全局转录工程法构建产S-腺苷蛋氨酸重组酿酒酵母的研究  

gTME for Construction of Recombinant Yeast for Improving the Production of S-adenosylmethionine (SAM)

作　　者：曹喜涛[1,2] 陈凯[1] 李扬[1] 窦洁[1] 王慧[1] 周长林[1] 奚涛[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009 [2]江苏科技大学蚕业研究所,江苏镇江212018

出　　处：《药物生物技术》2012年第5期386-391,共6页Pharmaceutical Biotechnology

基　　金：江苏省自然科学基金(No.SBK200921231);江苏省教育厅高校产业化促进项目(No.JH10-12);江苏省青蓝工程(2008)资助

摘　　要：利用全局转录工程(global transcription machinery engineering,gTME)方法对酿酒酵母RNA聚合酶II中的转录因子SPT15和TAF25编码的基因进行克隆并结合易错PCR随机突变,将SPT15和TAF25的易错PCR产物连接改造的pYES2.0表达载体。并用醋酸锂转化的方法转化酿酒酵母Saccharomyces cerevisiae CGMCC 2846中,研究了SPT15和TAF25的定向进化对酿酒酵母产S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)的影响。经过筛选分别获得了重组酿酒酵母菌株spt15-81和taf25-36。对其发酵性状分别进行了初步研究,在28℃、200 r/min条件下,250 mL摇瓶(50 mL O-medium)发酵48 h时,SAM的产量分别比对照菌株提高了2.0倍和1.8倍。由此表明。Global transcription machinery engineering(gTME),which was developed as new tools for technological applications,can be employed to effectively reprogram gene transcription for improving or eliciting new phenotypes.It was used to evaluate the effect on the Saccharomyces cerevisiae industrial strains for the improvement of S-adenosylmethionine(SAM).In this study,the error-prone PCR for the directed evolution of SPT15 and TAF25,which are the component of yeast RNA polymerase II responsible for the transcription of genes was used,and its effect on the improvement of SAM was also investigated.Mutant library was constructed by ligating the error-prone PCR products with a modified pYES 2.0 plasmid,and subsequently transformed to yeast industrial strain Saccharomyces cerevisiae CGMCC 2842.The results showed that the recombinant yeast strains spt15-81 and taf25-36 were selected from YPD plate with 120 mg/mL G418,and could produce 2.60 g/L and 2.40 g/L SAM in the O-medium under the condition of 28 ℃,200 r/min,and 48 h in the 250 mL flask,respectively.The productions of SAM were 2.0 and 1.8-fold higher than those of the control strain 1.30 g/L harboring the empty vector.This study revealed that global transcription machinery engineering was important tool for the metabolic engineering of S-adenosylmethionine,and provided a powerful approach to elicit phenotypes of industrial strains that were not readily accessible by conventional methods.

关 键 词：酿酒酵母 全转录工程(gTME) SPT15 TAF25 易错PCR S-腺苷甲硫氨酸 

分 类 号：Q812[生物学—生物工程]