WO2013171098A2

WO2013171098A2 - Method for the fermentative production of l-cystein and derivates of said amino acid

Info

Publication number: WO2013171098A2
Application number: PCT/EP2013/059477
Authority: WO
Inventors: Tobias Dassler; Marcel THÖN
Original assignee: Wacker Chemie Ag
Priority date: 2012-05-18
Filing date: 2013-05-07
Publication date: 2013-11-21
Also published as: DE102012208359A1; WO2013171098A3

Abstract

The invention relates to a method for producing L-cysteine and the L-cysteine and thiazolidine derivatives thereof by fermenting a micro-organism strain in a fermentation medium to which benzoic acid is added in a concentration range of 1-500 mg/L.

Description

Process for the fermentative production of L-cysteine and derivatives of these Aminos

1) Ls iHI over £ 11 ZL per t 6 n »int ^" Vs £ ciJfhtTGT- s ^" Li! Tf G tnGX tt. HL IP ocLmli. tion of L-cysteine and the derivatives of this amino acid L-cystine and 2-methyl-thiazolidine-2, 4-dicarboxylic acid.

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 (thiazolidine) is the condensation product of L-cysteine and pyruvate, which is formed in a purely chemical reaction (US5972663A), This reaction is also reversible. Thus, 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.

It is used, for example, as a food additive {especially in the baking industry), as a starting material in cosmetics, and as a starting material for the preparation of pharmaceuticals (in particular N-acetyl-cysteine and S-carboxyethyl-cysteine).

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. In addition, L-cysteine serves as a precursor for the biosynthesis of coenzyme A. The biosynthesis of L-cysteine has been reported in bacteria, especially in

Enterobacteria, studied in detail and described in detail in Kredich (1996, Biosynthesis of cysteines, p. 514-527, in Neidhardt FC, Curtiss III, JL Ingraham, ECC Lin, K. B. Low, B. Magasanik, WS Reznikoff, M. Riley, M. Schaechter, and HE ümbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed ASM Press, Washington, D. C. } describe.

In addition to the classical extraction of L-cysteine by extraction from keratin-containing material such as hair, bristles, horns, hooves and feathers or biotransformation by enzymatic conversion of precursors, a process for the fermentative production of L-cysteine was developed several years ago. The state of the art regarding the fermentative recovery of L-cysteine with microorganisms is e.g. in

US6218168B1, US5972663A, US20040038352A1, CA2386539A1,

US20090053778A1 and US20090226984A1 described. As bacterial host organisms u.a. Strains of the genus Corynebacterium as well as representatives of the family of Enterofoacte- riaceae, such as e.g. Escherichia coli or Pantoea ananatis used.

In addition to the traditional approach of mutating and selecting improved L-cysteine producers, targeted genetic modifications were also made to the strains to achieve effective L-cysteine overproduction.

Thus, introduction of a cysE allele encoding a secretin O-acetyl transferase with reduced feedback inhibition by L-cysteine resulted in an increase in cysteine production {US6218168B1; Nakamori et al. , 1998, Ap l. Env. Microbiol. 64: 1607-1611). A feedback-resistant CysE enzyme largely decouples the formation of O-acetyl-L-serine, the direct precursor of L-cysteine, from the cell's L-cysteine level.

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.

In addition, it is known that the 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-β-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}.

The export of L-cysteine from the cell to the fermentation medium has several advantages:

1) 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:

(1) L-cysteine (intracellular) ^ L-cysteine (medium)

2) The L-cysteine secreted into the medium is oxidized to the disulphide L-cystine in the presence of oxygen which is introduced into the medium during the cultivation (US Pat. No. 5,972,663A):

(2) 2 L-cysteine + 1/2 0 ₂ i * L-cystine + H ₂ 0

Since the solubility of L-cystine in aqueous solution at a neutral pH v. A. is very low compared to L-cysteine, the disulfide precipitates even at a low concentration and forms a white precipitate:

(3) 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.

3) The technical effort for the purification of the product is significantly lower if the amino acid can be obtained directly from the fermentation medium, as if the product accumulates intracellularly and must first be cell disrupted.

In the context of this invention, the term "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.

In addition to the genetic modification of the L ~ cysteine production strain, the optimization of the fermentation process, i. the way of cultivating the cells, an important role in the development of an efficient production process.

In this case, different cultivation parameters, such. For example, the type and dosage of the carbon and energy source, the temperature, the supply of oxygen (DE102011075656A1), the pH value and the composition of the culture medium, the product yield and / or the product spectrum in the fermentative production of L-cysteine influence. Due to constantly increasing raw material and energy costs, there is a continuing need to increase the product yield in the L-cysteine production, in order to improve the efficiency of the process in this way. 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.

In a screening of various chemical substances with respect to their effect on the production process, it was surprisingly found that the addition of benzoic acid in a concentration range of 1-500 mg / L can increase the overall cysteine yield. This finding is very surprising since benzoic acid is also used as a food preservative because of its antimicrobial effect (Beales, 2004, C.R.F.S.F.S. 3: 1-20) because it can inhibit the growth of microorganisms.

A positive effect of benzoic acid on the amino acid production of microorganisms has not previously been described.

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. Alternatively, benzoic acid can also be metered into the medium only during the fermentation. As 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,

CA2386539A1, US20090053778 or EP2138585.

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.

Of these microorganism strains, in turn, 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. Particularly preferred 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

(US2005009162A2) and in which at least one L-cysteine degrading enzyme is so far attenuated that in the cell only a maximum of 50% of this enzyme activity compared to a wild-type cell is present.

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

WO2004113373), bcr (see US2005221453) and emrAB (see

US2005221453) of E. coli or the corresponding homologous gene from another microorganism. By 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 β-lyase {MalY, Mete).

Particularly preferred are 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. Especially preferred are strains in which at least one of these enzymes is completely inactivated.

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. When ammonia is used as a correction agent for pH stabilization, this N source is regularly replenished during the fermentation.

As further media additives, salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, iron and trace (i.e., in μΜ concentrations) salts of the elements molybdenum, boron, cobalt, manganese, zinc and nickel may be added.

Furthermore, organic acids (eg acetate, citrate), amino acids (eg isoleucine) and vitamins (eg Bl, B6) can be added to the medium. As complex nutrient sources, for example, yeast extract, corn steep liquor, soybean meal or malt extract can be used.

The incubation temperature for mesophilic microorganisms, e.g. E. coli or P, ananatis 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.

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.

After fermentation, 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.

For further purification of the crude product, the following steps can be carried out:

- Dissolve the crude product with a mineral acid

- Clarify the crude product solution by centrifugation or filtration

- Decolorization of the solution

- Precipitation crystallization The reduction of 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.

Example 1: Production of cysteine production strains

Wild type strains E. coli W3110 (ATCC 27325} and P.ananatis (ATCC 11530} each with the plasmid

pACYC184 / cysEX-GAPDH-ORF306 (disclosed in Example 2 of

US5972663A) by electroporation as described in US5972663A. 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

(ORF306}, whose expression is controlled by the constitutive GAPDH promoter.

Selection for plasmid-carrying cells was on LB agar plates containing 15 mg / 1 tetracycline

After another plasmid isolation using the QIAprep Spin Plasmid Kit (Qiagen GmbH) and a restriction analysis, 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.

Example 2: Cysteine Production in the Presence of Benzoic Acid {Shake Flask)

Preculture:

3 ml of LB-edium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l NaCl, which additionally contained 15 mg / l tetracycline) with the respective strain (E. coli) were used as preculture for the culture in the shake flask W3110 pACYC184 / cysEX-GAPDH-ORF306 or P.ananatis pACYC184 / cysEX ~ GAPDH ORF306) and incubated at 30 ° C and 135 rpm for 16 h in a shaker. Main culture:

Subsequently, part of the respective preculture was transferred to a 300 ml Erlenmeyer coulter (with baffle) with 30 ml of SM1 medium

(12 g / 1 K ₂ HP0 _4, 3 g / 1 KH ₂ P0 _4, 5 g / 1 _{NH4} ₂ S0 _4, 0.3 g / 1 MgS0 ₄ x 7 H ₂ 0, 0.015 g / 1 CaCl ₂ × 2 H ₂ O, 0.002 g / 1 FeS0 ₄ × 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 B0 ₃ , 0.7 g / 1 CoCl ₂ x 6 H ₂ 0, 0.25 g / 1 CuS0 ₄ x 5 H ₂ 0, 1.6 g / 1 MnCl ₂ × 4 H ₂ O, 0.3 g / 1 ZnS0 ₄ × 7 H ₂ 0}, which contains 15 g / L glucose, 5 mg / L vitamin Bi and 15 mg / L Tetracycline was supplemented.

Starting from an initial optical density at 600 nm

(ODgoo) of 0.025, the entire 30 ml batch was incubated for a further 24 hours at 30 ° C and 135 rpm.

In each case 1 to 1000 mg / L of benzoic acid were additionally added to the medium of the corresponding comparison cultures.

After 24 h, samples were taken and the total cysteine content in the culture supernatant was determined (see Table 1). In contrast to the fermenter, precipitation of L-cystine does not occur in the shake flask due to the acidification of the medium (pH drop to approx. PH 5-5.5) and the overall lower cysteine yield.

The production of L-cysteine was monitored colorimetrically by the assay of Gaitonde (Gaitonde, MK (1967), Biochem J. 104, 627- 633), bearing in mind that the test under the strongly acidic reaction conditions does not distinguish between L-cysteine. Cysteine and the condensation product of L-cysteine and pyruvate described in EP 0885962 A1, which discriminates 2-methyl-thiazolidine-2,4-dicarboxylic acid (thiazolidine) L-cystine, which is formed by oxidation of two L-cysteine molecules, is also detected by reduction with dithiothreitol in dilute solution at pH 8.0 as L-cysteine. Table 1: Total cysteine content in the culture supernatant after 24 h

: Sum of the L-cysteine, L-cystine and thiazolidine dissolved in the supernatant {the average value incl. Standard deviation of four independent cultures is indicated in each case)

: each slight growth inhibition

^* : each strong growth inhibition

^** : each very strong growth inhibition

Example 3: Cysteine Production in the Presence of Benzoic Acid {Fermenter)

Preculture 1:

20 ml of LB medium containing 15 mg / l tetracycline were incubated in an Erlenmeyer flask (100 ml) with the respective strain {E. coli W3110 pACYC184 / cysEX-GAPDH-ORF306 or P.ananatis pACYC184 / cysEX-GAPDH-ORF306) and incubated for seven hours on a shaker {150 rpm, 30 ° C).

Preculture 2:

Subsequently, 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 / 1 ZnS0 ₄ × 7 H ₂ O) supplemented with 5 g / 1 glucose, 5 mg / 1 vitamin B1 and 15 mg / 1 tetracycline. The cultures were shaken in Erlenmeyer flasks (11) at 30 ° C. for 17 h at 150 rpm After this incubation, the optical density at 600 nm (OD _S00 ) was between 3 and 5,

Main culture:

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 was also 1, 2.5, 50, 400, 500 or 1000 mg / L benzoic acid added.

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. For inoculation, 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

Springs, Ohio, USA).

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). For this purpose, 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.

Table 2: Content of L-cysteine and L-cysteine derivatives in the culture broth after 48 h

¹ : sum of the dissolved in the supernatant L-cysteine, L-cystine and

thiazolidine

2: L-cystine in the precipitate

3: total cysteine {sum of supernatant and precipitate)

*: each significant growth inhibition

Claims

claims

1. A process for the preparation of L-cysteine and its derivatives L-cystine and thiazolidine by fermentation of a microorganism strain in a fermentation medium which is characterized in that the fermentation medium benzoic acid or a salt of benzoic acid in a concentration range of 1-500 mg / L is added ,

2. The method according to claim 1, characterized in that the benzoic acid or the salt of benzoic acid in a concentration range of 2.5-400 mg / L are added.

3. The method according to claim 1 or 2, characterized in that the microorganism strain is selected from the family of Enterobacteriaceae, preferably from the genera Escherichia or Pantoea, particularly preferably from the species E, coli or P. ananatis.

4. The method according to any one of claims 1 to 3, characterized in that the microorganism strain either have a modified 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 , or by overexpression of an efflux gene having a cysteine export from the cell increased by at least a factor of 2 compared to a wild-type cell.

5. The method according to claim 4, characterized in that the microorganism strain 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 a through Overexpressing an efflux gene by at least a factor of 2 increased cysteine export from the cell compared to a wild-type cell.

6. The method according to claim 4 or 5, characterized in that the microorganism strain additionally an altered

3 phosphoglycerate dehydrogenase with a reduced by at least a factor of 2 compared to the corresponding wild-type enzyme feedback inhibition by L-serine and in which at least one L ™ cysteine-degrading enzyme is so far attenuated that in the cell only a maximum of 50 % of this enzyme activity is present compared to a wild-type cell.

7. The method according to any one of claims 1 to 6, characterized in that the culture, a carbon source is metered so that the carbon source in the fermenter during the production phase does not exceed a concentration of 10 g / 1.