将克隆自拟南芥的4cl基因和巨峰葡萄的rs基因,利用降落重叠延伸PCR的方法,成功构建了含有G418抗性筛选标记的酵母表达载体pRS42K-4CL以及含有潮霉素抗性筛选标记的酵母表达载体pRS42H-RS。采用LiAc/SS carrier DNA/PEG法将含有目的基因的2个载体共同转化至酿酒酵母工业菌株EC1118中,通过PCR及酶切鉴定等方法验证重组工程菌。以对香豆酸为底物,将获得的酿酒酵母工程菌于YPD液体培养基中发酵(25℃,150 r/min,96 h),发酵液用乙酸乙酯抽提后采用高效液相色谱(HPLC)法进行检测,其结果显示发酵产物中白藜芦醇的含量为0.78 mg/L。这表明,拟南芥的4cl基因与巨峰葡萄的rs基因在酿酒酵母工程菌中成功得到了表达,并且表达产物利用对香豆酸为前体物质合成了目标产物白藜芦醇。该研究为进一步实现白藜芦醇在酵母中的工业化生产奠定了基础。

The 4-coumarate: coenzyme A ligase gene(4CL) from Arabidopsis thaliana and resveratrol synthase gene(RS) from Vitis vinifera were cloned into the 2μ-based multicopy drug resistance marker plasmids pRS42K(G418 resistance) and pRS42H(hygromycin resistance).The recombinant plasmids were transformed into EC1118 cells using the LiAc / SS carrier DNA / PEG method.A shaking flask fermentation(25 ℃,150 r / min) of the engineered strain in YPD medium supplemented with 0.1 mmol / L p-coumarate every 24 h was performed.Under these conditions,this strain produced 0.78 mg / L resveratrol within 96 h.The result showed that the A.thaliana 4CL gene and V.vinifera RS gene were expressed in this recombinant strain to produce resveratrol from p-coumarate successfully.