SK4972002A3 - Method for production of l-cysteine or l-cysteine derivatives by fermentation - Google Patents

Abstract

The invention relates to a method for production of l-cysteine or l-cysteine derivatives by means of a fermentation with micro-organisms and micro-organisms suitable for said method. The micro-organism strain adapted to fermentative production of l-cysteine or l-cysteine derivatives comprises a deregulated cysteine metabolism which is not dependent upon an altered cysB activity. Said micro-organism strain is furthermore characterized by an elevated cysB activity, whereby the cysB activity comprises a regulation pattern typical for a wild type cysB.

Description

METHOD OF MANUFACTURE OF L-CYSTEIN OR DERIVATIVES OF L-CYSTEIN

Field of the invention

The invention relates to a process for the production of L-cysteine or L-cysteine derivatives by fermentation with microorganisms, as well as suitable microorganisms for this process.

BACKGROUND OF THE INVENTION

The amino acid L-cysteine is of economic importance. It is used, for example, as a food additive (especially in the manufacture of bakery products), as a feedstock in cosmetics, as well as as a starting product in the production of pharmacologically active substances (especially N-acetyl-cysteine and S-carboxymethylcysteine).

L-cysteine derivatives are all sulfur-containing metabolites derived from cysteine in their synthesis, for example cystine, methionine, glutathione, biotin, thiazolidine, thiamine, liponic acid and coenzyme A.

Cysteine biosynthesis in bacteria is regulated at two levels (Fig. 1):

1. At the level of enzyme activity, serine acetyl transferase (a cysEgen product) is subject to inhibition by the end product L-cysteine. This means that the accumulation of L-cysteine leads directly to inhibition of the first specific cysteine biosynthesis reaction, which prevents further synthesis.

2. At the transcriptional level, the regulatory protein CysB (cysBgene encoded) acts as a transcriptional activator and provides for the regulated preparation (activation) of reduced sulfur. As an inducer, CysB needs N-acetyl-serine, which is formed in the cell from O-acetyl-serine when insufficiently reduced sulfur is available for the O-acetyl-serine-sulfhydrylase reaction. Consequently, all genes related to sulfur uptake, reduction and incorporation are controlled by CysB. These are the operons: cysPTWAM, cysDNC, cysJIH, and the cysK gene. While acetylserine acts for

31917 / T h cysB as an inducer, sulphide and thiosulphate exhibit a negative effect as a so-called "anti-inducer, because its existence indicates the availability of SH-groups.

The prior art regarding the recovery of L-cysteine and L-cysteine derivatives is discussed extensively in WO 97/15673 (corresponding to US application SN 09/065104). WO 97/15673 itself discloses a fermentation production process that utilizes the feedback resistance of serine acetyl transferase. The application further clarifies that it is possible to further increase cysteine yields by additional deregulation of the CysB regulatory protein at the gene level in terms of constitutive expression.

Patent application EP 885962 A1 (corresponding to US application SN 09/097759) discloses microorganisms which are suitable for the fermentative production of L-cysteine, L-cystine, N-acetylserine and thiazolidine derivatives, characterized in that they overexpress at least one gene encoding protein suitable for the production of antibiotics or other microorganisms of toxic substances from the cell.

CysB belongs to the group of LysR-Type-Transcriptional Regulators (LTTRs), of which more than 100 representatives are known (Schell M.A., 1993, Annu. Rev. Microbiol. 47: 597-626). They are characterized by an N-terminal DNA-binding domain with a helix-turn-helix-motif and a C-terminal inducer-binding domain. Detailed DNA-binding studies were presented for cysB. In addition, CysB is the first LTTR protein in which a crystal structure is also available (Tyrell et al., 1997, Structure 5: 1017-1032). As a rule, LTTR proteins act as positive gene regulators that are dependent on the existence of an inducer molecule. It has previously been found that only highly active cysB variants that are independent of effector molecules (the so-called constitutively active variants) allow for an increase in cysteine production, since such forms remain unchanged, high gene activation is eliminated. (Nakamori S. et al., 1998, Appl. Env. Microbiol. 64: 16071611).

Examples of constitutively active forms of CysB are described for Salmonella typhimurium. These variants exhibit a high activity, which activity is

31917 / T h completely independent of the inducer N-acetylserine and the negative effect of thiosulfate and sulfide. (ColyerT. E., Kredich N.M., 1994, Mol. Microbiol. 13: 797-805).

SUMMARY OF THE INVENTION

The present invention relates to a microorganism strain which is suitable for the fermentative production of L-cysteine and L-cysteine derivatives and has a deregulated cysteine metabolism, wherein this deregulation of the cysteine metabolism is not based on altered cysB activity but is characterized by an additionally increased cysB activity, wherein the cysB activity is a regulatory model typical of wild type cysB.

Strains of deregulated cysteine metabolism of microorganisms in which this deregulation is not based on altered cysB activity are known. These are strains with modified cysE alleles as described, for example, in WO 97/15673 (incorporated herein by reference) or Nakamori S. et. al., 1998, Appl. Env. Microbiol. 64: 1607-1611 (herein incorporated by reference), or strains in which efflux genes are used, as described, for example, in EP 0885962 A1 (corresponding to US application SN 09/097759, herein incorporated by reference), or strains which are obtained using non-specific mutagenesis methods combined with screening methods for cysteine overproduction or reduced cysteine degradation, as described, for example, in WO 97/15673 or in Nakamori S. et. al., 1998, Appl. Env. Microbiol. 64: 1607-1611.

According to the invention, it is an increased activity if the activity of cysB is at least 10% increased compared to cysB activity in the wild-type strain.

Preferably, the cysB activity is increased by at least 25%.

It is particularly preferred that the cysB activity is increased by at least 50%.

The Escherichia coli strain (MC4100:: λΚΖΙ_300), which is suitable for the determination of cysB activity, is described in Example 2. It was deposited on June 23, 1999 under DSM 12886 at DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, D-38142 Braunschweig). , under the Budapest Agreement. The present cysB31917 / T h containing construct was introduced into the strain MC4100: XKZL300 in a known manner.

The regulatory model of cysB activity typical of wild type cysB depends on the sulfur source, with the cysB activity of thiosulfate-grown cells versus sulfate-grown cells less than 75% and the cysB activity of cystine-grown cells versus sulfate-grown cells less than 75%. 20% (see Example 2).

The microorganism strains of the invention secrete L-cysteine or an L-cysteine derivative compared to a microorganism strain with deregulated cysteine metabolism without increased cysB activity to an increased extent.

Accordingly, the microorganism strain of the invention is a microorganism strain with a deregulated cysteine metabolism, wherein the deregulation of the cysteine metabolism does not consist in altered cysB activity but in which the homologous or heterologous cysB gene is expressed and encoded by the cysB regulatory model typical of the wild type .

Preferably, they are Escherichia coli strains with a deregulated cysteine exchange not based on altered cysB activity in which the wild-type cysB gene is overexpressed.

Particularly preferably, these are deregulated cysteine exchange strains of Escherichia coli, wherein the deregulation of the cysteine lath exchange does not rely on altered cysB activity in which the copy number of the wild-type cysB genes of Escherichia coli is increased and this gene is overexpressed.

Surprisingly, it has been found that not as postulated in the prior art, the use of cysB-alleles that encode constitutively active cysB regulatory proteins increases cysteine production, but quite the contrary, enhanced expression of the wild-type cysB gene increases cysteine production.

This finding is also surprising and unexpected because there is no known example in which an overexpression of a regulatory protein of the

LysR-Type-transcriptional regulators allowed overproduction of metabolites.

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The invention also relates to the use of regulatory proteins from the LysR-Typtranscriptional regulator family for metabolite overproduction, as well as to a method for metabolite overproduction characterized in that the regulatory gene from the LysR-Type-transcriptional regulator family is overexpressed in the microorganism and causes enhanced metabolite production in the microorganism. .

An increase in cysB activity in a microorganism while adopting a typical regulatory model can be achieved, for example:

1. by increasing the copy number of the cysB gene coding for cysB by a regulatory model typical of wild type cysB in a microorganism under the control of a promoter, or

2. Enhanced expression of the cysB gene coding for cysB by a regulatory model typical of wild type cysB by exchanging a promoter regulating the expression of wild type cysB gene for a stronger promoter.

The CysB genes are known from Escherichia coli, Salmonella typhimurium, Klebsiella aerogenes, Haemophilus influenzae, Pseudomonas aeruginosa and Thiocapsa roseopersicina. The cysB genes are preferably understood to be those whose gene products with the cysB protein from Escherichia coli exhibit an identity of at least 40%. The homology values refer to the results obtainable by the computer program "Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin". To do this, a data bank search is performed with the "blast" subroutine using standard parameters.

The CysB genes are further alleles of the wild-type cysB genes whose gene products are altered in their sequence without losing the typical CysB-type regulatory model. An example are cysB variants with a conservative amino acid exchange.

The microorganism according to the invention can be produced, for example, in such a way that in a microorganism strain with a deregulated cysteine metabolism, where the deregulation of the cysteine metabolism is not based on altered cysB activity, the number of cysB copies is increased by known methods.

31917 / T h wild-type or cysB-genes encoding cysB by a regulatory model typical of wild-type cysB genes, or enhanced expression of the cysB-wild-type gene or cysB-gene encoding cysB by a wild-type cysB regulatory model is known.

In the following, the term "cysB" means not only "wild-type cysB" but also "cysB variant with a regulatory model typical of wild-type cysB". The term "cysB gene" means in the following the "wild type cysB gene" as well as the "cysB gene encoding cysB by a regulatory model typical of the wild type cysB".

Increasing the copy number of cysB genes in a microorganism can be accomplished by methods known to those skilled in the art. For example, the cysB gene can be cloned in plasmid vectors with multiple copies per cell (e.g., pUC19, pBR322, pA-CYC184) and introduced into a microorganism with deregulated cysteine exchange. Alternatively, the cysB gene can be integrated multiple times in the chromosome of a microorganism with a deregulated cysteine metabolism. Known systems with mild bacteriophages, integrative plasmids or integration via homologous recombination can be used as integration procedures. (For example, Hamilton et al., 1989, J. Bacteriol. 171: 4617-4622).

It is preferred to increase the copy number by cloning the cysB gene in a plasmid vector under the control of a promoter. It is particularly preferred to increase the copy number by cloning the cysB gene in a pACYC derivative, such as e.g. pACYC184-LH (deposited under the Budapest Agreement in the German Collection of Microorganisms and Cell Cultures, Braunschweig, 18.8.1995 under number DSM 10172).

The natural promoter and operative regions of the gene can serve as a control region for expression of the plasmid encoding the cysB gene.

Enhanced expression of the cysB gene can also be accomplished through other promoters. Corresponding promoter systems are known to those skilled in the art (Makrides S. C., 1996, Microbiol. Rev. 60: 512-538). Such constructs can be used in known manner in plasmids or chromosomal.

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Cloning of the cysB gene in a plasmid vector is accomplished, for example, by specific amplification using a polymerase chain reaction using specific primers that capture a complete cysB gene with promoter and operative sequences and subsequent ligation of the vector with DNA fragmentary.

Preferred vectors for cloning the cysB gene are plasmids that already contain genetic elements for deregulating the cysteine metabolism, for example the cysEX gene (WO 97/15673) and the efflux gene (EP 0885962 A1). Such vectors allow the production of the microorganism strain of the invention from any microorganism strain, since such a vector also causes deregulation of the cysteine metabolism in the microorganism.

The invention also relates to a plasmid having genetic elements for the deregulation of a cysteine metabolism, wherein the genetic elements show no change in cysB activity and further comprises a promoter-controlled cysB gene.

By a conventional transformation method (e.g., electroporation), plasmids containing cysB are transferred to bacteria and selected, for example, through a antibiotic resistance, on a clone carrying a plasmid.

The invention also relates to a process for the production of a microorganism strain characterized in that a plasmid according to the invention is introduced into the microorganism strain.

The production of cysteine or cysteine derivatives using the microorganism strain according to the invention is carried out in a fermenter by known methods. As the carbon source, for example, glucose, lactose or other sugars can be used, as the nitrogen source ammonium or protein hydrolyzate. As the sulfur source, for example, sulphide, sulphite, sulphate or thiosulphate can be used. During fermentation, the L-cysteine formed can oxidize sparingly soluble cystine or condense with aldehydes or ketones to thiazolidines (e.g. pyruvic acid to 2-methyl-thiazolidine-2,4-dicarboxylic acid).

The invention also relates to a process for the production of L-cysteine or L-cysteine derivatives characterized in that the microorganism strain according to the invention is

31917 / T h is introduced into the fermenter in a known manner and L-cysteine or its derivatives are separated from the fermentation batch.

The following examples serve to further illustrate the invention.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Cloning of the wild-type cysB gene and cysB (T149M) allele

The wild-type cysB gene from Escherichia coli is cloned using a polymerase chain reaction (PCR). With the specific oligonucleotide primers (20 pmol per batch) of cysBPI (SEQ.ID.NO.1) and cysBP2 (SEQ.ID.NO: 2), a 3107 base pair genomic DNA fragment, including a wild-type, cysB-gene, was corrected. and terminal EcoRI- or SalI-restriction cleavage sites.

5'-GTT ACG AGA TCG AAG AGG-3 '(phosphorothioate linkage at the 3'-end) (SEQ.ID.NO: 1)

5'-GTC ACC GAG TGG TCA ATG-3 '(phosphorothioate linkage at the 3'-end) (SEQ.ID.NO: 2)

A polymerase chain reaction with a Pwo-DNA polymerase from Boehringer (Mannheim, D) was performed with 10 ng of genomic DNA as a matrix. The program included 29 cycles with an annealing temperature of 56 ° C (30 seconds per cycle), an extension temperature of 72 ° C (60 seconds per cycle), and a denaturation temperature of 94 ° C (30 seconds per cycle). The DNA fragment was additionally treated with EcoRI and SalI restriction enzyme and purified by gel electrophoresis and gene purification (Genclean Kit BIO101, P.O.Box 2284, La Jolla, Califirnia, USA, 92038-2284). This fragment was ligated in EcoRI-SalI-cleaved α

31917 / T h phosphatase engineered phagemid vector pTZ19U from Bio-Rad Laboratories (Hercules, California, USA). After transformation, positive clones were identified by restriction analysis.

A mutation (analogous to the mutation in the wild-type cysB gene from Salmonella typhimurium described by Colyer TE, Kredich NM, 1994, Mol. Microbiol. 13: 797-805) of the wild-type cysB gene codon 147 was made to construct a constitutively active cysB allele. to the methionine codon with "Mutagene In Vitro Mutagenezis" kit from Bio-Rad Laboratories (Hercules, California, USA). The oligonucleotide CysBMut4 (SEQ.ID.NO.3) was used. The underlined bases indicate a deviation from the wild-type sequence.

5'-TTC GCT ATC GCC ATG GAA GCG CTG CAT-3 '(SEQ.ID.NO: 3)

For the activity assay (see Example 2), both cysB alleles were cloned as EcoRI-SalI fragments according to Klenow treatment in an Ec / 136 µl phosphatase-treated pA-CYC184-LH vector.

Example 2

Determination of cysB activity in vivo

A reporter gene batch was selected to measure cysB activity. Here, the control region of the cysK gene was fused with the lacZ gene, which encodes the enzyme βgalactosidase. By integrating this fusion in an endogenous β-galactosidase-free strain, this enzyme was dependent on cysB activity and mediated indirectly the measurement of cysB activity in the form of β-galactosidase activity. The system described by Simons et al. (Simons, R.W. et al., 1987, Gene 53: 85-96). First, the promoter region of the cysK gene, including the first fifteen codons, was amplified by the polymerase chain reaction using the oligonucleotide primers cysKPI (SEQ.ID.NO: 4) and cysKP3 (SEQ.ID.NO: 5) and 10 ng of chromosomal DNA from Escherichia coli and Pwo. polymerase.

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5'-CCG GAA TTC CCG TTG CCG TTG GTG GCG-3 '(SEQ.ID.NO: 4) 5'-CGC GGA TCG GTG TGA CCG ATA GTC AGC-3' (SEQ.ID.NO: 5)

The conditions corresponded to those described in Example 1. The resulting 317 base pair product was digested with the restriction enzyme EcoRI and BamHI according to the manufacturer's data and purified by preparative gel electrophoresis and a gene purification method. Finally, the product was also ligated with EcoRI-BamHI digested and phosphatase-treated vector pRS552 (deposited September 14, 1999 under the Budapest Agreement in the German Collection of Microorganisms and Cell Cultures, Braunschweig under number DSM 13034). By electroporation, strain MC4100 (ATCC 35695) with ligation batch was transformed and positive clones were identified by restriction analysis. These contain a translational cysKlacZ-fusion. The plasmid obtained was subsequently recombined according to Simons et al. with bacteriophage XRS45 (deposited under the Budapest Agreement in the German Collection of Microorganisms and Cell Cultures, Braunschweig 14.9.1999 under DSM 13035) and a homogeneous lysate of recombinant phage designated λΚΖΙ_300 was produced. With this phage, the Alac-strain MC4100 was infected and a lysogenic clone was identified by kanamycin selection (MC4100: λΚΖΙ_300), which could now be used to measure cysB activity.

To compare the efficiency of the multiple copy of the cysB gene cloned in pACYC184-LH and the cysB (T149M) gene, the MC4100: λΚΖΙ_300 was transformed with the corresponding plasmids pACYC-cysB and pACYC-cysB (T149M).

Strains in VB-minimal medium (3.5 g / L Na (NH ₄ ) HPO ₄ ; 10 g / L KH ₂ PO ₄ ; 2 g / L citrate x H ₂ O; 0.078 g / L MgCl ₂ ; pH was set at 6.5 with NaOH, 5 g / l glucose; 5 mg / l vitamin B 1) with various sources of sulfur (1 mM sulfur each) cultured, with 15 mg / l tetracycline added. Determination of β-galactosidase activity was performed according to the method described by Miller (Miller JH 1972, Experiments in Molecular Genetics, Cold Spring Harbor, New York, 352-355).

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The results are shown in Table 1. The control showed (MC4100:: XKZL300 / pACYC184-LH) a typical model of cysBactivity-dependent regulation of cysBactivity for the cysB wild-type strain: cysB-activity of cells grown with thiosulfate over sulfate represented less than 75 % and cysB activity of cells grown with cystine versus sulfate was less than 20%. This model is maintained in the presence of the wild-type cysB gene in multiple copies at an overall elevated level of activity (for example, MC4100: λΚΖΙ_300 / pACYC-cysB according to the invention). In contrast, the CysB (T149M) -allel results in a constitutively high activity in the loss of a typical control model.

Table 1 - Determination of cysB-activity in the form of β-galactosidase activity of strains with chromosomal cysK-lacZ-fusion

tribe MC4100: λΚΖΙ_300 MC4100: λΚΖ1_300 MC4100: λΚΖ1_300 plasmid PACYC184-LH pACYC-cysB pACYC-cysB (T149M) genotype cysB one copy cysB multiple copies cysB (T149M) multiple copies S-source: β-galactosidase activity in Miller's units cystine 26 341 3791 sulphate 1906 2708 3824 thiosulfate 726 1094 3595

Example 3

Construction of the Plasmid of the Invention

The organisms of the invention are characterized by a deregulated cysteine metabolism and, for example, a multiple copy number of the cysB gene. For the production of such microorganisms, a plasmid (pACYC184-cysEX-GAPDH-ORF306) was chosen as the basic construct. This plasmid contained elements of the feedback-resistant cysE allele and an efflux gene for deregulation of the cysteine metabolism. It is described in detail in the patent

31917 / T h application EP 0885962 A1 (Example 2D). In this construct, the digested SnaBI restriction enzyme as well as the phosphatase engineered, a cysB fragment was inserted between the cysEXallel and the efflux gene. The latter was obtained by restriction with EcoRI and BstXI and subsequent splicing of the blunted DNA-termini with Klenow enzyme from plasmid pTZ19U-cysB. The plasmid is designated pHC34.

Transformation of strain W3110 Escherichia coli (ATCC 27325; Bachmann BJ, 1996, In: Neidhardt F. C: (ed.) Escherichia coli and Salmonella: Cellular and Molecular Biology, American Society for Microbiology, Washington DC, Chapter 133) invention. Transformation was performed by electroporation. The thick cell suspension in an ice-cold 10% glycerin solution was mixed with 0.1 pg of plasmid DNA and subjected to an electrical pulse at 2500 V, 200 Ohm and 12.5 pF. After transfer of the batch to sterile LB-medium (1% tryptone, 0.5% yeast extract, 1% NaCl) and incubation at 30 ° C for 1 hour, a plasmid carrying clones was selected on LB-agar plates with 15 µg / ml tetracycline.

To compare the efficacy of the wild-type cysB gene over the constitutive cysB (T149M) allele, an analogous construct (pHC30) was generated and also introduced into the W3100 strain. For further use, the organism W3110, transformed with plasmid pA-CYC184 / cysEX-GAPDHorf306, described in EP 885962 A1 served as a basic construct for comparison and at the same time to be defined against the prior art.

Since W3110 is a wild-type strain, all cysteine production effects relate to the plasmid-encoded gene.

Example 4

Cysteine production by microorganisms according to the invention

In order to demonstrate the production of cysteine, the microorganisms described in Example 3 were cultivated in a fed-batch fermenter by a continuous process.

31917 / T h by addition of glucose and thiosulfate. A Biostat M from Braun Biotech (Melsungen, D) with a maximum culture volume of 2 I was used as the apparatus.

20 ml of LB medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l NaCl), which additionally contained 15 mg / l tetracycline and incubated on a shaker at 30 ° C and 150, were inoculated as a starter culture. rpm. After seven hours the total batch was transferred to 100 ml SM1 medium (12 g / l K ₂ HPO ₄ ; 3 g / l KH ₂ PO ₄ ; 5 g / l (NH ₄ ) ₂ SO ₄ ; 0.3 g / l MgSO ₄ x 7 H ₂ O; 0.015 g / l CaCl ₂ x 2 H ₂ O; 0.002 g / l FeSO ₄ x 7 H ₂ O; 1 g / l Nascitrate x 2 H ₂ O; 0.1 g / l NaCl 1 ml / l trace element solution consisting of 0,15 g / l Na ₂ MoO ₄ x 2 H ₂ O; 2,5 g / l NaBO ₃ ; 0,7 g / l CO 2 x 6 H ₂ O; 25 g / l CuSO ₄ x 5 H ₂ O; 1.6 g / l MnCl ₂ x 4 H ₂ O; 0.3 g / l ZnSO ₄ x 7 H ₂ O) supplemented with 5 g / l glucose ; 0.5 mg / l vitamin Bi and 15 mg / l tetracycline. Further incubation was performed at 30 ° C for 17 hours at 150 rpm.

With this starter culture (optical density at 600 nm ca. 3), a fermenter was seeded with 900 ml of fermentation medium (15 g / l glucose, 10 g / l tryptone, 5 g / l yeast extract, 5 g / l (NH ₄ ) ₂ SO ₄ ; 1.5 g / l KH ₂ PO ₄ ; 0.5 g / l NaCl; 0.3 g / l MgSO ₄ x 7 H ₂ O; 0.015 g / l CaCl ₂ x 2 H ₂ O; 0.075 g / l FeSO ₄ x 7 H ₂ O; 1 g / l Nasitrate x 2 H ₂ O; and 1 ml / l trace element solution as above, 5 mg / l vitamin Βί and 15 mg / l tetracycline, adjusted to pH 7.0 with 25% ammonia).

During the fermentation, a temperature of 30 ° C was set and the pH was kept constant at 7.0 by adding 25% ammonia. The culture was aerated with sterilized compressed air at 1.5 vol / vol / min and stirred in a 200 rpm mixer. After the oxygen saturation dropped to 50%, the RPM of the control apparatus was increased up to 1200 rpm to maintain the oxygen saturation at 50%.

After two hours, a 30% sodium thiosulfate solution was added at a dosage of 3 ml / h. Glucose was added from a 56% stock solution while the content in the fermenter, which was initially 15 g / l, dropped to 5-10 g / l. Glucose addition was carried out at a flow rate of 8-14 ml / h, maintaining a constant concentration of 5-10g / l in the experiment. Determination of glucose

31917 / Th was performed with a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

L-cysteine production was monitored by the Gaitonde colorimetric assay (Gaitonde, M.K. 1967, Biochem. J. 104, 627-633). It should be noted that the test does not distinguish between L-cysteine and the condensation product of L-cysteine and pyruvate (2-methylthiazolidine-2,4-dicarboxylic acid) described in EP 0885962 A1. The sparingly soluble cystine formed by oxidation from L-cysteine, after dissolution in 8% hydrochloric acid and subsequent reduction with dithiothreitol (DTT) in a dilute solution at pH 8.0, proved to be the same as Lcysteine.

Table 2 shows the production progress of fermentation with organisms having the basic construct pA-CYC184 / cysEX-GAPDH-orf306 described in EP 0885962 A1 compared to the plasmid pHC34 of the invention and the corresponding construct with the cysB (T149M) allele which encodes the constitutively active cysB gene product. . It is clear that the batch of wild type cysB genes of the invention has a positive effect on production performance, while the constitutive allele of cysB (T149M) has a negative effect.

Table 2 - L-Cysteine Production with pHC34 Construct of the Invention control construct

plasmid construct pA-CYC184 / cysEX- GAPDH-ORF306 pHC34 pHC30 genotype cysEX ORF306 cysEX cysB ORF306 cysEX cysB (T149M) ORF306 ferment. time L-cysteine yield in g / L 24 hours 6.3 8,0+ 2,6 * 2.0 48 hours 10.2 + 7.0 * 10.0 + 12.6 * 3.4

* Starred data indicate L-cysteine, which is presented in oxidized form as sparingly soluble cystine

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SEQUENCE PROTOCOL <110> Consortium fuer elektrochemische Industrie GmbH <120> Method for producing L-cysteine or L-cysteine derivatives <130> Co9905 <140>

<141>

<160> 5 <170> Patentln Vers. 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> PCR primer <400> 1 gttacgagat cgaagagg

<210> 2 <211> 18 <212> DNA

31917 / T h <213> Artificial sequence <220>

<223> PCR primer <400> 2 gtcaccgagt ggtcaatg 18 <210> 3 <211> 27 <212> DNA <213> Artificial sequence <220>

<223> Oligonucleotide for in vitro mutagenesis <400> 3 ttcgctatcg ccatggaagc gctgcat 27 <210> 4 <211> 27 <212> DNA <213> Artificial sequence <220>

<223> PCR primer <400> 4 ccggaattcc cgttgccgtt tgtggcg 27

31917 / T h <210> 5 <211> 27 <212> DNA <213> Artificial sequence <220>

<223> PCR primer <400> 5 cgcggatccg tgtgaccgat agtcagc 27

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Claims (8)

A microorganism strain suitable for the fermentative production of Lcysteine or its derivatives and having a deregulated cysteine metabolism, wherein said deregulation of the cysteine metabolism is not based on an altered cysB activity, characterized in that it additionally has an increased cysB activity, wherein the cysB activity is a regulatory model typical of wild type cysB. 2. A microorganism strain suitable for the fermentative production of Lcysteine or its derivatives and having a deregulated cysteine metabolism, wherein the deregulation of the cysteine metabolism is not based on an altered cysB-activity in which homologous or heterologous cysB genes are expressed and encoded by cysB a regulatory model typical of wild type cysB. The microorganism strain according to claim 2, characterized in that it is a strain of Escherichia coli with a deregulated cysteine metabolism in which the cysB gene is overexpressed. Microorganism strain according to claim 3, characterized in that it is an Escherichia coli strain having a deregulated cysteine metabolism in which the copy number of the wild-type cysB gene from Escherichia coli is increased and the gene is overexpressed. Process for producing a microorganism according to any one of claims 1 to 4, characterized in that in the microorganism strain with deregulated cysteine metabolism, the number of copies of the wild-type cysB gene or the cysB-gene cysB gene is increased by a wild-type regulatory model. cysB gene or with known methods 31917 / T hampers the enhanced expression of the wild-type cysB gene or the cysB gene coding for the cysB gene by a regulatory model typical of the wild-type cysB gene. Process for the production of L-cysteine or derivatives thereof, characterized in that the microorganism according to any one of claims 1 to 4 is used in a known manner in fermentation and the L-cysteine or L-cysteine derivative is separated from the fermentation batch. 7. A plasmid having genetic elements for deregulating a cysteine metabolism, said genetic elements not causing a change in cysB activity, and also comprising a promoter-controlled cysB gene. Method for producing a microorganism strain according to any one of claims 1 to 4, characterized in that a plasmid according to claim 7 is introduced into the microorganism.