Study On The Effect Of Biosynthesis Of L-ascorbic Acid And Glutathione On Yeast Tolerance

Saccharomyces cerevisiae is one of the general chassis cells and is widely used in laboratory research and industrial production.Many industrial production processes cannot provide or maintain the optimal growth conditions for yeast,so the construction of highly tolerant yeast cells has always been a research hotspot.This research is dedicated to optimizing the synthesis of L-ascorbic acid(L-AA)and glutathione(GSH)to obtain the strains with higher tolerance.First of all,we analyzed the relevant gene transcriptional levels of the S.cerevisiae strains which can biosynthesize L-AA,which indicated that the key rate-limiting genes are vtc2 and gldh.After that,by optimizing the expression of endogenous and exogenous genes,the production of L-AA has further increased.Among them,the intracellular L-AA production of the strain YLAAG-5 with overexpression Gene alo1 and vtc2 reached 23.66±3.55 mg/L after 72 hours fermentation.We determined the optimal glucose concentration for L-AA production is 30 g/L,which can increase the yield by 1.43 times to 35.89±3.55 mg/L.In addition,the exogenous addition of Lgalactose-1,4-lactone,L-galactose and GSH will increase the accumulation of L-AA reaching nearly 40 mg/L.Secondly,we intended to further study the effect of biosynthetic L-AA on the tolerance of yeast strains.We characterized the tolerance under the stress of hydrogen peroxide(H2O2),high temperature(55℃ heat shock,40℃ high temperature cultivation)and penetration(5%NaCl)of six strains with a characteristic production range of 3.58±0.40～23.83±2.25 mg/L.The results showed that the lower-production strain YLAA has significantly better antioxidant capacity and tolerance than other strains.It is inferred that the relationship between L-AA production and the antioxidant capacity of yeasts is not a positive correlation.There is a positive correlation between the antioxidant capacity and tolerance of yeasts.Finally,this article constructed twelve yeast strains which expressed of GSHrelated gene modules in order to study the effect of optimizing GSH synthesis on its tolerance.Though analyzing their growth under oxidation(H2O2),high temperature(55℃heat shock,40℃ high temperature cultivation),penetration(5%NaCl),and inorganic acid(HCl,pH=3.0)stress,we investigated the effects of the expression of the abovementioned gene module under different stress conditions.The results indicated the following conclusions:Yeast cells under oxidative stress may need more GSH oxidation to provide reducing power to protect them;High temperature heat shock needs to improve the ability to remove O2-and maintain the reduced state of GSH;Maintaining the reduced state of GSH may be more important for yeast cells under high temperature and osmotic pressure;Optimization of the GSH cycle process is more conducive to the restoration of cell viability of yeast cells under acid stress.In summary,this study showed that the biosynthesis of L-AA and GSH could improve the tolerance of yeast by improving its antioxidant capacity.