摘要
利用聚合酶链式反应(PCR)技术从毕赤树干酵母的基因组中扩增得到木糖还原酶基因xyl1和木糖醇脱氢酶基因xyl2,它们分别或同时与高拷贝型酵母表达载体YEp24重组,再经醋酸锂转化酿酒酵母,得到了3种不同类型的重组酵母菌株,其中重组酿酒酵母菌株NLR04可利用单一的木糖为碳源进行生长,并能够在微氧或厌氧环境中缓慢发酵木糖生成乙醇,乙醇得率可达到理论得率的37.0%。该研究可为微生物的木糖代谢工程提供种质资源。检测结果表明,与毕赤树干酵母不同,在重组酿酒酵母中木糖还原酶基因以组成型表达,而木糖醇脱氢酶基因在某种程序上受木糖的诱导。与毕赤树干酵母相比,重组酿酒酵母菌株中的木糖醇脱氢酶的比活力明显较低,这说明在ScNLR04中该基因的表达存在某些抑制作用,这一缺陷可能是重组酵母菌株发酵木糖的瓶颈之一。
In order to establish the xylose-fermentation pathway in Saccharomyces cerevisiae, two key genes for xylose-metabolizing, xylose reductase xyl 1 and xylitol dehydrogenase xyl 2, were amplified from Pichia stipitis by polymerase chain reaction （PCR） technique and cloned into YEp 24, a high-copy expression vector in yeast, respectively and simultaneously, and then they were constructed into S. cerevisiae by transformation using lithium acetate. In three recombinant strains, the recombinant S. cerevisiae NLR04 could grow and ferment in media with xylose as sole carbon source while the two genes xyl 1 and xyl 2 were transferred simultaneously into an auxotrophic mutant of S. cerevisiae, and it showed an ineffective capacity of fermenting xylose to ethanol under trace oxygen or anaerobic conditions and its ethanol yield could reach 37.0 % of the theoretical value. These recombinant yeast strains enriched the strains library for the xylose-metabolic engineering. Detective results confirm that there was some difference between P. stipitis and the recombinant S. cerevisiae NLR04 in the gene xyl 1 and the gene xyl 2 expression mechanism, the gene xyl 1 is constitutely expressed in the latter but xylose-inducible in the former. However, the gene xyl 2 is reversed as it is constitutely expressed in the former but xylose-inducible to some extent. On the xylitol-dehydrogenase relative enzyme activity, the recombinant S. cerevisiae NLR04 is markedly lower than P. stiptis, which maybe the one of bottlenecks for xylose-utilization in recombinant strains.
出处
《林产化学与工业》
EI
CAS
CSCD
2008年第1期23-28,共6页
Chemistry and Industry of Forest Products
基金
江苏省高技术计划项目(BG2005327)
江苏省高校自然科学重大基础研究项目(06KJA22015)
国家973计划(2007CB70801)
关键词
木糖发酵
重组酿酒酵母
乙醇
xylose-fermentation
recombinant Saccharomyces cerevisiae
ethanol
