重组酿酒酵母合成白藜芦醇的研究  

Study on the Synthesis of Resveratrol by Recombinant Saccharomyces cerevisiae

作　　者：于跃 郭辉力 薛飞燕 彭程 姜梦嫣 马兰青 YU Yue;GUO Hui-li;XUE Fei-yan;PENG Cheng;JIANG Meng-yan;MA Lan-qing(Key Laboratory of Urban Agriculture (North China),Ministry of Agriculture,College of Biological Science and Engineering,Beijing University of Agriculture,Beijing 102206,China)

机构地区：[1]农业部华北都市农业重点实验室北京农学院生物科学与工程学院

出　　处：《现代食品科技》2019年第5期184-190,150,共8页Modern Food Science and Technology

基　　金：国家自然科学基金项目(21606020);大北农青年教师科研基金项目(16ZK001)

摘　　要：为了获得一种酵母快速合成白藜芦醇的体系,本研究分别从虎杖和烟草中克隆获得白藜芦醇合成途径的关键酶基因:芪合酶基因STS和4-香豆酰辅酶A连接酶基因4CL,引入3个连续重复的GSG作为连接肽,采用Overlap PCR扩增技术构建了融合基因4CL-(GSG)3-STS,进一步获得重组表达载体p ESC-TRP-4CL-(GSG)3-STS后转化至酿酒酵母中,之后利用HPLC分析检测重组酿酒酵母代谢产物,最后对重组菌合成白藜芦醇的诱导时间、底物添加浓度和添加方式进行了优化研究。结果表明:所构建的重组酿酒酵母菌体生长48 h后进行诱导表达,同时添加浓度为6 mg/L底物4-香豆酸,并且每隔2 h添加一次,8 h后白藜芦醇产量即可达7.01 mg/L。利用本体系合成白藜芦醇具有操作简单、生产周期短的特点,为进一步实现微生物大规模生产白藜芦醇提供了基础。In order to obtain a system for rapid synthesis of resveratrol by Saccharomyces cerevisiae,the key enzyme genes of the resveratrol synthesis pathway were cloned in this study,stilbene synthase gene(STS)from Polygonum cuspidatum and 4-coumaroyl-CoA ligase(4 CL)from Nicotiana tabacum,to construct the fusion gene of 4 CL-(GSG)3-STS by overlap PCR amplification technology through troducing three consecutive repeats of GSG as a linker peptide.The recombinant expression vector p ESC-TRP-4 CL-(GSG)3-STS obtained was then transformed into S.cerevisiae.The metabolites of the recombinant S.cerevisiae were analyzed by HPLC.Finally,the induction time,and substrate concentration and addition method for the synthesis of resveratrol by the recombinant S.cerevisiae were further optimized.The result showed that the yield of resveratrol could reach 7.01 mg/L 8 h after the established recombinant S.cerevisiae cells,which had grown for 48 h,were induced to express while the substrate 4-coumarate was added at 6 mg/L once every 2 h.The synthesis of resveratrol using this current system is simple for operation and requires only a short production cycle,thus,provides a basis for realizing a large-scale microbial production of resveratrol.

关 键 词：融合基因 酿酒酵母 白藜芦醇 发酵优化 

分 类 号：TQ28[化学工程—有机化工] Q78[生物学—分子生物学]