在酿酒酵母中共表达sXYLA和XKS1及木糖发酵的初步研究

为改善重组酵母发酵木糖生产乙醇的能力,将定点突变改造后的Thermus thermophilus木糖异构酶基因sXYLA克隆到酵母表达载体pYX212并用于转化酸酒酵母Saccharomyces cerevisiae YPH499进行表达研究。酶活检测表明,改造后的木糖异构酶活性是未改造的1.91倍。在此基础上将改造后具有良好特性的木糖异构酶基因sXYLA和来自酸酒酵母的木酮糖激酶基因XKS1耦联,构楚得到重组表达质粒pYX-sXYLA- XKS1,在酿酒酵母YPH499中实现组成型共表达。结果表明,在84 h时重组菌发酵液酶活达到最高,木糖异构酶为0.624 U/mg蛋白,木酮糖激酶为0.688 U/mg蛋白。以葡萄糖和木糖为混合碳源初步进行半通氧发酵,代谢产物分析表明酸酒酵母重组菌木糖的消耗为4.75 g/L,乙醇的产量为0.839 g/L,分别比出发菌提高20.9%和14.8%,为酿酒酵母利用木糖发酵乙醇奠定基础。

Construction of Saccharomyces cerevisiae Coexpressing sXYLA and XKS1 for Ethanol Production Using Xyolse as the Substrate

In order to construct a strain of Saccharomyces cerevisiae with good performance on utilizing the xylose for ethanol production,the molecule--engineered xylose isomerase gene sXYLA from Thermus thermophilus was inserted into the plasmid of pYX212,which was then expressed in the stain YPH499 of S. cerevisiae.The enzyme activity of engineered xylose isomerase is 1.91 times higher than that of the parent. The engineered gene sXYLA encoding xylose isomerase with high activity coupling xylulokinase gene XKS1 from S.cerevisiae were inserted into the plasmid pYX212 to produce the expression vector pYX-sXYLA- XKS1.Both genes were then constitutively expressed in the stain YPH499 of Saccharornyces cerevisiae.The best activities of sXYLA of 0.624U/mg protein and XKS1 of 0.688U/mg protein were achieved at 84h.The recombinant strain under the oxygen--limited condition was fermented using xylose as the substrate to pro- duce ethanol.Metabolic product analysis showed that the recombinant strain consumed 4.75g/L xylose and produced 0.839g/L ethanol,which were respectively 20.9%and 14.8%higher than the parent strain.This attempt established a firm foundation for the ethanol production from S.cerevisiae using xylose as the sub- strate.