紫穗槐-4,11-二烯合酶基因在酵母工程菌中具有协同作用

摘要

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.%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茵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-IB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茵的紫穗槐-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茵提供了一种思路.