Screening And Application Of Salt-stress Resistance Genes In Candida Glycerinogenes

Microorganisms face multiple environmental stresses in growth and industrial fermentation applications,and improving microbial salt stress tolerance is important for cost control,process optimization and fermentation yield improvement.Candida glycerinogenes is an industrial yeast strain with multiple stress tolerance properties,but its stress tolerance mechanism and stress tolerance gene elements are still not fully explored.In this thesis,based on the previous research,C.glycerinogenes was used as the target to mine salt stress resistance genes and to conduct a preliminary investigation on its resistance mechanism and application,mainly as follows:Exploring of C.glycerinogenes salt stress response genes.Based on C.glycerinogenes transcriptome data under stress,seven up-regulated genes were selected as potential resistance genes.Overexpression in Saccharomyces cerevisiae and antisense suppression of gene expression in C.glycerinogenes,respectively,revealed that the DNA damage checkpoint protein gene Cgrad9 and the RNA helicase gene Cgdbp7 had effects on salt stress tolerance.Under salt stress,S.cerevisiae biomass increased by 22.8%after overexpression of Cgrad9,and C.glycerinogenes biomass increased by 12.6%after inhibition of Cgdbp7 expression,indicating that Cgrad9 is salt stress resistance genes,and Cgdbp7 acts as a negative regulator in salt stress.A preliminary investigation of the salt stress response mechanism of the above genes was conducted.Analysis of membrane properties of recombinant strain revealed that S.cerevisiae membrane fluidity and permeability did not change significantly after overexpression of Cgrad9,and C.glycerinogenes membrane fluidity decreased after inhibition of Cgdbp7expression.RT-q PCR revealed that the transcript levels of Ion transporter protein nhx1 in recombinant S.cerevisiae and recombinant C.glycerinogenes were increased 2.2-fold and 26.6-fold under salt stress respectively.Yeast single hybridization screen revealed that the transcription factor Upc2 was able to bind the Cgrad9 promoter sequence.These results suggest that Cgrad9 and Cgdbp7 improve the salt stress tolerance of recombinant strains by affecting the membrane fluidity and ion transport protein transcript levels of recombinant bacteria.Meanwhile,the transcription factor Upc2 binds to the Cgrad9 promoter sequence and is a potential regulator of Cgrad9.The overexpression strain and antisense suppressor strain were applied to the fermentation of high-salt environment and undetoxified cellulose hydrolysate.The overexpression of Cgrad9under high salt environment increased the biomass of the overexpressing strain by 22.8%and ethanol yield by 17.3%compared to the control.Fermentation of antisense-suppressed recombinant strain showed a 25.8%increase in biomass and a 46.1%increase in ethanol yield of 39.0 g·L^-1 compared to the control.Application of antisense-suppressed recombinant C.glycerinogenes to the fermentation of undetoxified cellulose hydrolysate showed an 8.2%increase in biomass and a 13.7%increase in ethanol yield of 22.8 g·L^-1 compared to the control.Transcriptional analysis revealed that Ethanol dehydrogenase adh1 transcription was up-regulated under undetoxified cellulose hydrolysate,which may improve furfural tolerance of antisense-suppressed recombinant strain,resulting in higher biomass of antisense-suppressed recombinant strain and thus higher ethanol yield.The above results suggest that overexpression of Cgrad9 and inhibition of Cgdbp7 expression have positive effects on the improvement of fermentation level,which may provide a reference for the research and application of yeast resistance mechanism.