| Fossil fuels is the subject of today's energy structure, and it is a very valuable non-renewable resources. Faced with a growing shortage of fossil fuels, as well as its increasingly serious environmental pollution problems, it forces people to start looking for the sustainable use of cleaner alternative energy sources. Therefore, the development and utilization of biomass energy has become a strategic choice. At present, the scale of fuel ethanol is the fastest-growing in biomass energy industry, but the high cost of microbial fermentation for ethanol production is the bottlenecks in industry conditions. In order to research for yeast strains of high tolerance for ethanol production of simultaneous saccharification and fermentation technology to increase fuel ethanol production efficiency and reduce the costs, in this paper, with the yeast tolerance for high concentrations of sugar and ethanol gene——transcription factor SPT15 genetically engineered to carry out. The products that are random and site-directed mutagenesis of the gene SPT15 are connected to the plasmids PYES2 and Ycplac22, and over-express genes. The plasmids are the shuttle vector plasmid between E.coil and yeast and contain GALⅠpromoter,but they don't have the galactose genes. Adding Mn2+ in the standard PCR reaction system can reduce effectively the fidelity of Taq DNA polymerase, and cause random mutagenesis. The DNA fragments of different lengths are differently effective on the concentration of Mn2+. By exploring that effects, we can conclude that more than 500bp DNA has a rapid increase in mutation rate with the Mn2+ concentration improvement, but less than 250bp DNA is not particularly sensitive on the Mn2+ concentration. At the same time,when mutant library was established, we explored different transformation methods to determine the best conditions for electroporation. The mutant library of SPT15 creates the conditions for screening of high-tolerance yeast and provide valuable experimental experience. |
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