论文部分内容阅读
Ethanol production from lignocelluloses is a promising alternative liquid biofuel for sustainable energy supply.As the second abundant monosaccharide, xylose metabolism pathway needs to be introduced in Saccharomyces cerevisiae to improve the efficiency of lignocelluloses ethanol production.Heterologous expression of xylose isomerase (Ⅺ) is considered as an effective strategy to establish the xylose metabolic pathway in S.cerevisiae, since the converting of xylose to xylulose catalyzed by Ⅺ is cofactor-independent and not coupling xylitol formation.However, only a few Ⅺ genes (xylA)can be functionally expressed in S.cerevisiae at sufficient levels and their efficiency are still relatively low.In our study, a high efficiency xylose isomerase from cow rumen metagenomics (Ru-xylA) was cloned and expresssed in S.cerevisiae.In addition,further adaptive evolution increased the xylose metabolism capability and doubled the xylose isomerase activity in recombinant strain.However, there is no mutation in the xylA expression cassettes.Therefore, the protein-host matching mechanism for high efficiency xylose metabolism and higher Ⅺ activity were characterized.In order to study the reason of high activity when expressing Ru-xylA in S.cerevisiae, we compared the differences of four xylose isomerases when expressing them in E.coli and S.cerevisiae.We cloned Ru-xylA and the other three xylose isomerase genes from E.coli, Piromyces sp.E2, Sorangium cellulosum, respectively and expressed them in E.coli and S.cerevisiae.The specific activity of purified enzymes showed different activity trend in the two organisms.Pi-Ⅺ showed the highest activity when expressed in E.coli and followed by Ru-Ⅺ, Ec-Ⅺ, Sc-Ⅺ.But in S.cerevisiae, Ru-Ⅺ showed the highest activity and followed by Pi-Ⅺ, Sc-Ⅺ, Ec-Ⅺ.Further work is ongoing to analysis the reason of difference enzyme activity between different xylose isomerses.We also did transcriptome analysis to compare the host strain before and after adaptive evolution.It revealed up-regulation of enzymes involved in glycolysis and glutamate synthesis and down-regulation of trehalose and glycogen synthesis, which may have contributed to the improved xylose utilization of the strain.Further work is ongoing to study the reason of increased xylose activity.