Improved cysteine ​​producing strains

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 and thus suitable for the fermentative production of at least one substance selected from L-cysteine, L-cystine and thiazolidine, 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 with a wild-type promoter sequence. A particular advantage is that said microbial strain forms increased amounts of a substance selected from L-cysteine, L-cystine and thiazolidine compared to the corresponding microbial strain with the expression of the crp gene with a wild-type promoter. Thus, the present invention also provides a method for producing at least one compound selected from L-cysteine ​​and its derivatives L-cystine and thiazolidine using said microbial strain. [Background technology]

[0002] Cysteine, abbreviated as Cys or C, is an α-amino acid with the side chain -CH2-SH. Since the naturally occurring enantiomeric form is L-cysteine ​​and since L-cysteine ​​is the only proteinogenic amino acid, in the context of the present invention, when the term cysteine ​​is used without a descriptor, it is L-cysteine ​​that is meant. Oxidation of a sulfhydryl group can result in two cysteine ​​residues forming a disulfide bond together, resulting in the formation of cystine, to which the same description applies, i.e., in the absence of a descriptor, it is the L-enantiomer (or L-cystine, i.e. (R,R)-3,3'-dithiobis(2-aminopropionic acid)) that is meant in the present invention. Since L-cysteine ​​can be formed from the amino acid methionine, it 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 (EP 0 885 962).

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

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

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

[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, there are also methods for the fermentative production of cysteine. Prior art for the fermentative preparation of cysteine ​​using microorganisms is disclosed, for example, in EP 0 858 510, EP 0 885 962, EP 1 382 684, EP 1 220 940, EP 1 769 080, EP 2 138 585 and WO 2021 / 259491. Bacterial host organisms used include strains of the genus Corynebacterium and members of the Enterobacteriaceae family, such as Escherichia coli or Pantoea ananatis.

[0007] Wild-type host organisms without further modifications contain a cysteine ​​biosynthetic pathway (see, for example, the KEGG pathway database: "Cysteine ​​and methionine metabolism"), which is regulated so that the amount of cysteine ​​produced is only that required for cell growth. For example, as described in the review by Wada and Takagi 2006 (see above), cysteine ​​biosynthesis is regulated in WT strains by so-called feedback inhibition of key enzymes. 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 ​​biosynthetic pathway, and their feedback inhibition by L-serine and L-cysteine, respectively, prevents the formation of more cysteine ​​than is required by the cell. Such wild-type strains, as disclosed for example for E. coli strain K12 W3110 in Table 2 of the present invention, do not produce detectable cysteine ​​and are therefore not suitable for cysteine ​​production despite the presence of a cysteine ​​biosynthetic pathway. A wild-type microbial strain is made suitable for cysteine ​​production by deregulation of the cysteine ​​biosynthetic pathway. Various methods are available for producing microbial strains with deregulated cysteine ​​biosynthesis and characterized by improved cysteine ​​production. In addition to the traditional method of using mutation and selection to arrive at improved cysteine-producing strains, specific genetic modifications have also been made to strains to achieve effective overproduction of cysteine.

[0008] For example, introduction of a cysE allele encoding a serine O-acetyltransferase with reduced feedback inhibition by cysteine ​​resulted in increased cysteine ​​production (EP 0 858 510; Nakamori et al., Appl. Env. Microbiol. (1998) 64:1607-1611). The feedback-resistant CysE enzyme largely uncouples the formation of O-acetyl-L-serine, the direct precursor of cysteine, from intracellular cysteine ​​levels.

[0009] O-acetyl-L-serine is formed from L-serine and acetyl-CoA. It is therefore very important to provide sufficient amounts of L-serine for cysteine ​​production. This can be achieved by introducing a serA allele encoding 3-phosphoglycerate dehydrogenase with reduced feedback inhibition by L-serine. As a result, the formation of 3-phosphohydroxypyruvate, the biosynthetic precursor of L-serine, is largely uncoupled from the L-serine level in the cell. Examples of such SerA enzymes are described in EP 0 620 853 and EP 1 496 111. Alternatively, Bell et al., Eur. J. Biochem. (2002) 269: 4176-4184, discloses modifications to the serA gene to deregulate the enzyme activity.

[0010] Increasing the transport of cysteine ​​out of the cell is another way to increase the product yield in the medium. This can be achieved by overexpressing so-called export genes, which code for membrane-bound proteins that mediate the export of cysteine ​​out of the cell. Various export genes for cysteine ​​export have been described (EP 0 885 962, EP 1 382 684). Export of cysteine ​​from the cell into the fermentation medium has several advantages.

[0011] 1) L-cysteine ​​is constantly removed from the intracellular reaction equilibrium, so that intracellular levels of this amino acid are kept low and feedback inhibition of sensitive enzymes by L-cysteine ​​is halted. (1) L-cysteine ​​(intracellular) <-> L-cysteine ​​(culture medium)

[0012] 2) L-cysteine ​​secreted into the medium is oxidized in the presence of oxygen to form the disulfide L-cystine, which is introduced into the medium during cultivation (European Patent No. 0 885 962): (2) 2 L-cysteine ​​+ 1 / 2O2 -> L-cystine + H2O Since the solubility of L-cystine in aqueous solutions at neutral pH is extremely low compared to cysteine, this disulfide precipitates already 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 shifting the equilibrium of reactions (1) and (2) toward the product.

[0013] 3) If the amino acids can be obtained directly from the fermentation medium, purification of the product is significantly less technically complicated than if the product accumulated intracellularly and cell disruption was first required.

[0014] The cysteine ​​producing strains / microbial strains capable of producing cysteine / microbial strains with a deregulated cysteine ​​biosynthetic pathway / microbial strains with deregulated cysteine ​​biosynthesis are characterized by at least one modification / feature selected from feedback resistant SerA enzyme, feedback resistant CysE enzyme and overexpression of a cysteine ​​excretion 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 (EP 1 571 223).

[0016] In addition to the genetic modification of the cysteine ​​producing strains, optimization of the fermentation process, i.e. how the cells are cultivated, also plays an important role in the development of an efficient production process. Various culture parameters such as the nature and metering of the carbon and energy sources, temperature, oxygen supply (EP 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] Due to ever-rising raw material and energy costs, there is a constant need to increase product yields in cysteine ​​production to improve the economic viability of the process. [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 Summary of the Invention

[0020] The object of the present invention is to provide a microbial strain for the fermentative production of cysteine, L-cystine and / or thiazolidine, which makes it possible to achieve higher yields of L-cysteine, L-cystine and / or thiazolidine in fermentation compared to known strains from the prior art. [Means for solving the problem]

[0021] This object is achieved by a microbial strain which comprises a deregulated cysteine ​​biosynthetic pathway and is thereby suitable for the fermentative production of at least one substance selected from L-cysteine, L-cystine and thiazolidine, 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 with a wild-type promoter sequence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0023] From transcriptome analyses (e.g., Gosset et al., J. Bacteriol. (2004) 186:3516-3524), the expression of a large number of genes (>400 genes) is known to be affected by Crp or Crp-cAMP. Furthermore, Crp is part of a branched regulatory network of global transcription factors that influence each other (reviewed in Figure 1 of Frendorf et al., Comput. Structural Biotechnol. J. (2019) 17:730-736) and that affect the expression of target genes depending on the metabolic state of the cell.

[0024] Therefore, due to the large number of genes regulated by Crp-cAMP and due to the mutual influence of global transcription factors when expression of the crp gene is altered, it is not possible a priori to say how increased or attenuated crp expression will affect cellular metabolism in general or how increased or attenuated crp expression will affect biosynthetic metabolic pathways, such as the biosynthetic metabolic pathway for L-cysteine.

[0025] Frendorf et al., 2019 (see above) provide an overview of the numerous previously studied variants of the Crp protein. The variants studied so far are exclusively modifications of the Crp amino acid sequence caused by modifications of the crp cds. EP 3 686 214, EP 3 686 215 and EP 3 725 800 (all CJ Corp., Korea) likewise disclose variants of the Crp amino acid sequence and their use for producing L-amino acids, in particular L-threonine and L-tryptophan.

[0026] What has not been studied in the prior art is the effect on metabolism, particularly cysteine ​​biosynthesis, of the altered expression of the crp WT gene due to a mutation in the crp promoter in a microbial strain with a deregulated cysteine ​​biosynthetic pathway. The present invention differs from the prior art in that it is preferably not the amino acid sequence of Crp that is altered, but the nucleotide sequence of the crp promoter. That is, it is the amino acid sequence and thus the properties of the activity of the Crp protein as a transcription factor that has been altered in the prior art. In contrast, in the present invention, the amino acid sequence and thus the properties of the activity of the Crp protein (wild type Crp) preferably remain unchanged. However, it is the expression, i.e. the amount of protein, that is altered by modification of the crp promoter. From the prior art, it was not possible to predict how this treatment would affect cellular metabolism in general and cysteine ​​biosynthesis in particular in a microbial strain with a deregulated cysteine ​​biosynthetic pathway. For example, Liu et al., J. Agric. Food Chem 2020, 68:14928-14937, in Figure 2 of the publication, describes E. coli strain BW25113-pLH03, which showed both increased crp gene expression and improved L-cysteine ​​production compared to strain JM109-pLH03. Thus, it was completely unexpected that reduced expression of the crp gene in a microbial strain with a deregulated cysteine ​​biosynthetic pathway would result in improved cysteine ​​production, as disclosed in the present invention.

[0027] Mutation of the crp promoter sequence, preferably as a result of shortening the crp promoter sequence or as a result of a combination of insertion and shortening of the crp promoter sequence, results in weakened crp expression, with the crp cds particularly preferably remaining unchanged. The weakened crp expression results in improved production of L-cysteine ​​in microbial strains with a deregulated cysteine ​​biosynthetic pathway in a manner not previously known. In a particularly preferred embodiment, mutation of the crp promoter sequence results in no Crp protein being expressed at all.

[0028] Detection of crp gene expression: In order to be able to compare the expression of the crp gene in various strains, a method for quantitative detection of the expression of the crp gene is required. In general, various known test methods are available for the quantitative detection of gene expression. Immunological detection methods involve the binding of specific antibodies to the expressed Crp protein. Thus, the methods ELISA ("enzyme-linked immunosorbent assay") and Western blot, known to those skilled in the art, allow the quantitative determination of the expressed protein by a color reaction that occurs via the bound Crp-specific antibody. Another method of quantitative detection of gene expression involves determining gene-specific RNA of expressed genes, such as crp RNA, in total cellular RNA (total RNA). In the context of the present invention, the gene studied for its expression is called the target gene (e.g. crp gene). Methods known to the skilled artisan are Northern blot for direct detection of gene-specific RNA and preferably 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 carried out, for example, as follows.

[0030] 1) The cells to be analyzed (e.g., E. coli cells from a culture (as described in Example 4) of the strain to be analyzed (Examples 3 and 5)) are mixed with an RNA stabilizing reagent (e.g., RNA-Protect® "Bacteria Reagent" from Qiagen) after harvesting, and total RNA is extracted (e.g., using the RNeasy RNA extraction kit from Qiagen) and quantified (e.g., using the "Qubit™ 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) The cDNA is then 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 conventional techniques. Instruments for performing RT-PCR analysis are commercially available, including reagent kits for preparing RT-PCR reactions and evaluation software (e.g., from Qiagen). 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, which determined the relative expression of the crp gene in strains with modified crp promoters compared to the comparison strain E. coli W3110×pCys with WT crp promoter.

[0033] Relative expression: In the context of the present invention, -ΔΔCTThe relative gene expression determined as a value is defined as the expression of the crp gene in a microbial strain according to the invention with a deregulated cysteine ​​biosynthetic pathway in which the crp promoter sequence has been mutated (e.g. the cysteine ​​producing strains E. coli W3110-crp::kan-sacB×pCys, E. coli W3110-crpP-del×pCys, E. coli W3110-crp-Preg×pCys, E. coli W3110-crp-Preg2×pCys and E. coli W3110-crp-Preg3×pCys, as described in Example 3) compared to the expression of the crp gene in a corresponding comparison strain (e.g. in E. coli strain W3110×pCys with a WT crp promoter). The RT-PCR analysis method used in Example 5 of the present invention is quantitative RT-PCR and the so-called 2-fold PCR as described by Livak and Schmittgen, Methods (2001) 25:402-408. -ΔΔCT This involves the analysis of relative gene expression using the RotorGene Q control and evaluation software of 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 maximum CT value of the crp gene (CT crp ) and a reference gene. The reference gene serves for normalization of the 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 of the cysG gene as a reference gene (CT cysG ) was determined in Example 5, but the expression of the gene is known to change 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., the 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 with altered expression of the crp gene and the ΔCT value of a comparison strain with WT expression of the target gene (e.g., E. coli W3110×pCys). ΔΔCT=ΔCT−ΔCT W3110×pCys .

[0037] 2 -ΔΔCT Value:Value2 -ΔΔCT is formed from the ΔΔCT values ​​and is a measure of the expression of the crp gene in the modified strain compared to the expression of the crp gene in a comparison strain (e.g., 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 among various strains, the same amount of cDNA was used in RT-PCR, and 2- ΔΔCT The relative crp expression as a value is determined for each line. Comparison of strains -ΔΔCT The value has a value of 1. 2 -ΔΔCT A value >1 indicates increased expression compared to the comparison strain. 2 -ΔΔCT A value <1 indicates attenuated expression compared to the comparison strain.

[0039] Relative expression: -ΔΔCT From the comparison of values, it is possible to describe whether and to what extent a mutation in the promoter sequence of the crp gene has altered the expression of the crp gene. This change in gene expression (see Example 5) can be correlated with an altered characteristic of the strain, such as cell growth or yield of a metabolite, such as the production of the amino acid L-cysteine ​​(see Examples 6 and 7).

[0040] Open reading frame (ORF, synonymous with cds or coding sequence) refers to a region of DNA or RNA that begins with an initiation codon and ends with a termination codon and codes for the amino acid sequence of a protein. ORFs are also called coding regions or structural genes.

[0041] A gene refers to a segment of DNA that contains all the basic information to produce biologically active RNA. A gene contains a segment of DNA from which a single-stranded RNA copy is produced by transcription, and also contains expression signals involved in the regulation of this replication process. Expression signals include, for example, at least one promoter, transcription initiation, translation initiation, and ribosome binding site. In addition, expression signals can be terminators and one or more operators.

[0042] Promoter refers to the nucleotide sequence that is upstream of the 5' end of cds and allows the expression of a gene. Promoter is located in front of the RNA coding region in the direction of synthesis. Promoter mediates the initiation of transcription of a gene by RNA polymerase and contains a region that specifically interacts with DNA binding proteins called transcription factors.

[0043] Mutation refers to the modification of genetic material, including the modification of DNA sequences, and, in the case of sequences coding for proteins, also the amino acid sequence of the protein. In the context of the present invention, mutation includes 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 one or more amino acids in a protein for other amino acids. At the same time, the length of the DNA or protein sequence remains unchanged. Insertion refers to the incorporation of additional nucleotides in DNA, or additional amino acids in a protein. The term insertion also encompasses 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. If the modification results in the exchange of only individual nucleotides or individual amino acids for other nucleotides or amino acids, the term point mutation is used. Mutation also includes a combination of exchange, deletion and insertion.

[0044] In the context of the present invention, proteins such as Crp begin with a capital letter, whereas the genes for these proteins with their coding sequences are identified by lower case letters (eg, crp).

[0045] Thus, 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 crp gene from E. coli. E. coli Crp refers to the protein encoded by said cds and identified in SEQ ID NO:2. The protein is the Crp protein.

[0046] The abbreviation WT (Wt) refers to wild type. Wild type gene or wild type promoter refers to the form of the gene or promoter that naturally arises through evolution and is present in the wild type genome. The DNA sequence of the Wt gene or Wt promoter is publicly available in databases such as NCBI (National Center for Biotechnology Information, US National Library of Medicine).

[0047] Alleles define the states of a gene that can be converted into one another by mutation, i.e., by changes to the nucleotide sequence of DNA. A gene that occurs naturally in a microorganism is called a wild-type allele, and variants derived from the wild-type allele are called mutated alleles of that gene.

[0048] A homologous gene or sequence is to be understood as meaning that the DNA sequences of said genes or DNA portions are at least 80% identical, preferably at least 90% identical, particularly preferably at least 95% identical.

[0049] The degree of DNA identity is http: / / blast.ncbi.nlm.nih.gov / and is determined by the "nucleotide blast" program, which is based on the blastn algorithm. The algorithm parameters used to align two or more nucleotide sequences were the default parameters. The default general parameters are: max 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. The corresponding default scoring parameters are match / mismatch score = 1, -2; gap cost = linear.

[0050] The protein sequence is http: / / blast.ncbi.nlm.nih.gov / The sequences were compared using the "protein blast" program from BLOSUM62. This program uses the blastp algorithm. The algorithm parameters used to align two or more protein sequences were the default parameters. The default general parameters are: max 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. The default scoring parameters are: matrix = BLOSUM62; gap cost = presence: 11 extension: 1; composition adjustment = conditional composition score matrix adjustment.

[0051] In the case of a microorganism according to the invention having a deregulated cysteine ​​biosynthetic pathway, the mutation of the crp promoter sequence results in a relative expression of the crp gene of preferably at least 2.0 of 0.91. -ΔΔCT Particularly preferably, the value is at least 0.5. -ΔΔCT The value is particularly preferably 2 of 0.03. -ΔΔCT The expression of the crp gene in the microorganism with the wild-type promoter was reduced to a value of 2.00. -ΔΔCT The relative expression of the crp gene is preferably determined as described in Example 5.

[0052] 2 of 1.00 -ΔΔCT If expression of the crp gene having a wild-type promoter with a value of 0.01 is defined as 100% gene expression of the crp gene, the expression of the crp gene having a wild-type promoter with a value of 0.01 was reduced to at least 0.91. -ΔΔCT has a value of at least 0.5 -ΔΔCT and has a value of at least 0.03. -ΔΔCTThe crp gene expression in the microorganisms according to the invention having the values ​​of up to 91%, up to 50% and up to 3% of the expression of the crp gene with the wild-type promoter, respectively. That is to say, preferably, the method is characterized in that the crp expression in the microbial strain having the modified crp promoter sequence is reduced by at least 9% compared to the corresponding microbial strain having the Wt crp promoter sequence, and the yield of L-cysteine ​​in g / l is increased by at least 10% (w / v) when using this microbial strain.

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

[0054] Microbial strains suitable for the fermentative production of L-cysteine, L-cystine or thiazolidine include all microorganisms containing a deregulated cysteine ​​biosynthetic pathway leading to the synthesis of cysteine, cystine or thiazolidine. Such strains are disclosed, for example, in EP 0 885 962, EP 1 382 684, EP 1 220 940, EP 1 769 080 and EP 2 138 585 and WO 2021 / 259491.

[0055] A microbial strain having a deregulated cysteine ​​biosynthetic pathway is characterized by at least one of the following modifications:

[0056] a) The microbial strain is characterized by a modified serA gene encoding a 3-phosphoglycerate dehydrogenase (SerA) with feedback inhibition by L-serine that is at least two-fold reduced compared to the corresponding wild-type enzyme (e.g. as described in EP 1 950 287), and the SerA enzyme activity can be determined photometrically by oxidation of NADH dependent on the SerA substrate 3-phosphohydroxypyruvate, 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 a feedback inhibition by L-serine that is reduced by at least 5-fold, especially preferred by at least 10-fold, and in a more preferred embodiment by at least 50-fold.

[0058] and / or b) the microbial strain contains a modified cysE gene encoding a serine O-acetyltransferase (CysE) with feedback inhibition by cysteine ​​that is at least 2-fold reduced compared to the corresponding wild-type enzyme (e.g. as described in EP 0 858 510 or Nakamori et al. 1998 (see above)), and the CysE enzyme activity can be determined photometrically by the consumption of acetyl-CoA, a substrate for 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 a feedback inhibition by cysteine ​​that is reduced by at least 5-fold, especially preferred by at least 10-fold, and in a more preferred embodiment by at least 50-fold.

[0060] and / or c) the microbial strain exhibits at least a two-fold increased export of cysteine ​​to the outside of the cell due to overexpression of an efflux gene compared to the corresponding wild-type cell, the export of cysteine ​​being determined by photometric measurement of the extracellular content of cysteine ​​(including cysteine, cystine and the adduct 2-methylthiazolidine-2,4(R)-dicarboxylic acid formed from cysteine ​​and pyruvate) according to the method by Gaitonde, Biochem. J. (1967) 104:627-633, as described, for example, in EP 0 885 962.

[0061] Compared to wild-type cells, overexpression of the efflux genes preferably leads to an at least 5-fold, particularly preferably at least 10-fold, especially preferably at least 20-fold increased export of cysteine ​​out of the cell.

[0062] The efflux genes are preferably derived from the group consisting of ydeD (see EP 0 885 962), yfiK (see EP 1 382 684), cydDC (see WO 2004 / 113373), bcr (see US 2005-221453) and emrAB (see US 2005-221453) from E. coli or corresponding homologous genes from different microorganisms.

[0063] and / or d) The microbial strain can further be characterized in that at least one cysteine ​​degrading enzyme is attenuated to such an extent that the cells contain only at most 50% of this enzymatic activity compared to wild-type cells, the cysteine ​​degrading enzyme preferably coming from the group consisting of tryptophanase (TnaA) and cystathionine β-lyase (MalY, MetC).

[0064] Such strains are known, for example, from EP 0 858 510 and EP 0 885 962.

[0065] The microbial strains suitable for fermentative production of L-cysteine, L-cystine or thiazolidine described in the previous paragraph are deregulated in their cysteine ​​metabolism so as to produce an increased amount of L-cysteine ​​compared to a microbial strain that is not deregulated in cysteine ​​metabolism. This means that the cysteine ​​producing strain or the microbial strain capable of producing cysteine ​​is characterized by having a deregulated cysteine ​​biosynthetic pathway. In cells of a microbial strain that is not deregulated in cysteine ​​metabolism, the amount of L-cysteine ​​in the culture is about 0 g / l, so that the increased amount preferably means any amount exceeding 0.05 g / l of L-cysteine ​​measured in the culture after 24 hours of cultivation.

[0066] The amount of cysteine ​​can be quantified, for example, using the colorimetric assay according to Gaitonde 1967 (see above), 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 with a wild-type promoter (i.e. this microbial strain is also characterized by a deregulated cysteine ​​biosynthetic pathway), the microbial strain according to the invention with a deregulated cysteine ​​biosynthetic pathway, a mutated crp promoter sequence and a resulting reduced relative expression of the crp gene form, has an increased amount of substances selected from L-cysteine, L-cystine and thiazolidine, which is a great advantage. As demonstrated in Table 3 (Example 7), increasing the mutation of the crp promoter sequence up to a complete deletion of the crp promoter, i.e. a relative expression of the crp gene reduced by 9% to 100%, results in a significantly higher yield of total cysteine ​​in the fermentative process, i.e. the sum of the produced cysteine, cystine and thiazolidine. The microbial strain with the mutated promoter sequence in the crp gene is always compared to the corresponding microbial strain with the Wt promoter sequence in the crp gene, i.e. both microbial strains are deregulated with respect to their cysteine ​​metabolism.

[0068] When cysteine ​​is mentioned in the present invention, this always means L-cysteine ​​or one of its derivatives selected from L-cystine or thiazolidine. This means that the amount of cysteine ​​produced preferably means total cysteine, i.e. the sum of L-cysteine, L-cystine and thiazolidine produced. For example, the L-cystine formed can be reduced to form L-cysteine ​​and then detected simultaneously by measuring the produced cysteine. For example, when the determination is achieved by using the colorimetric assay by Gaitonde (see above), said assay cannot distinguish between L-cysteine ​​and 2-methylthiazolidine-2,4-dicarboxylic acid (thiazolidine), which is a condensation product of cysteine ​​and pyruvate, as described in EP 0 885 962, under highly acidic reaction conditions, and thiazolidine is also detected simultaneously by measuring the produced cysteine. According to the invention, this microbial strain forms increased amounts of substances selected from L-cysteine, L-cystine and thiazolidinedione, preferably L-cysteine ​​and L-cystine, particularly preferably L-cysteine.

[0069] Preferably, the microbial strain is characterized in that the amino acid sequence of the Crp protein is not mutated. That is, the amino acid sequence of the Crp protein is the Wt sequence specified in SEQ ID NO: 2. To achieve this, the coding sequence of crp contains only so-called silent mutations or does not contain any mutations at all. Silent mutations are defined as modifications of the cds that are due to the degeneracy of the genetic code and do not change the amino acid sequence resulting therefrom. This means that the crp cds are identical to the Wt DNA sequence available in the NCBI database for the crp of the corresponding organism or contain only silent mutations and code for an unmutated Crp protein having the Wt protein sequence. Particularly preferably, in the present invention, it is only the promoter sequence of the crp gene of the microbial strain that is mutated. Mutations of the promoter sequence of the crp gene from Escherichia coli (E. coli) comprising the sequence of SEQ ID NO: 1, nucleotides 565 to 865, are preferred, whereas the crp cds comprising the sequence of SEQ ID NO: 1, nucleotides 866 to 1498, are not mutated or contain only silent mutations and code for a protein having the protein sequence of SEQ ID NO: 2.

[0070] In the context of the present invention, a mutation in the crp promoter sequence is a) the crp promoter sequence of the crp gene has been partially or completely deleted, and / or b) the crp promoter sequence of the crp gene has been 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 Such modifications include: As a result, the expression of the crp gene is reduced, i.e. weakened or completely suppressed. As already mentioned above, by reduced is meant that the expression of the crp gene in the microorganism according to the invention is at most 91%, particularly preferably at most 50%, and especially preferably at most 3% of the expression of the crp gene with the wild-type promoter. In a particularly preferred embodiment, there is no detectable expression of the crp gene in the microorganism according to the invention.

[0071] Particularly preferably, the Crp protein does not carry a mutation in the aforementioned cases of reduced crp gene expression, ie the amino acid sequence is unchanged 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, the expression of the crp gene is weakened or completely suppressed by modification of the crp promoter. The weakening of the crp expression can also be achieved by completely or partially replacing the crp promoter with another weak promoter.

[0073] Particularly preferably, the modification of the crp promoter in the strain according to the invention comprises a complete or partial deletion of the crp promoter, or a modification of the crp promoter by one or more insertions or 5' and / or 3' extensions, or a combination of deletions and insertions.

[0074] Especially preferably, the modification of the crp promoter in the strain according to the invention comprises a complete or partial deletion of the crp promoter.

[0075] Preferably, the microbial strain is characterized in that the mutation in the crp promoter sequence comprises at least one deletion or insertion, particularly preferably at least one deletion.

[0076] In a particularly preferred embodiment, the mutation in the crp promoter sequence (which preferably comprises the sequence specified by nucleotides 565 to 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 in that at least nucleotides 565 to 624 are deleted from the crp promoter sequence defined by nucleotides 565 to 865 of SEQ ID NO: 1. In this case, the crp promoter sequence comprises at most nucleotides 625 to 865 from SEQ ID NO: 1.

[0078] Particularly preferably, the microbial strain is characterized in that the crp promoter sequence defined by nucleotides 565 to 865 of SEQ ID NO:1 is completely deleted.

[0079] Particularly preferred embodiments, including insertions, deletions and extensions, are given in the examples. For complete or partial deletion of the crp promoter sequence, see Example 2. For modification of the crp promoter sequence by one or more insertions, see Example 1; For modifications of the crp promoter sequence resulting in weakening or complete inhibition of promoter activity, see Example 5.

[0080] Preferably, the microbial strain is characterized in that it is a strain from the family Enterobacteriaceae or Corynebacteriaceae, particularly preferably from the family Enterobacteriaceae. Such strains are commercially available, for example, from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig), among others.

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

[0082] The crp gene from E. coli K12 is available in the NCBI gene database, for example, as an entry in the E. coli Genbank reference sequence with accession number NC_000913.3, nucleotides 3485255 to nucleotides 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 P. ananatis Genbank reference sequence with accession number NC_017554.1, nucleotides 430825 to nucleotides 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 the promoter sequences of the crp gene from Escherichia coli and the crp gene from Pantoea ananatis, and homologous sequences related to these sequences, the term homologous sequence having the definition specified above.

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

[0085] Thus, the microbial strain is preferably characterized in that the expressed Crp protein is SEQ ID NO: 2. This means that the expressed Crp protein has the Wt sequence (SEQ ID NO: 2) and that the mutation only concerns the crp promoter sequence (nucleotides 565 to 865 of SEQ ID NO: 1).

[0086] Furthermore, the production strain according to the invention can be further optimized for further improvement of cysteine ​​production. Optimization can be achieved, for example, genetically by further expressing one or more genes suitable for improving production properties. Said genes can be expressed in the production strain in a manner known per se, either as separate genetic constructs or in combination as an expression unit (so-called operon). Furthermore, the production strain can be optimized by inactivating further genes whose gene products have a negative effect on cysteine ​​production, in addition to reducing the expression of the crp gene. However, optimization is also possible in a manner known per se, by mutagenesis and selection of strains with improved cysteine ​​production.

[0087] An alternative approach envisages attenuating or completely suppressing the expression of the crp gene by adding an inhibitor, be it a chemical or a protein inhibitor, which 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 into 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 by mutagenic chemicals such as N-methyl-N'-nitro-N-nitrosoguanidine, or physically by UV irradiation), mutations are randomly generated in the genomic DNA, and then the desired mutants with modified crp promoter are selected from the large number of mutants generated, for example by quantitative determination of crp protein (e.g., immunologically by Western blot using a crp specific antibody) or quantitative determination of crp expression, for example by RT-PCR, after each mutant is isolated. Only mutants with 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 complex random mutagenesis and selection of the desired mutant with a modified crp promoter, the promoter of the crp gene can be subjected to targeted modification in a relatively simple manner, for example by the known mechanism of homologous recombination. Cloning systems for targeted gene inactivation by homologous recombination are known to those skilled in the art and are commercially available, for example as disclosed in the user manual of the "Quick and Easy E.coli Gene Deletion Kit" based on Red® / ET® technology from Gene Bridges GmbH (see "Technical Protocol, Quick&Easy E.coli Gene Deletion Kit by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012" and references cited therein).

[0090] According to the prior art, the crp promoter or a part of said promoter can be isolated and foreign DNA can be cloned into the crp promoter, thereby changing the sequence of the promoter. Thus, a DNA construct suitable for targeted modification of the crp promoter can consist of a 5' portion of DNA homologous to the genomic crp promoter, followed by a gene segment containing the foreign DNA, followed by a 3' portion of DNA also homologous to the genomic crp promoter.

[0091] Potential regions in the crp promoter for homologous recombination may include more than just the promoter sequence region. Potential regions may also include DNA sequences adjacent to the promoter, i.e. 5' flanking sequences before 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 relate to the cds of the crp gene (crp cds, nucleotides 866-1498 of SEQ ID NO: 1), in which case modification of the cds of the crp gene by homologous recombination is excluded. The foreign DNA is preferably a selection marker expression cassette, for example chosen from the 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 counterselection screening, a method for gene modification known to those skilled in the art and described in Examples 1 and 2. Said 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, which consists, starting from the 5' end, of 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 of a selection marker selected from the class of antibiotic resistance genes and b) an expression cassette of the sacB gene encoding the enzyme levansucrase, followed finally by further sequences homologous to the crp gene (including, for example, the sequence of the crp cds adjacent to the 3' of the crp promoter).

[0093] In a first step, the DNA construct is transformed into a production strain and antibiotic-resistant clones are isolated. The clones obtained are distinguished by the fact that they cannot grow on sucrose due to the co-integrated sacB gene. The two marker genes can be removed in a second step by the principle of counter-selection, where 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 then also the sensitivity to antibiotics. This method is used in examples 1 and 2 for the targeted shortening of the crp promoter of Escherichia coli (E. coli) (SEQ ID NO: 1, nucleotides 565-865). A DNA fragment suitable for this step comprises, starting from the 5' end, a sequence of at least 20 nucleotides long that is 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 long that is homologous to the target gene, e.g. the crp gene. DNA fragments can be prepared either chemically, for example by gene synthesis, or, as in Example 2, from individual DNA fragments by known so-called OE-PCR (e.g., Hilgarth and Lanigan, MethodsX (2020) 7:100759, https: / / doi.org / 10.1016 / j.mex.2019.12.001 The PCR products can be produced by overlap extension PCR as described in.

[0094] As an example of a strain according to the invention having attenuated crp expression due to a combination of insertion of a Kan-sacB cassette and a deletion in the crp promoter, the following E. coli strain: W3110-crp::kan-sacB is disclosed in the Examples, in which a 3.2 kb Kan-sacB cassette is inserted into the crp promoter between nucleotides 640 and 714 of SEQ ID NO: 1, resulting in a concomitant 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-fold, or only 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 E. coli strains are disclosed in the Examples (Example 2) as examples of strains according to the invention with attenuated crp expression due to truncation of the crp promoter: W3110-crpP-del: deletion of nucleotide 565 to nucleotide 865 from SEQ ID NO:1 W3110-crp-Preg: deletion of nucleotide 565 to nucleotide 713 from SEQ ID NO:1 W3110-crp-Preg2: deletion of nucleotides 565 to 675 from SEQ ID NO:1 W3110-crp-Preg3: deletion of nucleotide 565 to nucleotide 624 from SEQ ID NO:1

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

[0097] These results indicate that, on the one hand, increasing the truncation of the crp promoter reduced the relative expression of the crp gene from 0.91-fold to 0.03-fold that of the Wt promoter, or that the combination of insertion and deletion weakened the relative expression of the crp gene to 0.33-fold that of the Wt promoter.

[0098] For the E. coli crp gene, it is preferred that the deletion or a combination of insertion and deletion attenuates the relative expression of the crp gene from a value of 1 for the wild-type crp promoter to at least 0.91-fold, particularly preferably at least 0.5-fold, and especially preferably at least 0.33-fold, said value.

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

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

[0101] The present invention further provides a method comprising the production of at least one compound selected from L-cysteine, L-cystine and thiazolidine, characterized in that a microbial strain according to the invention is used. The method can comprise the cultivation of a microbial strain according to the invention in a shake flask (laboratory scale) or in a fermenter (production scale), with the fermenter (production scale) method being preferred. Shake flask cultivation also involves the specification of a specific medium and pH, as well as cultivation in the presence of oxygen and under constant movement (shaking), whereas in a fermenter it is possible to set and regulate more specific conditions regarding medium (e.g. regulation of the fermenter volume by feeding ingredients or partial discharge of the fermenter broth), temperature, pH, oxygen supply and mixing of the medium. Thus, both shake flask cultivation and fermenter cultivation are called fermentative methods, but differ in scale. In the fermentative method, a microbial producer strain is grown and metabolites are produced. A smaller culture can also be used as a preculture for inoculation of a larger culture.

[0102] The prior art does not disclose methods or production strains whereby attenuation of crp expression by modification of the crp promoter sequence can improve the production of cysteine.

[0103] The main product of the process according to the invention formed is L-cysteine. Oxidation gives the poorly soluble L-cystine according to formulas (1)-(3). L-cystine accumulates as a precipitate during fermentation (EP 0 885 962, EP 2 707 492). Formation of an adduct with pyruvate gives thiazolidine, which accumulates in the culture supernatant (EP 0 885 962).

[0104] Preferably, the process is characterized in that the L-cysteine, L-cystine or thiazolidine formed is isolated. Isolation of L-cysteine ​​is disclosed in EP 2 699 544 and EP 1 958 933. The precipitated L-cystine can be removed from the remaining constituents, for example using a decanter, followed by dissolution of the crude product with mineral acid, clarification of the crude product solution by centrifugation or filtration, decolorization of the solution, precipitation crystallization (EP 2 707 492).

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

[0106] As shown in the examples of the present application, attenuation of crp expression in microbial strains with deregulated cysteine ​​biosynthesis, suitable for cysteine, cystine or thiazolidine production, is suitable for significantly increasing the total cysteine ​​yield in fermentative processes, i.e. the sum of the produced cysteine, cystine and thiazolidine, which was completely unexpected from the perspective of the prior art.

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

[0108] This new and inventive means for improving cysteine ​​producing strains was confirmed by the results summarized in Table 2 of Example 6, where weakening the expression of the crp gene by deletions or a combination of insertions and deletions in the crp promoter already led to improved cysteine ​​yields in cultures in shake flasks. Moreover, Table 2 of Example 6 shows that in the case of strains with significantly weakened crp expression such as W3110-crpP-del×pCys (0.03-fold relative crp expression compared to W3110×pCys) and W3110-crp-Preg×pCys (0.05-fold relative expression compared to W3110×pCys), there was also impaired cell growth (OD in Table 2). 600 Nonetheless, there was a clear improvement in cysteine ​​production, which was even more pronounced when cysteine ​​production was based on growth (mg cysteine / OD in Table 2), corresponding to an increase of 285.5% or 360%.

[0109] Therefore, for those skilled in the art, the attenuation of 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 to improve cysteine ​​production in other cysteine ​​producing strains. Thus, 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 attenuated by deletion or a combination of insertion and deletion in the crp promoter, while cysteine ​​production is increased, and no modification of the crp cds is made.

[0110] Example 7 demonstrates that strains capable of cysteine ​​production, with deregulated cysteine ​​biosynthesis and with attenuated expression of the crp gene by deletion or a combination of insertions and deletions in the crp promoter, achieve significantly higher cysteine ​​yields in fermentation than strains containing the WT promoter of the crp gene, and that no modifications of the crp cds are present in all strains according to the invention.

[0111] In the fermentative process in question, it is not only the biomass of the production strain according to the invention that is formed, but also cysteine ​​and its oxidation product cystine. The formation of biomass and cysteine ​​can be correlated in time, or biomass and cysteine ​​can be formed over time in a mutually decoupled manner. The cultivation is carried out in a manner well known to the skilled artisan. For this purpose, the cultivation can be carried out in shake flasks (laboratory scale) or in fermenters (production scale).

[0112] The microbial strain is deregulated with respect to the cysteine ​​biosynthetic pathway and is characterized in that it contains at least one mutation in the promoter of the crp gene. At the same time, the strain forms an increased amount of L-cysteine ​​compared to a strain with a wild-type crp promoter. Preferably, the genetic modification in the promoter of the crp gene is a modification of the wild-type crp promoter (100% expression, 2 -ΔΔCT value = 1), the expression of the crp gene was increased 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).

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

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

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

[0116] Culture media are well known to those skilled in the art of microbial culture practice and 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 the cysteine ​​product. 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, starch, and monomers or oligomers released (enzymatically or chemically) from disaccharides and polysaccharides by hydrolysis. Other usable carbon sources besides sugars or carbohydrates are acetic acid (and acetates derived therefrom), ethanol, glycerol, citric acid (and its salts) or pyruvate (and its salts). However, gaseous carbon sources, such as carbon dioxide or carbon monoxide, can also be envisaged.

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

[0119] Nitrogen sources are nitrogen sources that can be used by the production strain for the formation of biomass. 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 extract, proteose peptone, malt extract, soybean peptone, casamino acids, corn steep liquor (liquid or dried as so-called CSD), as well as NZ Amine and yeast nitrogen base.

[0120] Metered addition of the sulfur source as a single addition in batch form or as continuous feeding is required for efficient production of cysteine ​​and cysteine ​​derivatives. Continuous metered 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 envisaged to use the respective acids with a given stability. Preferred sulfur sources are sulfates, sulfites, thiosulfates and sulfide salts, with sulfates and thiosulfates being particularly preferred, and thiosulfates such as sodium thiosulfate and ammonium thiosulfate being particularly preferred.

[0121] The cultivation may be carried out in the so-called batch mode, which involves inoculating the culture medium with a starter culture of the production strain and then growing the cells without further supply of nutrient sources. The cultivation may also be carried out in the so-called fed-batch mode, which involves additional supply of nutrient sources (feed) after the initial stage of growth in batch mode to compensate for the consumption of nutrient sources. 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 aforementioned. The feed components may be metered together as a mixture or separately in individual feed sections. In addition, other medium components may be added to the feed as additives that specifically increase cysteine ​​production. The feed may be supplied continuously or in portions (discontinuously) or in a combination of continuous and discontinuous feeding. Cultivation in fed-batch mode is preferred.

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

[0123] Preferably, the carbon source of the culture is metered in such a way that the content of the carbon source in the fermenter during the production phase does not exceed 10 g / L, with a maximum concentration of 2 g / L being preferred, a maximum concentration of 0.5 g / L being particularly preferred, and a maximum concentration of 0.1 g / L being especially preferred.

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

[0125] Preferred sources of sulfur in the feed are the salts of sulfates, sulfites, thiosulfates and sulfides, with sulfates and thiosulfates being particularly preferred, and thiosulfates such as sodium thiosulfate and ammonium thiosulfate being especially preferred.

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

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

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

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

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

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

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

[0133] The culture time is 10 to 200 hours, preferably 20 to 120 hours, and particularly preferably 30 to 100 hours.

[0134] The culture batch obtained by the above method contains L-cystine in precipitated form, which is formed from the primary product L-cysteine ​​according to formulas (1)-(3) (EP 0 885 962, EP 2 707 492). Depending on the fermentation conditions, L-cysteine ​​can also be the main product, which accumulates in dissolved form in the culture supernatant (EP 2 726 625). The cysteine ​​or cystine contained in the culture batch can be used further directly without further workup or can be isolated from the culture batch.

[0135] Preferably, the method is characterized in that the cysteine ​​or cystine formed is isolated. To isolate cysteine ​​and cystine, process steps known per se are available, including centrifugation, decantation, dissolution of the crude product with mineral acid, filtration, extraction, chromatography or crystallization or precipitation. Said process steps can be combined in any manner to isolate cysteine ​​with the desired purity, which depends on the further use. Methods for isolating L-cysteine ​​are disclosed in EP 2 699 544 and EP 1 958 933. A procedure for isolating L-cystine is described in EP 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 ​​in an electrochemical manner is disclosed in EP 0 235 908.

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

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

[0139] The figure shows the plasmids used in the examples. [Brief description of the drawings]

[0140] [Figure 1] 1 shows the 6.3 kb vector pKD46 used in Examples 1 and 2. [Diagram 2] 1 shows the 5 kb vector pKan-SacB used in Example 1. [Diagram 3] 1 shows the 7.1 kb vector pCys used in Example 3.

[0141] Abbreviations used in the diagram: bla: gene that confers resistance to ampicillin (β-lactamase) kanR: a gene conferring 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 recombination gene Bet: lambda phage Bet recombination gene Exo: lambda phage Exo recombination gene ORI101: Temperature-sensitive origin of replication RepA: the gene for plasmid replication protein A sacB: levansucrase gene pr-f: binding site for primer (forward) f pr-r: binding site for primer (reverse) r 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 to 317) cds cysE X:cysE (serine O-acetyltransferase gene, feedback resistant) cds ORF306: ydeD (cysteine ​​excretion gene) cds

[0142] The present invention is further illustrated by the following examples, without being limited thereto. EXAMPLES

[0143] [ Example 1 ] Construction of E. coli strain W3110-crp::kan-sacB The parent strain used for DNA isolation and for 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 genetic modification was the promoter region of the crp gene from E. coli. The DNA sequence of the crp gene region including the cds of the differentially expressed yhfA gene, the crp promoter region and the cds of the crp gene from E. coli K12 (Genbank NCBI Reference Sequence NC_000913.3, nucleotide 3485255 to nucleotide 3486950) is disclosed in SEQ ID NO:1.

[0145] Nucleotides 163 to 564 (specified by E. coli yhfA) contain the cds of the yhfA gene in reverse complement form.

[0146] Nucleotides 866 to 1495 (specified 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 intergenic region between the differentially expressed genes yhfA and crp, comprising nucleotides 565 to 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 E. coli strain W3110-crp::kan-sacB, characterized by integration of the kan-sacB cassette into the crp promoter, was produced by using a combination of Lambda Red recombination and counter-selection screens for genetic modifications known to those skilled in the art (see, e.g., Sun et al., 2008; supra).

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

[0150] 2. The 3.2 kb 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 5 kb plasmid pKan-sacB (Figure 2) contains expression cassettes for both the kanamycin (kanR) resistance gene and the sacB gene encoding the enzyme levansucrase. The E. coli kanamycin resistance gene encoding aminoglycoside phosphotransferase is disclosed in the NCBI database under the accession number SH02_03400. The B. subtilis sacB gene is disclosed in the NCBI database under the accession number 936413.

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

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

[0154] 4. Kanamycin-resistant clones were plated on LBSC plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar, and 15 mg / L kanamycin). Clones with an integrated sacB gene produced toxic levan from sucrose, which led to inhibition of growth.

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

[0156] 6. Integration of the Kan-sacB cassette was verified using genomic DNA in a PCR reaction ("Phusion™ High-Fidelity" DNA Polymerase, Thermo Scientific™) with primers crp-7f (SEQ ID NO:5) and crp-8r (SEQ ID NO:6).

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

[0158] 7. A clone with the 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 without the pKD46 plasmid were ampicillin sensitive and could no longer grow on LBamp plates. An ampicillin-sensitive clone was selected and named W3110-crp::kan-sacB.

[0160] [ Example 2 ] Generation of W3110 strains with different truncated crp promoters The following W3110 strains with truncated crp promoters were generated: W3110-crpP-del: deletion of nucleotide 565 to nucleotide 865 from SEQ ID NO:1 W3110-crp-Preg: deletion of nucleotide 565 to nucleotide 713 from SEQ ID NO:1 W3110-crp-Preg2: deletion of nucleotides 565 to 675 from SEQ ID NO:1 W3110-crp-Preg3: deletion of nucleotide 565 to nucleotide 624 from SEQ ID NO:1 The W3110 strain with a shortened crp promoter was generated 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 with the modified promoter sequence was generated in a known manner by fusion PCR (so-called OE-PCR, short for "Overlap Extension PCR") from two PCR products that define the modified promoter. To generate the PCR products described below, "Phusion™ High-Fidelity" DNA polymerase (Thermo Scientific™) was used in each case according to the manufacturer's instructions.

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

[0162] The following PCR products were generated: PCR 1: PCR using genomic DNA from E. coli W3110 and primers crp-13f and crp-14r resulted in the production of a 0.4 kb PCR product. PCR 2: PCR using genomic DNA from 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 E. coli W3110 and primers crp-18f and crp-12r resulted in the production of a 0.8 kb PCR product. PCR 4: PCR using genomic DNA from E. coli W3110 and primers crp-19f and crp-12r resulted in the production of a 0.8 kb PCR product. PCR 5: PCR using genomic DNA from E. coli W3110 and primers crp-20f and crp-12r resulted in the production of a 0.8 kb 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: 1 kb fusion PCR product as a result of OE-PCR of PCR 1 and PCR 2 and primers crp-13f and crp-12r for the generation of E. coli strain W3110-crpP-del. PCR 7: 1.2 kb fusion PCR product as a result of OE-PCR of PCR 1 and PCR 3 and primers crp-13f and crp-12r for the generation of E. coli strain W3110-crp-Preg. PCR 8: 1.2 kb fusion PCR product as a result of OE-PCR of PCR 1 and PCR 4 and primers crp-13f and crp-12r for the generation of E. coli strain W3110-crp-Preg2. PCR 9: 1.2 kb fusion PCR product as a result of OE-PCR of PCR 1 and PCR 5 and primers crp-13f and crp-12r for the generation of E. coli strain W3110-crp-Preg3.

[0164] Transformation of E. coli W3110-crp::kan-sacB x 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 LBS plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar) without kanamycin. Only clones that no longer contained an active sacB gene were able to grow on LBS plates. These clones were plated on LBkan plates to select clones that also no longer contained an active Kan gene and were inhibited in the presence of kanamycin with respect to growth. One clone in each case was selected and the temperature-sensitive plasmid pKD46 was removed by incubation at 42° C. Clones without the pKD46 plasmid were ampicillin sensitive and could no longer grow on LBamp plates. The clones were named E. coli W3110-crpP-del, E. coli W3110-crp-Preg, E. coli W3110-crp-Preg2 and E. coli W3110-crp-Preg3, respectively.

[0165] PCR analysis of transformants: Clones with 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] Genomic DNA was used in a PCR reaction ("Phusion™ High-Fidelity" DNA polymerase, Thermo Scientific™) with primers crp-7f and crp-8r to verify whether the Kan-sacB cassette was correctly replaced by the respective fusion PCR product. Clones with the expected size of the PCR product were selected in each case and analyzed for correct integration of the modified promoter sequence and the unmodified sequence of the crp cds by DNA sequencing of the PCR products (Eurofins Genomics).

[0167] The PCR products had the following expected sizes: E. coli strain W3110-crpP-del: 1397 nucleotides. Deletion of nucleotide 565 to nucleotide 865 from SEQ ID NO:1. E. coli strain W3110-crp-Preg: 1549 nucleotides. Deletion of nucleotide 565 to nucleotide 713 from SEQ ID NO:1. E. coli strain W3110-crp-Preg2: 1587 nucleotides. Deletion of nucleotide 565 to nucleotide 675 from SEQ ID NO:1. E. coli strain W3110-crp-Preg3 strain: 1637 nucleotides. Deletion of nucleotide 565 to nucleotide 624 from SEQ ID NO:1.

[0168] [ Example 3 Construction of cysteine-producing strains A cysteine-producing strain (a strain with deregulated cysteine ​​biosynthesis) was generated by using the cysteine-specific production plasmid pCys (Figure 3).

[0169] pCys is a derivative of the plasmid pACYC184-cysEX-GAPDH-ORF306 disclosed in EP 0 885 962.

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

[0171] Additionally, pCys further contains a serA317 gene fragment cloned after the ydeD (QRF306) efflux gene and encoding the N-terminal 317 amino acids of the SerA protein (full length of 410 amino acids). E. coli serA Gen is disclosed in the "GenBank" gene database under gene ID 945258. serA317 is disclosed in 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. Expression of serA317 is controlled by the serA promoter.

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

[0173] Transformants carrying the plasmid were selected on LBtet-agar plates (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 1.5% agar, 15 mg / L tetracycline). In each case, one clone was selected. 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 Cultivation in shake flasks Preculture: As a preculture for cultivation in shake flasks, 3 ml of LBtet medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 mg / L tetracycline) was inoculated with each cysteine-producing strain from Example 3 and incubated in a shaker (Infors) at 30° C. and 135 rpm for 16 h, and OD 600 / ml (optical density of culture per ml of culture measured at 600 nm) was determined.

[0175] Main culture: A volume of the preculture was then 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 to reach an OD of 0.05. 600 / ml was achieved.

[0176] Composition of SM1 medium: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L (NH4)2SO4, 0.3 g / L MgSO4 x 7H2O, 0.015 g / L CaCl2 x 2H2O, 0.002 g / L FeSO4 x 7H2O, 1 g / L Nacitrate x 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, a 30 ml batch was incubated at 30° C. and 140 rpm to obtain an OD of 0.5 / ml. 600 / ml (4-5 hour incubation time).

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

[0180] For comparative analysis of cysteine ​​production in Example 6 of the WT strain with non-deregulated cysteine ​​biosynthesis, when this strain with non-deregulated cysteine ​​biosynthesis was cultured, E. coli strain W3110 without the plasmid pCys was cultured in the same manner, but without tetracycline in all media.

[0181] [ Example 5 ] Analysis of crp expression by RT-PCR 1) RNA isolation: OD of 0.5 / ml from Example 4 6000.5 ml of the main culture with 0.5% ribosomal RNA was mixed with 1 ml of RNA-Protect® ("Bacteria Reagent", Qiagen) to stabilize the RNA. 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 the main culture. RNA concentration was determined by Qubit 3.0 fluorometer from Thermo Fisher Scientific using the "Qubit® RNA BR Assay Kit" according to the manufacturer's instructions.

[0182] 2) Preparation of complementary DNA (cDNA): 1.5 μg of isolated RNA was converted to cDNA by reverse transcription using the QuantiNova™ Reverse Transcription Kit (Qiagen). The cDNA concentration was determined by a Qubit 3.0 fluorometer from Thermo Fisher Scientific using the "Qubit™ dsDNA HS Assay Kit" according to the manufacturer's instructions. 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 the RotorGene Q control and evaluation software from the same manufacturer. In addition, QuantiNova™ Sybr® Green PCR Kit for Real-Time PCR (Qiagen) was used.

[0184] The expression of the crp gene and the cysG gene as a reference gene was analyzed. The reference gene cysG, whose expression hardly changes (Zhou et al., 2011, see above), served as an internal standard against which the expression of the crp gene was compared and thus served 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] Analyzed was cDNA from shake flask cultures of six strains transformed with plasmid pCys from Example 4, with crp expression in the W3110×pCys strain serving as a reference point for evaluating the relative expression of the crp gene (comparison strain; crp expression from the wild-type promoter) and crp expression in the other strains compared to said reference point. For statistical evidence, each RT-PCR reaction was carried out four times equally and simultaneously as replicates (quadruplicate determination). For the cDNA of the six strains, RT-PCR reactions were carried out simultaneously in one run for the cysG reference gene and the crp gene to be determined, which corresponded to a total of 48 RT-PCR reactions for the quadruplicate determination of the expression of two genes per strain.

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

[0189] After an initial incubation of 2 min at 95° C., the RT-PCR program consisted of 40 cycles of 5 s at 95° C. and 10 s at 60° C. Fluorescence signals caused by the binding of Sybr®Green to the newly formed double-stranded DNA were recorded by a detector in the RT-PCR instrument, and plots of said fluorescence signals versus time were analyzed by the evaluation software.

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

[0191] The plot of the fluorescent signal versus time was used by the evaluation software to determine the average of the so-called CT values ​​(CT "cycle threshold") for each sample determined in quadruplicate, and in a 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 as ΔΔCT. In the second step, ΔΔCT = ΔCT - ΔCT W3110×pCys As a result, the difference ΔΔCT between the ΔCT value of the strain to be analyzed and the ΔCT value of the comparison strain W3110×pCys was formed. In the third step, the value 2 was calculated, which was a measure of the relative expression of the crp gene in the strain compared to the expression of the crp gene in the comparison strain W3110×pCys (Wt crp promoter). -ΔΔCT The ΔΔCT values ​​were used to form a ΔΔCT ΔΔC T. Table 1 summarizes the relative crp expression in the strains examined compared to 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 supernatants from shake flask cultures After 24 hours of cultivation, samples were taken from the main culture from Example 4 and the cell density OD 600 The total cysteine ​​content in the culture supernatant 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 were used in a 2 ml assay. It should be noted that under highly acidic reaction conditions, this assay does not distinguish between cysteine ​​and 2-methylthiazolidine-2,4-dicarboxylic acid (thiazolidine), the condensation product of cysteine ​​and pyruvate described in EP 0 885 962. L-cystine, formed by oxidation of two cysteine ​​molecules according to formula (2), is likewise 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 / ml) and cysteine ​​content (cysteine ​​in mg / ml), and furthermore specific cysteine ​​production versus cell density (cysteine ​​in mg / OD) and specific cysteine ​​production in percentage compared to the comparison strain W3110×pCys (=100%) (cysteine ​​in % of W3110×pCys).

[0195] [Table 2]

[0196] [ Example 7 ] Cysteine ​​production in fermenters A comparison was made between E. coli W3110xpCys, W3110-crp::kan-sacBxpCys, W3110-crp-Preg2xpCys and W3110-crp-Preg3xpCys in production-scale fed-batch fermentations. The E. coli strains with the lowest crp expression, W3110-crpP-delxpCys and W3110-crp-PregxpCys (Table 1), were not investigated further due to poor fermenter growth.

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

[0198] Preculture 2: Each preculture 1 was then completely transferred to 100 ml of SM1 medium (for the composition of SM1 medium, see Example 4) supplemented with 5 g / L glucose, 5 mg / L vitamin B1 and 15 mg / L tetracycline.

[0199] The cultures were incubated in Erlenmeyer flasks (1 L volume) for 17 hours at 30° C. with shaking at 150 rpm (Infors incubator shaker). After this incubation, the cell density OD 600 / ml was 3-5.

[0200] Main culture: Fermentation was carried out in a "DASGIP® 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 x 7H2O, 0.015 g / L CaCl2 x 2H2O, 0.075 g / L FeSO4 x 7H2O, 1 g / L Nacitrate x 2H2O and 1 ml of trace element solution (see Example 6), 0.005 g / L vitamin B1 and 15 mg / L tetracycline.

[0201] Initially, the pH in the fermenter was adjusted to 7.0 by pumping in a 25% NH4OH solution. During the fermentation, the pH was maintained at a value of 7.0 by automatic correction with 25% NH4OH.

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

[0203] The target value of O2 saturation during fermentation was set at 30%. After O2 saturation fell below the target value, a cascade of adjustments was initiated to bring O2 saturation back to the target value. This involved first continuously increasing the gas supply (up to a maximum of 5 vvm) and then continuously increasing the agitation speed (up to a maximum of 1500 rpm).

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

[0205] When the glucose content in the fermenter dropped from the initial 15 g / L to about 2 g / L, a 56% (w / w) glucose solution was metered in continuously. The feed rate was then adjusted so that the glucose concentration in the fermenter did not exceed 2 g / L. Glucose was determined using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

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

[0207] The cell density OD of the examined strains as summarized in Table 3 600 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 having a deregulated cysteine ​​biosynthetic pathway and thereby suitable for the fermentative production of at least one substance selected from L-cysteine, L-cystine and thiazolidine, 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.

2. 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 claim 1 or 2, characterized in that the mutation in the crp promoter sequence comprises at least one deletion or insertion.

4. A microbial strain according to one or more of claims 1 to 3, characterized in that at least nucleotides 565 to 624 are deleted from the crp promoter sequence defined by nucleotides 565 to 865 of SEQ ID NO:

1.

5. The microbial strain according to one or more of claims 1 to 4, characterized in that the crp promoter sequence is completely deleted.

6. Microbial strain according to one or more of claims 1 to 5, characterized in that it is a strain from the family Enterobacteriaceae or Corynebacteriaceae.

7. 7. The microbial strain according to one or more of claims 1 to 6, characterized in that the microbial strain is selected from the group consisting of Escherichia coli, Pantoea ananatis and Corynebacterium glutamicum.

8. Microbial strain according to one or more of claims 1 to 7, characterized in that it is a strain of the species Escherichia coli.

9. A microbial strain according to one or more of claims 1 to 8, characterized in that the expressed Crp protein is SEQ ID NO:

2.

10. A method for producing at least one compound selected from L-cysteine, L-cystine and thiazolidine, characterized in that a microbial strain according to any one of claims 1 to 9 is used.

11. 11. The process according to claim 10, characterized in that the L-cysteine, L-cystine or thiazolidine formed is isolated.

12. 12. The method according to claim 11, characterized in that the 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 the yield of a substance selected from L-cysteine, L-cystine and thiazolidine in g / l is increased by at least 10% (w / v) when said microbial strain is used.

13. The method according to one or more of claims 10 to 12, characterized in that the method is a fermentative method and the fermentation volume is at least 1 L.

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