重组GAL基因型酿酒酵母菌株的生长、发酵及遗传稳定性研究  

Studies on the Growth, Fermentation and Genetic Stability of the Recombinant GAL Genotype Yeast(Saccharumyces cerevisiae)

作　　者：郭钦[1,2] 张伟[1] 阮晖[1] 何国庆[1] 

机构地区：[1]浙江大学食品与生物系统工程学院,杭州310029 [2]江苏大学食品与生物工程学院,镇江212013

出　　处：《农业生物技术学报》2015年第6期720-726,共7页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2006AA10Z316);国家自然基金项目(No.31130042)

摘　　要：半乳糖激酶基因(GAL1)启动子是酿酒酵母(Saccharumyces cerevisiae)表达外源蛋白的高效诱导性启动子,其表达效率受到GAL4的调控和半乳糖的诱导,改变酵母的GAL基因型有助于提高外源蛋白的表达水平,但重组GAL菌株的菌株特性仍需要进一步研究以确保蛋白表达的稳定性。本研究分别对敲除了不同GAL1和插入不同GAL4基因拷贝数的重组GAL基因型酵母进行了生长发酵性能和遗传稳定性研究。结果表明,重组菌株的GAL基因型在传代过程中较为稳定,没有发生回复突变和遗传霉素(G418)和博来霉素(Zeocin)抗性恢复的情况;与对照原始MS-1酵母相比,重组菌株(SG1、DG115、DPG65和GQ21)的生长性能在葡萄糖培养基中的生物量略有下降。与半乳糖诱导培养基(yeast peptone galactose,YPG)相比,GAL1基因的敲除在甘油培养基(yeast peptone galactose glycerin,YPGL)中对酿酒酵母半乳糖利用的影响更为显著。其中,在YPG和YPGL培养基内,对照MS-1、SG1、DG115和DPG65利用完所有半乳糖分别需要20、20、20、26、20、44、44和68 h,而GQ21均无法利用半乳糖。GAL基因的敲除和插入没有影响酿酒酵母的热致死温度,原始菌株和重组菌株皆为54℃。本研究将为工业利用酵母表达系统大规模、低成本生产重组蛋白提供研究基础。GALl promoter is one of high-efficient inducible promoters of yeast （Saccharumyces cerevisiae）, which is regulated by GAL4 and induced by galactose. GAL genotypes of yeast have significant effects on the expression levels of exogenous proteins controlled by GALl promoter, so the characteristics of recombinant GAL yeast strains need further study. In this study, the growth, fermentation performance and genetic stability of the recombinant GAL yeasts with different copies of GALl and GAlA genes were evaluated. The results indi- cated that, the changed GAL genotypes were stable and recovery mutation didn＇t occurred during cultivation. The deficiency of GALl gene slightly affected the abilities of yeast to use glucose and the different cultivation modes had also impacts on galactose utilization for the recombinant yeasts. In yeast peptone galactose （YPG） and yeast peptone galactose glycerin （YPGL） medium, the depletion time of galactose was 20, 20, 20, 26, 20, 44, 44 and 68 h for MS-1, SG1, DGll5 and DPG65, respectively. For GQ21, the galactose concentrations were always constant in medium, indicating that GALl genes＇ knockout. However, the heat-resistance temperature of yeast didn＇t change for the different GAL yeasts. This study will be helpful for large-scale and low-cost production of recombinant proteins using yeast expression system as bioreactor.

关 键 词：GAL1 GAL4 重组酵母 半乳糖 发酵性能 

分 类 号：S511[农业科学—作物学]