酿酒酵母工程菌生物合成白藜芦醇

Biosynthesis of Resveratrol in Engineered Saccharomyces cerevisiae Strain

目的:构建能以酪氨酸为底物产白藜芦醇的重组酿酒酵母。方法:利用降落-重叠延伸PCR法和一步等温法,将拟南芥的4-香豆酰辅酶A连接酶基因(4cl)、巨峰葡萄的白藜芦醇合酶基因(rs)以及粘红酵母的酪氨酸解氨酶基因(tal)分别构建含有不同抗性筛选标记的附加型酵母表达载体,采用Li Ac/SS carrier DNA/PEG法转化酿酒酵母工业型菌株EC1118,通过高效液相色谱法检测重组酵母发酵产物。结果:成功获得酵母工程菌EC1118-H-TTC-K-T4C-TRC,以3 mmol/L酪氨酸为底物,28℃,150 r/min摇床避光发酵5 d,发酵液中白藜芦醇的产量为6.1979 mg/L。结论:成功构建一株能以酪氨酸为底物产白藜芦醇的工业型重组酿酒酵母,为白藜芦醇的工业化生产进一步奠定了基础。

Objective： In this study, we constructed an engineered Saccharomyces cerevisiae strain with the ability to produce resveratrol from tyrosine. Methods： Two episomal expression vectors carrying 4-coumarate： coenzyme A ligase gene（4cl） from Arabidopsis thaliana, resveratrol synthase gene（rs） from Vitis vinifera and tyrosine ammonia lyase gene（tal） from Rhodotorula glutinis were built by Touchdown-overlap extension PCR and One-step isothermal DNA assembly protocol, then transformed into S. cerevisiae EC1118 by LiAc/SS carrier DNA/PEG method. HPLC analyses were carried out to measure the content of resveratrol accumulated in engineered strain. Results： When cultured with 3 mmol/L tyrosine at 28 ℃, 150 r/min for 5 days, engineered strain EC1118-H-TTC-K-T4C-TRC produced 6.1979 mg/L of resveratrol.Conclusions： An engineered industrial wine strain with the ability to produce resveratrol from tyrosine was obtained and the accumulation of resveratrol was 6.1979 mg/L. This study laid a foundation for further resveratrol industrial production.