Effects Of Various Glutathione Synthesases On The Glutathione Production By Engineered Pichia Pastoris

Glutathione (γ-glutamyl-cysteinyl-glycine) (GSH) is the predominant low molecular weight peptide thiol present in living organisms. It plays a pivotal role in many metabolic processes and has a number of diverse applications. To date, GSH is mainly produced via fermentation. Yeasts, especially Saccharomyces cerevisiae and Candida utilis, are the most commonly used microorganisms in GSH production in an industrial scale. Most recently, recombinant Pichia pastoris has shown great potential in GSH production due to its easiness to achieve high density culture at low-cost media.The GSH producing recombinant P. pastoris strains are generated by over-expressing glutathione synthesis enzymes, and the GSH yield is mainly depende- nton the catalytic characteristics of these enzymes. In previous studies,γ-GCS and GS derived from S.cerevisiae S288c were chosen to be expressed in P. pastoris. However, S.cerevisiae S288c did not accumulate significant amount of intracellular GSH possibly due to feedback inhibition of GSH onγ-GCS. Therefore, we hypothesized that GSH yield of recombinant P. pastoris could be further improved by optimizing the catalysis characteristics of glutathione synthesis enzymes. To this end, in this work, glutatithione synthesis enzymes derived from a variety of strains was tested for their catalytic activity in P. pastoris, which includes following aspects.1) GSHI(encodingγ-GCS) derived from S.cerevisiae S288c, S.cerevisiae Angel and C. utilis, were co-expressed with S.cerevisiae GSHII in P. pastoris, respectively. The effects of different GSHI on GSH yield by recombinant P. pastoris were determined. Results showed that the GSH yields of the three strains are 2.2, 4.3 and 4.4 times the level of GS115. After feeding with three substrate amino acids, the GSH yields of control and recombinant strains reached 116 mg/L,256 mg/L, 426 mg/L and 438 mg/L, respectively.2) The gram positive bacteria bifunctional enzyme (encoded by gshF) was tested for their potential in GSH production in the context of recombinant P. pastoris. gshF genes derived from L. monocytogenes,L. plantarum and S. agalactiae were over-expressed in P. pastoris, respectively, thus resulting in the recombinant P. pastoris strains GS115/Lm-gshF, GS115/Sa-gshF and GS115/ LpgshF. Results showed that GS115/Lm-gshF produced more than twice as much GSH as GS115. No significant difference of GSH production was found between GS115/Sa-gshF, GS115/ Lp-gshF and GS115. SDS-PAGE results revealed that gshF from L. monocytogenes rather than L. plantarum and S. agalactiae was successfully expressed. After feeding with three substrate amino acids, the highest GSH production of GS115/ Lm-gshF reached 336 mg/L.