CA2386539C - Method for production of l-cysteine or l-cysteine derivatives by fermentation - Google Patents
Method for production of l-cysteine or l-cysteine derivatives by fermentation Download PDFInfo
- Publication number
- CA2386539C CA2386539C CA002386539A CA2386539A CA2386539C CA 2386539 C CA2386539 C CA 2386539C CA 002386539 A CA002386539 A CA 002386539A CA 2386539 A CA2386539 A CA 2386539A CA 2386539 C CA2386539 C CA 2386539C
- Authority
- CA
- Canada
- Prior art keywords
- cysb
- cysteine
- gene
- activity
- wild
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/12—Methionine; Cysteine; Cystine
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
WO,Ol/27307 PCT/EP00/09720 Process for fermentatively producing L-cysteine or L
cysteine derivatives The invention relates to a process for producing L-cysteine or L-cysteine derivatives by fermenting microorganisms, and to microorganisms which are suitable for the process.
The amino acid L-cysteine is of economic importance.
For example, it is used as a foodstuff additive (in particular in the baking industry), as a substance which is employed in cosmetics, and as a starting material for producing pharmacological active compounds (in particular N-acetylcysteine and S
carboxymethylcysteine).
L-cysteine derivatives are all S-containing metabolites which, in their synthesis, are derived from cysteine, that is cystine, methionine, glutathione, biotin, thiazolidines, thiamine, lipoic acid and coenzyme A, for example.
In bacteria, cysteine biosynthesis is regulated at two levels (Fig. 1):
1. At the level of enzyme activity, serine acetyl-transferase (product of the cysE gene) is subject to end product inhibition by L-cysteine. This means that an accumulation of L-cysteine Leads directly to inhibition of the first specific reaction of cysteine biosynthesis and further synthesis is stopped.
cysteine derivatives The invention relates to a process for producing L-cysteine or L-cysteine derivatives by fermenting microorganisms, and to microorganisms which are suitable for the process.
The amino acid L-cysteine is of economic importance.
For example, it is used as a foodstuff additive (in particular in the baking industry), as a substance which is employed in cosmetics, and as a starting material for producing pharmacological active compounds (in particular N-acetylcysteine and S
carboxymethylcysteine).
L-cysteine derivatives are all S-containing metabolites which, in their synthesis, are derived from cysteine, that is cystine, methionine, glutathione, biotin, thiazolidines, thiamine, lipoic acid and coenzyme A, for example.
In bacteria, cysteine biosynthesis is regulated at two levels (Fig. 1):
1. At the level of enzyme activity, serine acetyl-transferase (product of the cysE gene) is subject to end product inhibition by L-cysteine. This means that an accumulation of L-cysteine Leads directly to inhibition of the first specific reaction of cysteine biosynthesis and further synthesis is stopped.
2. At the level of transcription, the regulatory protein CysB (encoded by the cysB gene) functions as a transcription activator and ensures that the provision of reduced sulphur is regulated. CysB
requires N-acetylserine, which is formed in the cell from 0-acetylserine when insufficient reduced sulphur is available for the 0-acetylserine sulphhydrylase reaction, as an inducer.
Consequently, all the genes which are connected with the uptake, reduction and incorporation of sulphur are under the control of CysB. These genes are the operons cysPTWAM, cysDNC and cysJIH and also the cysK gene. Whereas acetylserine acts as an inducer for CysB, sulphide and thiosulphate exhibit a negative effect as so-called "antiinducers", since their presence indicates the availability of SH groups.
The state of the art with regard to obtaining L-cysteine and L-cysteine derivatives is discussed in detail in WO 97/15673 (corresponds to US Patent 6,218,168). WO 97/15673 itself described a fermentative production process which uses feedback-resistant serine acetyltransferases. The application also discloses that it is possible to obtain a further increase in cysteine yield by additionally deregulating the regulatory protein CysB at the gene level such that the gene is constitutively expressed.
The patent application EP 885962 A1 (corresponds to the US Patent 5,972,663) discloses microorganisms which are suitable for fermentatively producing L-cysteine, L-cystine, N
acetyl serine and thiazolidine derivatives and which are characterized in that they overexpress at least one gene which encodes a protein which is directly suitable for secreting antibiotics, or other substances which are toxic for the microorganisms, out of the cell.
_ 3 _ CysB belongs to the family of LysR type transcription regulators (LTTR), more than 100 representatives of which are already known (Schell M.A., 1993, Annu. Rev.
Microbiol. 47: 597-626). They are distinguished by the fact that they possess an N-terminal DNA-binding domain, having a helix-turn-helix motif, and a C-terminal inducer-binding domain. Detailed DNA-binding studies are, available for CysB. Furthermore, CysB is the first LTTR protein for 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 which are dependent on the presence of an inducer molecule. It has previously been assumed that only highly active CysB variants, which are independent of effector molecules (so-called constitut.ively active variants), allow an increase in cysteine production since such forms display a constantly high degree of gene activation (Nakamori S. et al., 1998, Appl. Env.
Microbiol. 64:1607-1611).
Examples of constitutively active forms of CysB have been described in the case of Salmonella typhimurium.
These variants exhibit a high degree of activity, with this activity being completely independent of the inducer N-acetylserine and the negative effect of thiosulphate and sulphide (Colyer T. E., K:redich N. M., 1994, Mol. Microbiol. 13: 797-805).
The present invention relates to a microorganism strain which is suitable for fermentatively producing L-cysteine or L-cysteine derivatives and possesses a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB activity, characterized in that the strain additionally possesses an increased CysB activity, with the CysB activity having a regulatory pattern which is typical of a wild-type CysB.
In accordance with one embodiment of the present invention there is provided an isolated microorganism strain that is transformed with a polynucleotide encoding the transcription regulator protein CysB, for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives, the microorganism having a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB
activity, and the strain additionally having an increased CysB activity as compared to an unmodified microorganism strain, with the CysB activity having a regulatory pattern such that CysB activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide.
In accordance with another embodiment of the present invention there is provided an isolated microorganism strain that is transformed with a polynucleotide encoding the transcription regulator protein CysB for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives comprising a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB
activity, and in which strain homologous or heterologous cysB genes, which encode CysB having a regulatory pattern such that CysB activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide, are expressed to increased extent compared to an unmodified microorganism.
- 4a -Microorganism strains which possess a deregulated cysteine metabolism in which this deregulation is not due to an alteration in CysB activity are known. These are strains which possess modified cysE alleles, as described, for example, in WO 97/15673 or Nakamori S. et al., 1998, Appl.
Env. Microbiol. 64: 1607-1611, or strains in which efflux genes have been inserted, as described, for example, in EP
0885962 A1 (corresponds to U.S. Patent 5,972,663), or strains which are isolated using nonspecific mutagenesis methods combined with methods for screening for cysteine overproduction or decreased cysteine breakdown, as described, for example, in WO 97/15673 or in Nakamori S. et al., 1998, Appl. Env. Microbiol. 64:1607-1611.
Within the meaning of the invention, CysB activity is increased when this activity is at least 10'o higher than that in the wild-type strain.
Preference is given to the CysB activity being at least 250 higher.
Particular preference is given to the CysB activity being at least SOo higher.
An Escherichia coli strain (MC4100::AKZL300) which is suitable for determining the CysB activity is described in Example 2. This strain was deposited in the DSMZ (Deutsche Sammlung fur Mikroorganismen and Zellkulturen ' _ 5 _ [German collection of microorganisms and cell cultures]
GmbH, D-38142 Braunschweig) under number DSM 12886 on 23.6.99 in accordance with the Budapest Treaty. In addition, each respective cysB-containing construct is 5 introduced into strain MC4100::?~KZL300 in a manner known per se.
The CysB activity exhibits a regulatory pattern, in dependence on the S source, which is typical of a wild-10 type CysB, when the CysB activity of cells which are grown with thiosulphate is less than 750 of that of cells which are grown with sulphate and the CysB
activity of cells which are grown with cystine is less than 20~ of that of cells which are grown with sulphate 15 (see Example 2).
Microorganism strains according to the invention secrete L-cysteine or an L-cysteine derivative in greater amounts than does a microorganism strain whose 20 cysteine metabolism is deregulated without its CysB
activity being increased.
Consequently, a microorganism strain according to the invention is a microorganism strain which possesses a 25 deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB activity, and in which homologous or heterologous cysB genes, which encode CysB having a regulatory pattern which is typical of wild-type CysB, 30 are expressed to an increased extent.
Preference is given to the Escherichia coli strains being strains which possess a deregulated cysteine metabolism, with this deregulation of cysteine 35 metabolism not being based on a change in CysB
. ~ _ 6 _ activity, and in which a wild-type cysB gene is overexpressed.
Particular preference is given to the Escherichia coli strains being strains which possess a deregulated cysteine metabolism, with this deregulation of cysteine metabolism not being based on a change in. CysB
activity, and in which the copy number of the Escherichia coli wild-type cysB gene is increased and this gene is overexpressed.
Surprisingly, it was not found, as postulated in the abovementioned disclosures of the prior art, that cysteine production is increased by using cysB alleles which encode constitutively active CysB regulatory proteins, but that, quite on the contrary, cysteine production is increased by increasing the expression of a wild-type cysB gene.
This finding is also surprising and unexpected since there is no previously known example in which increased expression of a regulatory protein from the LysR-type transcription regulator family enables a metabolite to be overproduced.
The invention consequently also relates to the use of a regulatory protein from the LysR-type transcription regulator family for overproducing a metabolite and also to a process for overproducing a metabolite, characterized in that a regulatory gene from the LysR-type transcription regulator family is overexpressed in a microorganism and brings about an increased production of the metabolite in the microorganism.
The CysB activity in a microorganism can be increased, - 7 _ while at the same time retaining the typical regulatory pattern, by, far example:
1. increasing the copy number of a cysB gene, which encodes CysB having a regulatory pattern which is 5 typical of wild-type CysB, in the microorganism under the control of a promoter, or 2. increasing the expression of a cysB gene, which encodes CysB having a regulatory pattern which is typical of wild-type CysB, by replacing the 10 promoter which regulates the expression of the wild-type cysB gene with a stronger promoter.
cysB genes are known from Escherichia coli, Salmonella typhimurium, Klebsiella aerogenes, Haemophilus 15 influenzae, Pseudomonas aeruginosa and Thiocapsa roseopersicina. cysB genes are preferably understood as being genes whose gene products exhibit an identity of at least 40~ with the Escherichia coli CysB protein.
The homology values refer to results which are obtained 20 with the "Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin" computer program. In this connection, the database is searched with the "blast" subprogram using standard parameters.
25 In addition, cysB genes are those alleles of wild-type cysB genes the sequence of whose gene products is altered without the regulatory pattern which is typical of wild-type CysB thereby being lost. CysB variants containing conservative amino acid substitutions are an 30 example of this.
A microorganism according to the invention can be prepared, for example, by using methods which are known per se to increase the copy number of the wild-type 35 cysB gene or of a cysB gene which encodes CysB having _ g a regulatory pattern which is typical of wild-type CysB, or by using methods which are known per se to increase the expression of the wild-type cysB gene, or of a cysB gene which encodes a CysB having a regulatory pattern which is typical of wild-type CysB, in a microorganism strain which possesses a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB activity.
In accordance with one embodiment of the present invention there is provided a process for preparing a microorganism for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives comprising in a microorganism strain possessing a deregulated cysteine metabolism, increasing the copy number of a wild-type cysB gene, or of a cysB gene which encodes CysB having a regulatory pattern which is typical of wild-type CysB, or bringing about an increased expression of the wild-type cysB gene, or of a cysB gene which encodes a CysB
having a regulatory pattern which is typical of wild-type CysB.
In that which follows, the term "CysB" denotes both "wild-type CysB" and "CysB variant having a regulatory pattern which is typical of a wild-type CysB". In that which follows, the term "cysB gene" denotes "wild-type cysB gene"
and also "cysB gene which encodes CysB having a regulatory pattern which is typical of wild-type CysB".
Methods which are known to a skilled person can be used to increase the copy number of the cysB gene in a microorganism. Thus, the cysB gene can, for example, be cloned into plasmid vectors which are present in multiple - 8a -copies per cell (e.g. pUCl9, pBR322 and pACYC184) and introduced into a microorganism which possesses a deregulated cysteine metabolism. Alternatively, the cysB
gene can be integrated several times into the chromosome of a microorganism which possesses a deregulated cysteine metabolism. Integration methods which can be used are the known systems employing temperate bacteriophages or integrated plasmids, or else integration by way of homologous recombination (e.g. Hamilton et al., 1989, J.
Bacteriol. 171: 4617-4622).
Preference is given to increasing the copy number by . i _ g _ cloning a cysB gene into plasmid vectors under the control of a promoter. Particular preference is given to increasing the copy number by cloning a cysB gene into pACYC derivatives such as pACYC184-LH (deposited in the Deutsche Sammlung fur Mikroorganismen and Zellkulturen, Braunschweig, under number DSM 10172 on 18.8.95 in accordance with the Budapest Treaty).
The natural promoter and operator region of the gene can serve as the control region for expressing a plasmid-encoded cysB gene.
However, the expression of a cysB gene can also be increased using other promoters. Appropriate promoter systems are known to a skilled person (Makrides S.C., 1996, Microbiol. Rev. 60: 512-538). These constructs can be used on plasmids or chromosomally in a manner known per se.
For example, a cysB gene is cloned into plasmid vectors by being specifically amplified by means of the polymerase chain reaction using specific primers which encompass the complete cysB gene including the promoter and operator sequence and then being ligated to vector DNA fragments.
The preferred vectors used for cloning a cysB gene are plasmids which already contain genetic elements for deregulating the cysteine metabolism, for example a cysEX gene (W097/15673) and an efflux gene (EP 0885962 Al). Such vectors enable a microorganism strain according to the invention to be prepared from any arbitrary microorganism strain since such a vector also deregulates the metabolism of cysteine in a microorganism.
- l~ -The invention consequently also relates to a plasmid which is characterized in that it possesses genetic elements for deregulating cysteine metabolism, with these genetic elements not bringing about any change in the CysB activity, and also contains a cysB gene under the control of a promoter.
One embodiment of the invention provides plasmid having genetic elements for deregulating cysteine metabolism, with these genetic elements not bringing about any change in CysB
activity, and also containing a cysB gene under the control of a promoter, with the gene having a regulatory pattern such that CysB activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide.
The cysB-containing plasmids are introduced into bacteria using a current transformation method (e. g. electro-poration), and plasmid-harbouring clones are selected, for example by means of antibiotic resistance.
The invention consequently also relates to a process for preparing a microorganism strain according to the invention, characterized in that a plasmid according to the invention is introduced into a microorganism strain.
A microorganism strain according to the invention is used to produce cysteine or cysteine derivatives in a fermenter in accordance with methods which are known per se. The C
sources employed can, for example, be glucose or lactose, or other sugars, while the N source employed can be ammonium or protein hydrolyzate. The S source employed can, for example, be sulphide, sulphite, sulphates or thiosulphate.
- 10a -L-cysteine which is formed during fermentation can be oxidized to give difficulty soluble cysteine or be condensed with aldehydes or ketones to give thiazolidines (e. g. with pyruvic acid to give 2-methylthiazolidine-2,9-dicarboyxlic acid) .
The invention consequently also relates to a process for producing L-cysteine or L-cysteine derivatives ' - 11 -which is characterized in that a microorganism strain according to the invention is employed in the fermentation in a manner known per se and the L
cysteine or L-cysteine derivative is separated off from the fermentation mixture.
The following examples serve to clarify the invention.
Example 1: Cloning the v~ild-type cysB gene and the cysB(T149M) allele The Escherichia coli wild-type cysB gene was cloned using the polymerase chain reaction (PCR). The specific oligonucleotide primers cysBP1 (SEQ. ID.NO: 1) and cysBP2 (SEQ. ID. NO: 2) (20 pmol per mixture) were used to amplify a genomic DNA fragment of 3107 base pairs in length which encompasses the wild-type cysB gene, together with flanking regions, and possesses terminal EcoRI and Sall restriction cleavage sites, respectively.
5'-GTT ACG AGA TCG AAG AGG-3' (phosphorothioate bond at the 3'-end) (SEQ. ID.NO: 1) 5'-GTC ACC GAG TGG TCA ATG-3' (phosphorothioate bond at the 3'-end) (SEQ. ID.NO: 2) The PCR reaction was carried out using the Boehringer (Mannheim, Germany) Pwo DNA polymerase and employing 10 ng of genomic DNA as the template. The programme comprised 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 denaturing temperature of 94°C (30 seconds per cycle). The DNA
fragment was then treated with the restriction enzymes - 12 _ EcoRI and SalI and purified by way of a preparative gel electrophoresis and the Geneclean method (Geneclean° Kit BI0101 P.O. Box 2284, La Jolla, California, USA, 92038 2284). This fragment was ligated into the Bio-Rad Laboratories (Hercules, California, USA) phagemid vector pTZl9U, which had been cut with EcoRI/SalI and treated with phosphatase, thereby resulting in the plasmid pTZl9U-cysB (Fig. 2). Following transformation, positive clones were identified by means of restriction analysis.
In order to construct a constitutively active cysB
allele, codon 147 of the wild-type cysB gene was mutated (in analogy with the mutation in the Salmonella typhimurium wild-type cysB gene described in Colyer T.
E., Kredich N. M., 1994, Mol. Microbiol. 13: 797-805) into a methionine codon using the "Mutagene In Vitro Mutagenesis" kit from Bio-Rad Laboratories (Hercules, California, USA). The oligonucleotide CysBMut4 (SEQ.
ID. N0. 3) was used in this context. The underlined bases indicate the difference from the wild-type sequence.
S'-TTC GCT ATC GCC ATG GAA GCG CTG CAT-3' (SEQ. ID. NO.
requires N-acetylserine, which is formed in the cell from 0-acetylserine when insufficient reduced sulphur is available for the 0-acetylserine sulphhydrylase reaction, as an inducer.
Consequently, all the genes which are connected with the uptake, reduction and incorporation of sulphur are under the control of CysB. These genes are the operons cysPTWAM, cysDNC and cysJIH and also the cysK gene. Whereas acetylserine acts as an inducer for CysB, sulphide and thiosulphate exhibit a negative effect as so-called "antiinducers", since their presence indicates the availability of SH groups.
The state of the art with regard to obtaining L-cysteine and L-cysteine derivatives is discussed in detail in WO 97/15673 (corresponds to US Patent 6,218,168). WO 97/15673 itself described a fermentative production process which uses feedback-resistant serine acetyltransferases. The application also discloses that it is possible to obtain a further increase in cysteine yield by additionally deregulating the regulatory protein CysB at the gene level such that the gene is constitutively expressed.
The patent application EP 885962 A1 (corresponds to the US Patent 5,972,663) discloses microorganisms which are suitable for fermentatively producing L-cysteine, L-cystine, N
acetyl serine and thiazolidine derivatives and which are characterized in that they overexpress at least one gene which encodes a protein which is directly suitable for secreting antibiotics, or other substances which are toxic for the microorganisms, out of the cell.
_ 3 _ CysB belongs to the family of LysR type transcription regulators (LTTR), more than 100 representatives of which are already known (Schell M.A., 1993, Annu. Rev.
Microbiol. 47: 597-626). They are distinguished by the fact that they possess an N-terminal DNA-binding domain, having a helix-turn-helix motif, and a C-terminal inducer-binding domain. Detailed DNA-binding studies are, available for CysB. Furthermore, CysB is the first LTTR protein for 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 which are dependent on the presence of an inducer molecule. It has previously been assumed that only highly active CysB variants, which are independent of effector molecules (so-called constitut.ively active variants), allow an increase in cysteine production since such forms display a constantly high degree of gene activation (Nakamori S. et al., 1998, Appl. Env.
Microbiol. 64:1607-1611).
Examples of constitutively active forms of CysB have been described in the case of Salmonella typhimurium.
These variants exhibit a high degree of activity, with this activity being completely independent of the inducer N-acetylserine and the negative effect of thiosulphate and sulphide (Colyer T. E., K:redich N. M., 1994, Mol. Microbiol. 13: 797-805).
The present invention relates to a microorganism strain which is suitable for fermentatively producing L-cysteine or L-cysteine derivatives and possesses a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB activity, characterized in that the strain additionally possesses an increased CysB activity, with the CysB activity having a regulatory pattern which is typical of a wild-type CysB.
In accordance with one embodiment of the present invention there is provided an isolated microorganism strain that is transformed with a polynucleotide encoding the transcription regulator protein CysB, for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives, the microorganism having a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB
activity, and the strain additionally having an increased CysB activity as compared to an unmodified microorganism strain, with the CysB activity having a regulatory pattern such that CysB activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide.
In accordance with another embodiment of the present invention there is provided an isolated microorganism strain that is transformed with a polynucleotide encoding the transcription regulator protein CysB for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives comprising a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB
activity, and in which strain homologous or heterologous cysB genes, which encode CysB having a regulatory pattern such that CysB activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide, are expressed to increased extent compared to an unmodified microorganism.
- 4a -Microorganism strains which possess a deregulated cysteine metabolism in which this deregulation is not due to an alteration in CysB activity are known. These are strains which possess modified cysE alleles, as described, for example, in WO 97/15673 or Nakamori S. et al., 1998, Appl.
Env. Microbiol. 64: 1607-1611, or strains in which efflux genes have been inserted, as described, for example, in EP
0885962 A1 (corresponds to U.S. Patent 5,972,663), or strains which are isolated using nonspecific mutagenesis methods combined with methods for screening for cysteine overproduction or decreased cysteine breakdown, as described, for example, in WO 97/15673 or in Nakamori S. et al., 1998, Appl. Env. Microbiol. 64:1607-1611.
Within the meaning of the invention, CysB activity is increased when this activity is at least 10'o higher than that in the wild-type strain.
Preference is given to the CysB activity being at least 250 higher.
Particular preference is given to the CysB activity being at least SOo higher.
An Escherichia coli strain (MC4100::AKZL300) which is suitable for determining the CysB activity is described in Example 2. This strain was deposited in the DSMZ (Deutsche Sammlung fur Mikroorganismen and Zellkulturen ' _ 5 _ [German collection of microorganisms and cell cultures]
GmbH, D-38142 Braunschweig) under number DSM 12886 on 23.6.99 in accordance with the Budapest Treaty. In addition, each respective cysB-containing construct is 5 introduced into strain MC4100::?~KZL300 in a manner known per se.
The CysB activity exhibits a regulatory pattern, in dependence on the S source, which is typical of a wild-10 type CysB, when the CysB activity of cells which are grown with thiosulphate is less than 750 of that of cells which are grown with sulphate and the CysB
activity of cells which are grown with cystine is less than 20~ of that of cells which are grown with sulphate 15 (see Example 2).
Microorganism strains according to the invention secrete L-cysteine or an L-cysteine derivative in greater amounts than does a microorganism strain whose 20 cysteine metabolism is deregulated without its CysB
activity being increased.
Consequently, a microorganism strain according to the invention is a microorganism strain which possesses a 25 deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB activity, and in which homologous or heterologous cysB genes, which encode CysB having a regulatory pattern which is typical of wild-type CysB, 30 are expressed to an increased extent.
Preference is given to the Escherichia coli strains being strains which possess a deregulated cysteine metabolism, with this deregulation of cysteine 35 metabolism not being based on a change in CysB
. ~ _ 6 _ activity, and in which a wild-type cysB gene is overexpressed.
Particular preference is given to the Escherichia coli strains being strains which possess a deregulated cysteine metabolism, with this deregulation of cysteine metabolism not being based on a change in. CysB
activity, and in which the copy number of the Escherichia coli wild-type cysB gene is increased and this gene is overexpressed.
Surprisingly, it was not found, as postulated in the abovementioned disclosures of the prior art, that cysteine production is increased by using cysB alleles which encode constitutively active CysB regulatory proteins, but that, quite on the contrary, cysteine production is increased by increasing the expression of a wild-type cysB gene.
This finding is also surprising and unexpected since there is no previously known example in which increased expression of a regulatory protein from the LysR-type transcription regulator family enables a metabolite to be overproduced.
The invention consequently also relates to the use of a regulatory protein from the LysR-type transcription regulator family for overproducing a metabolite and also to a process for overproducing a metabolite, characterized in that a regulatory gene from the LysR-type transcription regulator family is overexpressed in a microorganism and brings about an increased production of the metabolite in the microorganism.
The CysB activity in a microorganism can be increased, - 7 _ while at the same time retaining the typical regulatory pattern, by, far example:
1. increasing the copy number of a cysB gene, which encodes CysB having a regulatory pattern which is 5 typical of wild-type CysB, in the microorganism under the control of a promoter, or 2. increasing the expression of a cysB gene, which encodes CysB having a regulatory pattern which is typical of wild-type CysB, by replacing the 10 promoter which regulates the expression of the wild-type cysB gene with a stronger promoter.
cysB genes are known from Escherichia coli, Salmonella typhimurium, Klebsiella aerogenes, Haemophilus 15 influenzae, Pseudomonas aeruginosa and Thiocapsa roseopersicina. cysB genes are preferably understood as being genes whose gene products exhibit an identity of at least 40~ with the Escherichia coli CysB protein.
The homology values refer to results which are obtained 20 with the "Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin" computer program. In this connection, the database is searched with the "blast" subprogram using standard parameters.
25 In addition, cysB genes are those alleles of wild-type cysB genes the sequence of whose gene products is altered without the regulatory pattern which is typical of wild-type CysB thereby being lost. CysB variants containing conservative amino acid substitutions are an 30 example of this.
A microorganism according to the invention can be prepared, for example, by using methods which are known per se to increase the copy number of the wild-type 35 cysB gene or of a cysB gene which encodes CysB having _ g a regulatory pattern which is typical of wild-type CysB, or by using methods which are known per se to increase the expression of the wild-type cysB gene, or of a cysB gene which encodes a CysB having a regulatory pattern which is typical of wild-type CysB, in a microorganism strain which possesses a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB activity.
In accordance with one embodiment of the present invention there is provided a process for preparing a microorganism for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives comprising in a microorganism strain possessing a deregulated cysteine metabolism, increasing the copy number of a wild-type cysB gene, or of a cysB gene which encodes CysB having a regulatory pattern which is typical of wild-type CysB, or bringing about an increased expression of the wild-type cysB gene, or of a cysB gene which encodes a CysB
having a regulatory pattern which is typical of wild-type CysB.
In that which follows, the term "CysB" denotes both "wild-type CysB" and "CysB variant having a regulatory pattern which is typical of a wild-type CysB". In that which follows, the term "cysB gene" denotes "wild-type cysB gene"
and also "cysB gene which encodes CysB having a regulatory pattern which is typical of wild-type CysB".
Methods which are known to a skilled person can be used to increase the copy number of the cysB gene in a microorganism. Thus, the cysB gene can, for example, be cloned into plasmid vectors which are present in multiple - 8a -copies per cell (e.g. pUCl9, pBR322 and pACYC184) and introduced into a microorganism which possesses a deregulated cysteine metabolism. Alternatively, the cysB
gene can be integrated several times into the chromosome of a microorganism which possesses a deregulated cysteine metabolism. Integration methods which can be used are the known systems employing temperate bacteriophages or integrated plasmids, or else integration by way of homologous recombination (e.g. Hamilton et al., 1989, J.
Bacteriol. 171: 4617-4622).
Preference is given to increasing the copy number by . i _ g _ cloning a cysB gene into plasmid vectors under the control of a promoter. Particular preference is given to increasing the copy number by cloning a cysB gene into pACYC derivatives such as pACYC184-LH (deposited in the Deutsche Sammlung fur Mikroorganismen and Zellkulturen, Braunschweig, under number DSM 10172 on 18.8.95 in accordance with the Budapest Treaty).
The natural promoter and operator region of the gene can serve as the control region for expressing a plasmid-encoded cysB gene.
However, the expression of a cysB gene can also be increased using other promoters. Appropriate promoter systems are known to a skilled person (Makrides S.C., 1996, Microbiol. Rev. 60: 512-538). These constructs can be used on plasmids or chromosomally in a manner known per se.
For example, a cysB gene is cloned into plasmid vectors by being specifically amplified by means of the polymerase chain reaction using specific primers which encompass the complete cysB gene including the promoter and operator sequence and then being ligated to vector DNA fragments.
The preferred vectors used for cloning a cysB gene are plasmids which already contain genetic elements for deregulating the cysteine metabolism, for example a cysEX gene (W097/15673) and an efflux gene (EP 0885962 Al). Such vectors enable a microorganism strain according to the invention to be prepared from any arbitrary microorganism strain since such a vector also deregulates the metabolism of cysteine in a microorganism.
- l~ -The invention consequently also relates to a plasmid which is characterized in that it possesses genetic elements for deregulating cysteine metabolism, with these genetic elements not bringing about any change in the CysB activity, and also contains a cysB gene under the control of a promoter.
One embodiment of the invention provides plasmid having genetic elements for deregulating cysteine metabolism, with these genetic elements not bringing about any change in CysB
activity, and also containing a cysB gene under the control of a promoter, with the gene having a regulatory pattern such that CysB activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide.
The cysB-containing plasmids are introduced into bacteria using a current transformation method (e. g. electro-poration), and plasmid-harbouring clones are selected, for example by means of antibiotic resistance.
The invention consequently also relates to a process for preparing a microorganism strain according to the invention, characterized in that a plasmid according to the invention is introduced into a microorganism strain.
A microorganism strain according to the invention is used to produce cysteine or cysteine derivatives in a fermenter in accordance with methods which are known per se. The C
sources employed can, for example, be glucose or lactose, or other sugars, while the N source employed can be ammonium or protein hydrolyzate. The S source employed can, for example, be sulphide, sulphite, sulphates or thiosulphate.
- 10a -L-cysteine which is formed during fermentation can be oxidized to give difficulty soluble cysteine or be condensed with aldehydes or ketones to give thiazolidines (e. g. with pyruvic acid to give 2-methylthiazolidine-2,9-dicarboyxlic acid) .
The invention consequently also relates to a process for producing L-cysteine or L-cysteine derivatives ' - 11 -which is characterized in that a microorganism strain according to the invention is employed in the fermentation in a manner known per se and the L
cysteine or L-cysteine derivative is separated off from the fermentation mixture.
The following examples serve to clarify the invention.
Example 1: Cloning the v~ild-type cysB gene and the cysB(T149M) allele The Escherichia coli wild-type cysB gene was cloned using the polymerase chain reaction (PCR). The specific oligonucleotide primers cysBP1 (SEQ. ID.NO: 1) and cysBP2 (SEQ. ID. NO: 2) (20 pmol per mixture) were used to amplify a genomic DNA fragment of 3107 base pairs in length which encompasses the wild-type cysB gene, together with flanking regions, and possesses terminal EcoRI and Sall restriction cleavage sites, respectively.
5'-GTT ACG AGA TCG AAG AGG-3' (phosphorothioate bond at the 3'-end) (SEQ. ID.NO: 1) 5'-GTC ACC GAG TGG TCA ATG-3' (phosphorothioate bond at the 3'-end) (SEQ. ID.NO: 2) The PCR reaction was carried out using the Boehringer (Mannheim, Germany) Pwo DNA polymerase and employing 10 ng of genomic DNA as the template. The programme comprised 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 denaturing temperature of 94°C (30 seconds per cycle). The DNA
fragment was then treated with the restriction enzymes - 12 _ EcoRI and SalI and purified by way of a preparative gel electrophoresis and the Geneclean method (Geneclean° Kit BI0101 P.O. Box 2284, La Jolla, California, USA, 92038 2284). This fragment was ligated into the Bio-Rad Laboratories (Hercules, California, USA) phagemid vector pTZl9U, which had been cut with EcoRI/SalI and treated with phosphatase, thereby resulting in the plasmid pTZl9U-cysB (Fig. 2). Following transformation, positive clones were identified by means of restriction analysis.
In order to construct a constitutively active cysB
allele, codon 147 of the wild-type cysB gene was mutated (in analogy with the mutation in the Salmonella typhimurium wild-type cysB gene described in Colyer T.
E., Kredich N. M., 1994, Mol. Microbiol. 13: 797-805) into a methionine codon using the "Mutagene In Vitro Mutagenesis" kit from Bio-Rad Laboratories (Hercules, California, USA). The oligonucleotide CysBMut4 (SEQ.
ID. N0. 3) was used in this context. The underlined bases indicate the difference from the wild-type sequence.
S'-TTC GCT ATC GCC ATG GAA GCG CTG CAT-3' (SEQ. ID. NO.
3) For activity tests (see Example 2), the two cysB
alleles were cloned, as EcoRI/SalI fragments, after treatment with Klenow, into the EcI136II-cut, phosphatase-treated vector pACYCI84-LH.
Example 2: Determining CysB activity in vivo A reporter gene assay was selected for measuring the activity of CysB. For this, the control region of the cysK gene was fused to the lacZ gene, which encodes the enzyme (3-galactosidase. When this fusion is integrated into strains which lack an endogenous a-galactosidase, this enzyme is formed in dependence on CysB activity S and consequently provides, in the form of ~i-galactosidase activity, an indirect measure of CysB
activity. The system described by Simons et al. (Simons R. W. et al., 1987, Gene 53: 85-96) was used for constructing the fusion. The promoter region of the cysK gene, including the first fifteen codons, was first of all amplified by means of the polymerase chain reaction using the oligonucleotide primers cysKP1 (SEQ.
ID. NO: 4) and cysKP3 (SEQ. ID. NO: 5) and 10 ng of Escherichia coli chromosomal DNA and Pwo polymerase.
5'-CCG GAA TTC CCG TTG CCG TTT GTG GCG-3' (SEQ. ID. N0:
alleles were cloned, as EcoRI/SalI fragments, after treatment with Klenow, into the EcI136II-cut, phosphatase-treated vector pACYCI84-LH.
Example 2: Determining CysB activity in vivo A reporter gene assay was selected for measuring the activity of CysB. For this, the control region of the cysK gene was fused to the lacZ gene, which encodes the enzyme (3-galactosidase. When this fusion is integrated into strains which lack an endogenous a-galactosidase, this enzyme is formed in dependence on CysB activity S and consequently provides, in the form of ~i-galactosidase activity, an indirect measure of CysB
activity. The system described by Simons et al. (Simons R. W. et al., 1987, Gene 53: 85-96) was used for constructing the fusion. The promoter region of the cysK gene, including the first fifteen codons, was first of all amplified by means of the polymerase chain reaction using the oligonucleotide primers cysKP1 (SEQ.
ID. NO: 4) and cysKP3 (SEQ. ID. NO: 5) and 10 ng of Escherichia coli chromosomal DNA and Pwo polymerase.
5'-CCG GAA TTC CCG TTG CCG TTT GTG GCG-3' (SEQ. ID. N0:
4) 5'-CGC GGA TCC GTG TGA CCG ATA GTC AGC-3' (SEQ. ID. NO:
5) The conditions corresponded to those which were described in Example 1. The resulting 317 base pair product was digested with the restriction enzymes EcoRI
and BamHI, in accordance with the manufacturer's instructions, and purified by means of preparative gel electrophoresis and the Geneclean method. The product was then ligated to the vector pRS552 (deposited in the Deutsche Sammlung fur Mikroorganismen and Zellkulturen, Braunschweig, under number DSM 13034 on 14.9.99 in accordance with the Budapest Treaty), which had likewise been digested with EcoRI-BamHI and treated with phosphatase. The strain MC4100 (ATCC 35695) was transformed with the ligation mixture using electroporation, and positive clones were identified f with the aid of restriction analyses. These positive clones contain a translational cysK-lacZ fusion. The resulting plasmid was recombined with the bacteriophage ARS45 (deposited in the Deutsche Sammlung fur Mikroorganismen and Zellkulturen, Braunschweig, under number DSM 13035 on 14.9.99 in accordance with the Budapest Treaty), following the instructions of Simons et al., and a homogeneous lysate of the recombinant phage, which was designated ~KZL300, was prepared. The ~lac strain MC4100 was infected with this phage and lysogenic clones (MC4100::J~KZL300), which it was now possible to use for measuring CysB activity, were identified by kanamycin selection.
Tn order to compare the effect of a multicopy cysB gene cloned into pACYC184-LH and of a cysB(T149M) gene, MC4100::?~KZL300 was transformed with the corresponding plasmids, i.e. pACYC-cysB and pACYC-cysB(T149M).
The strains were cultured in VB minimal medium (3.5 g of Na(NH4)HP04/l; 10 g of KHzP04/1; 2 g of citrate x Hz0/1; 0.078 g of MgClz/1; pH adjusted to 6.5 with NaOH, 5 g of glucose/1; 5 mg of vitamin B1/1) containing different sulphur sources (in each case 1 mM sulphur), with 15 mg of tetracycline being added per litre. (3 Galactosidase activity was determined in accordance with the method described by Miller (Miller J. H., 1972, Experiments in Molecular Genetics, Cold Spring Harbor, New York, 352-355).
The results are shown in Table 1. In the case of the control (MC4100::AKZL300/pACYC184-LH), the regulatory pattern of the CysB activity, in dependence on the S
source, is found to be that which is typical of a wild-type CysB; the CysB activity of cells which are grown with thiosulphate is less than 75~ of that when grown with sulphate, and the CysB activity of cells which are grown with cystine is less than 200 of that when grown with sulphate. This pattern is retained, at a level of activity which is raised overall, in the presence of multiple copies of a wild-type cysB gene (example MC4100::AKZL300/pACYC-cysB, in accordance with the invention). By contrast, the cysB(T149M) allele leads to a constitutively high level of activity with loss of the typical regulatory pattern.
Table 1 Determination of the CysB activity, in the form of ~i-galactosidase activity, of strains harbouring a chromosomal cysK-lacZ fusion Strain MC4100:: MC4100:: MC4100::
AKZL300 1~KZL300 AKZL300 Plasmid pACYC184-LH pACYC-cysB pACYC-cysB(T149M) Genotype cysB cysB cysB(T149M) single copy multicopy multicopy S source: (3-galactosidase activity in Miller units Cystine 26 341 3791 Sulphate 1906 2708 3824 Thiosulphate 726 1094 3595 - Example 3: Constructing plasmids according to the invention Organisms according to the invention are characterized by a deregulated cysteine metabolism and, for example, by a cysB gene which is present in multiple copies. The plasmid (pACYC184-cysEX-GAPDH-ORF306) was chosen as the basic construct for preparing these organisms. This plasmid contains the feedback-resistant cysE allele and the efflux gene as elements for deregulating cysteine metabolism. It is described in detail in patent application EP 0885962 A1 (Example 2D). A cysB fragment was inserted into this construct, which had been digested with the restriction enzyme SnaBI and treated with phosphatase, between the cysEX allele and the efflux gene. The cysB fragment was obtained from plasmid pTZl9U-cysB (Fig. 2) by restricting with the enzymes EcoRI and BstXI and subsequently smoothing the DNA ends with Klenow enzyme. The plasmid is designated pHC34.
An organism according to the invention is obtained by transforming the Escherichia coli strain W3110 (ATCC
27325; Bachmann B. J., 1996, in: Neidhardt F. C. (ed.) Escherichia coli and Salmonella: cellular and molecular biology, American Society for Microbiology, Washingivn D.C., Chapter 133). The transformation was carried out using electroporation. For transformation, 0.1 ug of plasmid DNA was added to a thick cell suspension in an ice-cold 10~ solution of glycerol and the suspension was then subjected to an electric pulse at. 2500 V, 200 Ohms and 12.5 uF. After the mixture had been transferred into sterile LB medium (1o tryptone, 0.5~
yeast extract, 1~ NaCl) and incubated at 30°C for one hour, plasmid-harbouring clones were selected on LB
. . _ 1'j _ agar plates containing 15 ug tetracycline/mi.
In order to compare the effect of the wild-type cysB
gene with that of the constitutive cysB(T149M) allele, an analogous construct (pHC30) was prepared and likewise introduced into the strain W3100. In addition, the organism W3110, which is described in EP 885962 A1, was used, transformed with the plasmid pACYC184/cysEX-GAPDH-orf306, as the basic construct for the comparison and, at the same time, for delimiting from the prior art.
Since W3110 is a wild-type strain, all the cysteine production effects are to be attributed to plasmid encoded genes.
Example 4: Cysteine production using microorganisms in accordance with the invention In order to detect cysteine production, the microorganisms described in Example 3 were cultured in fermenters in the fed batch mode with continuous feeding of glucose and thiosulphate. The device employed was a Braun Biotech (Melsungen, Germany) ~ Biostat M appliance having a maximum culture volume of 2 1.
20 ml of LB medium (10 g of tryptone/1, 5 g of yeast extract/l, 10 g of NaCl/1), which additionally contained 15 mg of tetracycline/l, were inoculated, as a preculture, and incubated at 30°C and 150 rpm in a shaker. After 7 hours, the entire mixture was transferred into 100 ml of SM1 medium (12 g of KZHPO9/1;
3 g of KHzP04/1; 5 g of (NH4)2S04/l; 0.3 g of MgS04 X
7Hz0/l; 0.015 g of CaCl2 X 2HZ0/1; 0.002 g of FeS04 X
7H20/l; 1 g of Na3 citrate X 2H20/l; 0.1 g of NaCl/l;
1 ml of trace element solution~,~consisting of 0.15 g of i Na.2Mo04 X 2H20/ 1; 2 . 5 g of NaB03 / 1; 0 . 7 g of CoClz X
6HZ0/1; 0.25 g of CuS04 X 5HZ0/l; 1.6 g of MnCl2 X
4H20/1; 0.3 g of ZnS04 X 7H20/1) which was supplemented with 5 g of glucose/1; 0.5 mg of vitamin Bl/1 and 15 mg of tetracycline/l. Subsequent incubation took place at 30°C for 17 hours at 150 rpm.
This preculture (optical density of approx. 3 at 600 nm) was used to inoculate the fermenter containing 900 ml of fermentation medium (15 g of glucose/1; 10 g of trypton/1; 5 g of yeast extract/l; 5 g of (NH4)zS04/l; 1.5 g of KHzP04/1; 0.5 g of NaCI/l; 0.3 g of MgS04 X 7Hz0/1; 0.015 g of CaCl2 X 2H20/1; 0.075 g of FeS04 X 7H20/1; 1 g of Na3 citrate X 2Hz0/1 and 1 ml of trace element solution, see above, per litre, 5 mg of vitamin B1/1 and 15 mg of tetracycline/1, adjusted to pH 7.0 with 25~ ammonia). During the fermentation, the temperature was set at 30°C and the pH was kept constant at a value of 7.0 by metering in 25~ ammonia.
The culture was gassed, at 1.5 vol/vol/min, with sterilized compressed air and stirred with a stirring device at a rotational speed of 200 rpm. After the oxygen saturation had decreased to a value of 50~, the rotational speed was increased, using a control device, to a value of 1200 rpm in order to maintain 50~ oxygen saturation.
After 2 hours, a 30o solution of Na thiosulphate was metered in at a rate of 3 ml/h. Glucose was fed in from a 56o stock solution as soon as the content in the fermenter, which was initially 15 g/l, had fallen to approx. 5-10 g/l. The glucose was fed in at a flow rate of 8-14 ml/h, with an attempt being made to maintain a constant glucose concentration of approx. 5-10 g/1. The . , _ 19 -glucose was determined using a YSI (Yellow Springs, Ohio, USA) glucose analyser.
The production of L-cysteine was monitored colorimetrically using the Gaitonde test (Gaitonde, M.K. (1967), Biochem. J. 104, 627-633). In this connection, account has to be taken of the fact that the test does not discriminate between L-cysteine and the condensation product of L-cysteine and pyruvate (2-methylthiazolidine-2,4-dicarboxylic acid) which is described in EP 0885962 A1. Difficultly soluble cystine, which is formed from L-cysteine by oxidation, was likewise detected as L-cysteine, in dilute solution at pH 8.0, after dissolving in 8~ hydrochloric acid and subsequently reducing with dithiothreitol (DTT).
Table 2 shows the production course of a fermentation of an organism harbouring the basic construct pACYC184/cysEX-GAPDH-ORF306, which is described in EP
0885962 A1, as compared with that of a fermentation of an organism harbouring a plasmid pHC34 according to the invention and of a fermentation of an organism harbouring a corresponding construct containing the cysB(T149M) allele, which encodes a constitutively active CysB gene product. It is clear that the use, according to the invention, of the wild-type cysB gene exerts a positive effect on productive performance whereas the constitutive allele cysB(T149M) exhibits a negative effect.
Table 2 Production of L-cysteine using the pHC34 constuct according to the invention and using control constructs.
r - 20 -Plasmid pACYC184/cys pHC34 pHC30 construct E X-GAPDH-Genotype cysEX cysEX c:ysEX
orf306 cysB cysB(T149M) orf306 orf306 Fermentatio yield of L-cysteine in g/1 n time ' 24 h 6.3 8.0 + 2.6 2.0 *
48 h 10.2 + 7.0* 10.0 + 3.4 12.6*
* the values which are given with an asterisk are values for L-cysteine which is present in oxidized form as difficultly soluble cystine.
SBQUENCE LISTING
<110> CONSORTIUM FUR ELERTROCHEMISCHE INDUSTRIE GHBH
<120> PROCESS FOR FERMENTATIVELY PRODUCING L-CYSTEINE OR L~CYSTEINE
DERIVATIVES
<130> 1546-348 <140> 2,386,539 <141> October 5, 2000 <150> PCT/EP00/09720 <151> October 5, 2000 <150> DE 199 49 579.3 <151> October 14, 1999 <160> 5 <170> Patentln version 3.1 <210> 1 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 1 gttacgagat cgaagagg 18 <210> 2 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 2 gtcaccgagt ggtcaatg 18 <210> 3 <211> 27 <212> DNA
<213> Artificial sequence <220>
<223> Oligonuclaotide for in vitro mutagenonsis <400> 3 ttcgctatcg ccatggaagc gctgcat 27 <210> 4 <211> 27 .
<212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 4 ccggaattcc cgttgccgtt tgtggcg 27 <210> 5 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 5 cgcggatccg tgtgaccgat agtcagc 27
5) The conditions corresponded to those which were described in Example 1. The resulting 317 base pair product was digested with the restriction enzymes EcoRI
and BamHI, in accordance with the manufacturer's instructions, and purified by means of preparative gel electrophoresis and the Geneclean method. The product was then ligated to the vector pRS552 (deposited in the Deutsche Sammlung fur Mikroorganismen and Zellkulturen, Braunschweig, under number DSM 13034 on 14.9.99 in accordance with the Budapest Treaty), which had likewise been digested with EcoRI-BamHI and treated with phosphatase. The strain MC4100 (ATCC 35695) was transformed with the ligation mixture using electroporation, and positive clones were identified f with the aid of restriction analyses. These positive clones contain a translational cysK-lacZ fusion. The resulting plasmid was recombined with the bacteriophage ARS45 (deposited in the Deutsche Sammlung fur Mikroorganismen and Zellkulturen, Braunschweig, under number DSM 13035 on 14.9.99 in accordance with the Budapest Treaty), following the instructions of Simons et al., and a homogeneous lysate of the recombinant phage, which was designated ~KZL300, was prepared. The ~lac strain MC4100 was infected with this phage and lysogenic clones (MC4100::J~KZL300), which it was now possible to use for measuring CysB activity, were identified by kanamycin selection.
Tn order to compare the effect of a multicopy cysB gene cloned into pACYC184-LH and of a cysB(T149M) gene, MC4100::?~KZL300 was transformed with the corresponding plasmids, i.e. pACYC-cysB and pACYC-cysB(T149M).
The strains were cultured in VB minimal medium (3.5 g of Na(NH4)HP04/l; 10 g of KHzP04/1; 2 g of citrate x Hz0/1; 0.078 g of MgClz/1; pH adjusted to 6.5 with NaOH, 5 g of glucose/1; 5 mg of vitamin B1/1) containing different sulphur sources (in each case 1 mM sulphur), with 15 mg of tetracycline being added per litre. (3 Galactosidase activity was determined in accordance with the method described by Miller (Miller J. H., 1972, Experiments in Molecular Genetics, Cold Spring Harbor, New York, 352-355).
The results are shown in Table 1. In the case of the control (MC4100::AKZL300/pACYC184-LH), the regulatory pattern of the CysB activity, in dependence on the S
source, is found to be that which is typical of a wild-type CysB; the CysB activity of cells which are grown with thiosulphate is less than 75~ of that when grown with sulphate, and the CysB activity of cells which are grown with cystine is less than 200 of that when grown with sulphate. This pattern is retained, at a level of activity which is raised overall, in the presence of multiple copies of a wild-type cysB gene (example MC4100::AKZL300/pACYC-cysB, in accordance with the invention). By contrast, the cysB(T149M) allele leads to a constitutively high level of activity with loss of the typical regulatory pattern.
Table 1 Determination of the CysB activity, in the form of ~i-galactosidase activity, of strains harbouring a chromosomal cysK-lacZ fusion Strain MC4100:: MC4100:: MC4100::
AKZL300 1~KZL300 AKZL300 Plasmid pACYC184-LH pACYC-cysB pACYC-cysB(T149M) Genotype cysB cysB cysB(T149M) single copy multicopy multicopy S source: (3-galactosidase activity in Miller units Cystine 26 341 3791 Sulphate 1906 2708 3824 Thiosulphate 726 1094 3595 - Example 3: Constructing plasmids according to the invention Organisms according to the invention are characterized by a deregulated cysteine metabolism and, for example, by a cysB gene which is present in multiple copies. The plasmid (pACYC184-cysEX-GAPDH-ORF306) was chosen as the basic construct for preparing these organisms. This plasmid contains the feedback-resistant cysE allele and the efflux gene as elements for deregulating cysteine metabolism. It is described in detail in patent application EP 0885962 A1 (Example 2D). A cysB fragment was inserted into this construct, which had been digested with the restriction enzyme SnaBI and treated with phosphatase, between the cysEX allele and the efflux gene. The cysB fragment was obtained from plasmid pTZl9U-cysB (Fig. 2) by restricting with the enzymes EcoRI and BstXI and subsequently smoothing the DNA ends with Klenow enzyme. The plasmid is designated pHC34.
An organism according to the invention is obtained by transforming the Escherichia coli strain W3110 (ATCC
27325; Bachmann B. J., 1996, in: Neidhardt F. C. (ed.) Escherichia coli and Salmonella: cellular and molecular biology, American Society for Microbiology, Washingivn D.C., Chapter 133). The transformation was carried out using electroporation. For transformation, 0.1 ug of plasmid DNA was added to a thick cell suspension in an ice-cold 10~ solution of glycerol and the suspension was then subjected to an electric pulse at. 2500 V, 200 Ohms and 12.5 uF. After the mixture had been transferred into sterile LB medium (1o tryptone, 0.5~
yeast extract, 1~ NaCl) and incubated at 30°C for one hour, plasmid-harbouring clones were selected on LB
. . _ 1'j _ agar plates containing 15 ug tetracycline/mi.
In order to compare the effect of the wild-type cysB
gene with that of the constitutive cysB(T149M) allele, an analogous construct (pHC30) was prepared and likewise introduced into the strain W3100. In addition, the organism W3110, which is described in EP 885962 A1, was used, transformed with the plasmid pACYC184/cysEX-GAPDH-orf306, as the basic construct for the comparison and, at the same time, for delimiting from the prior art.
Since W3110 is a wild-type strain, all the cysteine production effects are to be attributed to plasmid encoded genes.
Example 4: Cysteine production using microorganisms in accordance with the invention In order to detect cysteine production, the microorganisms described in Example 3 were cultured in fermenters in the fed batch mode with continuous feeding of glucose and thiosulphate. The device employed was a Braun Biotech (Melsungen, Germany) ~ Biostat M appliance having a maximum culture volume of 2 1.
20 ml of LB medium (10 g of tryptone/1, 5 g of yeast extract/l, 10 g of NaCl/1), which additionally contained 15 mg of tetracycline/l, were inoculated, as a preculture, and incubated at 30°C and 150 rpm in a shaker. After 7 hours, the entire mixture was transferred into 100 ml of SM1 medium (12 g of KZHPO9/1;
3 g of KHzP04/1; 5 g of (NH4)2S04/l; 0.3 g of MgS04 X
7Hz0/l; 0.015 g of CaCl2 X 2HZ0/1; 0.002 g of FeS04 X
7H20/l; 1 g of Na3 citrate X 2H20/l; 0.1 g of NaCl/l;
1 ml of trace element solution~,~consisting of 0.15 g of i Na.2Mo04 X 2H20/ 1; 2 . 5 g of NaB03 / 1; 0 . 7 g of CoClz X
6HZ0/1; 0.25 g of CuS04 X 5HZ0/l; 1.6 g of MnCl2 X
4H20/1; 0.3 g of ZnS04 X 7H20/1) which was supplemented with 5 g of glucose/1; 0.5 mg of vitamin Bl/1 and 15 mg of tetracycline/l. Subsequent incubation took place at 30°C for 17 hours at 150 rpm.
This preculture (optical density of approx. 3 at 600 nm) was used to inoculate the fermenter containing 900 ml of fermentation medium (15 g of glucose/1; 10 g of trypton/1; 5 g of yeast extract/l; 5 g of (NH4)zS04/l; 1.5 g of KHzP04/1; 0.5 g of NaCI/l; 0.3 g of MgS04 X 7Hz0/1; 0.015 g of CaCl2 X 2H20/1; 0.075 g of FeS04 X 7H20/1; 1 g of Na3 citrate X 2Hz0/1 and 1 ml of trace element solution, see above, per litre, 5 mg of vitamin B1/1 and 15 mg of tetracycline/1, adjusted to pH 7.0 with 25~ ammonia). During the fermentation, the temperature was set at 30°C and the pH was kept constant at a value of 7.0 by metering in 25~ ammonia.
The culture was gassed, at 1.5 vol/vol/min, with sterilized compressed air and stirred with a stirring device at a rotational speed of 200 rpm. After the oxygen saturation had decreased to a value of 50~, the rotational speed was increased, using a control device, to a value of 1200 rpm in order to maintain 50~ oxygen saturation.
After 2 hours, a 30o solution of Na thiosulphate was metered in at a rate of 3 ml/h. Glucose was fed in from a 56o stock solution as soon as the content in the fermenter, which was initially 15 g/l, had fallen to approx. 5-10 g/l. The glucose was fed in at a flow rate of 8-14 ml/h, with an attempt being made to maintain a constant glucose concentration of approx. 5-10 g/1. The . , _ 19 -glucose was determined using a YSI (Yellow Springs, Ohio, USA) glucose analyser.
The production of L-cysteine was monitored colorimetrically using the Gaitonde test (Gaitonde, M.K. (1967), Biochem. J. 104, 627-633). In this connection, account has to be taken of the fact that the test does not discriminate between L-cysteine and the condensation product of L-cysteine and pyruvate (2-methylthiazolidine-2,4-dicarboxylic acid) which is described in EP 0885962 A1. Difficultly soluble cystine, which is formed from L-cysteine by oxidation, was likewise detected as L-cysteine, in dilute solution at pH 8.0, after dissolving in 8~ hydrochloric acid and subsequently reducing with dithiothreitol (DTT).
Table 2 shows the production course of a fermentation of an organism harbouring the basic construct pACYC184/cysEX-GAPDH-ORF306, which is described in EP
0885962 A1, as compared with that of a fermentation of an organism harbouring a plasmid pHC34 according to the invention and of a fermentation of an organism harbouring a corresponding construct containing the cysB(T149M) allele, which encodes a constitutively active CysB gene product. It is clear that the use, according to the invention, of the wild-type cysB gene exerts a positive effect on productive performance whereas the constitutive allele cysB(T149M) exhibits a negative effect.
Table 2 Production of L-cysteine using the pHC34 constuct according to the invention and using control constructs.
r - 20 -Plasmid pACYC184/cys pHC34 pHC30 construct E X-GAPDH-Genotype cysEX cysEX c:ysEX
orf306 cysB cysB(T149M) orf306 orf306 Fermentatio yield of L-cysteine in g/1 n time ' 24 h 6.3 8.0 + 2.6 2.0 *
48 h 10.2 + 7.0* 10.0 + 3.4 12.6*
* the values which are given with an asterisk are values for L-cysteine which is present in oxidized form as difficultly soluble cystine.
SBQUENCE LISTING
<110> CONSORTIUM FUR ELERTROCHEMISCHE INDUSTRIE GHBH
<120> PROCESS FOR FERMENTATIVELY PRODUCING L-CYSTEINE OR L~CYSTEINE
DERIVATIVES
<130> 1546-348 <140> 2,386,539 <141> October 5, 2000 <150> PCT/EP00/09720 <151> October 5, 2000 <150> DE 199 49 579.3 <151> October 14, 1999 <160> 5 <170> Patentln version 3.1 <210> 1 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 1 gttacgagat cgaagagg 18 <210> 2 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 2 gtcaccgagt ggtcaatg 18 <210> 3 <211> 27 <212> DNA
<213> Artificial sequence <220>
<223> Oligonuclaotide for in vitro mutagenonsis <400> 3 ttcgctatcg ccatggaagc gctgcat 27 <210> 4 <211> 27 .
<212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 4 ccggaattcc cgttgccgtt tgtggcg 27 <210> 5 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Primer for PCR
<400> 5 cgcggatccg tgtgaccgat agtcagc 27
Claims (8)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An isolated microorganism strain that is transformed with a polynucleotide encoding the transcription regulator protein CysB, for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives, the microorganism having a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB activity, and the strain additionally having an increased CysB activity as compared to an unmodified microorganism strain, with the CysB activity having a regulatory pattern such that CysB
activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide.
activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide.
2. An isolated microorganism strain that is transformed with a polynucleotide encoding the transcription regulator protein CysB for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives comprising a deregulated cysteine metabolism, with this deregulation of the cysteine metabolism not being based on a change in CysB activity, and in which strain homologous or heterologous cysB genes, which encode CysB having a regulatory pattern such that CysB
activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide, are expressed to increased extent compared to an unmodified microorganism.
activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide, are expressed to increased extent compared to an unmodified microorganism.
3. Microorganism strain according to Claim 1, consisting of an Escherichia coli strain which possesses a deregulated cysteine metabolism and in which a wild-type cysB gene is overexpressed.
4. Microorganism strain according to Claim 2, consisting of an Escherichia coli strain which possesses a deregulated cysteine metabolism and in which the copy number of the Escherichia coli wild-type cysB gene is increased and said gene is overexpressed.
5. Process for preparing a microorganism for fermentatively producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives comprising in a microorganism strain possessing a deregulated cysteine metabolism, increasing the copy number of a wild-type cysB
gene, or of a cysB gene which encodes CysB having a regulatory pattern which is typical of wild-type CysB, or bringing about an increased expression of the wild-type cysB
gene, or of a cysB gene which encodes a CysB having a regulatory pattern which is typical of wild-type CysB.
gene, or of a cysB gene which encodes CysB having a regulatory pattern which is typical of wild-type CysB, or bringing about an increased expression of the wild-type cysB
gene, or of a cysB gene which encodes a CysB having a regulatory pattern which is typical of wild-type CysB.
6. Process for producing a substance selected from the group consisting of L-cysteine and L-cysteine derivatives, comprising employing a microorganism strain according to Claim 1 in a fermentation mixture, and separating off the L-cysteine or L-cysteine derivative from the fermentation mixture.
7. Plasmid having genetic elements for deregulating cysteine metabolism, with these genetic elements not bringing about any change in CysB activity, and also containing a cysB gene under the control of a promoter, with the gene having a regulatory pattern such that CysB activity is induced by the presence of N-acetylserine and reduced by the presence of sulphide or thiosulphide.
8. Process for preparing a microorganism strain comprising introducing a plasmid according to Claim 7 into a microorganism strain.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19949579.3 | 1999-10-14 | ||
| DE19949579A DE19949579C1 (en) | 1999-10-14 | 1999-10-14 | Microorganism with deregulated cysteine metabolism, useful for high-level production of cysteine and its derivatives, has increased activity of the CysB transcription regulator |
| PCT/EP2000/009720 WO2001027307A1 (en) | 1999-10-14 | 2000-10-05 | Method for production of l-cysteine or l-cysteine derivatives by fermentation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2386539A1 CA2386539A1 (en) | 2001-04-19 |
| CA2386539C true CA2386539C (en) | 2007-04-17 |
Family
ID=7925659
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002386539A Expired - Fee Related CA2386539C (en) | 1999-10-14 | 2000-10-05 | Method for production of l-cysteine or l-cysteine derivatives by fermentation |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP1220940B2 (en) |
| JP (1) | JP2003511086A (en) |
| KR (1) | KR100505336B1 (en) |
| CN (1) | CN1262646C (en) |
| AT (1) | ATE231918T1 (en) |
| CA (1) | CA2386539C (en) |
| DE (2) | DE19949579C1 (en) |
| DK (1) | DK1220940T3 (en) |
| SK (1) | SK4972002A3 (en) |
| TW (1) | TWI249575B (en) |
| WO (1) | WO2001027307A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9234223B2 (en) | 2011-04-01 | 2016-01-12 | Ajinomoto Co., Inc. | Method for producing L-cysteine |
Families Citing this family (77)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10046934A1 (en) * | 2000-09-21 | 2002-04-18 | Consortium Elektrochem Ind | Process for the fermentative production of non-proteinogenic L-amino acids |
| EP1404855B1 (en) * | 2001-07-11 | 2006-07-26 | Degussa AG | Process for the preparation of l-threonine using strains of the enterobacteriaceae family |
| DE10237479A1 (en) * | 2002-08-16 | 2004-02-26 | Degussa Ag | Animal feed additive containing sulfur |
| US7348037B2 (en) | 2002-08-16 | 2008-03-25 | Evonik Degussa Gmbh | Sulfur-containing animal-feed additives |
| JP2006517796A (en) | 2003-02-18 | 2006-08-03 | メタボリック エクスプローラー | Method for producing an evolved microorganism capable of generating or modifying a metabolic pathway |
| WO2004113373A1 (en) * | 2003-06-21 | 2004-12-29 | University Of Sheffield | Overexpression of the cyddc transporter |
| DE10331291A1 (en) | 2003-07-10 | 2005-02-17 | Consortium für elektrochemische Industrie GmbH | Variants of 3-phosphoglycerate dehydrogenase with reduced inhibition by L-serine and genes coding for it |
| RU2275425C2 (en) * | 2003-11-03 | 2006-04-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) | Bacterium belonging to genus escherichia as producer of l-cysteine and method for preparing l-cysteine |
| JP2009118740A (en) | 2006-03-03 | 2009-06-04 | Ajinomoto Co Inc | Method for producing l-amino acid |
| WO2007119574A2 (en) | 2006-03-23 | 2007-10-25 | Ajinomoto Co., Inc. | A method for producing an l-amino acid using bacterium of the enterobacteriaceae family with attenuated expression of a gene coding for small rna |
| JP2009165355A (en) | 2006-04-28 | 2009-07-30 | Ajinomoto Co Inc | L-amino acid-producing microorganism and method for producing l-amino acid |
| WO2007136133A1 (en) | 2006-05-23 | 2007-11-29 | Ajinomoto Co., Inc. | A method for producing an l-amino acid using a bacterium of the enterobacteriaceae family |
| EP2050816A4 (en) | 2006-07-25 | 2009-11-18 | Kyowa Hakko Bio Co Ltd | Method for production of amino acid |
| RU2006143864A (en) | 2006-12-12 | 2008-06-20 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) | METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE ENTEROBACTERIACEAE FAMILY IN WHICH THE EXPRESSION OF GENES cynT, cynS, cynX, OR cynR, OR THEIR COMBINATION IS DECREASED |
| JP2010041920A (en) | 2006-12-19 | 2010-02-25 | Ajinomoto Co Inc | Method for producing l-amino acid |
| CN101627110B (en) | 2007-01-22 | 2014-08-13 | 味之素株式会社 | Microorganism capable of producing l-amino acid, and method for production of l-amino acid |
| DE102007007333A1 (en) | 2007-02-14 | 2008-08-21 | Wacker Chemie Ag | Process for the purification of L-cysteine |
| JP2010110216A (en) | 2007-02-20 | 2010-05-20 | Ajinomoto Co Inc | Method for producing l-amino acid or nucleic acid |
| JP2010110217A (en) | 2007-02-22 | 2010-05-20 | Ajinomoto Co Inc | L-amino acid-producing microorganism and method for producing l-amino acid |
| WO2008126783A1 (en) | 2007-04-06 | 2008-10-23 | Kyowa Hakko Bio Co., Ltd. | Method for producing dipeptide |
| JP2011067095A (en) | 2008-01-10 | 2011-04-07 | Ajinomoto Co Inc | Method for producing target substance by fermentation process |
| EP2248906A4 (en) | 2008-01-23 | 2012-07-11 | Ajinomoto Kk | Method of producing l-amino acid |
| RU2008105793A (en) | 2008-02-19 | 2009-08-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) | METHOD FOR DESIGNING OPERONS CONTAINING TRANSLATION-CONJUGATED GENES, BACTERIA CONTAINING SUCH OPERON, METHOD FOR PRODUCING USEFUL METABOLITIS AND METHOD FOR EXPRESS MONITORING |
| JP5476545B2 (en) | 2008-02-21 | 2014-04-23 | 味の素株式会社 | L-cysteine producing bacterium and method for producing L-cysteine |
| DE602009000714D1 (en) | 2008-03-06 | 2011-03-24 | Ajinomoto Kk | L-cysteine-producing bacterium and process for producing L-cysteine |
| JP5332237B2 (en) | 2008-03-06 | 2013-11-06 | 味の素株式会社 | L-cysteine producing bacterium and method for producing L-cysteine |
| US20110111458A1 (en) | 2008-03-18 | 2011-05-12 | Kyowa Hakko Kirin Co., Ltd. | Industrially useful microorganism |
| WO2010024267A1 (en) | 2008-09-01 | 2010-03-04 | 国立大学法人信州大学 | Process for producing useful substance |
| KR101627095B1 (en) | 2008-09-08 | 2016-06-03 | 아지노모토 가부시키가이샤 | Microorganism capable of producing L-amino acid, and method for producing L-amino acid |
| BRPI1007069A2 (en) | 2009-01-23 | 2015-08-25 | Ajinomoto Kk | Method for producing an 1-amino acid. |
| US8623619B2 (en) | 2009-02-09 | 2014-01-07 | Kyowa Hakko Bio Co., Ltd. | Process for producing L-amino acid |
| EP2397545B1 (en) | 2009-02-10 | 2015-07-29 | Kyowa Hakko Bio Co., Ltd. | Method for producing amino acid |
| US20120034669A1 (en) | 2009-02-18 | 2012-02-09 | Kyowa Hakko Bio Co., Ltd. | Process for producing useful substance |
| JP5463528B2 (en) | 2009-02-25 | 2014-04-09 | 味の素株式会社 | L-cysteine producing bacterium and method for producing L-cysteine |
| JP5359409B2 (en) | 2009-03-12 | 2013-12-04 | 味の素株式会社 | L-cysteine producing bacterium and method for producing L-cysteine |
| EP2460883A4 (en) | 2009-07-29 | 2013-01-16 | Ajinomoto Kk | Method for producing l-amino acid |
| RU2009136544A (en) | 2009-10-05 | 2011-04-10 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) (RU) | METHOD FOR PRODUCING L-CISTEINE USING THE ENTEROBACTERIACEAE FAMILY BACTERIA |
| JP5817529B2 (en) | 2009-11-30 | 2015-11-18 | 味の素株式会社 | L-cysteine producing bacterium and method for producing L-cysteine |
| JP2013074795A (en) | 2010-02-08 | 2013-04-25 | Ajinomoto Co Inc | MUTANT rpsA GENE AND METHOD FOR PRODUCING L-AMINO ACID |
| RU2471868C2 (en) | 2010-02-18 | 2013-01-10 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) | Mutant adenylate cyclase, dna coding it, bacteria of enterobacteriaceae family containing said dan and method for preparing l-amino acids |
| RU2501858C2 (en) | 2010-07-21 | 2013-12-20 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) | METHOD FOR OBTAINING L-AMINOACID USING BACTERIUM OF Enterobacteriaceae FAMILY |
| RU2482188C2 (en) | 2010-07-21 | 2013-05-20 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) | METHOD FOR PREPARING L-ARGININE WITH USE OF BACTERIA OF GENUS Escherichia WHEREIN astCADBE OPERON IS INACTIVATED |
| BR112013006031A2 (en) | 2010-09-14 | 2016-06-07 | Ajinomoto Kk | bacteria, and method for producing a sulfur-containing amino acid, a related substance, or a mixture thereof. |
| JP2014036576A (en) | 2010-12-10 | 2014-02-27 | Ajinomoto Co Inc | Method for producing l-amino acids |
| EP2479279A1 (en) | 2011-01-20 | 2012-07-25 | Evonik Degussa GmbH | Method for producing sulphuric amino acids by means of fermentation |
| JP6301581B2 (en) | 2011-02-09 | 2018-03-28 | 協和発酵バイオ株式会社 | Production method of target substance by fermentation method |
| JP2014087259A (en) | 2011-02-22 | 2014-05-15 | Ajinomoto Co Inc | L-cysteine-producing bacterium, and production method of l-cysteine |
| BR112013023465B1 (en) * | 2011-04-01 | 2020-10-27 | Ajinomoto Co., Inc | method to produce l-cysteine |
| JP2014131487A (en) | 2011-04-18 | 2014-07-17 | Ajinomoto Co Inc | Method for producing l-cysteine |
| DE102011075656A1 (en) | 2011-05-11 | 2012-03-29 | Wacker Chemie Ag | Producing L-cystine useful as food additive, preferably in baking industry, as ingredient in cosmetics and as starting material for producing active pharmaceutical ingredient, comprises fermenting microorganism strain in fermentation medium |
| DE102011078481A1 (en) | 2011-06-30 | 2013-01-03 | Wacker Chemie Ag | Process for the fermentative production of natural L-cysteine |
| RU2011134436A (en) | 2011-08-18 | 2013-10-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | METHOD FOR PRODUCING L-AMINO ACID USING THE ENTEROBACTERIACEAE FAMILY POSSESSING AN INCREASED EXPRESSION OF GENES OF THE CASCADE OF THE FORMATION OF FLAGELLS AND CELL MOBILITY |
| EP2628792A1 (en) | 2012-02-17 | 2013-08-21 | Evonik Industries AG | Cell with reduced ppGppase activity |
| JPWO2013154182A1 (en) | 2012-04-13 | 2015-12-21 | 協和発酵バイオ株式会社 | Amino acid production method |
| DE102012208359A1 (en) | 2012-05-18 | 2013-11-21 | Wacker Chemie Ag | Process for the fermentative production of L-cysteine and derivatives of this amino acid |
| PL2700715T3 (en) | 2012-08-20 | 2019-03-29 | Evonik Degussa Gmbh | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family by means of fermentation |
| DE102012216527A1 (en) | 2012-09-17 | 2014-03-20 | Wacker Chemie Ag | Process for the fermentative production of L-cysteine and derivatives of this amino acid |
| EP2977442B1 (en) * | 2013-03-19 | 2018-02-28 | National University Corporation Nara Institute of Science and Technology | Enterobacteriaceae bacteria exhibiting increased l-cysteine producing ability |
| RU2013118637A (en) | 2013-04-23 | 2014-10-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE ENTEROBACTERIACEAE FAMILY IN WHICH THE yjjK GENE IS ADJUSTABLE |
| DE102013209274A1 (en) | 2013-05-17 | 2014-11-20 | Wacker Chemie Ag | Microorganism and method for fermentative overproduction of gamma-glutamylcysteine and derivatives of this dipeptide |
| JP2016165225A (en) | 2013-07-09 | 2016-09-15 | 味の素株式会社 | Method for producing useful substance |
| RU2013140115A (en) | 2013-08-30 | 2015-03-10 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE Enterobacteriaceae FAMILY IN WHICH THE EXPRESSION OF THE znuACB GENERAL CLUSTER IS DISORDERED |
| RU2013144250A (en) | 2013-10-02 | 2015-04-10 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") | METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE Enterobacteriaceae FAMILY IN WHICH THE EXPRESSION OF A GENE CODING A PHOSPHATE TRANSPORTER IS DECREASED |
| EP3053999B1 (en) | 2013-10-02 | 2019-11-20 | Ajinomoto Co., Inc. | Ammonia control apparatus and ammonia control method |
| JP6459962B2 (en) | 2013-10-21 | 2019-01-30 | 味の素株式会社 | Method for producing L-amino acid |
| RU2014105547A (en) | 2014-02-14 | 2015-08-20 | Адзиномото Ко., Инк. | METHOD FOR PRODUCING L-AMINO ACIDS USING THE ENTEROBACTERIACEAE FAMILY HAVING A SUPER EXPRESSED GENE yajL |
| RU2015120052A (en) | 2015-05-28 | 2016-12-20 | Аджиномото Ко., Инк. | A method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family in which gshA gene expression is weakened |
| EP3420096A1 (en) | 2016-02-25 | 2019-01-02 | Ajinomoto Co., Inc. | A method for producing l-amino acids using a bacterium of the family enterobacteriaceae overexpressing a gene encoding an iron exporter |
| JP7066977B2 (en) | 2017-04-03 | 2022-05-16 | 味の素株式会社 | Manufacturing method of L-amino acid |
| WO2020071538A1 (en) | 2018-10-05 | 2020-04-09 | Ajinomoto Co., Inc. | Method for producing target substance by bacterial fermentation |
| KR102112286B1 (en) * | 2018-12-05 | 2020-05-19 | 서울대학교산학협력단 | Gene related to acid resistance and methanotrophs comprising the same |
| WO2020171227A1 (en) | 2019-02-22 | 2020-08-27 | Ajinomoto Co., Inc. | METHOD FOR PRODUCING L-AMINO ACIDS USING A BACTERIUM BELONGING TO THE FAMILY Enterobacteriaceae HAVING OVEREXPRESSED ydiJ GENE |
| JP2022526181A (en) | 2019-04-05 | 2022-05-23 | 味の素株式会社 | Method for producing L-amino acid |
| WO2021060438A1 (en) | 2019-09-25 | 2021-04-01 | Ajinomoto Co., Inc. | Method for producing l-amino acids by bacterial fermentation |
| JP2023527107A (en) | 2020-04-03 | 2023-06-27 | ワッカー ケミー アクチエンゲゼルシャフト | Biocatalysts as core components of enzymatic catalytic redox systems for the biocatalytic reduction of cystine. |
| CN116018400A (en) | 2020-06-26 | 2023-04-25 | 瓦克化学股份公司 | Improved cysteine producing strains |
| WO2023165684A1 (en) | 2022-03-01 | 2023-09-07 | Wacker Chemie Ag | Improved cysteine-producing strains |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19539952A1 (en) * | 1995-10-26 | 1997-04-30 | Consortium Elektrochem Ind | Process for the preparation of O-acetylserine, L-cysteine and L-cysteine-related products |
| DE19726083A1 (en) * | 1997-06-19 | 1998-12-24 | Consortium Elektrochem Ind | Microorganisms and processes for the fermentative production of L-cysteine, L-cystine, N-acetyl-serine or thiazolidine derivatives |
-
1999
- 1999-10-14 DE DE19949579A patent/DE19949579C1/en not_active Expired - Fee Related
-
2000
- 2000-10-05 KR KR10-2002-7004742A patent/KR100505336B1/en not_active IP Right Cessation
- 2000-10-05 JP JP2001530510A patent/JP2003511086A/en active Pending
- 2000-10-05 WO PCT/EP2000/009720 patent/WO2001027307A1/en active IP Right Grant
- 2000-10-05 AT AT00969413T patent/ATE231918T1/en active
- 2000-10-05 EP EP00969413A patent/EP1220940B2/en not_active Expired - Lifetime
- 2000-10-05 DK DK00969413T patent/DK1220940T3/en active
- 2000-10-05 CN CNB008142726A patent/CN1262646C/en not_active Expired - Fee Related
- 2000-10-05 DE DE50001193T patent/DE50001193D1/en not_active Expired - Lifetime
- 2000-10-05 CA CA002386539A patent/CA2386539C/en not_active Expired - Fee Related
- 2000-10-05 SK SK497-2002A patent/SK4972002A3/en unknown
- 2000-10-16 TW TW089121607A patent/TWI249575B/en not_active IP Right Cessation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9234223B2 (en) | 2011-04-01 | 2016-01-12 | Ajinomoto Co., Inc. | Method for producing L-cysteine |
Also Published As
| Publication number | Publication date |
|---|---|
| ATE231918T1 (en) | 2003-02-15 |
| DE19949579C1 (en) | 2000-11-16 |
| EP1220940A1 (en) | 2002-07-10 |
| EP1220940B2 (en) | 2010-07-28 |
| WO2001027307A1 (en) | 2001-04-19 |
| DE50001193D1 (en) | 2003-03-06 |
| JP2003511086A (en) | 2003-03-25 |
| CA2386539A1 (en) | 2001-04-19 |
| CN1379823A (en) | 2002-11-13 |
| CN1262646C (en) | 2006-07-05 |
| EP1220940B1 (en) | 2003-01-29 |
| KR20020059620A (en) | 2002-07-13 |
| KR100505336B1 (en) | 2005-07-29 |
| DK1220940T3 (en) | 2003-06-23 |
| TWI249575B (en) | 2006-02-21 |
| SK4972002A3 (en) | 2002-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2386539C (en) | Method for production of l-cysteine or l-cysteine derivatives by fermentation | |
| CA2235752C (en) | Process for preparing o-acetylserine, l-cysteine and l-cysteine-related products | |
| JP4173777B2 (en) | Microbial strain, plasmid, method for producing microbial strain, and method for producing phosphoglycerate-family amino acid | |
| CA2235419C (en) | Microorganisms and processes for the fermentative preparation of l-cysteine, l-cystine, n-acetylserine or thiazolidine derivatives | |
| US20090298136A1 (en) | Methionine producing recombinant microorganisms | |
| Müller et al. | Metabolic engineering of the E. coli L-phenylalanine pathway for the production of D-phenylglycine (D-Phg) | |
| US7582460B2 (en) | 3-phosphoglycerate dehydrogenase variants whose inhibition by L-serine is reduced, and genes encoding them | |
| US20090298135A1 (en) | Method for fermentative production of L-methionine | |
| US7195897B2 (en) | Feedback-resistant homoserine transsuccinylases having a modified c-terminus | |
| AU773173B2 (en) | Process for preparing non-proteinogenic L-amino acids | |
| WO2004108894A2 (en) | Methods and compositions for amino acid production | |
| US7371551B1 (en) | Feedback-resistant homoserine transsuccinylases | |
| US20050089974A1 (en) | Fermentative production of d-hydroxyphenylglycine and d-phenylglycine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed | ||
| MKLA | Lapsed |
Effective date: 20081006 |