Isolation Of Glutathione-producing Yeast And The Construction Of Recombinant Yeast With High Yield Glutathione

Glutathione, bio-synthesized from L-glutamic acid, L-cysteine and glycine in two consecutive steps byγ-glutamyl-cysteine synthase and glutathione synthase, is one of the major non-protein thiol compounds. GSH (the reduced form of glutathione) is widely distributed in nature and plays many important physiological roles in living cells.Fermentative production of GSH is one of the most promising methods for GSH production at present and the strains for GSH production are yeast species mostly. 98 strains of yeast producing GSH were isolated from natural enviriment. The yield of intracellular GSH of 2053# and ALJ036 was 49.7 mg/L and 38.7 mg/L respectively after 30 h of fermentation. In addition to general physiological and biochemical properties, the strains were identified by 18S rDNA sequence and systematic analysis. The results showed that 18S rDNA sequence of the strain 2053# had similarity of 99.75% with Candida Parapsilosis suggesting that the strain 2053# is one of subspecies of Candida Parapsilosis. 18S rDNA of the strain ALJ036 had similarity of 99 % with Saccharomyces cereviseae W303.The gene encodingγ-glutamyl-cysteine synthase (gsh 1) was cloned to the vector pGAPZA then was transformated into Pichia pastoris x-33 strain after linearized by AvrⅡ. Transformants resistant to zeocin were obtained and then identified by PCR analysis. The resulting transformants were cultivated in flasks.The activity ofγ-glutamyl-cysteine synthase for the transformant named Pichia pastoris x-33(pGAPZA-gsh 1) was 98.5 U/mg protein which was 2.99 times of the host strain. While the yield of GSH for the transformant Pichia pastoris x-33(pGAPZA-gsh 1) was 42.2 mg/L which was 1.60 times of the Pichia pastoris x-33.The experiment was used to optimize parameters of the fermentation condition of the recombinant strain. The maximal yield of GSH and the biomass (98.5 mg/L, 19.6 g/L ) could be obtained when the concentrations of glycerol, yeast extract, tryptone , KH2PO4 and cysteine were set at 30 g/L, 9 g/L, 40 g/L, 3 g/L and 0.36 g/L, respectively at the optimal cultivation natural pH, rotate speed 200 r/min, inoculation size 10 %. In this condition the production of GSH was higher 1.33 times than that of the prior to optimization. The yield of GSH(97.9 mg/L) and the yield of biomass(18.7 g/L) were achieved respectively in a 5 L fermenter using the optimized medium.