Construction Of Engineering Pichia Pastoris Strains For High Glutathione Production

Glutathione (GSH), is comprised of L-glutamic acid, L-cysteine and glycine. GSH is the most abundant non-protein thiol compound widely distributed in living organisms and, predominantly, in eukaryotic cells. GSH plays pretty important rolls in the cell or the tissue. Recently, the mostly used method to produce GSH is fermentation with proper microorganisms, usually yeasts. Beyond traditional mutation breeding, gene modification is more and more extensively used.The yeast pichia pastoris has become the premier example of yeast species used for the production of recombinant proteins. Advantages of this yeast for expression include tightly regulated and efficient promoters and a strong tendency for respiratory growth as opposed to fermentative growth.In this study, an engineering pichia strain to produce high amount of GSH was constructed, in which the two GSH synthetases was highly expressed under the GAP promoter. Because of the accumulation of intracellular GSH, Gshlp is depressed by feedback inhibition, the consequence of which GSH production is decreased. Based on this, in this study the transporters of pichia pastoris was modified to increase the efflux of GSH, so that Gshlp was released from the feedback inhibition of GSH and the final GSH yield is improved.In this study an engineering strain JC312(arg4, sGSH1, sGSH2, opt1, sGEX1) was obtained based on the initial strain JC308(adel, arg4, his4, ura3), in which GSH synthetases sGSH1and sGSH2was highly expressed, and the GSH importer OPT1was knocked out, while the GSH exporter sGEXlwas over-expressed. With this newly constructed strain, the GSH production in shake-flask increased90%, and even2-fold increased in8L fed-batch fermentation.