紫穗槐-4,11-二烯合酶基因在酵母工程菌中具有协同作用  

Synergistic effect of amorpha-4,11-diene synthase gene in engineered Saccharomyces cerevisiae

作　　者：孔建强[1] 支晓慧[1] 王伟[1] 程克棣[1] 朱平[1] 

机构地区：[1]中国医学科学院&北京协和医学院药物研究所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,北京100050

出　　处：《生物工程学报》2011年第2期196-202,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30701061);国家高技术研究发展计划(863计划)(No.2007AA021501);北京市自然科学基金(No.7082063);教育部博士点新教师基金(No.20070023077)资助~~

摘　　要：为了构建高产的紫穗槐-4,11-二烯酵母工程菌,主要探究了含紫穗槐-4,11-二烯合酶基因的不同表达载体在酵母工程菌中是否存在协同效应。首先构建了含紫穗槐-4,11-二烯合酶基因的酵母表达载体pGADADS,分别将pGADADS和pYeDP60/G/ADS转入酿酒酵母W303-1B和WK1中,获得6种能产生紫穗槐-4,11-二烯的酵母工程菌:W303B[pGADADS]、W303B[pYGADS]、W303B[pYGADS+pGADADS]、WK1[pGADADS]、WK1[pYGADS]和WK1[pYGADS+pGADADS]。对上述6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。结果显示,这6种酵母工程菌均能产生紫穗槐-4,11-二烯,其产率随着紫穗槐-4,11-二烯合酶基因拷贝数的增加而上升,且产生的紫穗槐-4,11-二烯对酵母工程菌没有产生毒性。导入多种ADS重组质粒后酵母工程菌的紫穗槐-4,11-二烯产率显著高于多个单质粒工程菌产生的紫穗槐-4,11-二烯之和。上述结果表明,同一宿主细胞中多种含紫穗槐-4,11-二烯合酶基因的表达载体之间存在明显的协同效应,这为构建青蒿素前体高产工程菌提供了一种思路。To construct an engineered Saccharomyces cerevisiae producing high titres of amorpha-4,11-diene,we investigated the possible synergistic effect of different vectors containing amorpha-4,11-diene synthase（ADS） gene within one yeast cell.We constructed the ADS recombinant plasmid pGADADS.This plasmid and another ADS recombinant plasmid pYeDP60/G/ADS were alone,or co-transformed into yeast Saccharomyces cerevisiae W303-1B and WK1,respectively,resulting in the following engineered yeasts,W303B[pGADADS],W303B[pYGADS],W303B[pYGADS＋pGADADS],WK1[pGADADS],WK1[pYGADS] and WK1[pYGADS＋pGADADS].All of the six strains were cultured for GC-MS analysis of amorpha-4,11-diene.The results showed that all of the engineered yeasts could produce amorpha-4,11-diene.The yield of the product was improved with increasing ADS gene copies while no deleterious effect on the strain growth was found.Moreover,the product yield of the engineered yeast co-transformed with multiple plasmids was much higher than the total yield of the different engineered yeasts with only one plasmid,respectively.In conclusion,there was a distinct synergistic effect between different recombinant ADS plasmids within one cell.Our results facilitate the construction of the engineered yeast with high yield of amorpha-4,11-diene,the precursor of artemisinin.

关 键 词：酵母工程菌 紫穗槐-4 11-二烯 紫穗槐-4 11-二烯合酶 协同作用 青蒿素 

分 类 号：Q78[生物学—分子生物学]