多基因整合型载体转化酿酒酵母生物合成白藜芦醇

Multi-gene Integrated Vector Transforming Saccharomyces cerevisiae Biosynthesis Resveratrol

目的:以整合型载体p RS303K为框架,以来源于拟南芥的4-香豆酰辅酶A连接酶基因(4cl)和巨峰葡萄的白藜芦醇合酶基因(rs)为目标基因,采用一步等温法构建了表达白藜芦醇的整合型酵母表达载体p RS303KT4C-TRC。方法:采用Li Ac/SS carrier DNA/PEG转化法把表达载体成功转化酿酒酵母工业型菌株EC1118,经筛选和PCR鉴定获得酵母工程菌株EC1118-303K-T4C-TRC。酵母工程菌株采用添加和不添加抗生素的发酵培养。结果:在培养基添加抗生素和不加抗生素的发酵液中白藜芦醇的含量分别为3.4110 mg/L和3.4710 mg/L,两者差异不显著。结论:两个关键酶基因成功整合到酵母的染色体基因组中并得到表达,工程菌株在传代中不受选择压力影响,稳定组合表达白藜芦醇。

Objective： In this work, a resveratrol-generated and integrative yeast expression vecter p RS303 K-T4 C-TRC was constructed with 4-coumarate： coenzyme A ligase gene（4 cl） from Arabidopsis thaliana and resveratrol synthase gene（rs） from Vitis vinifera based on the plasmid p RS303 K. Methods： This vector was transformed into host cell Saccharomyces cerevisiae EC1118, and an recombined strain EC1118-303 K-T4 C-TRC was obtained based on PCR and restriction endonuclease analysis. Results： The resveratrol yield of recombined strain in antibiotic-added and control media were 3.4110 mg/L and 3.4710 mg/L respectively. There was no significant difference between these groups. Conclusions ：Results indicated that both the 4 cl gene and rs gene were integrated into the genome of S. cerevisiae, and antibiotic had no effect on the resveratrol production of recombined strain.