Mechanism Of ASK10 Mutation And Deletion Enhanced Xylose Isomerase Activity In Saccharomyces Cerevisiae

With the consumption of oil and other fossil fuels as well as the awareness of environmental protection,the second-generation fuel ethanol based on biomass as a renewable source has been paid gradually more and more recognized attention.Compared with the first-generation ethanol,the second-generation of ethanol production uses a variety of cellulosic material as feedstocks,and in can reduce air pollution.Saccharomyces cerevisiae has the advantages of high ethanol fermentation capacity,and it also shows high tolerance of sugar and ethanol,thus become the preferred strain of ethanol production.However,naturalS.cerevisiae strain only can utilize hexose fermentation,but cannot use xylose,which is the second abundant sugar in lignocellulose.Therefore the study of glucose and xylose co-fermentation in S.cerevisiae is particularly important for the economical of ethanol production.Our previous studies obtained a S.cerevisiae recombinant strain with highly xylose metabolism caoability by metabolic engineering and irrational evolution.The strain contained the xylose isomerase gene(xylA)derived from bovine rumen metagenome,and we also overexpressed endogenous xylulose kinase genes(XKS1)and key gene in non-oxidative pentose phosphate pathway to increase the flux of xylose metabolism,knocked-out GRE3 to reduce non-specific conversion of xylose to xylulose,and destroyed the respiratory metabolism by COX4 deletion.On this basis,we obtained a S.cerevisiae with high xylose ethanol production capability through irrational evolution in xylose as sole carbon source.We then did whole genome sequencing of evolved strain and compared it with CEN.PK113-7D,and found that the strain contained get 8 SNPs and 4 short InDels.But it remains unclear which mutant genes plays important role in improving ability of metabolization xylose.In this study,we constructed the strains with deletion or overexpression of 12 mutant genes.We found that most genes don't have positive effect for improving xylose metabolism,but the strain with mutation or deletion of ASK10 can improve the xylose isomerase activity and ability of growth on xylose.We also found that the deletion of ASK 10 increased the copy number of plasmids,and elevate transcription of Ru-xylA,thereby enhancing the xylose isomerase activity.Although ASK10m475R mutant gene also can increase the xylose isomerase activity,it did not affect the plasmid copy number.To further investigate the mechanism that ASK10M475R in improving xylose isomerase activity,we analyzed the transcriptome of ASK10M475R,ask10? mutant strains and evolved strain.We found ASK10M475R can increase the expression of molecular chaperones Hsp26p,thereby enhancing the xylose isomerase activity,so that enhance the ability of xylose metabolism.1)Overexpression or deletion of the mutant genes and the effect to xylose metabolismWe obtained 8 SNPs and 4 short InDels through whole genome sequencing of evolved strain and compared it with the genome sequence of CEN.PK113-7D.Then we constructed the strains with deletion and overexpression of 12 mutational genes.Growth assay in glucose and xylose shows that most genes don't have positive effect for improving xylose metabolism,but the strain with mutation or deletion of ASK10 can improve the xylose isomerase activity and ability of growth on xylose.2)Analysis of mutations in situ ASK10M475R function AsklOp function primarily as a regulatory factor involved in many stress responses,such as oxidative stress,etc.In order to verify whether ASK10M475R mutations plays an important role in maintaining Ask10p,We tested the ASK10M475R mutant strain growth in H2O2.ASK10M475R mutant strain sensitivity of H2O2 increased significantly,indicating that the mutation plays an important role in the function of ASK10 gene.3)ASK10 deletion can increase the copy number of pJFE3-Ru-xylA plasmids,and enhance the xylose isomerase activityWe found that the transcription of xylA in ASK10 deletion strain Ru-xylA transcript levels significantly increased significantly,and the plasmid copy number containing Ru-xylA increased in ASK 10 deletion strain.Therefore ASK1O deletion improve xylose isomerase activity by increasing the plasmid copy number.Although the transcription of Ru-xylA also increased in ASK10M475R mutant strains,but no significant change in plasmid copy number.4)ASK10M475R increases the expression of molecular chaperones Hsp26p,enhancing the xylose isomerase activityTo further study the mechanism that ASK10M475R improve xylose isomerase activity,we analyzed the transcriptome of ASK10M475R,ask10? mutant strains and control strain.We found ASK10M47R can increase the expression of molecular chaperones encoding gene HSP26,and further analysis revealed that overexpressing HSP26 gene has a positive role in improving xylose isomerase activity in the original strains.Thereby ASKI1047R mutations improved chaperone HSP26 gene encoding the transcriptional level in certain extent,enhancing the xylose isomerase activity,so that enhance the ability of xylose metabolism.