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

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pESC-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。

Abstract:

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 (4CL) from Nicotiana tabacum, to construct the fusion gene of 4CL-(GSG)3-STS by overlap PCR amplification technology through troducing three consecutive repeats of GSG as a linker peptide. The recombinant expression vector pESC-TRP-4CL-(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.