WO2015036301A1 - Method for the fermentative production of gamma glutamylcysteine and derivatives of said dipeptide - Google Patents

Abstract

The invention relates to a method for the fermentative production of γ-glutamylcysteine and its derivatives bis-γ-glutamylcystine and γ-glutamylcystine, in which a prokaryotic microorganism strain suitable for producing γ-glutamylcystein is cultivated in a fermentation medium, characterised in that thiosulfuric acid is added to the fermentation medium.

Description

 Process for the fermentative production of gamma-glutamyl cysteine and derivatives of this dipeptide

The invention relates to a process for the fermentative production of γ-glutamylcysteine (yGC) and the derivatives of this dipeptide, γ-glutamylcystine and bis-γ-glutamylcystine.

The state of the art for the fermentative preparation of these compounds is described in detail in DE102013209274.

As prokaryotic producers of γ-glutamylcysteine, various E. coli strains are described in US20100203592A1, all of which have increased gshA expression. In addition, these strains have normal or even increased glutathione synthetase activity compared to the corresponding parent strain. However, the yields with these strains are a maximum of 262 mg / L.

As further prokaryotic producers of γ-glutamylcysteine, DE102013209274 describes various E. coli strains which all have a reduced glutathione synthetase activity and additionally have a higher γ-glutamylcysteine synthetase activity than a comparable strain with a likewise reduced glutathione synthetase - Activity. This application further describes that by fermentation of the E. coli strains disclosed therein

γ-Glutamylcystein titer of 5.7 g / 1 up to 21.8 g / 1 can be achieved (see Tables 3 and 4 in DE102013209274). Due to continuously increasing raw material and energy costs, it is desirable to continuously increase the product yield of a microbial process in order to improve the economic efficiency of the corresponding process.

In addition to the genetic modification of microorganisms, which are used as a basis for the production of a particular natural product, the optimization of the corresponding fermentation process, ie the manner of cultivating The cells play an important role in the development and improvement of a production process.

The object of the present invention is therefore to provide an improved process for the fermentative production of γ-glutamylcysteine and its derivatives bis-γ-glutamylcystine and γ-glutamylcystine.

This object is achieved by adding to the fermentation medium, in which a prokaryotic microorganism strain suitable for γ-glutamylcysteine production is cultured, thio-sulfuric acid or a salt of thiosulphuric acid individually or as a mixture. The use of a salt of thiosulphuric acid is preferred.

The salt of the thiosulphuric acid is preferably ammonium thiosulphate or sodium thiosulphate. Particularly preferred is the use in the form of ammonium thiosulfate. Preferably, thiosulphuric acid or one of its salts is metered into the medium continuously during the fermentation at a rate of 8 to 17 mmol / l per hour. Particularly preferably, the continuous addition of thiosulphuric acid or one of its salts takes place at a dosage rate of 10 to 15 mmol / l per hour.

All prokaryotic microorganism strains which can be cultivated by fermentation and have the biosynthesis pathway for γ-glutamylcysteine are suitable for the process according to the invention, and the γ-glutamylcysteine and its derivatives release into the medium bis-γ-glutamylcystine and γ-glutamylcystine. Examples of such strains are disclosed in US20100203592A1 and DE102013209274, wherein the strains mentioned in DE102013209274 are preferably used.

Preferably, the cells are removed from the fermentation medium and the desired products purified from the culture medium. The cultivation (fermentation) of the prokaryotic microorganism strains preferably takes place on an industrial scale according to customary fermentation processes known to the person skilled in the art in a bioreactor (fermenter) having the feature that is essential to the invention that the fermentation medium as described

 Thiosulphuric acid or a salt of thiosulphuric acid are added.

The fermentation preferably takes place in a conventional bioreactor, for example a stirred tank, a bubble column fermenter or an airlift fermenter. Particularly preferred is a stirred tank fermentor. By industrial scale is to be understood a fermenter size of at least 2 1. Preference is given to fermenters having a volume of more than 5 l, more preferably fermenters having a volume of> 50 l.

The cultivation of the cells for γ-glutamylcysteine production is carried out under aerobic growth conditions, the oxygen content during fermentation being set 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. Particular preference is given in the process according to the invention as the carbon source to glucose, fructose, lactose, glycerol or mixtures which contain two or more of these compounds. The carbon source of the culture is preferably metered in such that the content of the carbon source in the fermenter does not exceed 10 g / l during the production phase. Preferred is a maximum concentration of 2 g / l.

The nitrogen source used in the process according to the invention is preferably ammonia, ammonium salts or protein hydrolyzates. When using ammonia as a correction agent for pH stabilization, this nitrogen source is regularly replenished during the fermentation. As further media additives salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, iron and in traces (ie in μΜ concentrations) salts of the elements molybdenum, boron, cobalt, manganese, zinc and nickel can be added.

Further, organic acids (e.g., acetate, citrate), amino acids (e.g., L-glutamic acid, L-cysteine) and vitamins (e.g., Bl, B6) may be added to the medium.

 L-glutamic acid can either be used directly as acid or in the form of one of its salts, e.g. Potassium or sodium glutamate, individually or as a mixture used. The use in the form of potassium glutamate is preferred.

As complex nutrient sources, e.g. Yeast extract, corn steep liquor, soy flour or malt extract are used.

The incubation temperature for mesophilic microorganisms, e.g. E. coli is preferably 15-45 ° C, more preferably 30-37 ° C.

The production phase of the fermentation process according to the invention begins with the time from which γ-glutamyl cysteine, bis-γ-glutamyl cystine or γ-glutamyl cystine can be detected in the culture broth for the first time and lasts until the end of the cultivation. The detection of γ-glutamylcysteine, bis-γ-glutamylcystine or γ-glutamylcystine in the culture broth takes place as described in Ex. Typically, this phase begins about 8-12 hours after inoculation of the production fermenter (main fermenter) with a preculture.

Prokaryotic microorganisms that are fermented by the method described, γ-glutamylcysteine secrete in a batch or fedbatch process after a growth phase in a period of at least 48 h hours and the compounds derived therefrom γ-glutamylcystin and bis-y-glutamylcystin with high Efficiency in the fermentation medium. The following examples serve to further illustrate the invention.

Example 1: yGC production (fermentation)

A. Preculture 1 (shake flask):

 100 ml of LB medium containing 15 mg / 1 tetracycline were incubated in a 1 L Erlenmeyer flask with the E. coli strains

W3110AgshB / ptufB _p -gshA ^ATG and W3110AgshB / ptufB _p -gshA ^ATG- cysE14 -serA2040-orf306 from one agar culture and seven

Hours on a shaker at 130 rpm and 32 ° C incubated. Generation of strains W3110AgshB / ptufB _p -gshA ^ATG and

W3110AgshB / ptufB _p -gshA ^ATG -cysE14-serA2040-orf306 is in

DE102013209274 (see DE10201320927; Examples 1 to 7),

B. Preculture 2 (Prefermenter):

A portion of the respective preculture 1 was inoculated into a fermenter filled with fermentation medium type Sixfors (Infors GmbH, Bottmingen, CH), so initially an optical density of about 0.01 in the fermenter was present (measured at 600 nm). The fermenter used had a total volume of 1 1 and an initial working volume of 0.7 1. The fermentation medium contained the following components: 3 g / 1 (NH ₄ ) ₂ S0 ₄ , 1.7 g / 1 KH ₂ P0, 0.25 g / 1 NaCl, 0.6 g / 1 MgSO ₄ × 7 H ₂ O, 0.03 g / 1 CaCl ₂ × 2 H ₂ O,

0.15 g / 1 FeS0 × 7 H ₂ O, 1 g / 1 Na ₃ citrate × 2 H ₂ 0, 5 g / 1 Cornsteep Dry (CSD) and 3 ml / 1 trace element solution (consisting of 2.5 g / 1 H ₃ BO ₃ , 0.7 g / 1 CoCl ₂ × 6 H ₂ O, 0.25 g / 1 CuS0 ₄ × 5 H ₂ O, 1.6 g / 1 MnCl ₂ × 4 H ₂ 0, 0.3 g / 1 ZnSO ₄ × 7 H ₂ O, 0.15 g / 1 Na ₂ Mo0 ₄ × 2 H ₂ O). After sterilization of this basal medium, the following constituents were added under sterile conditions: 40 g / 1 glucose, 0.018 g / 1 vitamin B1, 0.09 g / 1 vitamin B6 and 15 mg / 1 tetracycline. The pH in the pre-fermenter was adjusted to 7.0 by addition of a 25% NH ₄ OH solution. During the fermentation, the pH was maintained at 7.0 by automatic correction with a 25% NH 4 OH solution. The cultures were initially stirred at 400 rpm and at a gassing rate of 0.7 volume per volume per minute (vvm) of a Sterile filters degassed compressed air. 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 1.4 vvm) and then the stirring speed was increased continuously to a maximum of 1,200 rpm.

 The fermentation was carried out at a temperature of 32 ° C until an optical density of 30 to 40 was reached (measured at 600 nm). This was usually the case after about 17 h.

C. Main crop (production fermenter, main fermenter):

The fermentations were carried out in fermenters of the BIOSTAT B-DCU type from Sartorius Stedim GmbH (Göttingen, Germany). It was a culture vessel with 2 1 total volume used at an initial working volume of 1 1. The fermentation medium contains the following components: 3 g / 1 (NH ₄ ) ₂ S0 ₄ , 1.7 g / 1 KH ₂ P0 ₄ , 0.25 g / 1 NaCl, 0.6 g / 1 MgS0 ₄ x 7 H ₂ 0 , 0.03 g / l CaCl ₂ × 2 H ₂ O, 0.15 g / 1 FeS0 ₄ × 7 H ₂ O, 1 g / 1 Na ₃ citrate × 2 H ₂ O, 15 g / 1 Cornsteep Dry (CSD ) and 3 ml / 1 trace element solution (consisting of 2.5 g / 1 H ₃ BO ₃ , 0.7 g / 1 CoCl ₂ × 6 H ₂ O, 0.25 g / 1 CuS0 ₄ × 5 H ₂ 0, 1 , 6 g / 1 MnCl ₂ × 4 H ₂ O, 0.3 g / 1 ZnS0 ₄ × 7 H ₂ O, 0.15 g / 1 Na ₂ Mo0 ₄ × 2 H ₂ 0). After sterilization of this basal medium, the following constituents were added under sterile conditions: 10 g / 1 glucose, 0.018 g / 1 vitamin B1, 0.09 g / 1 vitamin B6 and 15 mg / 1 tetracycline.

To inoculate the main culture, 100 ml of preculture 2 were transferred to the fermenter vessel. The pH in the fermenter was initially adjusted to 7.0 by addition of a 25% NH ₄ OH solution and maintained at this value by automatic correction with 25% NH ₄ OH. 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 Service. The target value for the 0 ₂ saturation during the fermentation was set to 50%. After lowering the 0 ₂ saturation below the nominal value, a regulation cascade was started to restore the 0 ₂ saturation to the target value. The gas supply was initially increased continuously (to a maximum of 5 vvm) and then the stirring speed was increased continuously to a maximum of 1,500 rpm.

The fermentations were carried out at a temperature of 32 ° C. As soon as the glucose content in the fermenter had dropped from initially 10 g / l to approximately 2 g / l, a continuous addition of a 60% 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 YSI glucose analyzer (Yellow Springs, Ohio, USA).

The respective additional sulfur source became the main culture, starting from corresponding stock solutions 2.0 g SO ₄ × 7 H ₂ O (comparative example), 3.2 M (NH ₄ ) ₂ SO (comparative example), 2.0 M Na ₂ S ₂ O ₃ x 5 H ₂ 0 (example of the invention) or 1.5 M

(NH ₄ ) ₂ S ₂ O ₃ (example according to the invention), after 5 h fermentation time at a rate of 8-17 mmol / l per hour (average = 11 mmol / l per hour) added to the end of fermentation.

After a fermentation time of 16 h, the main culture was additionally supplemented with glutamate in the form of a sterile 2.3 M potassium glutamate stock solution at a rate of 7.4 mmol / l per hour until the end of fermentation.

 The fermentation time was 72 hours. After 24 h, 48 h and 72 h, samples were taken and, after reduction with dithiothereitol (DTT), the proportion of "total y-glutamylcysteine" in the culture supernatant was determined by means of HPLC analysis (procedure see Example 2).

The better yields of γ-glutamylcysteine by feeding the suitable for γ-glutamylcysteine production E. coli strains with thiosulfate (Na ₂ S ₂ 0 ₃ or (NH ₄ ) ₂ S ₂ 0 ₃ ) compared to sulfate (MgS0 ₄ or (NH ₄ ) ₂ S0 ₄ ) are summarized in Table 1 (for strain W3110ΔgshB / ptufB _p -gshA ^ATG ) and Table 2 (for strain W3110ΔgshB / ptufB _p -gshA ^AG -cysE14-serA2040-orf306). Example 2: Analysis of γ-glutamylcysteine and the derivatives

The term "total y-glutamylcysteine" is taken to mean γ-glutamylcysteine and the oxidation products formed therefrom bis-γ-glutamylcystine and γ-glutamylcystine, which are formed during the fermentation and accumulate in the culture supernatant.

 The levels of "total y-glutamylcysteine" in the fermentation supernatant were determined by HPLC analysis after the samples were completely reduced by dithiothreitol (DTT).

A. Pretreatment of the samples

 For the fermenter samples to be measured, the microorganisms were first separated from the fermentation broth by a centrifugation step and subsequent sterile filtration.

 For the exact determination of the "total y-glutamylcysteine concentration", the γ-glutamylcysteine derivatives in the sample to be measured were reduced for 1 hour at room temperature (22 ° C.) with a molar excess of dithiothreitol (DTT) DTT was only complete at neutral to alkaline pH, the pH of the sample was adjusted to a pH of> 7.0, if necessary, with concentrated potassium hydroxide (KOH).

B. HPLC analysis

 After preparation of appropriate dilutions with demineralized water, the HPLC analysis was carried out with the column Synergi 4μπι Hydro-RP 250 x 4.6 mm (Phenomenex Ltd., Aschaffenburg, D) at

20 ° C. The eluent used was 0.5% (v / v) phosphoric acid (A) or acetonitrile (B).

 For the separation of γ-glutamylcysteine from a cell-free and reduced substance mixture, the following method was used for HPLC.

 • Flow rate = 0.5 ml / min

 • Detection at 200 nm with diode array detector (DAD)

• The separation is isocratic at 100% A. By a Rinse at 100% B, the column is cleaned after each run.

The reference substance used to determine the retention time of γ-glutamylcysteine and the final concentration in the samples to be measured was reduced γ-glutamylcysteine from Sigma Aldrich GmbH (Steinheim, D). The concentration of "total γ-glutamylcysteine" was calculated on the basis of the peak areas in the chromatogram (see FIG. 1).

Table 1: Effect of sulfur source on γ-glutamylcysteine production with strain W3110AgshB / ptufB _p -gshA ^ATG . Content of "total y-glutamylcysteine" (yGC) after neutralization of the fermenter broth and subsequent reduction of the oxidation products with dithiothreitol.

 (V) Comparative Example, (E) Example According to the Invention

Table 2: Influence of sulfur source on γ-glutamylcysteine production with strain W3110AgshB / ptufB _p -gshA ^AG- cysE14-serA2040-orf306. Content of "total y-glutamylcysteine" (yGC) after neutralization of the fermenter broth and subsequent reduction of the oxidation products with dithiothreitol.

 (V) Comparative Example, (E) Example According to the Invention

Claims

 claims

A process for the fermentative production of γ-glutamylcysteine and its derivatives bis-γ-glutamylcystine and γ-glutamylcystin in which a microorganism strain suitable for γ-glutamylcysteine production is cultivated in a fermentation medium, characterized in that the fermentation medium comprises thiosulphuric acid or a salt of Thiosulphuric be added individually or as a mixture.

A method according to claim 1, characterized in that the fermentation medium, a salt of thiosulfuric acid is added.

A method according to claim 2, characterized in that is added to the fermentation medium as the salt of thiosulfuric ammonium thiosulfate or sodium thiosulfate.

A method according to claim 3, characterized in that ammonium thiosulfate is added to the fermentation medium as the salt of thiosulfuric acid.

A method according to claim 1 to 4, characterized in that thiosulphuric acid or a salt of thiosulphuric acid are metered into the medium during the fermentation continuously at a rate of 7 to 18 mmol / l per hour.

A method according to claim 5, characterized in that thiosulphuric acid or a salt of thiosulphuric acid during the fermentation are continuously metered into the medium at a rate of 10 to 15 mmol / l per hour.

Method according to one of claims 1 to 6, characterized in that the cells of the microorganism strain are removed from the fermentation medium and the desired products are purified from the culture medium.