| With the consumption of fossil fuel petroleum,the price of petroleum has been roaring,which has prompted researchers to turn to other alternative fuels.The 2nd generation bioethanol based on lignocellulose feedstock has been studied and developed widely.Saccharomyces cerevisiae is widely used ethanol producer,and it shows high tolerance to toxic componds,high density sugars and ethanol.However,Saccharomyces cerevisiae cannot use xylose,which is the second abundant sugur in lignocellulose feedstock,although it can utilize hexose.It is important to co-ferment C5 and C6 for the economical ethanol production.In our previous work,we selected and isolated a xylose isomerase with higher activity at 30? from bovine rumen metagenomics,introduced this xylose isomerase to yeast,overexpress endougenous XKS1 and the genes in non-oxidative pentose phosphate pathway to redirect carbon flux towards xylose fermenting pathways,knocked out GRE3 to minimize the xylitol production,and interrupt respiratory metabolism by COX4 gene deletion.However,the recombinant strains cannot use xylose effectively as expected,and the respiratory-deficient strain cannot even grow on the xylose the sole carbon source.Evolutionary engineering was then employed in the medium with xylose as the sole carbon source.After adaption evulation,the evolved strain can ferment xylose effectively.However,the mechanism of improved xylose metabolism was not investigated clearly using globle transcription profile.In this study,we has performed genome sequencing with the evolved strain,compared it with the unevolved strain,analyzed the mutations to reveal the molecular mechanism of effective xylose metabolism.1)Determination of XI activity and the xylA gene transcription level in the evolved and unevolved strainsCompared to the unevolved strain,the enzyme activity of XI and transcription level of Ru-xylA increased by 100%and 80%,respectively.However,the sequence of xylA gene did not changed.We also investigated the enzyme activity of heterologous XR,XDH or Pi-XI and found that their activiity were all improved.However,we did not observe higher enzyme activity when testing the report gene lacZ.2)Genetic varation determination by genome resequencingBased on the genomic data,we found out 117 nonsyn SNPs in the evolved strain,which lead to 66 mutant genes,and totally 738 ShortIndels effected 43 genes.We removed the SNPs and ShortIndels that were synonymy SNPs,or located in non-coding regions,or without annotation,and resequenced the 55 remaining genes.Finally,gene function enrichment analysis were performed with 12 mutant identified genes by Gene Ontology.3)Screen the the candidate mutation which affects the xylose metabolismWe did the gene deletion or over-expression to test the effect of the gene mutation on xylose.The Growth assay showed that most of the mutant genes did not have positive effects on xylose utilization.However,compared with the control strains,the strains ASK10M475R,ask10? and ask10?+ASK10M475R showed better growth.The result shown that ASK10 deletion can improve yeast growth on xylose.4)The effect of Ask10p to xylose metabolismAsk10p was identified as a important component to protect cell from various stress including oxidative stress.We therefore determined the effect of ASK10deletion or ASK10M475R mutation to oxidative stress.The results showed ASK10M475R was much more sensitive to H2O2 than ASK10wt,indicating that M475R was very important to maintain the function of Ask10p.We then test how ASK10M475R and ask10A influened xylose utilization.The result showed that both of them could promote xylose utilization obviously.We found out that XI enzyme activity and trancription level of xylA were increased to 2-fold in strain ASK10M475R and ask10?.Transcriptome anylysis showed that compared with the ASK 10 wild type,the transcription of genes encoding molecular chaperones changed significantly in both strain ask10? and ASK10M475R.These genes involved protein folding may contribute to the higher XI enzyme actiivty. |
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