Abstract

The SPT15 gene of Saccharomyces cerevisiae obtained using site-saturation mutagenesis was connected to the expression vector pYES2NTc. Then, the mutant library was constructed. Twelve single point mutant genes were obtained, and the recombinant plasmids were constructed with wild gene SPT15 and control gene. The S. cerevisiae strains were obtained by converting recombinant plasmids into the S. cerevisiae INVSC1 by lithium acetate method. Using glucose as substrate, the recombinant S. cerevisiae was inoculated and fermented to determine its ethanol yield. The ethanol yields of recombinant S. cerevisiae INVSC1-SPT15-K127M and S. cerevisiae INVSC1-SPT15-K127N were greatly increased by 43.0 ± 0.9 and 43.7 ± 0.2 g/L, i.e., 26.5 and 28.5%, respectively, compared with that of the control strain S. cerevisiae INVSC1-Neo. The site-saturation mutagenesis technique was used instead of random mutagenesis, and 2 mutant strains with phenotypic improvements were screened in the limited mutant library, which indicated that 127 amino acids had an important effect on the binding efficiency of Spt15 and TATA.

中文翻译：

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使用全球转录机械工程（GTME）和位点饱和诱变技术来提高酿酒酵母的乙醇收率

摘要

利用位点饱和诱变获得的酿酒酵母的SPT15基因与表达载体pYES2NTc连接。然后，构建突变体文库。获得了12个单点突变基因，并用野生基因SPT15和对照基因构建了重组质粒。在酿酒酵母的菌株通过将重组质粒到所获得的酿酒酵母通过醋酸锂法INVSC1。以葡萄糖为底物，将重组酿酒酵母接种并发酵以确定其乙醇产量。重组酿酒酵母INVSC1-SPT15-K127M和酿酒酵母的乙醇产量与对照菌株酿酒酵母INVSC1-Neo相比，INVSC1-SPT15-K127N分别增加了43.0±0.9和43.7±0.2 g / L，即分别为26.5和28.5％。用位点饱和诱变技术代替随机诱变，在有限的突变体文库中筛选了2个具有表型改良的突变菌株，这表明127个氨基酸对Spt15和TATA的结合效率有重要影响。