产白藜芦醇大肠杆菌基因工程菌的构建

Construction of genetically engineering Escherichia coli for resveratrol production

摘要: 利用降落重叠延伸PCR和连接酶连接的方法,构建了组成型表达载体pMD18-VvRs-At4cl,该表达载体带有大肠杆菌甘油醛三磷酸脱氢酶启动子（pGAP）,拟南芥（Arabidopsis thalianna）4-香豆酸辅酶A连接酶（At4CL）基因和葡萄（Vitis vinifera）白藜芦醇合酶（VvRS）基因。表达载体转化大肠杆菌BW27784,并通过PCR鉴定和酶切鉴定后,获得重组菌株。对重组菌株进行发酵培养,对发酵液进行HPLC检测,发酵液中白藜芦醇含量为4.51mg/L。这表明,拟南芥的4cl基因与金手指葡萄的rs基因在重组大肠杆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白藜芦醇。

Abstract: To construction of expression plasmid pMD18- Vvrs- At4cl by touchdown overlap extension PCR and ligase connection.The constitutive vector containing a gap promoter (pGAP) of Escherichia coli and 4- coumaroyl:CoA ligase gene of Arabidopsis thalianna (At4CL) and resveratrol synthase gene of Vitis vinifera, (VvRS) , which transformed into host strain E.coli BW27784.Next, with fermentation and HPLC detection, created an recombination E.coli strain caple of bioproducing resveratrol by additionally supplemented with p-coumaric acid and resulted in a final titer of 4.51mg /L resveratrol.