway in a manner not previously known. In a particularly preferred embodiment, mutations in the CRP promoter sequence result in no expression of the Crp protein at all.

[0028] Detection of CRP gene expression: To enable comparison of CRP gene expression across various strains, a method for quantitatively detecting CRP gene expression is necessary. Generally, various known test methods are available for the quantitative detection of gene expression. • Immunological detection methods involve the binding of a specific antibody to the expressed Crp protein. Thus, methods known to those skilled in the art, such as ELISA ("enzyme-linked immunosorbent assay") and Western blotting, allow for the quantitative determination of the expressed protein by a color reaction that occurs via the bound Crp-specific antibody. Another method for quantitatively detecting gene expression involves determining the gene-specific RNA of an expressed gene, such as crp RNA, within the total cellular RNA (total RNA). In the context of this invention, the gene studied for its expression is referred to as the target gene (e.g., the crp gene). Methods known to those skilled in the art include Northern blotting for direct detection of gene-specific RNA and preferred real-time PCR (RT-PCR), also known as qPCR (quantitative PCR), for indirect detection of gene-specific RNA (e.g., crp RNA).

[0029] RT-PCR analysis can be performed, for example, as follows:

[0030] 1) After collecting the cells to be analyzed (e.g., Escherichia coli (E. coli) cells from cultures of the strains to be analyzed (Examples 3 and 5) (as described in Example 4)), mix them with an RNA stabilization reagent (e.g., RNA-Protect(R) "Bacteria Reagent" from Qiagen), extract the total RNA (e.g., using the RNeasy RNA Extraction Kit from Qiagen), and quantify it (e.g., using the "Qubit(trademark) RNA BR Assay Kit" from Thermo Fisher Scientific).

[0031] 2) Use the total RNA of these strains to produce complementary DNA (cDNA) by reverse transcription (e.g., using the QuantiNova® Reverse Transcription Kit from Qiagen), and quantify the cDNA (e.g., using the Qubit® dsDNA HS Assay Kit from Thermo Fisher Scientific).

[0032] 3) Next, the cDNA is used in an RT-PCR reaction containing a gene-specific primer for the target gene crp and a gene-specific primer for a reference gene (e.g., the cysG gene) according to prior art. Instruments for performing RT-PCR analysis, including reagent kits and evaluation software for preparing the RT-PCR reaction (e.g., from Qiagen), are commercially available. For the analysis of crp expression described in Example 5, an RT-PCR instrument from Qiagen (RotorGene Q 2plex RT-PCR instrument, operated by Rotor-Gene Q control and evaluation software from the same manufacturer) was used to determine the relative expression of the crp gene in strains with a modified crp promoter compared to a comparative strain of Escherichia coli (E. coli) W3110×pCys with a WT crp promoter.

[0033] Relative manifestation: In the context of the present invention, 2 -ΔΔCTThe relative gene expression determined as a value is defined as the expression of the crp gene in the microbial strains according to the present invention (e.g., the cysteine-producing strains Escherichia coli (E. coli) W3110-crp::kan-sacB×pCys, Escherichia coli (E. coli) W3110-crpP-del×pCys, Escherichia coli (E. coli) W3110-crp-Preg×pCys, Escherichia coli (E. coli) W3110-crp-Preg2×pCys and Escherichia coli (E. coli) W3110-crp-Preg3×pCys described in Example 3), which have a mutated crp promoter sequence and a deregulated cysteine ​​biosynthesis pathway, compared to the expression of the crp gene in the corresponding comparison strain (e.g., Escherichia coli (E. coli) strain W3110×pCys having a WT crp promoter). The RT-PCR analysis method used in Example 5 of the present invention is a so-called 2-degree RT-PCR, as described in Quantitative RT-PCR and Livak and Schmittgen, Methods (2001) 25:402-408. -ΔΔCT This includes the analysis of relative gene expression using the method. This evaluation method forms the basis of the RotorGene Q control and evaluation software for the RotorGene Q 2plex RT-PCR instrument from Qiagen.

[0034] CT value: The basis for determining relative gene expression is the so-called CT value (CT: "cycle threshold"), which is the value obtained in RT-PCR of cDNA of a certain strain of the crp gene (CT crp The CT value (CT) of the cysG gene as the reference gene is determined. The reference gene plays a role in normalizing RT-PCR and is selected from known constitutively expressed genes that are not subject to any regulation. In the context of the present invention, the CT value (CT) of the cysG gene as the reference gene is determined. cysG Although the expression of the gene was determined in Example 5, it is known that the expression of the gene changes very slightly during the culture process (Zhou et al., BMC Molecular Biology (2011) 12:18).

[0035] ΔCT value: In the context of the present invention, the ΔCT value is defined as the difference between the CT values of the crp gene of a strain and a reference gene (e.g., cysG reference gene). ΔCT = CT crp - CT cycG .

[0036] ΔΔCT value: In the context of the present invention, ΔΔCT is defined as the difference between the ΔCT value of a strain having an altered expression of the crp gene and the ΔCT value of a comparative strain having the WT expression of the target gene (e.g., Escherichia coli (E. coli) W3110 × pCys). ΔΔCT = ΔCT - ΔCT W3110×pCys .

[0037] 2 -ΔΔCT value: The value 2 -ΔΔCT is formed from the ΔΔCT value and is a measure of the expression of the crp gene in a modified strain compared to the expression of the crp gene in a comparative strain (e.g., Escherichia coli (E. coli) W3110 × pCys), and is also referred to as the relative expression of the target gene.

[0038] 4) When comparing the expression of the crp gene between various strains, the same amount of cDNA is used in RT-PCR, and the relative crp expression as a 2 - ΔΔCT value is determined for each strain. · The 2 -ΔΔCT value of the comparative strain has a value of 1. · 2 -ΔΔCT value > 1 indicates increased expression compared to the comparative strain. · 2 -ΔΔCT value < 1 indicates attenuated expression compared to the comparative strain.

[0039] By comparing the 2 -ΔΔCT values for relative expression, it is possible to describe whether a mutation in the promoter sequence of the crp gene has changed the expression of the crp gene and the extent to which the mutation has changed the expression of the crp gene. This change in gene expression (see Example 5) can correlate with altered characteristics of the strain, such as cell growth or the yield of a metabolite, such as the production of the amino acid L - cysteine (see Examples 6 and 7).

[0040] An open reading frame (ORF, cds, or coding sequence) is a region of DNA or RNA that begins with a start codon and ends with a stop codon, encoding the amino acid sequence of a protein. ORFs are also called coding regions or structural genes.

[0041] A gene refers to a portion of DNA that contains all the fundamental information necessary to produce biologically active RNA. A gene contains the portion of DNA from which single-stranded RNA copies are produced by transcription, and also contains expression signals involved in regulating this replication process. Expression signals include, for example, at least one promoter, transcription start, translation start, and ribosome binding site. Furthermore, expression signals can also include terminators and one or more operators.

[0042] The promoter is located upstream of the 5' end of a cds and refers to a nucleotide sequence that enables gene expression. In the direction of synthesis, the promoter is located before the RNA coding region. The promoter mediates the initiation of gene transcription by RNA polymerase and contains regions that specifically interact with DNA-binding proteins called transcription factors.

[0043] Mutation refers to modifications of genetic material, including modifications to DNA sequences, and, if a protein-coding sequence is involved, also modifies the amino acid sequence of the protein. In the context of this invention, mutations include the exchange, insertion, and / or deletion of one or more nucleotides or one or more amino acids. Exchange refers to the exchange of one or more nucleotides in DNA for other nucleotides, or the exchange of one or more amino acids in a protein for other amino acids. At the same time, the length of the DNA sequence or protein sequence remains unchanged. Insertion refers to the incorporation of an additional nucleotide in DNA, or the incorporation of an additional amino acid in a protein. The term insertion also includes elongation. In the case of deletion, one or more nucleotides or parts of a nucleotide sequence, or one or more amino acids or parts of an amino acid sequence are missing. When the modification results in only individual nucleotides or individual amino acids being exchanged for another nucleotide or another amino acid, the term point mutation is used. Mutations also include combinations of exchanges, deletions, and insertions.

[0044] In the context of this invention, proteins, such as Crp, begin with a capital letter, while the genes for these proteins having coding sequences are identified by lowercase letters (e.g., crp).

[0045] Therefore, the *E. coli* crp gene refers to nucleotides 565-865 of SEQ ID NO: 1, which is the promoter region of the crp gene, and nucleotides 866-1495 of SEQ ID NO: 1, which is the cds of the *E. coli* crp gene. *E. coli* Crp refers to the protein encoded by the aforementioned cds and identified by SEQ ID NO: 2. This protein is the Crp protein.

[0046] The abbreviation WT (Wt) refers to the wild type. A wild-type gene or wild-type promoter refers to the form of a gene or promoter that arose naturally through evolution and is present in the wild-type genome. The DNA sequences of the Wt gene or Wt promoter are publicly available in databases such as the NCBI (National Center for Biotechnology Information, U.S. National Library of Medicine).

[0047] Alleles define the states of a gene that can be converted to one another through mutation, that is, through changes in the nucleotide sequence of DNA. Genes that occur naturally in microorganisms are called wild-type alleles, and mutants derived from wild-type alleles are called mutated alleles of that gene.

[0048] Homologous genes or homologous sequences should be understood to mean that the DNA sequences of the gene or DNA portion are at least 80% identical, preferably at least 90% identical, and particularly preferably at least 95% identical.

[0049] The degree of DNA identity is, http: / / blast.ncbi.nlm.nih.gov / It can be found and determined by the "nucleotide blast" program based on the blastn algorithm. The algorithm parameters used to align two or more nucleotide sequences were the initial parameters. Common initial parameters are: Maximum target sequence = 100; Short query = "Automatically adjust parameters for short input sequences"; E value threshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. Corresponding initial scoring parameters are: Match / mismatch score = 1, -2; Gap cost = linear.

[0050] The protein sequence is, http: / / blast.ncbi.nlm.nih.gov / The comparison is performed using the "protein blast" program. This program uses the blastp algorithm. The algorithm parameters used to align two or more protein sequences were the default parameters. Common default parameters are: Maximum target sequence = 100; Short query = "Automatically adjust parameters for short input sequences"; E value threshold = 10; Word size = 3; Automatically adjust parameters for short input sequences = 0. Default scoring parameters are: Matrix = BLOSUM62; Gap cost = Presence: 11 Extension: 1; Composition adjustment = Conditional composition score matrix adjustment.

[0051] In the case of the microorganism according to the present invention having a deregulated cysteine ​​biosynthesis pathway, mutations in the CRP promoter sequence result in a relative expression of the CRP gene of preferably at least 0.91. -ΔΔCT The value is, in particular, at least 0.5. -ΔΔCT The value is, in particular, preferably 0.03. -ΔΔCT This results in a decrease in the value, and the expression of the CRP gene in microorganisms with a wild-type promoter is 2 to 1.00. -ΔΔCT The values ​​are normalized. The relative expression of the CRP gene is preferably determined as described in Example 5.

[0052] 1.00 of 2 -ΔΔCT If the expression of the CRP gene with a wild-type promoter having a value is defined as 100% gene expression of the CRP gene, then a value reduced to at least 0.91 is considered to be 2. -ΔΔCT 2 have a value that has decreased to at least 0.5 -ΔΔCT 2 have a value and have decreased to at least 0.03 -ΔΔCTThe crp gene expression in the microorganism according to the present invention having the specified values ​​is at most 91%, 50%, and 3% of the expression of the crp gene with the wild-type promoter, respectively. That is, preferably, this method is characterized in that crp expression in a microbial strain having a modified crp promoter sequence is at least 9% lower compared to the corresponding microbial strain having a Wt crp promoter sequence, and when this microbial strain is used, the yield of L-cysteine ​​at g / l increases by at least 10% (w / v).

[0053] In the context of the present invention, "compared to / in comparison with / in comparison with the expression (corresponding) of the crp gene having a wild-type promoter (activity of the crp wild-type promoter, or WT expression)" means compared to the activity of the crp promoter corresponding to an unmutated type of the crp promoter derived from a microorganism, i.e., the crp promoter that arose naturally through evolution and is present in the wild-type genome of the microorganism.

[0054] Microbial strains suitable for the fermentation production of L-cysteine, L-cystine, or thiazolidinediones include all microorganisms that contain a deregulated cysteine ​​biosynthetic pathway resulting in the synthesis of cysteine, cystine, or thiazolidinediones. Such strains are disclosed, for example, in European Patent No. 0885962, European Patent No. 1382684, European Patent No. 1220940, European Patent No. 1769080, and European Patent No. 2138585, as well as International Publication No. 2021 / 259491.

[0055] Microbial strains possessing a deregulated cysteine ​​biosynthesis pathway are characterized by at least one of the following modifications:

[0056] a) The microbial strain is characterized by a modified serA gene encoding 3-phosphoglycerate dehydrogenase (SerA) with L-serine-mediated feedback inhibition, which is at least 2-fold lower compared to the corresponding wild-type enzyme (as described, for example, in European Patent No. 1950 287), and the SerA enzyme activity can be determined photometrically by oxidation of NADH dependent on the SerA substrate 3-phosphohydroxypirubate, as described, for example, by McKitrick and Pizer, J. Bacteriol (1980) 141:235-245.

[0057] Compared to the corresponding wild-type enzyme, particularly preferred mutants of 3-phosphoglycerate dehydrogenase (serA) have L-serine-mediated feedback inhibition reduced to at least 1 / 5, especially preferably at least 1 / 10, and in a more preferred embodiment, at least 1 / 50.

[0058] and / or b) The microbial strain contains a modified cysE gene encoding serine O-acetyltransferase (CysE) with cysteine-mediated feedback inhibition, which is reduced by at least 1 / 2 compared to the corresponding wild-type enzyme (as described, for example, in European Patent No. 0858510 or Nakamori et al. 1998 (see above)), and the CysE enzyme activity can be determined photometrically by the consumption of acetyl-CoA, a substrate of CysE, by reaction with L-serine to form O-acetyl-L-serine, as described, for example, by Nakamori et al. 1998 (see above).

[0059] Compared to the corresponding wild-type enzyme, particularly preferred mutants of serine O-acetyltransferase (cysE) have cysteine-mediated feedback inhibition reduced to at least 1 / 5, especially preferably at least 1 / 10, and more preferably at least 1 / 50 in a more preferred embodiment.

[0060] and / or c) The microbial strains exhibit at least a twofold increase in extracellular cysteine ​​export compared to the corresponding wild-type cells due to overexpression of the efflux gene, and cysteine ​​export can be determined by photometric measurement of the extracellular content of cysteine ​​(including cysteine, cystine, and the adduct 2-methylthiazolidined-2,4(R)-dicarboxylic acid formed from cysteine ​​and pirubate) according to the method of Gaitonde, Biochem. J. (1967) 104:627-633, as described, for example, in European Patent No. 0885962.

[0061] Compared to wild-type cells, overexpression of the efflux gene preferably results in an increase of at least 5 times, particularly preferably at least 10 times, and especially preferably at least 20 times, in the extracellular transport of cysteine.

[0062] The extrusion genes are preferably derived from the following genes from Escherichia coli (E. coli): ydeD (see European Patent No. 0885962), yfiK (see European Patent No. 1382684), cydDC (see International Publication No. 2004 / 113373), bcr (see U.S. Patent Application Publication No. 2005-221453), and emrAB (see U.S. Patent Application Publication No. 2005-221453), or from the group consisting of corresponding homologous genes from different microorganisms.

[0063] and / or d) The microbial strain may further be characterized in that at least one cysteine-degrading enzyme is attenuated to such an extent that the cells contain up to 50% of the enzyme activity 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).

[0064] Such strains are known, for example, from European Patent No. 0858510 and European Patent No. 0885962.

[0065] Microbial strains suitable for the fermentation production of L-cysteine, L-cystine, or thiazolidinediones, as described in the previous paragraph, are deregulated with respect to their cysteine ​​metabolism to form an increased amount of L-cysteine ​​compared to microbial strains that are not deregulated with respect to cysteine ​​metabolism. This means that cysteine-producing strains, or microbial strains capable of producing cysteine, are characterized by having a deregulated cysteine ​​biosynthesis pathway. In cells of microbial strains that are not deregulated with respect to cysteine ​​metabolism, the amount of L-cysteine ​​in the culture is approximately 0 g / l, so the increased amount preferably means any amount exceeding 0.05 g / l of L-cysteine ​​measured in the culture after 24 hours of incubation.

[0066] The amount of cysteine ​​can be quantified using a colorimetric assay by Gaitonde 1967 (see above), for example, as described in Example 6 (see Table 2, strains W3110 and W3110×pCys).

[0067] Compared to the corresponding microbial strain with expression of the crp gene having a wild-type promoter (i.e., this strain is also characterized by a dysregulated cysteine ​​biosynthesis pathway), the microbial strain according to the present invention, having a dysregulated cysteine ​​biosynthesis pathway, a mutated crp promoter sequence, and consequently reduced relative expression of the crp gene morphology, has increased amounts of substances selected from L-cysteine, L-cystine, and thiazolidinediones, which is a significant advantage. As demonstrated in Table 3 (Example 7), increasing the mutation in the crp promoter sequence to a complete deletion of the crp promoter, i.e., a 9% to 100% reduction in relative expression of the crp gene, results in a significantly higher yield of total cysteine ​​in the fermentative method, i.e., the total amount of cysteine, cystine, and thiazolidinediones produced. Microbial strains with a mutated promoter sequence in the crp gene are always compared to the corresponding microbial strains with a Wt promoter sequence in the crp gene, i.e., both microbial strains are dysregulated with respect to their cysteine ​​metabolism.

[0068] Where cysteine ​​is referred to in this invention, it always means L-cysteine ​​or one of its derivatives selected from L-cystine or thiazolidinedione. This means that the amount of cysteine ​​produced preferably means total cysteine, i.e., the sum of L-cysteine, L-cystine, and thiazolidinedione produced. For example, the formed L-cystine can be reduced to form L-cysteine ​​and then simultaneously detected by measurement against the produced cysteine. For example, when the determination is achieved by using the Gaitonde colorimetric assay (see above), the assay cannot distinguish L-cysteine ​​from 2-methylthiazolidinedione-2,4-dicarboxylic acid (thiazolidinedione), which is a condensation product of cysteine ​​and pirubate as described in European Patent No. 0885962, and thiazolidinedione is also simultaneously detected by measurement against the produced cysteine. According to the present invention, this microbial strain forms increased amounts of L-cysteine, L-cystine, and thiazolidinediones, preferably L-cysteine ​​and L-cystine, and particularly preferably L-cysteine.

[0069] Preferably, the microbial strain is characterized by an unmutated amino acid sequence of the Crp protein. That is, the amino acid sequence of the Crp protein is the Wt sequence identified by SEQ ID NO: 2. To achieve this, the coding sequence of crp contains only so-called silent mutations or no mutations at all. Silent mutations are defined as modifications of cds that are due to degeneracy of the genetic code and do not alter the resulting amino acid sequence. This means that the crp cds are either identical to the Wt DNA sequence available in the NCBI database for the crp of the corresponding organism, or contain only silent mutations, encoding an unmutated Crp protein having a Wt protein sequence. Particularly preferably, in this invention, only the promoter sequence of the crp gene of the microbial strain is mutated. Mutations in the promoter sequence of the crp gene derived from Escherichia coli (E. coli), containing the sequence of nucleotides 565-865, are preferred, whereas crp cds containing the sequence of nucleotides 866-1498, containing only silent mutations, are unmutated or encode a protein having the protein sequence of SEQ ID NO: 2.

[0070] In the context of the present invention, mutations in the CRP promoter sequence are a) The crp promoter sequence of the crp gene is partially or completely deleted, and / or b) The crp promoter sequence of the crp gene is modified by one or more insertions or 5' and / or 3' extensions, and / or c) The crp promoter sequence of the crp gene contains one or more point mutations. Includes such modifications, As a result, the expression of the CRP gene is reduced, i.e., weakened or completely suppressed. As already mentioned above, reduced means that the CRP gene expression in the microorganism according to the present invention is at most 91%, particularly preferably at most 50%, and especially preferably at most 3%, of the expression of the CRP gene with a wild-type promoter. In a particularly preferred embodiment, there is no detectable CRP gene expression in the microorganism according to the present invention.

[0071] Particularly preferable is that the Crp protein does not have mutations in the aforementioned case of reduced crp gene expression; that is, its amino acid sequence is invariant compared to Wt.

[0072] In the context of the present invention, any desired combination of gene modifications in the promoter of the CRP gene listed in a) to c) is also possible. In short, in the context of the present invention, CRP gene expression is weakened or completely suppressed by modification of the CRP promoter. Weakening of CRP expression can also be achieved by completely or partially replacing the CRP promoter with another weaker promoter.

[0073] Particularly preferred, the modification of the CRP promoter in the strain according to the present invention includes the complete or partial deletion of the CRP promoter, modification of the CRP promoter by one or more insertions or 5' and / or 3' extensions, or a combination of deletion and insertion.

[0074] Particularly preferred, the modification of the CRP promoter in the strain according to the present invention includes a complete or partial deletion of the CRP promoter.

[0075] Preferably, the microbial strain is characterized by having at least one deletion or insertion, particularly preferably at least one deletion, in the CRP promoter sequence.

[0076] In a particularly preferred embodiment, the mutation in the CRP promoter sequence (which preferably includes the sequence identified at nucleotides 565-865 of SEQ ID NO: 1) is at least one deletion or insertion, and the CRP coding sequence is not mutated.

[0077] Preferably, the microbial strain is characterized by the deletion of at least nucleotides 565-624 from the CRP promoter sequence identified by nucleotides 565-865 of SEQ ID NO: 1. In this case, the CRP promoter sequence contains at most nucleotides 625-865 from SEQ ID NO: 1.

[0078] Particularly preferred is the microbial strain characterized by a complete deletion of the CRP promoter sequence identified at nucleotides 565-865 of Sequence ID No. 1.

[0079] Particularly preferred embodiments, including insertion, deletion, and extension, are described in the examples. For complete or partial deletions of the CRP promoter sequence, see Example 2. For modifications of the CRP promoter sequence by one or more insertions, see Example 1. For modifications of the CRP promoter sequence that result in weakening or complete inhibition of promoter activity, see Example 5.

[0080] Preferably, the microbial strain is characterized by being a strain from the Enterobacteriaceae or Corynebacteriaceae family, and 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).

[0081] Preferably, the microbial strain is selected from the group consisting of Escherichia coli, Pantoea ananatis, and Corynebacterium glutamicum, and particularly preferably from the group consisting of Escherichia coli and Pantoea ananatis. Particularly preferably, the microbial strain is a strain of the Escherichia coli species. Preferably, the E. coli strain is selected from E. coli K12, and particularly preferably from E. coli K12 W3110. Such strains, in particular, including Escherichia coli (E. coli) K12 W3110 DSM 5911 (id. ATCC 27325) and Pantoea ananatis DSM 30070 (id. ATCC 11530), are commercially available from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig).

[0082] The crp gene from Escherichia coli (E. coli) K12 is available in the NCBI gene database, for example, as an entry in the E. coli (E. coli) Genbank reference sequence with accession number NC_000913.3 and nucleotides 3485255 to 3486950 (SEQ ID NO: 1). The crp gene from Pantoea ananatis is available in the NCBI gene database, for example, as an entry in the Pantoea ananatis (P. ananatis) Genbank reference sequence with accession number NC_017554.1 and nucleotides 430825 to 431818 (crp cds: nucleotides 430883 to nucleotides 431515; gene identification number 57266449).

[0083] In a preferred embodiment, the microbial strain is characterized in that the mutated crp promoter sequence is selected from the group consisting of promoter sequences of the crp gene from Escherichia coli and the crp gene from Pantoea ananatis, and homologous sequences associated with these sequences, the term homologous sequence having the definitions defined above.

[0084] The crp gene is preferably a crp gene from Escherichia coli (E. coli) that encodes a Crp protein having a promoter region identified at nucleotides 565-865 of SEQ ID NO: 1 and crp cds identified at nucleotides 866-1495 of SEQ ID NO: 1, and the amino acid sequence identified in SEQ ID NO: 2.

[0085] Therefore, the microbial strain is preferably characterized by the expressed Crp protein being sequence number 2. This means that the expressed Crp protein has a Wt sequence (sequence number 2) and the mutation is related only to the crp promoter sequence (nucleotides 565-865 of sequence number 1).

[0086] Furthermore, the production strain according to the present invention can be further optimized for further improvement of cysteine ​​production. Optimization can be achieved genetically, for example, by further expressing one or more genes suitable for improving production characteristics. These genes can be expressed in the production strain in a manner known to the present, either as separate gene constructs or combined as expression units (so-called operons). Furthermore, the production strain can be optimized by reducing the expression of the CRP gene, as well as by inactivating further genes whose gene products adversely affect cysteine ​​production. However, optimization can also be achieved in a manner known to the present, by mutagenesis and selection of strains having improved cysteine ​​production.

[0087] An alternative approach would involve weakening or completely suppressing the expression of the Crp gene by adding an inhibitor, whether a chemical or protein inhibitor, such that the inhibitor has an inhibitory effect on the activity of the Crp promoter but not on the activity of the Crp protein.

[0088] Various methods for introducing modifications to the promoter of the crp gene are known to those skilled in the art. In the simplest case, the parent strain can be subjected to mutagenesis in a known manner (e.g., chemically with mutagenic chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine, or physically by UV irradiation), and mutations are randomly generated in the genomic DNA. Then, the desired mutants having the modified crp promoter are selected from a number of mutants generated, for example, by quantitative determination of crp protein (e.g., immunologically by Western blotting using a crp-specific antibody) or quantitative determination of crp expression by RT-PCR, for example, after each mutant has been isolated. Only mutants with the modified crp promoter are selected in each case, while the crp cds remain unchanged and correspond to the wild-type sequence.

[0089] In contrast to the selection of desired mutants with complex random mutagenesis and modified crp promoters, the promoter of the crp gene can be subjected to targeted modification in a relatively simple manner, for example, by known mechanisms of homologous recombination. Cloning systems for targeted gene inactivation by homologous recombination are known to those skilled in the art and are commercially available, for example, as disclosed in the user manual for the “Quick and Easy E. coli Gene Deletion Kit” based on Red(R) / ET(R) technology from Gene Bridges GmbH (see “Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, Catalog No. K006, Version 2.3, June 2012” and the references cited therein).

[0090] According to prior art, the CRP promoter or a part thereof can be isolated, foreign DNA can be cloned into the CRP promoter, and the promoter sequence can be altered. Therefore, a DNA construct suitable for targeted modification of the CRP promoter may consist of a 5' portion of DNA homologous to the genomic CRP promoter, a gene segment containing foreign DNA thereafter, and a 3' portion of DNA also homologous to the genomic CRP promoter thereafter.

[0091] Possible regions in the CRP promoter for homologous recombination may include areas other than the promoter sequence region. Possible regions may also include DNA sequences adjacent to the promoter, i.e., the 5' adjacent sequence prior to the start of the CRP promoter (e.g., nucleotides 1-564 of SEQ ID NO: 1). DNA sequences in the 3' region of the CRP promoter are related to the CRP gene's cds (crp cds, nucleotides 866-1498 of SEQ ID NO: 1), in which case modification of the CRP gene's cds by homologous recombination is excluded. The exogenous DNA is preferably a selection marker expression cassette selected from, for example, a class of antibiotic resistance genes.

[0092] Another such system for targeted gene inactivation based on homologous recombination is based on a combination of Lambda Red recombination and counter-selection screening, is known to those skilled in the art, and is the method for gene modification described in Examples 1 and 2. The system is described, for example, in Sun et al., Appl. Env. Microbiol. (2008) 74:4241-4245. For example, a DNA construct for inactivating the crp promoter is used, comprising, starting from the 5' end, a sequence homologous to the crp gene (including the 5' region of the crp promoter), followed by two expression cassettes in any order: a) an expression cassette for a selection marker selected from a class of antibiotic resistance genes and b) an expression cassette for the sacB gene encoding the enzyme levansculas, and finally, a further sequence homologous to the crp gene (e.g., including the sequence of crp cds adjacent to the 3' of the crp promoter).

[0093] In the first step, a DNA construct is transformed into a producing strain to isolate antibiotic-resistant clones. The resulting clones 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 in the second step by the principle of rival selection, in which a suitable DNA fragment replaces the two marker genes by homologous recombination. The clones obtained in this step then regain the ability to grow on sucrose and subsequently regain their susceptibility to antibiotics. This method is used in Examples 1 and 2 for the targeted shortening of the crp promoter (SEQ ID NO: 1, nucleotides 565-865) of Escherichia coli (E. coli). A DNA fragment suitable for this step includes, starting from the 5' end, a sequence of at least 20 nucleotides homologous to the target gene, e.g., the crp gene, followed by a portion of DNA containing the desired modified DNA sequence, e.g., the shortened crp promoter, and finally, a further sequence of at least 20 nucleotides homologous to the target gene, e.g., the crp gene. DNA fragments can be obtained, for example, chemically by gene synthesis, or from individual DNA fragments by known so-called OE-PCR (e.g., Hilgarth and Lanigan, MethodsX (2020) 7:100759), as in Example 2. https: / / doi.org / 10.1016 / j.mex.2019.12.001 It can be produced by overlap extension PCR as described above.

[0094] As an example of a strain according to the present invention having weakened crp expression due to a combination of insertion of a Kan-sacB cassette and deletion in the crp promoter, the following Escherichia coli (E. coli) strain:W3110-crp::kan-sacB is disclosed in the examples. In W3110-crp::kan-sacB, a 3.2kb Kan-sacB cassette is inserted into the crp promoter between nucleotide 640 and nucleotide 714 of SEQ ID NO: 1, resulting in the simultaneous deletion of 73 nucleotides of the crp promoter (SEQ ID NO: 1, nucleotides 641 to 713) (see Example 1). As a result of this modification of the crp promoter, the relative expression of the crp gene in the W3110-crp::kan-sacB×pCys strain transformed with plasmid pCys was only 0.33 times, or 33%, of the expression in the W3110×pCys strain with the wild-type crp promoter, normalized to 1 or 100% (see Example 5, Table 1).

[0095] The following Escherichia coli (E. coli) strain is disclosed in Example (Example 2) as an example of a strain according to the present invention having weakened CRP expression due to shortening of the CRP promoter. W3110-crpP-del: Deletion of nucleotides 565-865 from SEQ ID NO: 1 W3110-crp-Preg: Deletion of nucleotides 565-713 from SEQ ID NO: 1 W3110-crp-Preg2: Deletion of nucleotides 565-675 from SEQ ID NO: 1 W3110-crp-Preg3: Deletion of nucleotides 565-624 from SEQ ID NO: 1

[0096] As a result of these examples of shortened crp promoters, the relative expression of the crp gene in strains transformed with plasmid pCys was only the following proportions of expression in the W3110×pCys strain with a wild-type crp promoter normalized to 1 (see Example 5, Table 1): In E. coli (E. coli) W3110-crpP-del×pCys with a completely deleted crp promoter (deletion of nucleotides 565-865 from SEQ ID NO: 1), the relative crp expression was only 0.03 times that of crp expression with a wild-type crp promoter. In E. coli (E. coli) W3110-crp-Preg×pCys, the crp promoter was shortened by 149 nucleotides (deletion of nucleotides 565-713 from SEQ ID NO: 1). The relative crp expression was only 0.05 times that of crp expression with a wild-type crp promoter. In *E. coli* W3110-crp-Preg2×pCys, the crp promoter was shortened by 111 nucleotides (deletion of nucleotides 565-675 from SEQ ID NO: 1). Relative crp expression was only 0.5 times that of crp expression with the wild-type crp promoter. In *E. coli* W3110-crp-Preg3×pCys, the crp promoter was shortened by 60 nucleotides (deletion of nucleotides 565-624 from SEQ ID NO: 1). Relative crp expression was only 0.91 times that of crp expression with the wild-type crp promoter.

[0097] These results suggest that, on the one hand, increasing the shortening of the crp promoter reduced the relative expression of the crp gene to 0.91 to 0.03 times that of expression under the Wt promoter, or that a combination of insertion and deletion weakened the relative expression of the crp gene to 0.33 times that of expression under the Wt promoter.

[0098] With respect to the E. coli crp gene, it is preferable to reduce the relative expression of the crp gene from a value of 1 relative to the wild-type crp promoter to at least 0.91 times, particularly preferably at least 0.5 times, and especially preferably at least 0.33 times, by a combination of deletion or insertion and deletion.

[0099] Strains according to the present invention, such as Escherichia coli (E. coli) strains W3110-crp::kan-sacB, W3110-crpP-del, W3110-crp-Preg, W3110-crp-Preg2, or W3110-crp-Preg3, characterized by modifications of the crp promoter in a manner that results in weakened crp expression, can be produced by using the above combination of Lambda Red recombination and counter-selection screening for gene modifications, as disclosed in Examples 1 and 2 (see, for example, Sun et al., 2008, see above).

[0100] Particularly preferred strains are Escherichia coli (E. coli) W3110-crp-Preg2 and Escherichia coli (E. coli) W3110-crp-Preg3 (described in Example 2).

[0101] The present invention further provides a method for producing at least one compound selected from L-cysteine, L-cystine, and thiazolidinediones, characterized by the use of a microbial strain according to the present invention. The method may include culturing the microbial strain according to the present invention in a shaking flask (laboratory scale) or a fermenter (production scale), with the fermenter (production scale) method being preferred. Shaking flask culture includes specifying a particular medium and pH, as well as culturing in the presence of oxygen and under constant motion (shaking), while in a fermenter, it is possible to set and adjust more specific conditions regarding the medium (e.g., adjusting the fermenter volume by supplying components or partially draining the fermenter broth), temperature, pH, oxygen supply, and medium mixing. In other words, both shaking flask culture and fermenter culture are called fermentative methods, differing in scale. In fermentative methods, a microbial-producing strain is grown and metabolites are produced. Smaller cultures can also be used as pre-cultures for inoculation of larger cultures.

[0102] The prior art does not disclose a method or strain that can improve cysteine ​​production by weakening CRP expression through modification of the CRP promoter sequence.

[0103] The main product formed by the method according to the present invention is L-cysteine. By oxidation, sparingly soluble L-cystine is obtained according to formulas (1) to (3). L-cystine accumulates as a precipitate during fermentation (European Patent No. 0885962, European Patent No. 2707492). Thiazolidine is obtained by adduct formation with pirubate. Thiazolidine accumulates in the culture supernatant (European Patent No. 0885962).

[0104] Preferably, the method is characterized by the isolation of the formed L-cysteine, L-cystine, or thiazolidinedione. The isolation of L-cysteine ​​is disclosed in European Patent No. 2,699,544 and European Patent No. 1,958,933. The precipitated L-cystine can be removed from the remaining components, for example, using a decanter, followed by dissolution of the crude product with a mineral acid, clarification of the crude product solution by centrifugation or filtration, decolorization of the solution, and precipitation and crystallization (European Patent No. 2,707,492).

[0105] In the context of the present invention, the total cysteine ​​yield is defined as the sum of the cysteine, cystine, and thiazolidinedions produced. This is determined from the entire culture, as described in Example 7. The total cysteine ​​yield can be quantified, for example, using a colorimetric assay by Gaitonde (Gaitonde, MK (1967), Biochem. J. 104, 627-633).

[0106] As illustrated in the examples of this application, attenuation of CRP expression in microbial strains having deregulated cysteine ​​biosynthesis suitable for cysteine, cystine, or thiazolidinedione production is suitable for significantly increasing the total cysteine ​​yield, i.e., the sum of cysteine, cystine, and thiazolidinedione produced, by fermentation. This was entirely unexpected from the prior art.

[0107] It is surprising that fermentation of microbial strains having a deregulated cysteine ​​biosynthetic pathway and reduced crp gene expression due to mutations in the crp promoter sequence yields significantly higher cysteine ​​yields compared to the expression of the crp gene with a wild-type promoter sequence. The evidence summarized in Table 3 of Example 7 shows that fermentation of Escherichia coli (E. coli) strain W3110-crp::kan-sacB×pCys with a relative crp gene expression of 0.33 times, Escherichia coli (E. coli) strain W3110-crp-Preg2×pCys with a relative crp gene expression of 0.5 times, and Escherichia coli (E. coli) strain W3110-crp-Preg3×pCys with a relative crp gene expression of 0.91 times achieved significantly higher cysteine ​​yields compared to the corresponding wild-type strain W3110×pCys with a relative crp expression of 1. In contrast to the prior art, and unexpected to those skilled in the art, attenuation of CRP gene expression by complete or partial deletion of the CRP promoter, or a combination of insertions and deletions, resulted in improved cysteine-producing strains.

[0108] This novel and inventive means for improving cysteine-producing strains has already been confirmed by the results summarized in Table 2 of Example 6, where attenuation of crp gene expression by deletion or a combination of insertion and deletion in the crp promoter resulted in improved cysteine ​​yield in culture in a shaking flask. Furthermore, Table 2 of Example 6 also shows that cell proliferation was impaired in strains with significantly attenuated crp expression, such as W3110-crpP-del×pCys (relative crp expression of 0.03 times compared to W3110×pCys) and W3110-crp-Preg×pCys (relative expression of 0.05 times compared to W3110×pCys) (Table 2, OD). 600 This indicates ( / ml). Nevertheless, a clear improvement in cysteine ​​production exists, and this improvement is even more pronounced when cysteine ​​production is based on proliferation (mg / OD of cysteine ​​in Table 2), corresponding to an increase of 285.5% or 360%.

[0109] Therefore, to those skilled in the art, weakening the expression of the CRP gene by deletion or a combination of insertion and deletion in the CRP promoter is also a novel and useful means for improving cysteine ​​production in other cysteine-producing strains. Accordingly, in the microbial strain according to the present invention, which is suitable for cysteine ​​production and has deregulated cysteine ​​biosynthesis, the expression of the CRP gene is weakened by deletion or a combination of insertion and deletion in the CRP promoter, while cysteine ​​production is increased, and CRP CDS is not modified.

[0110] Example 7 demonstrates that strains capable of cysteine ​​production, possessing deregulated cysteine ​​biosynthesis and having weakened expression of the crp gene due to deletion or a combination of insertion and deletion in the crp promoter, achieve significantly higher cysteine ​​yields in fermentation than strains containing the WT promoter of the crp gene, demonstrating that no modifications to crp cds exist in any of the strains according to the present invention.

[0111] In the fermentation method in question, not only the biomass of the production strain according to the present invention is formed, but also cysteine ​​and its oxidation product, cystine. The formation of biomass and cysteine ​​may be temporally correlated, or they may be formed over time in a manner that is separate from each other. Cultivation is carried out in a manner well known to those skilled in the art. For this purpose, cultivation may be carried out in a shaking flask (laboratory scale) or a fermenter (production scale).

[0112] The microbial strain is characterized by being deregulated with respect to the cysteine ​​biosynthesis pathway and containing at least one mutation in the promoter of the crp gene. At the same time, this strain produces an increased amount of L-cysteine ​​compared to strains with a wild-type crp promoter. Preferably, the gene modification in the promoter of the crp gene is wild-type crp promoter (100% expression, 2 -ΔΔCT Compared to value = 1), the expression of the crp gene was reduced by at least 9% (2 -ΔΔCT Value ≤ 0.91), particularly preferably at least 50% (2 -ΔΔCTValue ≤ 0.5), particularly preferably at least 67% (2 -ΔΔCT (Value ≤ 0.33) Reduce.

[0113] Preferably, the method is characterized in that the expression of crp in a microbial strain having a modified crp promoter sequence is reduced by at least 9%, particularly preferably at least 50%, and especially preferably at least 67%, compared to the corresponding microbial strain having a Wt crp promoter sequence, and the yield of a substance selected from L-cysteine, L-cystine, and thiazolidinedione in g / l is increased by at least 10% (w / v), particularly preferably at least 20% (w / v), and especially preferably at least 50% (w / v) when using this microbial strain. According to the present invention, this microbial strain forms an increased amount of a substance selected from L-cysteine, L-cystine, and thiazolidinedione, preferably L-cysteine ​​and L-cystine, and particularly preferably L-cysteine.

[0114] As a result of reduced CRP expression, total cysteine ​​production (amount produced per unit volume in g / L) when cultured in a shaking flask or fermenter, i.e., the production of cysteine, cystine, and thiazolidinediones, is preferably increased by at least 10% (w / v), particularly preferably at least 20% (w / v), and especially preferably at least 50% (w / v) compared to a comparative strain with a WT CRP promoter. The amount produced per unit volume in shaking flask culture within 24 hours is preferably at least 0.37 g / L (Table 2), and the amount produced per unit volume in fermentation within 48 hours is preferably at least 15.9 g / L (Table 3).

[0115] Preferably, the method is a fermentation method, characterized in that the fermentation volume is at least 1 L, particularly preferably more than 10 L, especially preferably more than 1000 L, and especially preferably more than 10000 L. Particularly preferably, the fermentation method is a method in a fermentation tank.

[0116] Culture media are well known to those skilled in the art in the field of microbial culture. Culture media typically consist of a carbon source, a nitrogen source, and additives such as vitamins, salts, and trace elements, as well as a sulfur source, which optimize cell growth and cysteine ​​production.

[0117] Carbon sources are those that can be used by the producing strain for the formation of 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, as well as all conceivable disaccharides and polysaccharides formed from monosaccharides, such as sucrose, lactose, maltose, maltodextrin, and starch, and monomers or oligomers released (enzymatically or chemically) from disaccharides and polysaccharides by hydrolysis. Other usable carbon sources other than sugars or carbohydrates are acetic acid (and its derived acetates), ethanol, glycerol, citric acid (and its salts), or pirubate (and its salts). However, gaseous carbon sources such as carbon dioxide or carbon monoxide may also be conceivable.

[0118] Preferred carbon sources for culturing the production strain are glucose, fructose, sucrose, mannose, xylose, and arabinose, with glucose and sucrose being particularly preferred.

[0119] Nitrogen sources are those that can be used by the producing strain for biomass formation. These include ammonia in gaseous form or in aqueous solution as NH4OH, or salts of ammonia such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, or ammonium nitrate. Furthermore, suitable nitrogen sources are known nitrates such as KNO3, NaNO3, ammonium nitrate, Ca(NO3)2, Mg(NO3)2, or other nitrogen sources such as urea. Nitrogen sources also include complex mixtures of amino acids such as yeast extracts, proteose peptone, malt extract, soy peptone, casamino acids, corn steep liquor (in liquid or dried as so-called CSD), as well as NZ Amine and yeast nitrogen bases.

[0120] The weighed addition of a sulfur source, either as a single addition in batch form or as continuous feeding, is required for the efficient production of cysteine ​​and cysteine ​​derivatives. Continuous weighed addition can be carried out as a pure feed solution or in a mixture with further feed components such as glucose. Suitable sulfur sources are sulfates, sulfites, dithionites, thiosulfates, or sulfide salts, and it is also conceivable that each acid be used with given stability. Preferred sulfur sources are sulfates, sulfites, thiosulfates, and sulfide salts, particularly preferably sulfates and thiosulfates, especially preferably thiosulfates such as sodium thiosulfate and ammonium thiosulfate.

[0121] Culturing may be carried out in a so-called batch mode, which involves inoculating a starter culture of the producing strain into a culture medium and then allowing the cells to grow without further supply of nutrients. Culturing may also be carried out in a so-called fed-batch mode, which involves the additional supply of nutrients (feed) after the initial stage of growth in batch mode to compensate for nutrient consumption. The feed may consist of a carbon source, a nitrogen source, a sulfur source, one or more vitamins or trace elements important for production, or a combination of the above. The feed components may be weighed together as a mixture or separately in individual feed sections. In addition, other culture medium components may be added to the feed as additives that specifically increase cysteine ​​production. The feed may be supplied continuously, in small batches (discontinuously), or in a combination of continuous and discontinuous feeding. Culturing in fed-batch mode is preferred.

[0122] Preferred carbon sources in feed are glucose, sucrose, and glucose or sucrose-containing plant hydrolysates, as well as mixtures of preferred carbon sources in any mixing ratio. A particularly preferred carbon source in feed is glucose.

[0123] Preferably, the carbon source of the culture is measured 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, a maximum concentration of 0.5 g / L is particularly preferred, and a maximum concentration of 0.1 g / L is especially preferred.

[0124] Preferred nitrogen sources in feed include ammonia in gaseous form or aqueous solution as NH4OH, particularly preferably ammonia or ammonium salts, yeast extract, soy peptone or corn steep liquor (in liquid or dry form), as well as salts of ammonia such as ammonium sulfate, ammonium phosphate, ammonium acetate and ammonium chloride, and further urea, KNO3, NaNO3 and ammonium nitrate, yeast extract, proteose peptone, malt extract, soy peptone, casamino acids, corn steep liquor, and NZ Amine and yeast nitrogen bases.

[0125] Preferred sulfur sources in feed are sulfates, sulfites, thiosulfates, and sulfide salts, particularly preferably sulfates and thiosulfates, and especially preferably thiosulfates such as sodium thiosulfate and ammonium thiosulfate.

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

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

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

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

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

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

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

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

[0134] The culture batch obtained by the above method contains L-cystine in precipitated form. This is formed from the primary product L-cysteine ​​according to formulas (1) to (3) (European Patent No. 0885962, European Patent No. 2707492). Depending on the fermentation conditions, L-cysteine ​​may also be a major product, accumulating in a dissolved form in the culture supernatant (European Patent No. 2726625). The cysteine ​​or cystine contained in the culture batch can be used directly for further processing without further treatment, or it can be isolated from the culture batch.

[0135] Preferably, the method is characterized by the isolation of the formed cysteine ​​or cystine. Methods known to themselves, including centrifugation, decantation, dissolution of the crude product with mineral acid, filtration, extraction, chromatography, or crystallization or precipitation, are available for isolating cysteine ​​and cystine. These method steps can be combined in any form to isolate cysteine ​​to a desired purity. The desired purity depends on further use. Methods for isolating L-cysteine ​​are disclosed in European Patent No. 2,699,544 and European Patent No. 1,958,933. A procedure for isolating L-cystine is described in European Patent No. 2,707,492.

[0136] The cystine obtained by post-treatment can be reduced to cysteine ​​for further use. A method for reducing L-cystine to L-cysteine ​​by electrochemical means is disclosed in European Patent No. 0235908.

[0137] Various analytical methods are available for identifying, quantifying, and determining the purity of cysteine ​​or cystine products, including spectrophotometric analysis, NMR, gas chromatography, HPLC, mass spectrometry, gravimetric analysis, or combinations thereof.

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

[0139] The figure shows the plasmids used in the examples. [Brief explanation of the drawing]

[0140] [Figure 1] The 6.3kb vector pKD46 used in Example 1 and Example 2 is shown. [Figure 2] The 5kb vector pKan-SacB used in Example 1 is shown. [Figure 3] The 7.1kb vector pCys used in Example 3 is shown.

[0141] Abbreviations used in the diagram: bla: A gene that confers resistance to ampicillin (β-lactamase). kanR: A gene that confers resistance to kanamycin. araC: araC gene (repressor gene) P araC: Promoter of the araC gene P araB: Promoter of the araB gene Gam: Lambda phage Gam recombinant gene Bet: Lambda phage Bet recombinant gene Exo: Lambda phage Exo recombinant gene ORI101: Temperature-sensitive origin of replication RepA: Gene for plasmid replication protein A sacB: Levans sucrase gene pr-f: Binding site f for primer (forward) pr-r: Binding site r for primer (reverse) OriC: origin of replication C TetR: A gene that confers resistance to tetracycline. P15A ORI: Origin of replication serA317:serA (3-phosphoglycerate dehydrogenase gene, encoding amino acids 1-317)cds cysE X:cysE (serine O-acetyltransferase gene, feedback resistance) cds ORF306:ydeD (cysteine ​​efflux gene) cds

[0142] The present invention is not limited by the following embodiments, but is further illustrated by the following embodiments. [Examples]

[0143] [ Example 1 Preparation of Escherichia coli (E. coli) strain W3110-crp::kan-sacB The parent strain used for DNA isolation and strain development was Escherichia coli K12 W3110 (commercially available from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM 5911).

[0144] The target of the gene modification was the promoter region of the crp gene from Escherichia coli (E. coli). The DNA sequence of the crp gene region, including the cds of the yhfA gene, the crp promoter region, and the cds of the crp gene, from E. coli K12 (Genbank NCBI reference sequence NC_000913.3, nucleotides 3485255 to 3486950) is disclosed in Sequence ID No. 1.

[0145] Nucleotides 163-564 (identified by *E. coli* yhfA) contain the cds of the yhfA gene in a reverse-complementary form.

[0146] Nucleotides 866-1495 (identified by *E. coli* crp) contain the cds of the crp gene, which encodes a protein having the amino acid sequence of SEQ ID NO: 2.

[0147] The intergenetic region between the differently expressed genes yhfA and crp, including nucleotides 565-865 in SEQ ID NO: 1, contains the promoter sequence of the crp gene. Analysis of the crp promoter region is described in Hanamura and Ajba 1991 (see above).

[0148] The Escherichia coli (E. coli) strain W3110-crp::kan-sacB, characterized by the incorporation of the kan-sacB cassette into the crp promoter, was produced by using a combination of Lambda Red recombination and counter-selection screening for gene modifications known to those skilled in the art (see, e.g., Sun et al., 2008; see above).

[0149] The procedure was as follows: 1. Escherichia coli (E. coli) W3110 was transformed with plasmid pKD46, and transformants were selected on an LBamp plate (10 g / L tryptone from GIBCO®, 5 g / L yeast extract from BD Biosciences, 5 g / L NaCl, 1.5% agar, and 100 mg / L ampicillin from Sigma-Aldrich). Ampicillin-resistant clones were selected and named E. coli (E. coli) W3110 × pKD46. The 6.3 kb plasmid pKD46 (the so-called "Red Recombinase" plasmid, Figure 1) is disclosed in the GenBank gene database under accession number AY048746.1.

[0150] 2. A 3.2kb Kan-sacB cassette was isolated from plasmid pKan-SacB by PCR using primers crp-9f (SEQ ID NO: 3) and crp-10r (SEQ ID NO: 4).

[0151] The 5kb plasmid pKan-sacB (Figure 2) contains expression cassettes for both the kanamycin (kanR) resistance gene and the sacB gene encoding the enzyme levansculas. The Escherichia coli (E. coli) kanamycin resistance gene encoding aminoglycoside phosphotransferase is disclosed in the NCBI database under accession number SH02_03400. The B. subtilis (B. subtilis) sacB gene is disclosed in the NCBI database under accession number 936413.

[0152] Primer crp-9f contained 50 nucleotides from the CRP promoter region (nucleotides 591-640 of SEQ ID NO: 1) and 20 nucleotides specific to plasmid pKan-SacB, linked to these nucleotides (labeled "pr-f" in Figure 2). Primer crp-10r contained 51 nucleotides from the CRP promoter region (nucleotides 714-764 of SEQ ID NO: 1, in reverse complementary form) and 21 nucleotides specific to plasmid pKan-SacB, linked to these nucleotides (labeled "pr-r" in Figure 2).

[0153] 3. Escherichia coli (E. coli) W3110×pKD46 was transformed with a 3.2kb PCR product specific to the CRP promoter region, and kanamycin-resistant clones were isolated on an LBkan plate (10g / L tryptone, 5g / L yeast extract, 5g / L NaCl, 1.5% agar, 15mg / L kanamycin).

[0154] 4. Kanamycin-resistant clones were seeded on LBSC plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar, and 15 mg / L kanamycin). Clones containing the incorporated sacB gene produced toxic levan from sucrose, which inhibited growth.

[0155] 5. Genomic DNA was isolated from cells cultured in LBkan medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L kanamycin) from kanamycin-resistant and sucrose-sensitive clones using a DNA isolation kit (Qiagen).

[0156] 6. The integration of the Kan-sacB cassette was verified using genomic DNA in PCR reactions using primers crp-7f (SEQ ID NO: 5) and crp-8r (SEQ ID NO: 6) ("Phusion® High-Fidelity" DNA polymerase, Thermo Scientific®).

[0157] In the PCR reaction, wild-type E. coli (E. coli) W3110 DNA produced a 1696-nucleotide DNA fragment (corresponding to the sequence from SEQ ID NO: 1), as expected for the complete gene structure consisting of yhfA cds, a crp promoter, and crp cds. In contrast, the kanamycin-resistant clone produced a DNA fragment of approximately 4800 nucleotides in the PCR reaction, as expected for the incorporation of a 3.2 kb PCR product at a site within the crp promoter defined by primers crp-9f and crp-10r. DNA sequencing of the 4800-nucleotide PCR product confirmed the removal of 74 nucleotides from the crp promoter (SEQ ID NO: 1, nucleotides 641-713) as a result of the incorporation of a 3.2 kb Kan-sacB cassette at the site defined by primers crp-9f and crp-10r.

[0158] 7. A clone with an integrated Kan-sacB cassette was selected and named W3110-crp::kan-sacB×pKD46.

[0159] 8. The temperature-sensitive plasmid pKD46 was removed by incubation at 42°C. Clones lacking the pKD46 plasmid were ampicillin-sensitive and could no longer be grown on LBamp plates. An ampicillin-sensitive clone was selected and named W3110-crp::kan-sacB.

[0160] [ Example 2 Creation of the W3110 strain with differently shortened CRP promoters. The following W3110 strain with a shortened CRP promoter was created: W3110-crpP-del: Deletion of nucleotides 565-865 from SEQ ID NO: 1 W3110-crp-Preg: Deletion of nucleotides 565-713 from SEQ ID NO: 1 W3110-crp-Preg2: Deletion of nucleotides 565-675 from SEQ ID NO: 1 W3110-crp-Preg3: Deletion of nucleotides 565-624 from SEQ ID NO: 1 A W3110 strain with a shortened crp promoter was constructed by using homologous recombination to replace the kan-sacB cassette of the W3110-crp::kan-sacB strain with a DNA fragment containing a modified promoter sequence. The DNA fragment containing the modified promoter sequence was prepared in a known manner by fusion PCR (so-called OE-PCR, or "Overlap Extension PCR") from two PCR products defining the modified promoter. Phusion® High-Fidelity DNA polymerase (Thermo Scientific®) was used in each case to prepare the PCR products described below, according to the manufacturer's instructions.

[0161] The following primers were used: CRP-13F (SEQ ID NO: 7): Corresponds to nucleotides 160-180 of SEQ ID NO: 1. CRP-14R (SEQ ID NO: 8): Corresponds to nucleotides 545-564 of SEQ ID NO: 1; an inverse complementary form. CRP-17F (SEQ ID NO: 9): Corresponds to nucleotides 537-565 (nucleotides 1-29 of CRP-17F) and nucleotides 866-885 (nucleotides 30-49 of CRP-17F) of SEQ ID NO: 1. CRP-12R (SEQ ID NO: 10): Corresponds to nucleotides 1478-1498 of SEQ ID NO: 1; an inverse complementary form. CRP-18F (SEQ ID NO: 11): Corresponds to nucleotides 537-564 (nucleotides 1-28 of CRP-18F) and nucleotides 714-734 (nucleotides 29-49 of CRP-18F) of SEQ ID NO: 1. CRP-19F (SEQ ID NO: 12): Corresponds to nucleotides 537-564 (nucleotides 1-28 of CRP-19F) and nucleotides 676-700 (nucleotides 29-53 of CRP-19F) of SEQ ID NO: 1. CRP-20F (SEQ ID NO: 13): Corresponds to nucleotides 537-564 (nucleotides 1-28 of CRP-20F) and nucleotides 625-644 (nucleotides 29-48 of CRP-20F) of SEQ ID NO: 1.

[0162] The following PCR products were produced: PCR 1: PCR using genomic DNA from Escherichia coli (E. coli) W3110 and primers crp-13f and crp-14r resulted in the production of a 0.4kb PCR product. PCR 2: PCR using genomic DNA from Escherichia coli (E. coli) W3110 and primers crp-17f and crp-12r resulted in the production of a 0.65 kb PCR product. PCR 3: PCR using genomic DNA from Escherichia coli (E. coli) W3110 and primers crp-18f and crp-12r resulted in the production of a 0.8kb PCR product. PCR 4: PCR using genomic DNA from Escherichia coli (E. coli) W3110 and primers crp-19f and crp-12r resulted in the production of a 0.8kb PCR product. PCR 5: PCR using genomic DNA from Escherichia coli (E. coli) W3110 and primers crp-20f and crp-12r resulted in the production of a 0.8kb PCR product.

[0163] For example, OE-PCR (Overlap Extension PCR), as described in Hilgarth and Lanigan, MethodsX (2020), 7:100759, resulted in the production of the following fusion PCR products: PCR 6: A 1kb fusion PCR product for the preparation of Escherichia coli (E. coli) strain W3110-crpP-del as a result of OE-PCR of PCR 1 and PCR 2 and primers crp-13f and crp-12r. PCR 7: A 1.2kb fusion PCR product for the preparation of Escherichia coli (E. coli) strain W3110-crp-Preg as a result of OE-PCR of PCR 1 and PCR 3 and primers crp-13f and crp-12r. PCR 8: A 1.2kb fusion PCR product for the preparation of Escherichia coli (E. coli) strain W3110-crp-Preg2 as a result of OE-PCR of PCR 1 and PCR 4 and primers crp-13f and crp-12r. PCR 9: A 1.2kb fusion PCR product for the preparation of Escherichia coli (E. coli) strain W3110-crp-Preg3 as a result of OE-PCR of PCR 1 and PCR 5 and primers crp-13f and crp-12r.

[0164] Transformation of Escherichia coli (E. coli) W3110-crp::kan-sacB×pKD46: The fusion PCR products PCR 6, PCR 7, PCR 8, and PCR 9 were transformed into E. coli (W3110-crp::kan-sacB×pKD46), respectively, and clones were selected on kanamycin-free LBS plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar). Only clones that no longer contained the active sacB gene could grow on the LBS plate. To select clones that no longer contained the active Kan gene and whose growth was inhibited in the presence of kanamycin, these clones were seeded on LBkan plates. One clone was selected in each case, and the temperature-sensitive plasmid pKD46 was removed by incubation at 42°C. Clones lacking the pKD46 plasmid were ampicillin-sensitive and could no longer grow on LBamp plates. Each clone was named Escherichia coli (E. coli)W3110-crpP-del, Escherichia coli (E. coli)W3110-crp-Preg, Escherichia coli (E. coli)W3110-crp-Preg2, and Escherichia coli (E. coli)W3110-crp-Preg3, respectively.

[0165] PCR analysis of transformed organisms: Clones exhibiting positive growth in the presence of sucrose and negative growth in the presence of kanamycin were selected, and 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).

[0166] In PCR reactions using primers crp-7f and crp-8r ("Phusion® High-Fidelity" DNA polymerase, Thermo Scientific®), genomic DNA was used to verify whether the Kan-sacB cassette was correctly replaced by the respective fusion PCR product. Clones with PCR products of the expected size were selected for each case, and DNA sequencing of the PCR products (Eurofins Genomics) was performed to analyze the correct incorporation of the modified promoter sequence and the unmodified crp cds sequence.

[0167] The PCR product had the following predicted size: Escherichia coli (E. coli) strain W3110-crpP-del: 1397 nucleotides. Deletion of nucleotides 565 to 865 from SEQ ID NO: 1. Escherichia coli (E. coli) strain W3110-crp-Preg: 1549 nucleotides. Deletion of nucleotides 565 to 713 from SEQ ID NO: 1. Escherichia coli (E. coli) strain W3110-crp-Preg2: 1587 nucleotides. Deletion of nucleotides 565 to 675 from SEQ ID NO: 1. Escherichia coli (E. coli) strain W3110-crp-Preg3: 1637 nucleotides. Deletion of nucleotides 565 to 624 from SEQ ID NO: 1.

[0168] [ Example 3] Creation of cysteine-producing strains By using the cysteine-specific plasmid pCys, we created cysteine-producing strains (strains with undiced cysteine ​​biosynthesis) (Figure 3).

[0169] pCys is a derivative of the plasmid pACYC184-cysEX-GAPDH-ORF306 disclosed in European Patent No. 0885962.

[0170] In addition to the origin of replication and the tetracycline resistance gene (parent vector pACYC184), plasmid pACYC184-cysEX-GAPDH-ORF306 contains the cysEX allele, which encodes a reduced serine O-acetyltransferase with cysteine-mediated feedback inhibition, and the efflux gene ydeD(ORF306), whose expression is regulated by the constitutive GAPDH promoter.

[0171] Furthermore, pCys is cloned after the ydeD(QRF306) efflux gene and further contains the serA317 gene fragment, which encodes the N-terminal 317 amino acids of the SerA protein (full length of 410 amino acids). The *E. coli* serA gene is disclosed in the "GenBank" gene database under gene ID 945258. serA317 was disclosed by Bell et al., 2002 (see above, where it is referred to as "NSD:317"), and encodes a serine feedback-resistant mutant of 3-phosphoglycerate dehydrogenase. The expression of serA317 is regulated by the serA promoter.

[0172] E. coli strains W3110, W3110-crp::kan-sacB, W3110-crpP-del, W3110-crp-Preg, W3110-crp-Preg2, and W3110-crp-Preg3 were each transformed with plasmid pCys.

[0173] Transformants containing plasmids were selected on LBtet-agar plates (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 1.5% agar, 15 mg / L tetracycline). One clone was selected in each case. The strains used in the following examples were named as follows: Escherichia coli (E.coli) W3110×pCys, Escherichia coli (E.coli)W3110-crp::kan-sacB×pCys Escherichia coli (E.coli)W3110-crpP-del×pCys Escherichia coli (E.coli)W3110-crp-Preg×pCys E. coli W3110-crp-Preg2×pCys E. coli W3110-crp-Preg3×pCys

[0174] [ Example 4] Culture in a shaking flask Pre-culture: As a pre-culture for culturing in a shaking flask, each cysteine-producing strain from Example 3 was inoculated into 3 ml of LBtet medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 mg / L tetracycline), and incubated in a shaker (Infors) at 30°C and 135 rpm for 16 hours. 600 The optical density of the culture per 1 ml of culture, measured at 600 nm, was determined.

[0175] Main culture: Subsequently, a certain volume of the preculture was inoculated into a 300 ml Erlenmeyer flask (with baffles) containing 30 ml of SM1 medium containing 15 g / L glucose, 5 mg / L vitamin B1, 2 g / L sodium thiosulfate, and 15 mg / L tetracycline, and the OD was adjusted to 0.05. 600 / ml achieved.

[0176] Composition of SM1 medium: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L (NH4)2SO4, 0.3 g / L MgSO4×7H2O, 0.015 g / L CaCl2×2H2O, 0.002 g / L FeSO4×7H2O, 1 g / L Sodium citrate 3×2H2O, 0.1 g / L NaCl; 1 ml / L trace element solution.

[0177] Composition of trace element solution: 0.15g / L Na2MoO4×2H2O, 2.5g / L H3BO3, 0.7g / L CoCl2×6H2O, 0.25g / L CuSO4×5H2O, 1.6g / L MnCl2×4H2O, 0.3g / L ZnSO4×7H2O.

[0178] To isolate RNA for RT-PCR experiments, 30 ml batches were incubated at 30°C and 140 rpm, and the OD was reduced to 0.5 / ml. 600 The sample was left to stand until it reached / ml (4-5 hours of incubation).

[0179] To determine cysteine ​​production, 30 ml batches were left at 30°C and 140 rpm for 24 hours.

[0180] For a comparative analysis of cysteine ​​production in the WT strain with unregulated cysteine ​​biosynthesis in Example 6, E. coli strain W3110, which does not contain plasmid pCys, was cultured in the same manner as the strain with unregulated cysteine ​​biosynthesis, but without tetracycline in all media.

[0181] [ Example 5 Analysis of CRP expression by RT-PCR 1) RNA isolation: 0.5 / ml OD from Example 4 600RNA was stabilized by mixing 0.5 ml of main culture containing 0.5 ml of RNA with a concentration of 0.5 ml / ml with 1 ml of RNA-Protect(R) ("Bacteria Reagent", Qiagen). RNA was isolated from the samples using the RNeasy RNA Isolation Kit (RNeasy Mini Kit, Qiagen) according to the manufacturer's instructions. Depending on the strain, 8–10 μg of RNA was isolated from 0.5 ml of main culture. RNA concentration was determined using the "Qubit® RNA BR Assay Kit" according to the manufacturer's instructions and a Qubit 3.0 fluorometer from Thermo Fisher Scientific.

[0182] 2) Preparation of complementary DNA (cDNA): Using the QuantiNova® Reverse Transcription Kit (Qiagen), 1.5 μg of isolated RNA was converted to cDNA by reverse transcription. The cDNA concentration was determined using the Qubit® dsDNA HS Assay Kit according to the manufacturer's instructions, and a Qubit 3.0 fluorometer from Thermo Fisher Scientific. The cDNA yield from 1.5 μg of RNA was 350-390 ng.

[0183] 3) RT-PCR: A RotorGene Q 2plex RT-PCR instrument from Qiagen was used, operated by RotorGene Q control and evaluation software from the same manufacturer. In addition, the QuantiNova® Sybr® Green PCR Kit for Real-Time PCR (Qiagen) was used.

[0184] We analyzed the expression of the crp gene and the cysG gene as a reference gene. The expression of the reference gene cysG remained largely unchanged (Zhou et al., 2011 (see above)), while the expression of the crp gene served as a useful internal standard for normalization.

[0185] The following primers were used: CRP gene: Primers CRP-1F (SEQ ID NO: 14) and CRP-2R (SEQ ID NO: 15).

[0186] cysG gene: Primers cysg-1f (SEQ ID NO: 16) and cysg-2r (SEQ ID NO: 17).

[0187] The cDNAs analyzed were from shaking flask cultures of six strains transformed with plasmid pCys from Example 4. CRP expression in the W3110×pCys strain served as a reference point for evaluating the relative expression of the crp gene (comparison strain; crp expression with wild-type promoter), and crp expression in the other strains was compared against this reference point. For statistical evidence, each RT-PCR reaction was replicated equally and simultaneously four times (quadruplication). For the cDNAs of the six strains, RT-PCR reactions were performed simultaneously in a single run for the cysG reference gene and the crp gene to be determined, which corresponded to a total of 48 RT-PCR reactions to determine the expression of two genes per strain in four pairs.

[0188] RT-PCR reaction: The RT-PCR reaction for the analysis of crp gene expression (final volume 20 μl) consisted of 10 μl of H2O containing 20 ng of cDNA and 14 pmol each of crp-specific primers crp-1f and crp-2r, and 10 μl of QuantiNova® Sybr® Green Mastermix for Real-Time PCR (Qiagen) containing the fluorescent dye Sybr® Green for the detection of DNA polymerase and newly formed DNA. The RT-PCR reaction for the analysis of cysG gene expression (final volume 20 μl) consisted of 10 μl of H2O containing 20 ng of cDNA and 14 pmol each of cysG-specific primers cysg-1f and cysg-2r, and 10 μl of QuantiNova® Sybr® Green Mastermix for Real-Time PCR (Qiagen) containing the fluorescent dye Sybr® Green for the detection of DNA polymerase and newly formed DNA.

[0189] After an initial incubation of 2 minutes at 95°C, the RT-PCR program consisted of 40 cycles of 5 seconds at 95°C and 10 seconds at 60°C, respectively. The fluorescence signal caused by the binding of Sybr(R)Green to the newly formed double-stranded DNA was recorded by a detector in the RT-PCR instrument, and the plot of the fluorescence signal against time was analyzed using evaluation software.

[0190] 4) Evaluation of RT-PCR: The evaluation software determined the CRP expression in the strain compared to the CRP expression in the comparative strain W3110×pCys. The basis of the analysis was the so-called 2 -ΔΔCT This is the method, and its mathematical derivation is described by Livak and Schmittgen 2001 (see above). For comparison strains, this analysis is 1 / 2- ΔΔCT A value was given. Therefore, a value > 1 means increased CRP expression, and a value < 1 means decreased CRP expression accordingly.

[0191] The plot of the fluorescence signal against time is used by evaluation software to determine the average of the so-called CT values ​​(CT "cycle threshold") for each sample determined in a set of four, and in the first step, for each strain, ΔCT = CT crp -CT cysG This was used to form the difference ΔCT between the CT value of the crp gene and the CT value of the cysG reference gene. In the second step, ΔΔCT = ΔCT - ΔCT W3110×pCys As a result, the difference ΔΔCT was formed between the ΔCT value of the strain to be analyzed and the ΔCT value of the comparison strain W3110×pCys. In the third step, the value 2 was a measure of the relative expression of the crp gene in the strain, compared with the expression of the crp gene in the comparison strain W3110×pCys (Wt crp promoter). -ΔΔCT The ΔΔCT value was used to form the formula. Table 1 summarizes the relative CRP expression in the strains examined compared to the expression in the comparison strain W3110×pCys (expression normalized to a value of 1).

[0192] [Table 1]

[0193] [ Example 6 Determination of cysteine ​​production from supernatant of shaking flask cultures After 24 hours of incubation, a sample was taken from the main culture from Example 4, and the cell density OD was measured. 600 The total cysteine ​​content in the culture supernatant and per ml was determined, and a colorimetric assay according to Gaitonde 1967 (see above) was used for the quantitative determination of cysteine. 20 μl of culture supernatant was used in 2 ml of the assay. It should be noted that under highly acidic reaction conditions, this assay does not distinguish between cysteine ​​and 2-methylthiazolidinedion-2,4-dicarboxylic acid (thiazolidinedion), which is a condensation product of cysteine ​​and piruvate as described in European Patent No. 0885962. L-cystine, formed by the oxidation of two cysteine ​​molecules according to formula (2), is similarly detected as cysteine ​​in this assay by reduction with dithiothreitol in a dilute solution at pH 8.0.

[0194] Table 2 shows the cell density (OD) of the main culture. 600 This includes the cysteine ​​content (mg / ml) and cysteine ​​content (cysteine ​​in mg / ml), as well as specific cysteine ​​production relative to cell density (cysteine ​​in mg / OD) and specific cysteine ​​production in percentage compared to the comparative strain W3110×pCys (=100%) (cysteine ​​in percentage of W3110×pCys).

[0195] [Table 2]

[0196] [ Example 7 Cysteine ​​production in fermenters In production-scale fed-batch fermentation, comparisons were made among the following Escherichia coli (E. coli) strains: W3110×pCys, W3110-crp::kan-sacB×pCys, W3110-crp-Preg2×pCys, and W3110-crp-Preg3×pCys. The E. coli strains with the lowest CRP expression, W3110-crpP-del×pCys and W3110-crp-Preg×pCys (Table 1), could not be further investigated due to poor growth in the fermenters.

[0197] Preculture 1: Each strain was inoculated into 20 ml of LBtet medium in a 100 ml Erlenmeyer flask and incubated on a shaker (150 rpm, 30°C) for 7 hours.

[0198] Preculture 2: Subsequently, each pre-culture 1 was completely 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 4 for the composition of SM1 medium).

[0199] The culture was shaken at 150 rpm for 17 hours at 30°C in a 1L Erlenmeyer flask (Infors incubator shaker). After this incubation period, the cell density OD was measured. 600 The / ml ratio was 3-5.

[0200] Main culture: Fermentation was carried out in a DASGIP(R) Parallel Bioreactor System for Microbiology fermenter from Eppendorf. 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 MgSO4×7H2O, 0.015 g / L CaCl2×2H2O, 0.075 g / L FeSO4×7H2O, 1 g / L sodium citrate3×2H2O, and 1 ml of trace element solution (see Example 6), 0.005 g / L vitamin B1, and 15 mg / L tetracycline.

[0201] First, the pH in the fermenter was adjusted to 7.0 by adding a 25% NH4OH solution using a pump. During fermentation, the pH was maintained at 7.0 by automatic correction with 25% NH4OH.

[0202] For inoculation, 100 ml of pre-culture 2 was pumped into the fermenter vessel. Therefore, the initial volume was approximately 1 L. The culture was initially stirred at 400 rpm and aerated with sterile compressed air through a sterile filter at an aeration rate of 2 vvm (vvm: liters of compressed air per liter of fermentation volume per minute, specified as the amount of compressed air added to the fermentation batch). Under these initial conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0203] The target value for O2 saturation during fermentation was set at 30%. After the O2 saturation fell below the target value, an adjustment cascade was initiated to restore the O2 saturation to the target value. This involved 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).

[0204] Fermentation was carried out at a temperature of 30°C. After 2 hours of fermentation, a sulfur source in the form of a sterile 60% (w / v) stock solution of sodium thiosulfate × 5H2O was supplied at a rate of 1.5 ml / hour.

[0205] When the glucose content in the fermenter decreased from the initial 15 g / L to approximately 2 g / L, a 56% (w / w) glucose solution was continuously added by weighing. The supply rate was then adjusted to ensure that the glucose concentration in the fermenter did not exceed 2 g / L. The glucose content was determined using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0206] The fermentation time was 48 hours. Subsequently, samples were taken from the fermentation batches, and the content of L-cysteine ​​and its derivatives in the culture supernatant (mainly L-cysteine ​​and thiazolidinediones) and its derivatives in the precipitate (L-cystine) was determined separately. For this purpose, a colorimetric assay according to Gaitonde 1967 was used in each case (see above). L-cystine present in the precipitate had to be dissolved in 8% (v / v) hydrochloric acid before it could be quantified in the same manner. Finally, the total amount of cysteine ​​was determined as the sum of cysteine ​​in the pellet and supernatant.

[0207] As summarized in Table 3, the cell density OD of the examined strains 600 The levels were comparable in g / ml. In contrast, the amount of cysteine ​​produced per volume (in g / L) was higher in W3110-crp::kan-sacB×pCys, W3110-crp-Preg2×pCys, and W3110-crp-Preg3×pCys than in the control strain W3110×pCys, which has an unmodified crp promoter.

[0208] [Table 3]

Claims

1. A microbial strain of the Escherichia coli species having a deregulated cysteine ​​biosynthesis pathway and thereby suitable for the fermentation production of at least one substance selected from L-cysteine, L-cystine, and thiazolidinediones, characterized in that, as a result of a mutation in the crp promoter sequence, the relative expression of the crp gene is reduced by at least 9% compared to the expression of the crp gene having a wild-type promoter sequence. Mutations in the CRP promoter sequence i) Deletion of at least nucleotides 565–624 from the CRP promoter sequence identified at nucleotides 565–865 of SEQ ID NO: 1; ii) Insertion of at least one nucleotide in the region between nucleotides 640 to 714 of the CRP promoter sequence identified by nucleotides 565 to 865 of SEQ ID NO: 1; and iii) Insertion of at least one nucleotide in the region between nucleotides 640 and 714 of the CRP promoter sequence identified at nucleotides 565 to 865 of SEQ ID NO: 1, and simultaneous deletion of 73 nucleotides of the CRP promoter (SEQ ID NO: 1, nucleotides 641 to 713) A microbial strain selected from a group consisting of the following.

2. The microbial strain according to claim 1, characterized in that the amino acid sequence of the Crp protein is not mutated.

3. A microbial strain according to one or more claims 1 and 2, characterized in that the CRP promoter sequence is completely deleted.

4. A microbial strain according to one or more of claims 1 to 3, characterized in that the expressed Crp protein is sequence number 2.

5. A method for producing at least one compound selected from L-cysteine, L-cystine, and thiazolidinedione, characterized in that a microbial strain described in any one of claims 1 to 4 is used.

6. The method according to claim 5, characterized in that the formed L-cysteine, L-cystine, or thiazolidinedione is isolated.

7. The method according to claim 6, characterized in that crp expression in a microbial strain having a modified crp promoter sequence is reduced by at least 9% compared to a corresponding microbial strain having a Wt crp promoter sequence, and when the microbial strain is used, the yield of a substance selected from L-cysteine, L-cystine, and thiazolidinedione in g / l increases by at least 10% (w / v).

8. The method according to one or more of claims 5 to 7, characterized in that the method is a fermentation method and the fermentation volume is at least 1 L.

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