Manipulation Of Endogenous Gene Expression To Improve Xylose Utilization And Acetic Acid Tolerance Of Saccharomyces Cerevisiae

Xylose is the major pentose in lignocellulosic hydrolysate.However,the commonly used yeast Saccharomyces cerevisiae for production of biofuels and biochemicals from lignocellulosic hydrolysate has poor xylose utilization capability.In addition,a variety of inhibitors are generated during pretreatment of lignocellulosic feedstocks which repress growth and fermentation ability of yeast cells.Acetic acid is one of the major inhibitors in lignocellulosic hydrolysate,and yeast cell growth can be severely repressed by acetic acid stress during xylose fermentation.Therefore,it is of great significance to increase xylose metabolism ability and acetic acid tolerance for breeding of highly efficient S.cerevisiae strains for bioconversion of lignocellulosic biomass.Recently,it was found that the expression of multiple endogenous genes of S.cerevisiae involved in transcription regulation,mitochondrial function and MAPK signaling pathway has great influence on xylose utilization,but related studies on regulation of xylose utilization by endogenous mechanisms are still limited.Studies on key endogenous genes involved in xylose metabolism benefit exploreation of global regulation mechanism of xylose utilization and improve the efficiency of metabolic engineering.In this study,multiple strains including laboratory,industrial and naturally isolated yeasts were compared in fermentation efficiency,mixed-sugar utilization and stress tolerance abilities.A natural yeast isolated from bagasse,named YB-2625,was selected due to its superior xylose-fermenting performance.YB-2625 consumed 15.2 g/1 xylose in 96 h when fermenting with 80 g/1 glucose and 20 g/1 xylose,whereas the model yeast S288c only consumed 7.6 g/1 xylose,implying that YB-2625 is able to adjust its innate metabolic network for xylose utilization.RNA-seq analysis was performed using YB-2625 grown in a mixture of glucose and xylose,and the model yeast strain S288c served as a control.Global gene transcription was compared at both the early mixed-sugar utilization stage and the latter xylose-utilization stage.Genes involved in endogenous xylose-assimilation(XYL2 and XKS1),gluconeogenesis,and TCA cycle showed higher transcription levels in YB-2625 at the xylose-utilization stage,when compared to the reference strain.On the other hand,transcription factors involved in regulation of glucose repression(MIG1,MIG2,and MIG3)and HXK2 displayed decreased transcriptional levels in YB-2625.suggesting the alleviation of glucose repression of S.cerevisiae YB-2625.Notably,genes encoding antioxidant enzymes(CTT1,CTA1,SOD2 and PRX1)showed higher transcription levels in S.cerevisiae YB-2625 in the xylose-utilization stage than that of the reference strain.Consistently,catalase activity of YB-2625 was 1.9-fold higher than that of S.cerevisiae S288c during the xylose utilization stage.As a result,intracellular reactive oxygen species(ROS)levels of S.cerevisiae YB-2625 were 43.3%and 58.6%lower than that of S288c at both sugar utilization stages.Overexpression of CTT1 and PRX1 in xylose-fermenting recombinant increased cell growth when xylose was used as the sole carbon source,leading to 13.5%and 18.1%,respectively,more xylose consumption.The genome of YB-2625 was further sequenced and analyzed to confirm the mutated genes related to xylose utilization and stress tolerance ability.When analyzing the 11.84 Mb assembled genome sequence,multiple YB-specific genes and mutated genes were found compared to laboratory strain.There was a non-synonymous mutation in the histone acetylase-encoding gene RTT109 and it showed significantly lower transcription level in YB-2625 according to the RNA-seq analysis.In laboratory yeast BY4741,acetic acid tolerance could be improved obviously by deletion of RTT109.During fermentation with 100 g/1 glucose,the lag phase was shortened from 60 h in the wild type strain to 12 h in BY4741-RTT109? under the stress of 5.5 g/1 acetic acid.What's more,ethanol production rate of RTT 109-deleting mutant reached 0.60 g/1/h in the presence of 5.5 g/1 acetic acid,in contrast to 0.39 g/1/h for the control strain BY4741.It was found that expression of the genes related to stress response,such as CTT1,GSH1,SOD1,and GPX1,increased in this mutant.In the stationary phase,the SOD activity of BY4741-RTT109? was 115.45 U/mg protein,higher than that in BY4741(79.63 U/mg protein),while the GSH-Px activities of BY4741-RTT109? was 141.14 U/mg protein,77.1%higher than the control level.However,no significant change was observed in the RTT109-deleting mutant under the genetic background of YB-2625,indicating that other genes in YB-2625 may impact the function of this gene.A xylose co-fermenting yeast was constructed with YB-2625 as host strain and its fermentation performance was evaluated.To solve the problem of inefficient xylose utilization and high yield of xylitol of initially xylose-fermenting strain YB-2625 CCX,xylitol dehydrogenase(XDH)was overexpressed by integrated in rDNA site,and a recombined yeast named YB-73 was obtained.Compared to YB-2625 CCX,xylitol yield of YB-73 was decreased by 64.6%and the ethanol production was increased by 13.9%during mixed sugar fermentation.Combined with genome sequencing and comparative transcriptomic analysis,it was found that there were point mutations in the sequence of chromatin remodeling related gene NGG1 and it showed significantly decreased expression in the stage of xylose utilization.Tolerance of xylose-fermenting recombinant under 5 mM H2O2 condition was enhanced by deletion of NGG1.What's more,xylose-fermenting ability was improved when deleting NGG1.The residual sugar of ?NGG1 mutant was 4.9 g/1 after fermenting for 96 h in 40 g/l xylose and 12.7 g/l xylose was detected in the fermentation broth of the control.Simultaneously,ethanol yield of the mutant was 44.4%higher than that of the control strain.It was found that the transcription level of xylose dehydrogenase encoding gene XYL1 and antioxidant enzymes encoding genes,SOD]and CTT1,were improved in NGG1-deleting mutant,which means that the function of NGG1 was involved in inhibitors tolerance and xylose metabolism.Further exploration of the underlying mechanisms will facilitate improvement of xylose fermentation efficiency.The results in this thesis provide basis for unveiling the molecular mechanisms underlying xylose metabolism in S.cerevisiae and influences of host genetic background,and also provide novel targets for the construction of xylose utilizing strains with improved stress tolerance,which benefits efficient bioconversion of lignocellulose.