STRAIN OF MICROORGANISMS AND PROCESS FOR THE FERMENTATIVE PRODUCTION OF GAMMA-GLUTAMYLTYROSINE AND GAMMA-GLUTAMYLPHENYLALANINE

Abstract

A micro-organism strain and process for fermentative production of the same. The fermentative production of the substances gamma-glutamyltyrosine and gamma-glutamylphenylalanine by a micro-organism strain which is cultured in a fermentation medium. The fermentation medium is removed from the micro-organisms after the fermentation and gamma-glutamyltyrosine and gamma-glutamylphenylalanine are isolated from the fermentation medium. The microorganism strain contains at least one further gene encoding a glutamate-cysteine ligase and is deficient in glutathione synthetase. For the culture, the fermentation medium is admixed with L-tyrosine and L-phenylalanine.

Claims

1-7. (canceled)

8. A process for fermentative production of gamma-glutamyltyrosine and gamma-glutamylphenylalanine: comprising: providing a bacterial strain of the Enterobacteriaceae family that is cultured in a fermentation medium, wherein the fermentation medium is removed from the cells after the fermentation, and gamma-glutamyltyrosine and gamma-glutamylphenylalanine are isolated from the fermentation medium, wherein the bacterial strain a) either contains an open reading frame encoded in the wild-type genome that encodes glutamate-cysteine ligase and, in addition, at least one further open reading frame (ORF) encoding a glutamate-cysteine ligase, or b) contains no ORF encoded in the wild-type genome that encodes a glutamate-cysteine ligase, but at least one further ORF encoding a glutamate-cysteine ligase; wherein the bacterial strain is deficient in glutathione synthetase, i.e., no functional glutathione synthetase is produced by the corresponding gene; and wherein the fermentation medium contains at least 50 mg/L L-tyrosine and 50 mg/L L-phenylalanine.

9. The process of claim 8, wherein the bacterial strain is a strain of the species Escherichia coli.

10. The process of claim 8, wherein the at least one further open reading frame encoding a glutamate-cysteine ligase is SEQ ID NO. 1 or homologs of this sequence; and wherein the homologs are those ORFs that have a sequence identity greater than 70% in relation to SEQ ID NO. 1 and the protein expressed by this DNA sequence has a glutamate-cysteine ligase function.

11. The process of claim 8, wherein the bacterial strain contains at least one plasmid which contains at least one ORF encoding a glutamate-cysteine ligase under the control of a functional promoter.

12. The process of claim 8, wherein the gamma-glutamyltyrosine and gamma-glutamylphenylalanine are purified from the fermentation medium after the removal of the fermentation medium.

Description

EXAMPLES

[0096] The invention is described in more detail hereinbelow with reference to example embodiments, without being limited thereby.

[0097] In the examples, use was made of the E. coli strain W3110gshB/ptufB.sub.p-gshA.sup.ATG-cysE14-serA2040-orf306 as disclosed in US 2014/0342399 A. The strain name refers to the E. coli W3110 strain (ATCC 27325) with deletion of the gshB gene, the bacterial cells bearing the plasmid shown in US 2014/0342399 in FIG. 10 and comprising: [0098] the gshA gene with the start codon ATG, wherein the gene is under the control of the tufB promoter, [0099] the serA gene (encoding D-3-phosphoglycerate dehydrogenase), [0100] the cysE gene (encoding serine acetyltransferase) and [0101] ORF306 (encoding the cysteine/O-acetylserine exporter EamA).

Example 1: Shake-Flask Preculture of a -Glu-Tyr and -Glu-Phe Production Strain for Fermentation

[0102] 100 mL of sterile LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) were prepared with 5 g/L D(+)-glucose monohydrate and then admixed with tetracyclineHCl in a sterile 500 mL Erlenmeyer flask (final concentration of tetracyclineHCl in the shake flask: 0.01 g/L, sterile-filtered).

[0103] Using an inoculation loop, sufficient cells of the production strain W3110gshB/ptufB.sub.p-gshA.sup.ATG-cysE14-serA2040-orf306 were removed from the agar plate and transferred into the supplemented LB medium until at most a weak turbidity of the supplemented LB medium was discernible. This preculture was cultured for 5 h at 32 C. and 130 rpm on a CERTOMAT IS incubation shaker (Sartorius Stedium Biotech, Gttingen, Germany).

Example 2: Pre-Fermenter for the Production of -Glu-Tyr and -Glu-Phe

[0104] The pre-fermentation was carried out in a 2 L DASGIP fermenter from Eppendorf (Hamburg, Germany).

[0105] 100 mL of LB preculture from Example 1 were used for inoculation of the 900 mL of production medium, consisting of 30 g/L D(+)-glucose monohydrate, 0.03 g/L pyridoxineHCl,0.005 g/L thiamineHCl, 0.01 g/L tetracyclineHCl, 5 g/L (NH.sub.4).sub.2SO.sub.4, 0.5 g/L NaCl, 0.9 g/L L-isoleucine, 0.6 g/L D/L-methionine, 5 g/L corn steep solids (e.g., from Sigma-Alldrich), 0.225 g/L CaCl.sub.22 H.sub.2O, 0.6 g/L MgSO.sub.47 H.sub.2O, 0.15 g/L Na.sub.2MO.sub.42 H.sub.2O, 0.3 g/L H.sub.3BO.sub.3, 0.2 g/L CoCl.sub.26 H.sub.2O, 0.25 g/L CuSO.sub.45 H.sub.2O, 1.6 g/L MnCl.sub.24 H.sub.2O, 1.35 g/L ZnSO.sub.47 H.sub.2O, 0.075 g/L FeSO.sub.47 H.sub.2O, 1 g/L Na.sub.3 citrate2 H.sub.2O and 1.71 g/L KH.sub.2PO.sub.4. The media constituents were initially charged in a 2 L fermenter under sterile conditions and adjusted to pH=7.0 using sterile correction agents (25% ammonia & 6.8 N H.sub.3PO.sub.4). During culturing for 48 h, the temperature was kept constant at 32 C. and the pH was kept constant at 7.0 by the correction agent (25% ammonia). Struktol J673 (Schill+Seilacher, Hamburg) in a 1:10 dilution in sterile water was used as antifoam agent. The culture was aerated with sterile compressed air at 100 L/h and stirred at a stirrer speed of 450 rpm. After the oxygen saturation had fallen to a value of 50%, the speed was increased via the control unit of the fermenter up to a value of 1450 rpm in order to obtain 30% oxygen saturation. The oxygen saturation was determined using a pO.sub.2 probe which was calibrated to 100% saturation at 450 rpm. Once the glucose content in the fermenter had dropped to approx. 1-2 g/L, a 56% (w/v) D(+)-glucose monohydrate solution was metered in. The feeding of glucose was effected at a flow rate of 10-15 mL/h, with the glucose concentration in the fermenter being kept constant between 0.1 g/L and 10 g/L. The determination of glucose was carried out using the YSI 7100 MBS analyzer (YSI, Yellow Springs, Ohio, USA).

Example 3: Fermentative Production of -Glu-Tyr and -Glu-Phe

[0106] The production of -Glu-Tyr and -Glu-Phe in a 2 L DASGIP fermenter from Eppendorf (Hamburg, Germany) was carried out in two independent batches.

[0107] The following description of the experimental procedure applies to each of the two batches 1 and 2:

[0108] 100 mL of the preculture from Example 2 were used for inoculation of the 900 mL production medium, consisting of 5 g/L D(+)-glucose monohydrate, 0.03 g/L pyridoxineHCl, 0.005 g/L thiamineHCl, 0.01 g/L tetracyclineHCl, 5 g/L (NH.sub.4).sub.2SO.sub.4, 0.5 g/L NaCl, 0.9 g/L L- isoleucine, 0.6 g/L D/L-methionine, 30 g/L corn steep solids, 0.225 g/L CaCl.sub.22 H.sub.2O, 0.6 g/L MgSO.sub.47 H.sub.2O, 0.15 g/L Na.sub.2MO.sub.42 H.sub.2O, 0.3 g/L H.sub.3BO.sub.3, 0.2 g/L CoCl.sub.26 H.sub.2O, 0.25 g/L CuSO.sub.45 H.sub.2O, 1.6 g/L MnCl.sub.24 H.sub.2O, 1.35 g/L ZnSO.sub.47 H.sub.2O, 0.075 g/L FeSO.sub.47 H.sub.2O, 1 g/L Na.sub.3 citrate2 H.sub.2O and 1.71 g/L KH.sub.2PO.sub.4. The media constituents were initially charged in a 2 L fermenter under sterile conditions and adjusted to pH=6.9 using sterile correction agents (25% ammonia & 6.8 N H.sub.3PO.sub.4). During culturing for 48 h, the temperature was kept constant at 32 C. and the pH was kept constant at 7.0 by the correction agent (25% ammonia). Struktol J673 (Schill+Seilacher, Hamburg) in a 1:10 dilution in sterile water was used as antifoam agent. The culture was aerated with sterile compressed air at 100 L/h and stirred at a stirrer speed of 450 rpm. After the oxygen saturation had fallen to a value of 50%, the speed was increased via the control unit of the fermenter up to a value of 1450 rpm in order to obtain 30% oxygen saturation. The oxygen saturation was determined using a pO.sub.2 probe which was calibrated to 100% saturation at 450 rpm. Once the glucose content in the fermenter had dropped to approx. 1-2 g/L, a 56% (w/v) glucose solution was metered in. The feeding of glucose was effected at a flow rate of 10-15 mL/h, with the glucose concentration in the fermenter being kept constant between 0.1 g/L and 10 g/L. The determination of glucose was carried out using the YSI 7100 MBS analyzer (YSI, Yellow Springs, Ohio, USA). For efficient production of the products -Glu-Tyr and -Glu-Phe, L-Tyr and L-Phe were metered in via the glucose feed. The supplementary feeding was effected 2 h after the start of culturing at a rate of 10-15 mL/h, with the feed solution having a concentration of 6 g/L L-Tyr and 6 g/L L-Phe.

[0109] In addition to the feeding of glucose, potassium glutamate and ammonium thiosulfate [(NH4).sub.2S.sub.2O.sub.3] were metered in via a combined feed. The supplementary feeding was effected 2 h after the start of culturing at a rate of 4-4.5 mL/h, with the concentration of ammonium thiosulfate and the concentration of potassium glutamate in the feed solution being 73 g/L and 21 g/L, respectively.

[0110] The results of the -Glu-Tyr and -Glu-Phe fermentations using the production strain W3110gshB/ptufB.sub.p-gshA.sup.ATG-cysE14-serA2040-orf306 are combined in Tables 1 (1st batch) and 2 (2nd batch).

TABLE-US-00001 TABLE 1 Content of -Glu-Tyr and -Glu-Phe in the culture supernatant of the 1st batch after fermentation of the strain W3110gshB/ptufB.sub.p-gshA.sup.ATG-cysE14-serA2040-orf306 Culturing time Product 24 h 48 h -Glu-Tyr [mg/L] 29 32 -Glu-Phe [mg/L] 190 610

TABLE-US-00002 TABLE 2 Content of -Glu-Tyr and -Glu-Phe in the culture supernatant of the 2nd batch after fermentation of the strain W3110gshB/ptufB.sub.p-gshA.sup.ATG-cysE14-serA2040-orf306 Culturing time Product 24 h 48 h -Glu-Tyr [mg/L] 23 240 -Glu-Phe [mg/L] 22 118

Detection of -Glu-Tyr and -Glu-Phe

[0111] The determination of -Glu-Tyr and -Glu-Phe was carried out by means of an HPLC-MS method. The analysis was carried out on an Ultimate 3000 HPLC system from Thermo Fisher Scientific (Dreieich, Germany) coupled to an Amazon SL mass spectrometer from Bruker Daltonik (Bremen, Germany). The separation column used was a Nucleoshell Bluebird RP 18 column, 2.7 m (100 mm3.0 mm) from Macherey Nagel (Dren, Germany). The samples were subjected to gradient elution (mobile phase A: water containing 0.1% (v/v) acetic acid, mobile phase B: methanol) at 30 C. and a flow rate of 0.7 mL/min. The detection by mass spectrometry was effected in ESI positive mode on the basis of the [M+H].sup.+ ions (m/z for -Glu-Tyr: 311, m/z for -Glu-Phe: 295).

Example 5: Production of a Kokumi Product

[0112] To produce a Kokumi product, 1.5 L of a fermentation as described in Example 3 were worked up. After centrifugation to remove the cells, the supernatant was sterile-filtered (Thermo Scientific Nalgene Rapid-Flow 90 mm 0.2 Filter Unit, Dreieich, Germany) and then dried in a drying cabinet (Binder ED115 E2, Tuttlingen, Germany). For homogenization, the dried raw product was homogenized in a mill (IKA All basic, Staufen, Germany).