Preliminary Study On Improving Inhibitors Stress Tolerance Of Yeast Cells Self-flocculation

Cellulosic ethanol is a new renewable and clean energy that can mitigate oil depletionand reduce environmental pollution.During pretreatment of cellulosic biomass,organic acids and furfans are released and can have an inhibitory effect on yeast cell growth and ethanol fermentation.For achieving an efficient cellulosic ethanol production,it is crucial to develop yeast strains that are tolerant to multiple inhibitors.In preliminary studies,it was found that self-flocculation can improve the tolerance of yeast cells to inhibitors.In order to explore the mechanism of improvement of tolerance by self-flocculation,the knockout strain SPSC01-?FLO1 was obtained from a flocculating yeast,SPSC01,by deleting the flocculation gene FLO1.The transcriptome of SPSC01 under inhibiting stress was measured and analyzed by using the SPSC01-?FLO1 as a control.Previous studies showed that the expression of 2-deoxyglucose-6-phosphate phosphatase gene DOG1,NADH-ubiquinone reductase gene TIR1 and the gene with a possible role in phospholipid biosynthesis YOL032 W was up-regulated in SPSC01 under furfural-vanillic acid stress.Overexpression strains,4126-DOG1,4126-TIR1 and S288C-YOL032 W,were constructed and functions these three genes were studied in terms of tolerance to inhibitors.Under stresses of 4 g/L 5-HMF and 5 g/L vanillic acid,fermentation time of 4126-DOG1 and4126-TIR1 was more than 12 h shorter than that of 4126-HO.Ethanol yields of these two recombinant strains were 41.9% and 97.3% higher than that of the control strain,4126-HO,during ethanol fermentation processes using a simulated hydrolysate.Under stresses of 3 g/L furfural and 2 g/L vanillin,S288C-YOL032 W showed 66.5% and 42.9% higher cell concentrations,and 167.9% and 82.9% higher ethanol yields than S288C-HO,respectively.Overexpression of DOG1,TIR1 or,YOL032 W can effectively enhance the tolerance of yeast to 5-HMF/furfural and vanillic acid.In order to explore the relationship between inhibitors tolerance and yeast cells self-flocculation at both molecular level and process engineering level,the FLO1 expression was regulated by different promoters.Recombinant strains with different flocculation ability were obtained: 6525 PTDH3-FLO1 > 6525 PPGK1-FLO1 > 6525 PRPL4A-FLO1(without inhibitor).The strongest flocculating mutant 6525 PTDH3-FLO1 obtained the most biomass with thesupplementation of 5-HMF,acetic acid or AFP(Acetic acid,furfural and phenol).Under stress of other inhibitors,6525 PPGK1-FLO1 obtained more biomass than other strains.6525PRPL4A-FLO1 was the worst in terms of cell growth and ethanol fermentation.Phenol,furfural and 5-HMF had different effects on ethanol production of different flocculating strains.The results showed that stronger flocculating mutant had higher tolerance to inhibitors and better ethanol fermentation performance.Speeds had no significant effects on 6525 PTDH3-FLO1 and6525 PPGK1-FLO1 with real hydrolysate,although the ethanol fermentation performance of6525 PRPL4A-FLO1 became worse with the speed increase.At the same speed,there was no significant difference in cell growth and ethanol fermentation between 6525 PPGK1-FLO1 and6525 PTDH3-FLO,although 6525 PRPL4A-FLO1 showed the lowest biomass and ethanol production.Compared with the speed controlling,regulation of FLO1 expression with different promoters had greater effect on the ethanol fermentation and tolerance of flocculation yeast to inhibitors.According to AFP transcriptome of different flocculating mutants,the expression of glutamine permease gene GNP1 and NDE1 was significantly up-regulated in strongly flocculating mutant.Using SPSC01 as a host,SPSC01-GNP1 and SPSC01-NDE1 mutant were constructed.These two mutants showed significantly high phenol tolerance,and 40%higher biomass than SPSC01-HO.Under the new-AFP mixed stress,they obtained 28.6% and109.1% higher ethanol yield than the control strain SPSC01-HO.Overexpression of GNP1 and NDE1 gene could significantly improve ethanol fermentation in flocculating yeast SPSC01 under stresses of acetic acid,furfural,and phenol.