孔建强，支晓慧，王伟，程克棣，朱平

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

紫穗槐-4,11-二烯合酶基因在酵母工程菌中具有协同作用

孔建强，支晓慧，王伟，程克棣，朱平

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

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。

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

Abstract:To construct an engineered Saccharomyces cerevisiae producing high titres of amorpha-4,11-diene, we investigated thepossible 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.

Keywords:engineered yeast, amorpha-4,11-diene, amorpha-4,11-diene synthase, synergistic effect, artemisinin

近几年，随着青蒿素生物合成途径的逐渐明晰[1]，以及青蒿素生物合成相关基因的成功克隆[2-7]，极大地促进了青蒿素合成生物学的发展[6,8]。……