紫穗槐-4,11-二烯合酶基因在酵母工程菌中具有协同作用

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：W303BpGADADS]、W303BpYGADS]、W303BpYGADS+pGADADS]、WK1pGADADS]、WK1pYGADS]和WK1pYG

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

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, W303BpGADADS], W303BpYGADS], W303BpYGADS+pGADADS], WK1pGADADS], WK1pYGADS] and WK1pYGADS+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.