乳酸克鲁维酵母Cu/Zn-SOD基因的克隆及在酿酒酵母中的表达研究

根据Genbank中乳酸克鲁维酵母(Kluyveromyces lactis)的Cu/Zn-SOD基因序列设计引物,通过PCR扩增得到Cu/Zn-SOD基因。在PGK1启动子驱动下,将该基因与荧光报告基因GFP融合,分别构建重组质粒YEplac195-PSGA和YCplac33-PSGA,并转化酿酒酵母(Saccharomyces cerevisiae)W303α菌株。通过菌落PCR和荧光显微观察证实乳酸克鲁维酵母(Kluyveromyces lactis)的Cu/Zn-SOD基因在W303α中成功表达。将获得的阳性转化子在添加20mmol/L百草枯的发酵培养基中进行发酵,SOD的比活力和总活力分别是不添加百草枯培养基中发酵菌体的6.7倍和4.7倍。通过热激胁迫处理进一步探讨Cu/Zn-SOD对宿主sod1Δ酿酒酵母菌株EG118耐受力的影响,结果显示抗热击能力的顺序为EG118(YEplac195-PSGA)EG118(YCplac33-PSGA)EG118。以上结果为发酵工业中防止菌体老化和增强菌体的发酵能力提供一定的理论指导,也为后续的Cu/Zn-SOD体外分子定向进化改造奠定必要的基础。

Clone of Cu/Zn-Superoxide Dismutase Gene from Kluyveromces lactis and Its Expression in Saccharomyces cerevisiae

According to Genbank Kluyveromyces lactis Cu/Zn-SOD gene sequence primers were designed；the Cu / Zn-SOD gene were obtained by PCR amplification. Driven by the PGK1 promoter, the gene fused to the fluorescent reporter gene GFP were used to construct recombinant plasmid YEplac195-PSGA and YCplac33-PSGA, and transformed into yeast (Saccharomyces cerevisiae) W303α strain. By colony PCR and fluorescence microscopic observation we confirmed that the Cu / Zn-SOD gene of Kluyveromyces lactis was successfully expressed in W303α strain.20 mM paraquat were added to the positive transformants before fermentation. SOD activity and total activity were respectively 6.7 times and 4.7 times as high as those without paraquat in the biomass of the fermentation medium. To further investigate the impact of the Cu / Zn-SOD gene on the host sod1Δ strain EG118, heat shock treatment was applied. Results show that heat strikes capability of host tolerance in the following order of EG118 (YEplac195-PSGA)> EG118 (YCplac33-PSGA)> EG118. The results is not only necessary for the fermentation industry to prevent bacterial fermentation in the aging strains and furthermore, an enhanced capacity to provide some theoretical guidance, but also a foundamental of in vitro directed evolution of the Cu/Zn-SOD.