Cloning of xylose reductase gene and site-specific integrating it into Saccharomyces cerevisiae genome

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°C. Ten fast 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.