酿酒酵母(Saccharomyces cerevisiae)是重要的乙醇生产菌株，但因缺少戊糖代谢途径而不能利用木糖，为了改良工业酿酒酵母利用半纤维素发酵生产乙醇的性能，利用分子生物学技术构建能够利用木糖的基因工程酵母。选取酿酒酵母染色体的rDNA重复序列作为外源基因整合位点，依此构建多拷贝染色体整合型载体pUG-LR。采用融合表达策略扩增得到含有酿酒酵母乙醇脱氢酶启动子PADH和树干毕赤酵母木糖还原酶基因xyl1的融合序列，并将其插入pUG-LR载体中，构建成含遗传霉素G418抗性标记的同源重组质粒pUG-LR-XYL1。以工业酿酒酵母ZU-01为宿主，通过优化后的电穿孔法将重组质粒导入经缓冲液处理的酵母细胞，30℃培养。通过提高YEPX复筛培养基G418浓度，得到10株生长较快的优良性状转化子。在不含G418的YEPX培养基上传代8次以上，以转化子基因组DNA为模板，进行PCR检测，均可获得目的基因片段。研究结果表明：木糖还原酶基因xyl1已定向整合于ZU-01染色体DNA上并稳定遗传，为后续构建工业酿酒酵母的木糖代谢通路、利用木糖产酒精的重组菌株奠定了基础。% Saccharomyces cerevisiae is an important ethanol producing fungus, but it cannot be used in large-scale bioethanol production from hemicelluloses due to its lack of the metabolism pathway of pentose, and therefore the xylose can not be utilized. In order to improve its ability of utilizing hemicelluloses to produce ethand, molecular biology technology was adopted to obtain recombinant strain with ability of xylose-using in this study. The repeat region of rDNA in the S. cerevisiae chromosome was chosen as the insert site of foreign genes. Plasmid pUG6, with KanMX resistance marker, was connected with rDNA fragments to construct site-specific integration vector pUG-LR. Then a Pichia stipitis xylose reductase gene xyl1 was cloned. Further the xyl1 gene was fused with PADH (alcohol dehydrogenase promoter cloned from host S. cerevisiae) by Overlap-PCR method, and inserted into pUG-LR vector to construction a recombination plasmid pUG-LR-xyl 1 with rDNA sequence as homologous arms of the host. The buffer-treated industrial S. cerevisiae ZU-01 was transformed with pUG-LR-xyl 1 by optimization electroporation method, and then cultured at 30 growth transformants with good traits were identified by rescreening on the culture plates using high concentrations of geneticin-dissulfate (G-418). It was further confirmed that the xyl1 gene has been site-specific integrated into the chromosomal DNA of ZU-01 by PCR analysis, and performs good genetic stability. This result is significant for the further work of industrial S. cerevisiae and its industrial process for bioethanol.℃.Ten fast
展开▼
