【作者】 孔建强； 王丽娜； 支晓慧； 程克棣； 王伟； 朱平；

【Author】 Kong Jianqiang Wang Lina Zhi Xiaohui Cheng Kedi Wei Wang Ping Zhu (Institute of Materia Medica,Chinese Academy of Medical Sciences & Peking Union Medical College(Key Laboratory of Biosynthesis of Natural Products,Ministry of Health of PRC & Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine,Ministry of Education of PRC),Beijing,100050)

【机构】 中国医学科学院； 北京协和医学院(卫生部天然药物生物合成重点实验室&中草药物质基础与资源利用教育部重点实验室)；

【摘要】 克隆了四种鲨烯合酶基因片段,将其反向克隆到酿酒酵母表达载体pYeDP60/G中,获得四种含反义鲨烯合酶基因片段的酿酒酵母表达载体:pYeDP60/G/SQS12,pYeDP60/G/SQS34,pYeDP60/G/SQS56和pYeDP60/G/SQS78。将上述四个表达载体分别和pGADADS,pGBT9/A/HMG/G/FPP共转化酿酒酵母W303-1B和WK1,获得含反义鲨烯合酶基因片段的紫穗槐-4,11-二烯酵母工程菌。对上述四种酵母工程菌进行发酵培养,通过GC-MS对发酵产物进行检测,结果发现,这四种酵母工程菌均能产生紫穗槐-4,11-二烯,含反义鲨烯合酶基因片段的酵母工程菌中紫穗槐-4,11-二烯的产量比对照要高,鲨烯含量略有下降。实验表明反义鲨烯合酶基因的反义RNA确实能抑制鲨烯合酶基因的表达,从而促进代谢流流向紫穗槐-4,11-二烯合成途径,增加了紫穗槐-4,11-二烯的产量。更多还原

【Abstract】 Fragments of yeast squalene synthase(SQS) gene were amplified and cloned reversly into the expression vector pYeDP60/G resulting in four plasmids,pYeDP60/G/SQS12,pYeDP60/G/SQS34, pYeDP60/G/SQS56 and pYeDP60/G/SQS78.The four plasmids were cotransformed into Saccharomyces cerevisiae W303-1B and WK1,respectively,with plasmids pGADADS and pGBT9/A/HMG/G/FPP to form ten kinds of engineered Saccharomyces cerevisiae containing antisense RNA of SQS gene.GC-MS results identified all of the engineered yeasts can produce amorpha-4,11-diene and the production in engineered yeasts containing antisense-RNA of SQS gene were higher than that of the control yeasts.The squalene production in engineered yeasts harboring antisense RNA of SQS gene was a litter lower than that of the control yeasts.These results showed the antisense RNA of SQS gene do improve the amorpha-4,11-diene production in engineered yeasts.更多还原