MXPA98004927A - Microorganisms and procedures for the fermentative obtaining of l-cysteine, l-cistine, n-acetyl-serine or derivatives of tiazolid - Google Patents
Microorganisms and procedures for the fermentative obtaining of l-cysteine, l-cistine, n-acetyl-serine or derivatives of tiazolidInfo
- Publication number
- MXPA98004927A MXPA98004927A MXPA/A/1998/004927A MX9804927A MXPA98004927A MX PA98004927 A MXPA98004927 A MX PA98004927A MX 9804927 A MX9804927 A MX 9804927A MX PA98004927 A MXPA98004927 A MX PA98004927A
- Authority
- MX
- Mexico
- Prior art keywords
- cysteine
- leu
- seq
- sequence
- microorganism
- Prior art date
Links
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- 238000000034 method Methods 0.000 title claims abstract description 23
- JJIHLJJYMXLCOY-BYPYZUCNSA-N N-acetyl-L-serine Chemical compound CC(=O)N[C@@H](CO)C(O)=O JJIHLJJYMXLCOY-BYPYZUCNSA-N 0.000 title claims abstract description 21
- -1 l-cistine Chemical compound 0.000 title description 4
- 229960002433 Cysteine Drugs 0.000 claims abstract description 76
- 239000004201 L-cysteine Substances 0.000 claims abstract description 51
- 235000013878 L-cysteine Nutrition 0.000 claims abstract description 51
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- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine zwitterion Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 claims abstract description 23
- 239000004158 L-cystine Substances 0.000 claims abstract description 14
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- 239000000126 substance Substances 0.000 claims abstract description 14
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- 229940042786 Antitubercular Antibiotics Drugs 0.000 claims abstract description 8
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- 238000005755 formation reaction Methods 0.000 description 16
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Abstract
The present invention relates to microorganisms and processes for the fermentative production of L-cysteine, L-cystine, N-acetyl-serine or thiazolidine derivatives, the strain of microorganisms according to the invention, which is suitable for the fermentative preparation of L-cysteine, L-cystine, N-acetyl-serine and / or thiazolidine derivatives, is characterized in that it overexpresses at least one gene encoding a suitable protein directly for the exclusion of the cell from antibiotics or other substances toxic to the microorganism
Description
MICROORGANISMS AND PROCEDURES FOR THE FERMENTATIVE OBTAINING OF L-CYSTEINE, L-CISTINE, N-ACETYL-SERINE OR TIAZOLIDINE DERIVATIVES
The invention relates to microorganisms and processes for the fermentative production of L-cysteine, L-cystine, N-acetyl-serine or thiazolidine derivatives. Fermentative amino acid production is, in the meantime, part of the current technique for many amino acids. However, until now there is no industrial fermentation process to obtain
L-cysteine. The thiazolidine derivatives and the corresponding hemithiocetals are generally formed during the condensation of cysteine with ketones or aldehydes. The chemical condensation of cysteine with various ketones or aldehydes, particularly with α-keto acids, has been known for a long time. The condensation is carried out through the heiocetal as an intermediate stage, which is formed by the attack of the free electron pair of the sulfur to the positive carbon atom of the aldehyde or keto group. A cyclization under the dissociation of water then leads to the corresponding thiazolidine derivative.
In the following scheme, the formation of thiazolidine derivatives is generally shown:
of carbonyl Hemi ioce al
Thiazolidine derivative
R1 and R2 can mean any organic radical. Thus, the educts are in equilibrium through the hemithioketal with the thiazolidine derivative. For this reason, in addition to the thiazolidine derivative, in aqueous solution the hemithioketal is also generally present.
In the sense of the present invention, "thiazolidine derivative" is also understood as a balance of these substances with the corresponding hemithioketal. Thiazolidines have not been described so far as direct metabolites of cells. All reports of thiazolidine formations by cells are based on the external excess addition of one of the educts, usually L-cysteine, which is then transformed by desulphurization and deamination into pyruvate, which in turn reacts with the cysteine added. (Ronald C. Simpson et al., Biochimica et Biophysic Acta, 496 (1977), 12-19). Kredich and collaborators described in J. of Biol. Chem. 248, 17: 6187-6196, the in vitro formation of 2-methyl-2,4-thiazolidinedicarbonic acid in an enzymatic desulfhydrogenation of L-cysteine, however they consider the formation of This substance in vivo as very unlikely. It is known to employ thiazolidines as racemic precursors for the preparation of L-cysteine by biotransformation (EP-A 0 101 052, Ok Hee Ryu et al., Biotechnology Letters 17 No. 3, 275-280
(March 1995)). When the racemate is used to obtain L-cysteine, it must be transformed stereoselectively by enzymes or whole cells to L-cysteine. The remaining diastereomers must then be racemised again. For these reasons, said biotransformation is linked to high costs. The chemical synthesis of thiazolidines from racemic cysteine and a corresponding ketone or aldehyde leads to four different diastereomers. A chemical synthesis from pure L-cysteine of enantiomers is expensive and is not suitable for the purpose of the subsequent production of L-cysteine. Therefore, a process for obtaining thiazolidine diastereomers which have the R configuration at the C4 atom is hampered by the high costs of the educts. The present invention relates to microorganisms that are suitable for the fermentative production of L-cysteine, L-cystine, N-acetyl-serine and / or thiazolidine derivatives. A strain of microorganisms according to the invention is characterized in that it overexpresses at least one gene coding for a protein, which is directly suitable for the escape from the cell of antibiotics or other substances toxic to the organism. By substances toxic to the body, in the sense of the invention, preference is given to compounds that negatively influence the growth of the organism. These compounds are, for example, carbonic acids or carbonic acid derivatives in high intracellular concentrations. The genes that encode proteins, which are suitable directly for the cell's secretion of antibiotics and other toxic substances, or which cause the formation of this type of proteins, are referred to hereinafter as efflux genes. Thus, the invention also relates to the use of efflux genes for the enhanced expression of amino acids or amino acid derivatives formed intracellularly in fermentation. As an efflux gene, in the microorganism according to the invention, preferably at least one gene chosen from the sea-Locus group was overexpressed (SP Cohen et al., Journal of Bacteriology, Mar. 1993, 175: 5, 1484-1492), Locus, acr-Locus, cmr-Locus (see PF Miller and MC Sulavik, Molecular Microbiology (1996) 21 (3), 441-448), mex genes (T. Kóhler et al., Molecular Microbiology (1997) 23 (2) , 345-354), gene br (AA Neyfakh et al., Proc. Nati, Acad. Sci. USA 88: 4781-4785 (1991), qacA gene (JM Tennent et al., J. Gen. Microbiol. 10 (1989) Preferably, in the microorganism according to the invention, Sea-Locus efflux genes are overexpressed, particularly preferably in the microorganism according to the invention a gene coding for a protein including the protein is overexpressed. MSR sequence KDGVLALLW WWGLNFWI KVGLHNMPRL MLAGLRFMLV (SEQ ID: 1) or a sequence with a sequence homology to SEQ ID: 1 greater than 50%. Preferably, the sequence homology to SEQ ID: 1 is greater than 75%, particularly preferably sequence homology to SEQ. ID. NO: 1 is greater than 90%. Thus, the invention also relates to genes that encode a protein that includes the MSR sequence KDGVLALLW WWGLNFWI KVGLHNMPRL MLAGLRFMLV (SEQ ID NO: 1) or a sequence with a sequence homology to SEQ. ID NO: 1 greater than 50%. The invention also relates to proteins that include the MSR sequence KDGVLALLW WWGLNFWI KVGLHNMPRL MLAGLRFMLV (SEQ ID NO: 1) or a sequence with a sequence homology to SEQ. ID. NO: 1 greater than 50%. Preferably, sequence homology to SEQ
ID: 1 is greater than 75%, particularly preferably sequence homology to SEQ. ID. NO: 1 is greater than 90%. For example, the proteins according to the invention can possess the following sequence: 1 MKFRGGRMSR KDGVLALLW VVWGLNFVVT KVGLHNMPRL MLAGLRFMLV
51 AFPAIFFVAR PKVPLNLLLG YGLTISFAQF AFLFCAINFG MPAGLASLVL
101 QAQAFFTIML GAFTFGERLH GKQLAGIALA IFGVLVLIED SLNGQHVAML
151 GFMLTLAAAF SWACGNIFNK KIMSHSTRP? VMSLVT SAL IPIIPFFVAS
201 LILDGSATMI HSLVTIDMTT ILSLMYLAFV ATIVGYGIWG TLLGRYETWR
251 VAPLSLLVPV VGLASAALLL DERLTGLQFL GAVLIMTGLY INVFGLRWR
301 AVKVGS * (SEQ ID NO: 2)
The open reading frame that encodes the protein with the amino acid sequence according to SEQ. ID. NO: 2 is hereinafter also referred to as ORF 306. Another example of a protein according to the invention is shown by the following sequence. 1 MSR KDGVLALLW WWGLNFWI KVGLHNMPRL MLAGLRFMLV
44 AFPAIFFVAR PKVPLNLLLG YGLTISFAQF AFLFCAINFG MPAGLASLVL
94 QAQAFFTIML GAFTFGERLH GKQLAGIALA IFGVLVLIED SLNGQHVAML
144 GFMLTLAAAF SWACGNIFNK KIMSHSTRPA VMSLVI SAL IPIIPFFVAS
194 LILDGSATMI HSLVTIDMTT ILSLMYLAFV ATIVGYGI G TLLGRYETWR
244 VAPLSLLVPV VGLASAALLL DERLTGLQFL GAVLIMTGLY INVFGLRWRK
294 AVKVGS * (SEQ ID NO: 3)
The proteins according to the invention are also those proteins that possess an amino acid sequence with amino acid sequence homology higher than 50% to the amino acid sequence according to SEQ. ID. NO: 2 or SEQ. ID. NO: 3. Preferably, the sequence homology of the proteins according to the invention to SEQ ID: 2 or SEQ. ID.
NO: 3 is greater than 75%, particularly preferably sequence homology to SEQ. ID. NO: 2 or SEQ. ID. DO NOT:
3 is greater than 90%. The genes according to the invention are therefore also those genes that encode proteins with an amino acid sequence according to SEQ. ID. NO: 2 or SEQ. ID. NO: 3 or an amino acid sequence with sequence homology at amino acid level greater than 50%, preferably 75%, particularly preferably 90%, to the amino acid sequence according to SEQ. ID. NO: 2 or SEQ. ID. NO: 3. In the present invention, all homology values mentioned refer to results obtained with the computer program "Wisconsin Package Version 9.0, Genetics Computer Group (GLG), Madison, Wisconsin". The determination of the homology is carried out by means of a search in the data bank with the subprogram "fasta" and the previously adjusted values (word size 2). The most similar sequences are then examined with the "gap" subprogram with respect to homology. For this, the previously adjusted parameters "gap creation penalty 12" and "gap extension penalty 4" are used. Another example of the overexpression of a gene according to the invention for the increase of cysteine formation is the overexpression of a DNA fragment of 5.5 kb in length, which also encodes the sea-Locus. This plasmid with the designation 100-1-1 was deposited in the DSMZ Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, D-38124 Braunschweig in E. coli K12 W3110 under the number DSM 11545. Figure 1 shows a plasmid letter of said plasmid . This can be used for the amplification of genes according to the invention by PCR. Another controlled modification of these genes in the respectively desired position of the sequence by known methods, for example the technique of site directed mutagenesis, is known to the person skilled in the art. Also microorganisms that contain genes modified in this way are part of the invention as long as the genes modified in this way contribute to the production of L-cysteine, L-cystine, N-acetyl-serine and / or thiazolidine derivatives. By overexpression it is to be understood in the sense of the invention that the expression of the protein in the microorganism according to the invention is effected at least twice as strong as in the wild type from which it comes from the protein. Preferably, the expression of the protein in the microorganism according to the invention is carried out at least five times stronger than in the wild type, particularly preferably at least ten times stronger than in the wild type from which the protein comes. Without an overexpression of the mentioned genes, for example by independent transcription by a promoter separately or, for example, without the presence of the MarA-encoding gene, in several copies on a plasmid, no significant increase in yield is observed for L -cysteine or thiazolidine derivatives with respect to the starting strain. The increase in yield due to the overexpression of the mentioned sequences was more surprising, as the gene product described in the specialized literature of the open reading frame ORF266 corresponds to the sequence SEQ. ID. NO: 4 in its sequence from methionine at position 41 in SEQ. ID. NO: 2, does not lead in an overexpression to an increase in the yield of L-cysteine. In the amino acid sequence indicated above, derived from ORF306, the starting methionine of ORF266 is written in bold and underlined. The person skilled in the art knows a series of procedures that achieve an overexpression of a gene. One possibility is, for example, the expression of the gene in a plasmid that is present in the cell with an increased number of copies. Said plasmids are known. Examples pACYC177, pACYC184, derivatives of pACYC184, pBR322, other derivatives of pBR, pBlueskript, pUC18, pUC19, as well as other plasmids usually employed in Escherichia coli are mentioned. The preferred plasmids for the overexpression according to the invention are pACYC177, pACYCl84, derivatives of pACYC184, pBR322 and other derivatives of pBR. Particularly preferred are pACYC184 and its derivatives, such as pACYC184-LH (deposited at DSMZ Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, D-38124 Braunschweig, under number DSM 10172). The invention thus also relates to plasmids containing genes according to the invention. Other possibilities for an amplification of the expression is the increase of the copy number of an efflux gene by means of an amplification of the section of the gene in the chromosome or the use of strong promoters for the best transcription of the efflux gene. Suitable promoters are, for example, the GAPDH promoter, the tac promoter (ptac), the Lac promoter (p? Ac), the trp promoter (prP), lambda PL or Lambda PR. The GAPDH promoter or the tac (pac) promoter are preferably suitable. • Particularly preferred is the GAPDH promoter. Another possibility for an amplification of expression is the inactivation of repressor genes, which act in an inhibitory manner on the expression of an efflux gene. For the sea gene, this would be, for example, the inactivation of the sea gene R. Also the elements that positively influence the translation contribute to an overexpression of the efflux gene. These elements are, for example, a good place for binding ribosomes (for example, Shine-Dalgarno sequence) or Downstream Box.
A preferred element that positively influences the translation is the good ribosome binding point of the GAPDH gene. For the expression of efflux genes, they are transformed into a microorganism that produces L-cysteine. Preferably, the efflux genes are transformed into microorganisms chosen from the Bacillus group, such as B. subtilis, Corynebacterium as C. glutamicum, Streptomyces and E. coli. Preferably, the efflux genes are transformed into organisms which, in their cysteine metabolism, are so deregulated, that large amounts of L-cysteine are formed and, if necessary, subsequently to the formation of a thiazolidine derivative. of L-cystine or N-acetyl-serine. Examples of microorganisms that produce high amounts of L-cysteine are microorganisms with CysE allele resistant to feedback. In another preferred embodiment, microorganisms that form a thiazolidine derivative are formed intracellularly by the condensation of L-cysteine and a ketone or an aldehyde, particularly pyruvate. Microorganisms that produce high amounts of L-cysteine are described, for example, in the patent application DE 19539952 (DE 19539952 is incorporated by reference). The person skilled in the art knows processes for the transformation of a microorganism, for example, through standard textbooks. All known methods can be applied to obtain a microorganism according to the invention. By means of a reinforced expression of the efflux genes in microorganisms, which produce amino acids or derivatives of amino acids formed intracellularly thereof, such as for example L-cysteine, L-cystine, N-acetyl-serine or thiazolidine derivatives, one arrives surprisingly to a reinforced blockage of amino acids or amino acid derivatives formed intracellularly thereof from the cell, such as for example L-cysteine, L-cystine, N-acetyl-serine and thiazolidine derivatives. In this way, clearly higher yields of these products are achieved in the fermentation. Thus, the invention also relates to processes for the preparation of L-cysteine, L-cystine, N-acetyl-serine or thiazolidine derivatives thereof, characterized in that a microorganism of the overexpressed efflux genes is used in the fermentation. way known. The process according to the invention for the fermentative preparation of L-cysteine, L-cystine, N-acetyl-serine or thiazolidine derivatives has several advantages: Only thiazolidine diastereomers are formed which have only the R configuration at the C4 carbon atom , because by the provision of enzyme from the cell, L-cysteine is formed stereoselectively, which can then react with the ketone or aldehyde respectively available to exclusively result in the aforementioned thiazolidine diastereomers. Starting from the thiazolidines with the R configuration at the C4 carbon atom, applying usual chemical and biological methods and techniques, L-cysteine can be obtained only by the equilibrium displacement in the direction of the educts. Surprisingly, it has also been found to be advantageous to obtain L-cysteine from a thiazolidine derivative formed intracellularly in the fermentation. A more detailed examination of this surprising fact led to the finding that the toxicity of thiazolidine to the cell is considerably lower than the toxicity of L-cysteine. Thus, the invention also relates to processes for obtaining L-cysteine, which are characterized in that it is reacted intracellularly in a microorganism L-cysteine formed intracellularly of the microorganism with the ketone or aldehyde present intracellularly in the microorganism to obtain a derivative of thiazolidine, this thiazolidine derivative is shut off from the microorganism by a protein, which is directly suitable for blocking from the cell antibiotics or other substances toxic to the microorganism, and, eventually after separating the thiazolidine derivative, L-cysteine is obtained by shifting the equilibrium of the reaction between L-cysteine and the derivative of thiazolidine in the direction of L-cysteine. One possibility for the intracellular formation of a thiazolidine derivative is the reaction of L-cysteine with a ketone or an aldehyde present respectively intracellularly. Many ketones and aldehydes are known in the metabolism of organisms that come into consideration for condensation. In bacterial metabolisms these are, among others, for example pyruvate, oxaloacetate, α-ketoglutarate or glyoxylate. Preferably, L-cysteine reacts with pyruvate or glyoxylate. Preferably, for the thiazolidine derivatives which are formed in the process according to the invention and according to the above, in the scheme of page 2, at least one radical Rl or R2 means a carboxyl group, particularly preferably in the formula I Rl means COOH and R2 CH3. The educts for the condensation to thiazolidine derivative, either that both can be formed by the microorganism or that only one educt is formed by the microorganism and the second educt is added during the fermentation. In a preferred embodiment of the invention, both educts for condensation to thiazolidine derivative are formed by the microorganism. As a derivatization agent for the derivation of pyruvate, which can thus be removed from equilibrium, hydroxylamine or 2,4-dinitrophenylhydrazine can be used among others. Advantageously, in the process according to the invention, the thiazolidine derivative (and the corresponding hemithioketal) can be obtained from simple and inexpensive C and N and S sources. In the process according to the invention, in the fermentation the usual C sources can be used in a fermentation, such as glucose, lactose, fructose, starch and the like, sources of N, such as for example ammonium or protein hydrolysates and the like , and sources of S, such as sulfides, sulphites, sulfates, thiosulphates or dithionite. The thiazolidine derivatives obtained fermentatively can not only be used to obtain cysteine. Many application possibilities are known to be able to use the thiazolidine derivatives obtained fermentatively with the R configuration at the C4 carbon atom, as a starting material for more extensive synthesis (building block). The following examples serve to illustrate the invention in more detail. The quantitative determination of thiazolidine / hemithioketal derivative is only possible indirectly. In the examples, it was carried out by the determination of cysteine according to Gaitonde, M. K. (1967), Biochem. J. 104, 627-633. By the derivation of cysteine in strong acid with ninhydrin, it comes out of equilibrium. Thus, the hemithioketal reacts and finally also the thiazolidine derivative. After approximately 10 minutes at 100 ° C, all the thiazolidine derivative and the corresponding hemithioketal is transformed into the cysteine-ninhydrin derivative, which can then be quantified at 560 nm. Free cysteine is also included in the determination. The amount of free SH groups and, thus, free cysteine alone was determined by the test described by Sang-Han Lee et al., Biochemical and Biophysical Research Communications, Vol. 213, No. 3 (1995), pages 837 et seq. by 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). In the case of a formation of free L-cysteine, it is oxidized by the oxygen in the air fed during the fermentation to obtain L-cystine. Cystine is difficult to dissolve in aqueous medium at pH 7.0 and precipitates as a white bead. In the case of an insoluble cystine bead formation, it is dissolved in semiconcentrated HCl and also measured under reducing conditions with dithiothreite (DTT) in the aforementioned test. In example 3, as fermentation results indicate the quantities measured according to the test according to Gaitonde of "total cysteine". They are mainly 2-methyl-thiazolidin-2,4-dicarbonic acid, the corresponding hemithioketal, free L-cysteine and dissolved cystine. The precipitated cystine was quantified and indicated separately. The great precipitation capacity of 2-methyl-thiazolidin-2, -dicarbonic acid, which is formed in the present invention, by means of bivalent metal ions, can be used to check the formation of said derivative. So far only the precipitation capacity with zinc acetate has been described
(Schubert et al., See previous indication of specialized literature). However, precipitation with other bivalent metal ions, such as magnesium, iron, copper, zinc, manganese, cobalt and the like, is also possible. The precipitation and subsequent identification of the thiazolidine product formed is described in example 4. It is also shown that after 24 hours of fermentation time, the main product is 2-methyl-thiazolidin-2,4-dicarbonic acid. The great capacity of precipitation is not of great help only in the analysis of the product of the fermentation, but also in the purification of the same.
EXAMPLE 1 Amplification of the alleles by PCR
A. Amplification of the cysE alleles
The cysE, cysEIV and cysEX alleles used below are described in DE 19539952, example 2/10. Obtaining the mutations mentioned there is possible with the use of site-specific mutagenesis. It is possible to obtain equipment for carrying out mutagenesis in commerce, for example from Stratagene
(Stratagene GmbH, Postfach 105466, D-69044 Heidelberg) under the trade name ExSite or Chameleon. After performing the site-specific mutagenesis, the alleles obtained are amplified by the polymerase chain reaction (PCR) (Saiki et al. 1988, Science 239: 487-491) of the corresponding DNA, by the following primers.
cysE-fw: (SEQ ID NO: 5)
'-TGG ACC AGA GCT CTG GCT GGC GCA TCG CTT CGG CGT TG-3 * Sacl
cysE-rev: (SEQ ID NO: 6)
'-CTC GAT GCA TTA CGT AGG GGT ATC CGG GAG CGG TAT TG-3 'Nsil
The PCR experiments were carried out in 30 cycles in the presence of 200 μM of deoxynucleotide triphosphates
(dATP, dCTP, dGTP, dTTP), respectively 1 μM of the corresponding oligonucleotide, 100 ng of DNA arrays with the corresponding cysE allele, 1/10 reaction buffer x 10 (100 mM KCl, 100 mM (NH4) 2S04, 200 mM tris-HCl (pH 8.8), 20 mM MgSO4, 1% triton X-100 and
1000 μg / ml of BSA) and 2.5 units of a heat-stable recombinant Pfu-DNA polymerase (Stratagene), in a
Thermocycler (Gene-ATAG-Controler, Pharmacia) under the following conditions: 94 ° C for 1 min, 60 ° C for 1 min and 72 ° C for 3 min. The product of the amplification was hydrolyzed with Sacl and Nsil (both from Boehringer Mannheim GmbH) under the conditions indicated by the manufacturer, separated by a 1% agarose gel and isolated with the help of the Geneclean method (Geneclean Kit BIO101, PO Box 2284, La Jolla, California, 92038-2284), according to the manufacturer's instructions, of the agarose gel as a fragment of approximately 1.0 kb. Until its use, the fragment was stored at -20 ° C.
B. Amplification of the sea-Locus
The sea-Locus of Escherichia coli was amplified by PCR. The procedure for obtaining the amplifications is the same as that described in Example 1, section A. As the DNA of the matrices, the chromosomal DNA of Escherichia coli W3110 (ATCC 27325) was used. As
Matrix DNA is also suitable plasmid 100-1-1 (DSM 11545). The lysis of the cells and the purification of the
Chromosomal DNA according to the protocol described in Ausubel et al., 1987, 2.4.1 - 2.4.2, Current Protocols in
Molecular Biology, Greene Publishing Associates and Wiley-Interscience. The following primers were used for the sea-Locus amplification: mar-fw: (SEQ ID NO: 7)
'-TTT GGC GCG CCG ATC AGC GGC GGC GCA ACC ATC AG-3' AscI
mar-rev: (SEQ ID NO: 8)
'-GCC TTA ATT AAG ATC GAC ACT CAG GCT GTA CTG GCG AC-3 'Pací
The amplification of the sea-Locus led to a fragment of approximately 3 kb, which was purified as described in Example 1, section A. The following restriction digestion is carried out with the enzymes AscI and Pací (both from New England Biolabs GmbH , Postfach 2750, D-65820 Schwalbach / Taunus), according to the manufacturer's instructions and shock absorbers. After purification of the fragment by agarose gel electrophoresis, it was stored at -20 ° C.
C. ORF306 DNA amplification
The DNA encoding ORF306 was amplified by PCR as described in Example 1, section A. As matrix DNA, the chromosomal DNA isolated in Example 1, section B, of E. coli W3110 (ATCC 27325) was used.
Plasmid 100-1-1 (DSM 11545) is also suitable as matrix DNA. The initiators employed are the following:
ORF306-fw: (SEQ- ID NO: 9)
'-GGA ATT CAT TAA TCC GGC GAC TAA CGA ATC AAC TG-3' Asnl
ORF306-rev: (SEQ ID NO: 10)
'-GCC TTA ATT AAC GCT ATG TAG TTT GTT CTG GCC CCG-3' Pací
The amplified DNA fragment is approximately 1.05 kb and, as described, was purified by agarose gel electrophoresis. A subsequent restriction digestion with Asnl enzymes
(Boehringer Mannheim) and Pací (New England Biolabs) led after removing the enzymes to the desired DNA fragment. This was stored until use at -20 ° C.
D. Amplification of the DNA fragment encoding the GAPDH promoter
For the effective transcription of ORG306, the promoter of the glycerinaldehyd-3-phosphatidehydrogenase gene was used. This desired DNA fragment was also obtained by PCR. The chromosomal DNA of Escherichia coli W3110 (ATCC 27325) was used as template DNA. Plasmid 100-1-1 is also suitable as matrix DNA. The following initiators were used:
GAPDH-fw: (SEQ ID NO: 11) 5 '-GTC GAC GCG TGA GGC GAG TCA GTC GCG TAA TGC-3' Mlul
GAPDH-rev: (SEQ ID NO: 12)
'-GAC CTT AAT TAA GAT CTC ATA TGT TCC ACC AGC TAT TTG TTA G-3 »Pací Ndel
The DNA fragment obtained, with approximately 0.3 kb, was isolated and purified by agarose gel electrophoresis, as described in Example 1, section A. A subsequent restriction digestion with the enzymes Mlul and Pací led to the desired DNA fragment. . After removing the restriction enzymes, the DNA was stored at -20 ° C.
Example 2 Construction of the plasmids according to the invention.
Plasmid pACYC184 was used as the base plasmid for the construction of the plasmids according to the invention. This plasmid was modified as described in DE 19539952 and deposited as plasmid pACYC184-LH under deposit number DSM 10172 at the Deutsche Sammlung für Mikroorganismen in Braunschweig. A restriction and function chart of the plasmid pACYC184-LH is shown in FIG. Said plasmid carries a polylinker. This polylinker has the following restriction intersections:
Notl - Ncol - Sacl - Nsil - Mlul - Pací - Notl
In these linkers, the DNA fragments obtained in Example 1 were ligated by PCR and subsequent restriction digestion.
A. Construction of the control plasmids pACYC184 / cysEIV and pACYC184 / cysEX
The preparation of plasmids pACYC184 / cysEIV and pACYC184 / cysEX is described in DE 19539952, example 3 and is summarized below:
Approximately 1 μg of the plasmid pACYC184-LH (DSM 10172) was digested with the restriction enzymes Sacl and Nsil, according to the manufacturer's instructions (Boehringer Mannheim). The digested DNA was then purified by agarose gel electrophoresis to remove the enzymes, as described above. The DNA fragments obtained in Example 1, section A, which encode the corresponding cysE allele, were then mixed in an equimolar way with the plasmid pACYC184-LH digested with Sacl and Nsil and 1 μl of T4 DNA ligase and 2 μl were added. of ligase buffer 10 (both from Boehringer Mannheim) and filled with sterile H20, twice distilled to a total volume of 20 μl. The mixture was incubated overnight at 4 ° C and used for the transformation of Escherichia coli W3110 (ATCC 27325). The transformation procedure described below was used in all the transformations mentioned in the examples. The transformation of E. coli W3110 was effected by electroporation. For this, 500 ml of LB medium (10 g of triplet) was inoculated, 5 g of yeast extract, 5 g of NaCl) in a 1 1 Erlenmeyer flask with 1% (V / V) of a culture overnight in the same medium. After an incubation in the round agitator at 37 ° C to an optical density of 0.5 - 0.6 at 600 nm, the cells were cultured by centrifugation at 4 ° C in a sterile container. All other steps were performed on ice and keeping sterile conditions. The cell bead was washed twice with 500 ml of H20 distilled twice, sterile, ice-cold and finally resuspended in 30 ml 10% (V / V) of sterile glycerin. After further centrifugation, the cell bead was incorporated in 500 μl 10% (V / V) of glycerin and stored in 200 μl of aliquot at -80 ° C. For transformation, the cells were thawed on ice, approximately 10-100 ng of DNA were added and placed in a sterile electroporation cuvette (BioRad). The cuvette was placed in the Gene Pulser (BioRad) and electroporated at a voltage of 2500 Volts, a parallel resistance of 200 Ohms and a capacity of 25 μtF. The cells were then resuspended in 1 ml of SOC medium (caseinpeptone 20.0 g / 1, yeast extract 5.0 g / 1, NaCl 0.58 g / 1, KCl 0.19 g / 1, MgCl2 2.03 g / 1, MgSO4 2.46 g / 1, glucose 3.60 g / 1, pH = 7.0) and stirred for one hour at 37 ° C. Afterwards, the cells were diluted correspondingly, placed on LB agar plates (10 g / 1 of trypton, 5 g / 1 of yeast extract, 5 g / 1 of NaCl, 15 g / 1 of agar, pH = 7.2) and incubated overnight at 37 ° C, until isolated colonies were observed. The desired transformants were identified by restriction analysis after plasmid isolation by QIAprep Spin Plasmid Kit (Qiagen GmbH, Max-Volmer-Strasse 4, D-0724 Hilden). They were used as control in the fermentation in Example 3. Plasmids pACYC18 / cysEIV and pACYC184 / cysEX are shown in Figure 3.
B. Construction of plasmids pACYC184 / cysEIV-mar and pACYC184 / cysEX-mar
1 μg of the plasmids pACYC184 / cysEIV and pACYC184 / cysEX constructed in Example 2, section A were digested respectively, successively with the restriction enzymes Mlul (Boehringer) and Pací (New England Biolabs), according to the manufacturer's instructions. After this restriction digestion, the DNA was isolated and purified by an agarose gel electrophoresis, as described in Example 1, section A. About 20 ng of the vectors pACYC184 / cysEIV, or pACYC184 / cysEX were mixed. digested with Mlul-Pacl respectively with 200 ng of the DNA fragment obtained in Example 1, section A, 1 μl of T4 DNA ligase (Boehringer Mannheim) and 2 μl of ligase buffer x 10 (Boehringer Mannheim) and the amount needed of H20 distilled twice, sterile, in a final volume of 20 μl. After an overnight incubation at 4 ° C, the two DNA mixtures were used for the transformation of Escherichia coli W3110 (ATCC 27325). After plasmid isolation by means of QIAprep Spin Plasmid Kit (Qiagen GmbH) and a restriction analysis, the desired transformants were isolated and used in the fermentation, as described in example 3. A chart of restriction and function of plasmids pACYC18 / cysEIV-mar, or, pACYC184 / cysEX-mar.
C. Construction of plasmids pACYC184 / cysEIV-GAPDH and pACYC184 / cysEX-GAPDH
Plasmids pACYC184 / cysEIV and pACYC184 / cysEX constructed in Example 2, section A were respectively digested with the restriction enzymes Mlul
(Boehringer Mannheim) and Pací (New England Biolabs), according to the manufacturer's instructions. After purification of these plasmids treated in this manner, two ligands were respectively charged with the DNA fragment obtained in Example 1, section D, as described in Example 2, section B. After an overnight incubation to
4 ° C, the binding charges were transformed into E. coli W3110. The correct transformants were identified after the isolation of the plasmid DNA, by an analysis with the appropriate restriction enzymes. Plasmids pACYC184 / cysEIV-GAPDH and pACYC184 / cysEX-GAPDH were used as starting materials for the construction of plasmids pACYC184 / cysEIV-GAPDH-ORF306 and pACYC184 / cysEX-GAPDH-ORF306, as described in the following section D.
D. Construction of plasmids pACYC184 / cysEIV-GAPDH-ORG306 and pACYC184 / cysEX-GAPDH-ORF306
The plasmids obtained in section C were digested with Ndel (Boehringer Mannheim) and Pací (New
England Biolabs), according to the manufacturer's instructions. After purifying the DNA plasmids, two ligands were loaded with the DNA fragment of the example
1, section C, which encodes ORF306, cut with AsnI-PacI. After an overnight incubation a
4 ° C, the DNA mixture was transformed into E. coli W3110 respectively and placed on LB plates. After isolated colonies appeared, they were examined by plasmid isolation and restriction digestion as to their accuracy. Figure 5 shows a restriction and function chart of the plasmids pACYC184 / cysEIV-GAPDH-ORG306 and pACYC184 / cysEX-GAPDH-ORF306.
Example 3 Comparison of the yields of the structures according to the invention and the known structures in the fermentation
All the plasmids compared in the fermentation were fermented in E. coli W3110. In this way it is guaranteed that the respectively observed increases in yields result exclusively from the use according to the invention of the genes. 20 ml of LB medium were inoculated with 15 mg / l of tetracycline in an Erlenmeyer flask (100 ml) with the corresponding structure of E. coli. After a seven hour incubation in the shaking incubator (150 r.p.m., 30 ° C), the respective cultures were transferred to 100 ml of SM1 medium (12 g / 1 of K2HP04)., 3 g / 1 of KH2P04, 5 g / 1 of (NH4) 2S04, 0.3 g / 1 of MgSO4 x 7H20, 0.015 g / 1 of CaC12 x 2H20, 0.002 g / 1 of FeS04 x 7H20, 1 g / 1 of Na2citrate x 2H20, 0.1 g / 1 of NaCl, 1 ml / l of trace element solution, composed of 0.15 g / 1 of Na2Mo04 x 2H20, 2.5 g / 1 of H3B03, 0.7 g / 1 of CoC12 x 6 H20, 0.25 g / 1 of CuS04 x 5H20, 1.6 g / 1 of MnC12 x 4H20, 0.3 g / 1 of ZnS04 x 7H20), which had as a supplement 5 g / 1 of glucose, 5 mg / l of vitamin Bl and 15 mg / l of tetracycline. The cultures were shaken with 150 r.p.m. in Erlen eyer flasks (11) at 30 ° C for 17 h. After this incubation, the optical density at 600 nm (OD600) was between 3 and 5. The fermentation was carried out in the BIOSTAT M fermentor from the Braun-Melsungen company. A culture vessel with 2 1 total volume was used. The fermentation medium contains 15 g / 1 of glucose, 10 g / 1 of trypton (Difco), 5 g / 1 of yeast extract (Difco), 5 g / 1 of (NH4) 2 S04, 1.5 g / 1 of KH2P04 , 0.5 g / 1 of NaCl, 0.3 g / 1 of MgSO4 x 7H20, 0.015 g / 1 of CaCl2 x 2H20, 0.075 g / 1 of FeS04 x 7H20, 1 g / 1 of Na3citrate x 2H20 and 1 ml of solution of elements trace (see above), 0.005 g / 1 of vitamin Bl and 15 mg / l of tetracycline. The pH in the fermenter was adjusted at the start to 7.0 by pumping a 25% NH40H solution. During the fermentation, the pH was maintained by automatic correction with 25% NH40H at a value of 7.0. To inoculate, 100 ml of preculture was pumped into the fermenter vessel. The initial volume was approximately 1 1. The cultures were shaken first with 200 r.p.m. and gasified with 1.5 vvm of air at degerminated pressure through a sterile filter. The oxygen saturation of the air was adjusted during the fermentation to 50%. The control was performed automatically by the agitation speed. The fermentation was carried out at a temperature of 30 ° C. After 2 h of fermentation time, a feed was made from a 30% sterile stock solution of Na-thiosulfate x 5H20, with a flow of 3 ml per hour. After reaching an OD600 of 10, a sterile stock at 56% glucose was added, with a flow of about 8 to 14 ml per hour. The determination of the glucose content was carried out enzymatically with the help of a glucose analyzer from the company YSI. The glucose concentration was adjusted during fermentation by continuous feeding at between 10 and 20 g / 1. The total cysteine content of the medium was determined from the excess cell free of the sample, in a chlorimetric manner according to Gaitonde, M. K. (1967), Biochem. J. 104, 627-633. It should be noted that the cysteine that remains in solution during fermentation is present mainly as a thiazolidine derivative, but was also included in the test. If for the reaction of the cysteine that was formed to obtain the thiazolidine derivative, a sufficient quantity of the necessary ketone or aldehyde is no longer available (in this case pyruvate), free L-cysteine is formed, which also it is included in the test. In the case of a formation of free L-cysteine, it is slowly oxidized by the oxygen of air fed during fermentation to L-cystine. Cystine is difficult to dissolve in aqueous medium at pH 7.0 and precipitates as a white bead. In the case of an insoluble cystine bead formation, it dissolves after removing an excess of a sample taken, after centrifugation, in semi-concentrated HCl and is also measured under reducing conditions (DTT) in the aforementioned test. Under these conditions, after a fermentation duration of 24 hours, or 48 hours, the yields shown in tables 1 and 2 were achieved. It is clear that the genes used according to the invention, namely the sea-Locus of E. coli and particularly the section encoding ORF306, greatly increase the yields of cysteine and / or thiazolidine derivative (total cysteine). If a cystine precipitation was formed, this is indicated extra in the tables.
Table 1 Yields of total cysteine with the cysEIV allele
* amount of cystine present as a pearl, in grams per liter Table 2 Total cysteine yields with the cysEX allele
* quantity of cystine present as a pearl, in grams per liter
Example 4 Verification of the formation of 2-methyl-thiazolidin-2,4-dicarbonic acid
To check the formation of 2-methyl-thiazolidin-2,4-dicarbonic acid as the main product of the fermentation described in Example 3, the E. coli W3110 x pACYC184 / cysEX-GAPDH-ORF306 structure was fermented as described in Example 3. After 24 hours, the protrusion from the fermentation was separated from the cells by centrifugation. The described cysteine measurement resulted in 12.8 g of total cysteine in the overhang. The fermentation overhang was then mixed with MgSO4 at a final concentration of 0.3 M. After incubation of this overhang overnight at 4 ° C under stirring, a white precipitate formed. It was the hardly soluble magnesium salt of 2-methyl-thiazolidin-2,4-dicarbonic acid. After a separation of said precipitate by centrifugation, only a residual amount of cysteine of 2.5 g / 1 was measured in the overhang. The precipitate was dissolved in semiconcentrated HCl and also subjected to a cysteine test. A concentration of 9.5 g / 1 of cysteine was found. After an NMR ÍH and 13C examination of the precipitate dissolved in D20 + HCl, it was identified against a reference substance (MP Schubert, J. Biol. Chem. 121, 539-548 (1937)) as 2-methyl-thiazolidin acid -2, 4-dicarbonic.
Example 5 Various toxicities of L-cysteine and 2-methyl-thiazolidin-2,4 (R) -dicarbonic acid
For this test 2-methyl-thiazolidin-2,4 (R) -dicarbonic acid was synthesized according to the method of Schubert (MP Schubert, J. Biol. Chem. 121, 539-548 (1937)), from L- cysteine and pyruvate. A one-night culture of E. coli W3110 in LB medium was inoculated in 20 ml of SM1 medium (see example 3), which had as a supplement 10 g / 1 glucose, 10% LB medium, 5 mg / l vitamin Bl, 15 mg / l of tetracycline and respective amounts of L-cysteine or 2-methyl-thiazolidin-2,4 (R) -dicarbonic acid. After an incubation of 7 hours at 37 ° C, in the medium added with L-cysteine no growth was observed from 1 mM, while in the medium added with 2-methyl-thiazolidin-2-, 4 ( R) -dicarbonic growth was observed up to 50 mM. Due to the easy oxidation of cysteine, longer incubation times were not possible. Thus, for L-cysteine a clearly higher toxicity results for E. coli than for 2-methyl-thiazolidin-2,4 (R) -dicarbonic acid. Therefore, 2-methyl-thiazolidin-2,4 (R) -dicarbonic acid is clearly better suited for obtaining L-cysteine by fermentative processes, even when a chemical step is still needed for the release of L -cysteine.
Example 6 Reinforced formation of N-acetyl-L-serine
The formation of N-acetyl-L-serine is effected by spontaneous repositioning from 0-acetyl-L-serine. This O-acetyl-L-serine is the immediate previous stage of L-cysteine in the path of biosynthesis in the case of bacteria. With an inclusion of insufficient or missing sulfur in O-acetyl-L-serine, the final product of a fermentation of this type is thus N-acetyl-L-serine. If the sulfur feed is missing, the genes according to the invention also increase the yield of this fermentation product. The fermentation described in example 3 was carried out without thiosulfate feed. The structures used, which should show the effectiveness of the genes present, particularly of ORF306, were pACYC184 / cysEX and pACYC184 / cysEX-GAPDH-ORF306. As the results in Table 3 show, the genes according to the invention, particularly ORF306, clearly increase the yield of N-acetyl-L-serine in the fermentation.
Table 3 Yields of N-acetyl-L-serine after 24 h of fermentation
In the case of using cysE alleles that are more strongly resistant to feedback (for example, cysEXIV, cysEXI and cysEXXII of DE 19539952) in combination with the genes according to the invention, yields of N-acetyl-L-serine can be achieved. more than 30 g / 1.
SEQUENCE PROTOCOL
(1) GENERAL DATA (i) APPLICANT: (A) NAME: Consortium für elektrochemische
Industrie GmbH (B) STREET: Zielstattstr. 20 (C) PLACE: Munich (D) FEDERATED STATE: Bavaria (E) COUNTRY: Germany (F) POSTAL CODE: 81379 (G) TELEPHONE: 089-74844-0 (H) TELEFAX: 089-74844-350 (II) TITLE OF THE INVENTION: MICROORGANISMS AND PROCEDURES FOR THE FERMENTATIVE OBTAINING OF L-CYSTEINE, L-CISTINE, N-ACETYL-SERINE OR DERIVATIVES OF TLAZOLIDINE (iii) NUMBER OF SEQUENCES: 12 (iv) LEGIBLE VERSION WITH COMPUTER (A) DATA CARRIER: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, version # 1.30 (EPA) (2) DATA OF THE SEQ ID NO: 1 (i) CHARACTERIZATION OF THE SEQUENCE (A) LENGTH: 43 amino acids (B) TYPE: Amino Acid (C) BAND FORM: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal fragment (vi) ORIGINAL PROVENANCE (A) ORGANISM: Escherichia coli (B) CEPA: K12 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1:
Mee Ser? Rg Lys? Sp Gly Val Leu? The Leu Leu Val Val Val Val Tzp 1 S 10 15
Gly Leu? An Phe Val Val He Lys Val Gly Leu His? ßn Mee Pro Arg 20 25 30
Leu Mee Leu? The Gly Leu? Rg Phe Mee Leu Val 35 40 (2) DATA OF SEQ ID NO: 2 (i) CHARACTERIZATION OF THE SEQUENCE (A) LENGTH: 306 amino acids (B) TYPE: Amino Acid (C) FORM BAND: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL PROVENANCE (A) ORGANISM: Escherichia coli (B) CEPA: K12 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Mee Lys Phe? Rg Gly ßly? Rg Mee Ser Arg Lys? Sp Gly Val Leu Ala
1 5 10 15
Leu Leu Val val Val Val Tzp Gly Leu Asa he val Val He Lys at 20 25 30
ßly Leu His? sn Mee Pro Arg Leu Mee Leu Ala ßly Leu Arg Phe Mee
40 45
Leu val? The Phe Pro Ala lie Phe Phe Val? The Arg Pro Lys Val Pro 50 55 60
Leu Asn Leu Leu Leu Gly Tyr Gly Leu Thr He Ser Phe? La Gln Phe 65 70 75 80
Ala Phe Leu Phe Cys Ala He Asn Phe Gly Mee Pro? La ßly Leu Ala 85 90 95
Ser Leu Val Lau ßln Ala Gn Ala Phe Phe Thr He Mee Leu ßly Ala 100 105 110
Phe Thr Phe ßly ßlu Arg Leu His ßly Lya ßln Leu Ala ßly He? The 115 120 125
Leu? La He Phe ßly Val Leu Val Leu He ßlu? ßp Ser Leu? ßn Gly 130 135 140
ßln His Val? the Hee Leu ßly Phe Mee Leu Thr Leu? the? the? the Phe 145 150 155 160
Being Trp? The Cyß ßly Aßn He Phe Aßn Lyß Lyß He Mee Ser Hiß Ser 165 170 175
Thr Arg Ro? The Val Hee Ser Leu Val He Trp Ser? The Leu He Pro 110 185 190
Xle He Pro Phe Phe Val Wing Ser Leu Xle Leu Aßp ßly Ser Wing Thr 195 200 205
Mee He Hi Be Ser Leu Val Thr He Asp Mee Thr Thr He Leu Ser Leu 210 215 220
Mee Tyr Leu? The Phe Val Ala Thr He Val Gly Tyr Gly He Trp Gly 225 230 235 240
Thr Leu Leu Gly Arg Tyr ßlu Thr Tzp Arg Val? The Pro Leu Ser Leu 245 250 255
Leu Val Pro Val Val ßly Leu Ala Ser Ala Ala Leu Leu Leu Aap Glu 260 265 270
Arg Leu Thr ßly Leu ßln Phe Leu ßly and Wing Val Leu He Mee Thr ßly 275 280 285
Leu Tyr He Aßn val Phe ßly Leu Arg Trp Arg Lyß Wing val Lys val 290 295 300
ßly Ser 305
(2) DATA OF SEQ ID NO: 3 (i) CHARACTERIZATION OF THE SEQUENCE (A) LENGTH: 299 amino acids (B) TYPE: Amino Acid (C) BAND FORM: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (Vi) ORIGINAL PROVENANCE (A) ORGANISM: Escherichia coli (B) CEPA: K12 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
Hee Ser? Rg Lys? ßp ßly Val Leu Ala Leu Leu Val Val Val Val Trp
1 5 10 15
ßly Leu Aan Phe Val Val He Lys Val Gly Leu His Asn Mee Pro Arg 20 25 30
Leu Mee Leu Ala Gly Leu Arg Phe Mee Leu Val? The Phe Pro? La He 35 40 45
Phe Phe Val? The? Rg Pro Lyß Val Pro Leu? ßn Leu Leu Leu ßly Tyr 50 55 60
ßly Leu Thr He Ser Phe Ala ßln Phe Ala Phe Leu Phe Cyß? la He 65 70 75 80
? ßn Phe ßly Mee Pro? la ßly Leu Ala Ser Leu Val Leu ßln? the Gln 85 90 95
? the Phe Phe Thr He Mee Leu ßly? the Phe Thr Phe ßly ßlu? rg Leu 100 105 110
His Gly Lyß Gln Leu? The Gly He? The Leu? The He Phe ßly Val Leu 115 120 125
val Leu He ßlu Aßp Ser Leu Aßn ßly ßln Hiß Val? la Mee Leu sly 130 135 140
Phe Mee Leu Thr Leu? The? La? The Phe Ser Trp? The Cy? Oly? ßn He 145 150 155 160
Phe? ßn Lyß Lyß He Mee Ser Ser Ser Thr? Xg Pro? La al Mee Ser 165 170 175
Leu Val He Trp Be Wing Leu He Pro He He Pro Phe Phe Val Wing 180 185 190 Ser Leu He Leu? ßp Gly Ser? Thr Mee He Hi? Ser Leu Val Thr 195 200 205
He? ßp Mee Thr Thr He Leu Ser Leu Mee Tyr Leu? The Phe Val Wing 210 215 220
Tnr He Val Oly Tyr Gly He Trp ßly Thr Leu Leu Oly Arg Tyr ßlu 225 230 235 240
Thr Trp? Rg Val? The Pro Leu Ser Leu Leu Val Pro Val Val? Sly Leu 245 250 255
The Be? la? the Leu Leu Leu? ap ßlu? rg Leu Thr ßly Leu Oln Phe 260 265 270
Leu Oly? The Val Leu He Mee Thr ßly Leu Tyr He? ßn Val Phe Gly 275 280 285
Leu? Rg Trp? Rg Lyß? The Val Ly? Val Oly Ser 290 295
(2) DATA OF SEQ ID NO: 4 (i) CHARACTERI ZATION OF THE SEQUENCE (A) LENGTH: 266 amino acids (B) TYPE: Amino Acid (C) BAND FORM: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL PROVENANCE (A) ORGANISM: Escherichia coli (B) CEPA: K12 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4:
Mee Leu? The Gly Leu Arg Phe Mee Leu val Wing Phe Pro? La He Phe 1 5 10 15
Phe Val? The? Rg Pro Lyß Val Pro Leu? ßn Leu Leu Leu Oly Tyr Gly 20 25 30
Leu Thr Xle Being Phe? The Gln Phe? The Phe Leu Phe Cy?? The He A? N 35 40 45
Phe Oly Mee Pro? The Gly Leu? The Ser Leu Val Leu ßln? The ßln Ala 50 55 60
Phe Phe Thr He Mee Leu ßly Wing Phe Thr Phe ßly ßlu Arg Leu His 65 70 75 80
ßly Lys Oln Leu Ala ßly He? the Leu? the Xle Phe ßly and Val Leu val 85 90 95
Leu Xle Olu? ßp Ser Leu? Sn ßly ßln His Val? La Mee Leu ßly Phe 100 105 110
Mee Leu Thr Leu? The? The? The Phe Ser Tzp? The Cyß ßly? An He Phe 115 120 125
? ßn Lyß Lyß He Mee be Hiß Ser Thr? rg Pro? the Val Mee Ser Leu 130 135 140
Val? Le Trp Ser? The Leu He Pro He Xle Pro Phe Phe Val? The Ser 145 150 155 160
Leu He Leu? ßp ßly Ser? The Thr Mee He Hi? Ser Leu Val Thr He 165 170 175
Aßp Mee Thr Thr He Leu Ser Leu Mee Tyr Leu? The Phe Val? The Thr 180 185 190 He Val Gly Tyr Gly He Trp Gly Thr Leu Leu Oly? Rg Tyr ßlu Thr 195 200 205
Trp? Rg Val? Pro Leu Ser Leu Leu Val Pro Val Val Qly Leu Ala 210 215 220
Be Ala? The Leu Leu Leu? Ap Glu? Rg Leu Thr ßly Leu Oln Phe Leu 225 230 235 240
Gly? The Val Leu He Mee Thr Gly Leu Tyr He? An Val Phe ßly Leu 245 250 255
? rg Trp? rg Lyß? the al Lyß Val ßly Ser 260 265
(2) DATA OF SEQ ID NO: 5 (i) CHARACTERI ZATION OF SEQUENCE (A) LENGTH: 38 base pairs (B) TYPE: Nucleotide (C) BAND FORM: Individual band (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: TGGACCAGAG CTCTGGCTGG CGCATCGCTT CGGCGTTG 38
(2) DATA OF SEQ ID NO: 6 (i) CHARACTERIZATION OF SEQUENCE (A) LENGTH: 38 base pairs (B) TYPE: Nucleotide (C) BAND FORM: Individual band (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: CTCGATGCAT TACGTAGGGG TATCCGGGAG CGGTATTG 38 (2) DATA OF SEQ ID NO: 7 (i) CHARACTERIZATION OF THE SEQUENCE (A) LENGTH: 35 base pairs (B) TYPE: Nucleotide (C) BAND FORM: Individual band (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: TTTGGCGCGC CGATCAGCGG CGGCGCAACC ATCAG 35 SEQ ID NO: 8 (i) CHARACTERIZATION OF SEQUENCE (A) LENGTH: 38 base pairs (B) TYPE: Nucleotide (C) BAND FORM: Individual band (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 8: GCCTTAATTA AGATCGACAC TCAGGCTGTA CTGGCGAC 38 (2) DATA OF SEQ ID NO: 9 (i) CHARACTERIZATION OF THE SEQUENCE (A) LENGTH: 35 base pairs (B) TYPE: Nucleotide (C) BAND FORM: Individual band (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9: GGAATTCATT AATCCGGCGA CTAACGAATC AACTG 35
(2) DATA OF SEQ ID NO: 10 (i) CHARACTERIZATION OF THE SEQUENCE (A) LENGTH: 36 base pairs (B) TYPE: Nucleotide (C) BAND FORM: Individual band (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: GCCTTAATTA ACGCTATGTA GTTTGTTCTG GCCCCG 36
(2) DATA OF SEQ ID NO: 11 (i) CHARACTERIZATION OF THE SEQUENCE (A) LENGTH: 33 base pairs (B) TYPE: Nucleotide (C) BAND FORM: Individual band (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 11 GTCGACGCGT GAGGCGAGTC AGTCGCGTAA TGC 33
(2) DATA OF SEQ ID NO: 12 (i) CHARACTERIZATION OF SEQUENCE (A) LENGTH: 43 base pairs (B) TYPE: Nucleotide (C) BAND FORM: Individual band (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic" (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: GACCTTAATT AAGATCTCAT ATGTTCCACC AGCTATTTGT TAG_43_
Claims (10)
1. A strain of microorganisms suitable for the fermentative production of L-cysteine, L-cystine, N-acetyl-serine and / or thiazolidine derivatives, characterized in that it overexpresses at least one gene that encodes a suitable protein directly for the cell's secretion. antibiotics or other substances toxic to the microorganism.
2. A microorganism strain according to claim 1, characterized in that as a gene coding for a protein suitable directly for the cell blockage of antibiotics or other substances toxic to the microorganism, at least one gene chosen from the sea group is overexpressed. -Locus, emr-Locus, acr-Locus, cmr-Locus, mex genes, bmr gene, qacA gene.
3. A microorganism strain according to claim 1, characterized in that a gene coding for a protein that includes the sequence is overexpressed as a gene coding for a protein suitable directly for cell blockage of antibiotics or other substances toxic to the microorganism. MSR KDGVLALLW WWGLNFWI KVGLHNMPRL MLAGLRFMLV (SEQ ID NO: 1) or a sequence with sequence homology greater than 50% with SEQ. ID. NO: 1.
4. A gene encoding a protein that includes the MSR sequence KDGVLALLW WWGLNFWI KVGLHNMPRL MLAGLRFMLV (SEQ ID NO: 1) or a sequence with sequence homology greater than 50% with SEQ. ID. NO: 1.
5. A gene that encodes a protein that includes the sequence 1 MKFRGGRMSR KDGVLALLW WWGLNFWI KVGLHNMPRL MLAGLRFMLV 51 AFPAIFFVAR PKVPLNLLLG YGLTISFAQF AFLFCAINFG MPAGLASLVL 101 QAQAFFTIML GAFTFGERLH GQLAGIALA IFGVLVLZEO SLNGQHVAML 151 GFMLTLAAAF SWACGNIFNK KIMSHSTRPA VMSLVX SAL IPIIPFFVAS 201 LILDGSATMI HSLVTIDMTT ILSLMYLAFV ATrVGYGIWG TLLGRYETWR 251 VAPLSLLVPV VGLASAALLL DERLTGLQFL GAVLIMTGLY INVFGLRWRK 301 AVKVGS * (SEQ ID NO: 2) or a sequence with sequence homology greater than 50% with SEQ. ID. NO: 2.
6. A protein that includes the MSR sequence KDGVLALLW WWGLNFWI KVGLHNMPRL MLAGLRFMLV (SEQ ID NO: 1) or a sequence with sequence homology greater than 50% with the SEQ. ID. NO: 1.
7. A plasmid containing at least one gene according to claim 4 or 5.
8. A process for obtaining L-cysteine, L-cystine, N-acetyl-serine or thiazolidine derivatives thereof, characterized in that a strain of microorganisms according to claim 1 is used in the fermentation in a manner known per se. 6 3.
9. The use of efflux genes for the amplified expression of amino acids or derivatives of amino acids formed intracellularly in fermentation.
10. Procedures for obtaining L-cysteine, characterized in that it is reacted intracellularly in a microorganism L-cysteine formed intracellularly of the microorganism with the ketone or aldehyde present intracellularly in the microorganism to obtain a thiazolidine derivative, this thiazolidine derivative is locked of the microorganism by means of a protein, which is directly suitable for blocking from the cell antibiotics or other substances toxic to the microorganism, and, eventually after separating the thiazolidine derivative, L-cysteine is obtained by shifting the equilibrium of the reaction between L-cysteine and the thiazolidine derivative in the direction of L-cysteine.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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DE19726083.7 | 1997-06-19 |
Publications (1)
Publication Number | Publication Date |
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MXPA98004927A true MXPA98004927A (en) | 1999-09-20 |
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