酵母工程菌制备紫穗槐-4,11-二烯的研究  

Production of amorpha-4,11-diene in engineered yeasts

作　　者：孔建强[1] 沈君豪[1] 黄勇[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：《药学学报》2009年第11期1297-1303,共7页Acta Pharmaceutica Sinica

基　　金：科技部"863"重点项目资助项目(2007AA021501);国家自然科学基金资助项目(30701061);北京市自然科学基金资助项目(7082063);教育部博士点新教师基金资助项目(20070023077);药物所基本科研业务费资助项目(2006QN05)

摘　　要：构建了两种能产生紫穗槐-4,11-二烯的酿酒酵母工程菌,其中附加体型工程菌W303-1B[pYeDP60/G/ADS]含有表达载体pYeDP60/G/ADS。整合体型工程菌W303-1B[rDNA:ADS]是将ADS基因的表达盒序列通过同源重组的方式整合到酿酒酵母W303-1B基因组中。GC-MS检测发酵产物,结果表明这两种工程菌均能产生紫穗槐-4,11-二烯,但附加体型工程菌产生的紫穗槐-4,11-二烯的产量要高于整合体型工程菌的产量。Southern杂交检测表明,ADS基因以单拷贝的形式整合到W303-1B基因组中,低于附加体型工程酵母中的ADS基因拷贝数。这些结果表明,ADS基因的拷贝数与酵母工程菌中紫穗槐-4,11-二烯的产量呈正相关。Plasmid-carrying Saccharomyces cerevisia （W303-1B[pYeDP60/G/ADS]） and genome-transformed S. cerevisia （W303-1B[rDNA：ADS]）, both harboring amorpha-4,11-diene synthase （ADS） gene were constructed to investigate the production of amorpha-4,11-diene. The recombinant plasmid pYeDP60/G/ADS that harbors the ADS gene was transformed into S. cerevisiae W303-1B, resulting in the engineered yeast W303-1B[pYeDP60/G/ ADS], which contains multi-copies of the plasmid. The ADS gene expression cassette was obtained by PCR amplification of the pYeDP60/G/ADS template, and then introduced into S. cerevisiae W303-1B to obtain the engineered yeast W303-1B[rDNA：ADS], in which the ADS gene was integrated into the rDNA locus of the yeast genome through the homologous recombination. GC-MS analysis confirmed that both of the engineered yeasts could produce amorpha-4,11-diene. Moreover, the amorpha-4,11-diene yield of W303-1B[pYeDP60/G/ADS] was higher than that of W303-1B[rDNA：ADS]. Southern blot analysis showed that there is only one copy of ADS gene in the genome of W303-1B[rDNA：ADS]. It implied that the amorpha-4,11-diene yield can be improved by increasing the ADS gene copies.

关 键 词：紫穗槐-4 11-二烯合酶 酵母工程菌 紫穗槐-4 11-二烯 

分 类 号：R915[医药卫生—微生物与生化药学]