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WO2013171098A2 - Procédé de production de l-cystéine et de dérivés de cet acide aminé par fermentation - Google Patents

Procédé de production de l-cystéine et de dérivés de cet acide aminé par fermentation Download PDF

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Publication number
WO2013171098A2
WO2013171098A2 PCT/EP2013/059477 EP2013059477W WO2013171098A2 WO 2013171098 A2 WO2013171098 A2 WO 2013171098A2 EP 2013059477 W EP2013059477 W EP 2013059477W WO 2013171098 A2 WO2013171098 A2 WO 2013171098A2
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WO
WIPO (PCT)
Prior art keywords
cysteine
factor
cell
compared
benzoic acid
Prior art date
Application number
PCT/EP2013/059477
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German (de)
English (en)
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WO2013171098A3 (fr
Inventor
Tobias Dassler
Marcel THÖN
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Wacker Chemie Ag
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Application filed by Wacker Chemie Ag filed Critical Wacker Chemie Ag
Publication of WO2013171098A2 publication Critical patent/WO2013171098A2/fr
Publication of WO2013171098A3 publication Critical patent/WO2013171098A3/fr

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    • 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

Definitions

  • L ⁇ cystine is a disulfide that results from the oxidation of two molecules of L-cysteine. This reaction is reversible, which means that L-cystine can be converted back into L-cysteine by reduction.
  • 2-Methyl-thiazolidine-2, 4-dicarboxylic acid is the condensation product of L-cysteine and pyruvate, which is formed in a purely chemical reaction (US5972663A), This reaction is also reversible.
  • the thiazolidine may be e.g. be decomposed in acid at elevated temperature back into its starting components.
  • the amino acid L-cysteine is of economic importance.
  • L-cysteine occupies a key position in sulfur metabolism in all organisms and is used in the synthesis of proteins, glutathione, biotin, lipoic acid, methionine and other sulfur-containing metabolites.
  • L-cysteine serves as a precursor for the biosynthesis of coenzyme A. The biosynthesis of L-cysteine has been reported in bacteria, especially in
  • O-acetyl-L-serine is formed from L-serine and acetyl-CoA. Therefore, the supply of L-serine in sufficient quantity is of great importance for L-cysteine production.
  • This can be achieved by introducing a serA allele encoding a 3-phosphoglycerate dehydrogenase with reduced feedback inhibitability by L-serine. Thereby for example, the formation of 3-hydroxypyruvate, a precursor of L-serine, is largely decoupled from the cell's L-serine level. Examples of such SerA enzymes are described in EP0620853 and US2005009162A2.
  • L-cysteine yield in the fermentation can be increased by attenuating or destroying genes coding for L-cysteine degrading enzymes, e.g. the tryptophanase TnaA or the cystathionin- ⁇ -lyases MalY or etC (EP1571223).
  • L-cysteine degrading enzymes e.g. the tryptophanase TnaA or the cystathionin- ⁇ -lyases MalY or etC (EP1571223).
  • Increasing the transport of L-cysteine out of the cell is another way to increase the product yield in the medium. This can be achieved by overexpression of so-called efflux genes. These genes code for membrane-bound proteins that mediate the export of L-cysteine from the cell. Various efflux genes for L-cysteine export have been described (US5972663A, US20040038352A1 ⁇ .
  • L-cysteine is continuously withdrawn from the intracellular reaction equilibrium, with the result that the level of this amino acid in the cell is kept low and thus the feedback inhibition of sensitive enzymes by L-cysteine is omitted:
  • L-cystine dissolved
  • L-cystine precipitate
  • the precipitation of L-cystine lowers the level of product dissolved in the medium, which also causes the reaction equilibrium of (1) and (2) to be drawn onto the product side.
  • total cysteine comprises L-cysteine and the compounds L-cystine and thiazolidine formed therefrom, which are formed during the fermentation and accumulate in the culture supernatant and in the precipitate.
  • the object of the present invention is to provide an improved process for the preparation of L-cysteine and its derivatives L-cystine and thiazolidine by fermentation of a microorganism. nismenstatnmes to provide in a fermentation medium.
  • the object is achieved by a method which is characterized in that benzoic acid or a salt of benzoic acid in a concentration range of 1-500 mg / L is added to the fermentation medium.
  • Benzoic acid can be used either directly as an acid or in the form of one of its salts, e.g. Calcium, potassium or sodium benzoate - used singly or as a mixture ⁇ .
  • Benzoic acid or its salts are preferably added to the medium in a concentration range of 2.5-400 mg / L.
  • the supplementation of the medium with benzoic acid or its salts can be carried out as a batch addition at the beginning of the cultivation.
  • benzoic acid can also be metered into the medium only during the fermentation.
  • microorganisms for the process according to the invention it is possible to use all cysteine-producing strains described in the prior art. Such strains are for example disclosed in US6218168B1, US5972663A, US20040038352A1,
  • Preferred microorganisms are representatives of the family of Enterobacteriaceae, more preferably representatives of the genera Escherichia and Pantoea, very particularly preferred are strains of the species E. coli and P. ananatis.
  • strains are preferred which either have an altered serine O-acetyltransferase which, compared to the corresponding wild-type enzyme, has at least a factor of 2 reduced feedback inhibition by L-cysteine or by overexpression of an efflux gene have an increased by at least a factor of 2 cysteine export from the cell compared to a wild-type cell.
  • strains of microorganisms have both a serine O-acetyl transferase, which has a reduced by at least a factor of 2 feedback inhibition by L-cysteine compared to the corresponding wild-type enzyme, as well as increased by at least a factor of 2 by overexpression of an efflux gene Cysteine export from the cell compared to a wild-type cell.
  • Such strains are known, for example, from US6218168B1 and US5972663A.
  • Very particularly preferred strains are those which additionally possess an altered 3-phosphoglycerate dehydrogenase with a feedback inhibition by L-serine which has been reduced by at least a factor of 2 compared with the corresponding wild-type enzyme
  • Preferred variants of the serine O-acetyl transferase have a feedback inhibition by L-cysteine which is reduced by at least the factor 5, particularly preferably by at least the factor 10, very particularly preferably by at least the factor 50, compared to the corresponding wild-type enzyme on.
  • the efflux gene is preferably from the group ydeD (see US5972663A), yfiK (see US20040038352A1), cydDC (see
  • E. coli or the corresponding homologous gene from another microorganism.
  • a homologous gene is meant that the sequence of this gene is at least 80% identical to the DNA sequence of the corresponding E. coli gene.
  • the overexpression of an efflux gene preferably leads to a cysteine export from the cell which is increased by at least the factor 5, particularly preferably by at least the factor 10, very particularly preferably by at least the factor 20, compared to a wild-type cell.
  • Preferred variants of the 3-phosphoglycerate dehydrogenase have in comparison to the corresponding wild-type enzyme on at least a factor of 5, more preferably at least a factor of 10, most preferably at least a factor of 50 reduced feedback inhibition by L-serine.
  • the L-cysteine degrading enzyme preferably originates from the group tryptophanase (TnaA) and cystathionine ⁇ -lyase ⁇ MalY, Mete).
  • microorganism strains in which at least one of these enzymes is so far attenuated that in the cell only a maximum of 10% of the enzyme activity compared to a wild-type strain is present.
  • strains in which at least one of these enzymes is completely inactivated are particularly preferred.
  • the cultivation of the cells in the L-cysteine production is carried out under aerobic growth conditions, wherein the oxygen content is set during fermentation at a maximum of 50% saturation.
  • the regulation of the oxygen saturation in the culture takes place automatically via the gas supply and the stirring speed.
  • the carbon source used are preferably sugars, sugar alcohols, organic acids or sugar-containing plant hydrolysates. Glucose, fructose, lactose, glycerol or mixtures containing two or more of these compounds are particularly preferred in the process according to the invention as the carbon source.
  • the production phase of the fermentation process according to the invention begins with the time from which L-cysteine, L-cystine or thiazolidine can be detected for the first time in the culture broth and lasts until the end of the cultivation. Typically, this phase begins about 8-10 hours after inoculation of the production fermenter.
  • the carbon source of the culture is preferably metered in so that the content of the carbon source in the fermenter does not exceed 10 g / l during the production phase. Preference is given to a maximum concentration of 2 g / l, more preferably of 0.5 g / l, most preferably of 0.1 g / l.
  • the N-source used in the process according to the invention is preferably ammonia, ammonium salts or protein hydrolysates.
  • ammonia is used as a correction agent for pH stabilization, this N source is regularly replenished during the fermentation.
  • salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, iron and trace (i.e., in ⁇ concentrations) salts of the elements molybdenum, boron, cobalt, manganese, zinc and nickel may be added.
  • organic acids eg acetate, citrate
  • amino acids eg isoleucine
  • vitamins eg Bl, B6
  • yeast extract for example, yeast extract, corn steep liquor, soybean meal or malt extract can be used.
  • the incubation temperature for mesophilic microorganisms is preferably 15-45 ° C, more preferably 30-37 ° C.
  • the pH of the fermentation medium during the fermentation is preferably in the pH range from 5.5 to 7.5, more preferably a pH of 6.0-7.0, very particularly preferably a pH from 6.5.
  • a sulfur source For the production of L-cysteine and L-cysteine derivatives, a sulfur source must be added during the fermentation. Sulfates or thiosulfates are preferably used here.
  • Microorganisms that are fermented by the method described, secrete L-cysteine and derived compounds in high efficiency in the fermentation medium in a batch or fedbatch process after a growth phase in a period of eight to 150 hours.
  • the precipitated L-cystine can be separated by known methods from the remaining components of the culture broth, for example with the aid of a decanter.
  • L-cystine to L-cysteine can be carried out, for example, via an electrochemical process as described in EP0235908.
  • the following examples serve to further illustrate the invention.
  • the plasmid pACYC184 / cysEX ⁇ GAPDH ⁇ ORF306 contains, in addition to the origin of replication and a tetracycline resistance gene, the cysEX allele which codes for a serine O-acetyltransferase with a reduced feedback inhibition by L-cysteine and the effluxgene ydeD
  • the desired transformants i. Cells which have taken up the plasmid pACYC184 / cysEX-GAPDH ORF306 isolated and used in the cultivation described in Examples 2 and 3.
  • the preculture 1 was completely converted into 100 ml of S l medium ⁇ 12 g / 1 K 2 HP0 4 , 3 g / 1 KH 2 PO 4 , 5 g / 1 ⁇ NH 4 ) 2 SO 4 , 0.3 g / 1 MgSO 4 ⁇ 7 H 2 O, 0.015 g / 1 CaCl 2 ⁇ 2 H 2 O, 0.002 g / 1 FeSCX ⁇ 7 H 2 O, 1 g / 1 Na 3 citrate ⁇ 2 H 2 O, 0.1 g / 1 NaCl, 1 ml / 1 trace element solution, consisting of 0.15 g / 1 Na 2 Mo0 4 x 2H 2 0, 2.5 g / 1 H 3 BO 3 , 0.7 g / 1 CoCl 2 x 6 H 2 0 , 0.25 g / 1 CuSO 4 ⁇ 5 H 2 O, 1.6 g / 1 MnCl 2 ⁇ 4 H 2 O, 0.3 g
  • the fermentation was carried out in fermenters of the BIOSTAT B type from Sartorius Stedim at various benzoic acid concentrations (added as a batch). It was used a culture vessel with 2 1 total volume.
  • the fermentation medium (900 ml) contains 15 g / 1 glucose, 10 g / 1 tryptone (Difco), 5 g / 1 yeast extract (Difco), 5 g / 1 (NH 4 ) 2 S0 4 , 1.5 g / 1 KH 2 P0 4 , 0.5 g / 1 NaCl, 0.3 g / 1 MgSO 4 ⁇ 7 H 2 O, 0.015 g / 1 CaCl 2 ⁇ 2 H 2 O, 0.075 g / 1 FeS0 4 ⁇ 7 H 2 O, 1 g / 1 Na 3 citrate ⁇ 2 H 2 O and 1 ml trace element solution (see above), 0.005 g / 1 vitamin B1 and 15 mg / 1 tetracycline In various experimental settings, the medium
  • the pH in the fermenter was initially adjusted to 6.5 by pumping a 25% NH 4 OH solution. During the fermentation, the pH was maintained at a value of 6.5 by automatic correction with 25% NH 4 OH.
  • 100 ml of preculture 2 were pumped into the fermenter vessel. The initial volume was thus about 1 1.
  • the cultures were initially stirred at 400 rpm and gassed with 2 vvm compressed air sterilized via a sterile filter. Under these start conditions, the oxygen probe was calibrated to 100% saturation prior to inoculation.
  • the target value for the 0 2 saturation during the fermentation was set to 50%. After lowering the 0 2 saturation below the target value, a regulatory cascade was started to bring the 0 2 saturation back to the target value.
  • the gas supply was initially increased continuously (to a maximum of 5 vvm) and then the stirring speed was continuously increased (to a maximum of 1,500 rpm).
  • the fermentation was carried out at a temperature of 30 ° C. After 2 h fermentation time, the feed of a sulfur source in the form of a sterile 60% sodium Thiosulfate x 5 H 2 O stock solution at a rate of 1.5 ml per hour. As soon as the glucose content in the fermenter had fallen from initially 15 g / l to approximately 2 g / l, a continuous addition of a 56% glucose solution took place. The feeding rate was adjusted so that the glucose concentration in the fermenter no longer exceeded 2 g / 1. The glucose determination was carried out with a glucose analyzer from YSI (Yellow
  • the fermentation time was 48 hours. Thereafter, samples were taken and the content of L-cysteine and the derivatives derived therefrom in the culture supernatant (mainly L-cysteine and thiazolidine) and in the precipitate (L-cystine) were determined separately from each other ⁇ s. Table 2).
  • the colorimetric assay of Gaitonde was used (Gaitonde, M.K. (1967), Biochem J. 104, 627-633).
  • the precipitated L-cystine must first be dissolved in 8% hydrochloric acid before it could be quantitated in the same way.

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  • Organic Chemistry (AREA)
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  • Engineering & Computer Science (AREA)
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Abstract

L'invention concerne un procédé de production de L-cystéine et de ses dérivés L-cystine et thiazolidine par fermentation d'une souche de microorganismes dans un milieu de fermentation auquel est ajouté de l'acide benzoïque dans une plage de concentration de 1-500 mg/L.
PCT/EP2013/059477 2012-05-18 2013-05-07 Procédé de production de l-cystéine et de dérivés de cet acide aminé par fermentation WO2013171098A2 (fr)

Applications Claiming Priority (2)

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DE102012208359A DE102012208359A1 (de) 2012-05-18 2012-05-18 Verfahren zur fermentativen Produktion von L-Cystein und Derivaten dieser Aminosäure
DE102012208359.6 2012-05-18

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WO2013171098A2 true WO2013171098A2 (fr) 2013-11-21
WO2013171098A3 WO2013171098A3 (fr) 2014-01-09

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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0235908A2 (fr) 1986-01-23 1987-09-09 The Electricity Council Procédé de production de L-cysteine
EP0620853A1 (fr) 1991-12-12 1994-10-26 Wacker Chemie Gmbh Matieres et procedes utilises pour la biosynthese de serine et de produits apparentes a la serine.
EP0885962A1 (fr) 1997-06-19 1998-12-23 Consortium für elektrochemische Industrie GmbH Microorganismes et procédé de production de L-cystéine, L-cystine, N-acetyl-sérine ou dérivés de thiazolidine par fermentation
US6218168B1 (en) 1995-10-26 2001-04-17 CONSORTIUM FüR ELEKTROCHEMISCHE INUDSTRIE GMBH Process for preparing O-acetylserine, L-cysteine and L-cysteine-related products
CA2386539A1 (fr) 1999-10-14 2001-04-19 Thomas Maier Procedes pour produire de la l-cysteine ou des derives de cette derniere par fermentation
US20040038352A1 (en) 2002-07-19 2004-02-26 Consortium Fur Elektrochemische Industrie Gmbh Method for fermentative production of amino acids and amino acid derivatives of the phosphoglycerate family
WO2004113373A1 (fr) 2003-06-21 2004-12-29 University Of Sheffield Surexpression du transporteur cyddc
US20050009162A1 (en) 2003-07-10 2005-01-13 Thomas Maier 3-phosphoglycerate dehydrogenase variants whose inhibition by L-serine is reduced, and genes encoding them
EP1571223A2 (fr) 2004-03-04 2005-09-07 Ajinomoto Co., Inc. Microorganisme producteur de L-cystéine, et procédé de préparation de L-cystéine
US20050221453A1 (en) 2004-03-31 2005-10-06 Ajinomoto Co., Inc L-cysteine producing microorganism and method for producing L-cysteine
US20090053778A1 (en) 2004-07-20 2009-02-26 France Telecom Microorganisms for producing sulfur-containing compounds
US20090226984A1 (en) 2008-03-06 2009-09-10 Gen Nonaka L-cysteine producing bacterium and a method for producing l-cysteine
DE102011075656A1 (de) 2011-05-11 2012-03-29 Wacker Chemie Ag Verfahren zur fermentativen Produktion von L-Cystin

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
JPS5010397B1 (fr) * 1964-08-24 1975-04-21

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0235908A2 (fr) 1986-01-23 1987-09-09 The Electricity Council Procédé de production de L-cysteine
EP0620853A1 (fr) 1991-12-12 1994-10-26 Wacker Chemie Gmbh Matieres et procedes utilises pour la biosynthese de serine et de produits apparentes a la serine.
US6218168B1 (en) 1995-10-26 2001-04-17 CONSORTIUM FüR ELEKTROCHEMISCHE INUDSTRIE GMBH Process for preparing O-acetylserine, L-cysteine and L-cysteine-related products
EP0885962A1 (fr) 1997-06-19 1998-12-23 Consortium für elektrochemische Industrie GmbH Microorganismes et procédé de production de L-cystéine, L-cystine, N-acetyl-sérine ou dérivés de thiazolidine par fermentation
US5972663A (en) 1997-06-19 1999-10-26 Consortium Fur Elektrochemische Industrie Gmbh Microorganisms and processes for the fermentative preparation of L-cysteine, L-cystine, N-acetylserine or thiazolidine derivatives
CA2386539A1 (fr) 1999-10-14 2001-04-19 Thomas Maier Procedes pour produire de la l-cysteine ou des derives de cette derniere par fermentation
US20040038352A1 (en) 2002-07-19 2004-02-26 Consortium Fur Elektrochemische Industrie Gmbh Method for fermentative production of amino acids and amino acid derivatives of the phosphoglycerate family
WO2004113373A1 (fr) 2003-06-21 2004-12-29 University Of Sheffield Surexpression du transporteur cyddc
US20050009162A1 (en) 2003-07-10 2005-01-13 Thomas Maier 3-phosphoglycerate dehydrogenase variants whose inhibition by L-serine is reduced, and genes encoding them
EP1571223A2 (fr) 2004-03-04 2005-09-07 Ajinomoto Co., Inc. Microorganisme producteur de L-cystéine, et procédé de préparation de L-cystéine
US20050221453A1 (en) 2004-03-31 2005-10-06 Ajinomoto Co., Inc L-cysteine producing microorganism and method for producing L-cysteine
US20090053778A1 (en) 2004-07-20 2009-02-26 France Telecom Microorganisms for producing sulfur-containing compounds
US20090226984A1 (en) 2008-03-06 2009-09-10 Gen Nonaka L-cysteine producing bacterium and a method for producing l-cysteine
EP2138585A1 (fr) 2008-03-06 2009-12-30 Ajinomoto Co., Inc. Bactérie productrice de L-cystéine et procédé de production de L-cystéine
DE102011075656A1 (de) 2011-05-11 2012-03-29 Wacker Chemie Ag Verfahren zur fermentativen Produktion von L-Cystin

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Title
BEALES, C.R.F.S.F.S., vol. 3, 2004, pages 1 - 20
F. C. NEIDHARDT; R. CURTISS III; J. L. INGRAHAM; E. C. C. LIN; K. B. LOW; B. MAGASANIK; W. S. REZNIKOFF; M. RILEY; M. SCHAECHTER;: "Escherichia coli and Salmonella: cellular and molecular biology", ASM PRESS
GAITONDE, M. K., BIOCHEM. J., vol. 104, 1967, pages 627 - 633
KREDICH, BIOSYNTHESIS OF CYSTEINE, 1996, pages 514 - 527
NAKAMORI ET AL., APPL. ENV. MICROBIOL., vol. 64, 1998, pages 1607 - 1611

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WO2013171098A3 (fr) 2014-01-09

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