酵母工程菌制备紫穗槐-4,11-二烯的研究

孔建强 沈君豪 黄勇 王伟 程克棣 朱平

酵母工程菌制备紫穗槐-4,11-二烯的研究

孔建强沈君豪黄勇王伟程克棣朱平

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

KONG Jian-Jiang

摘要

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W303-1BpYeDP60/G/ ADS含有表达载体pYeDP60/G/ADS。整合体型工程菌W303-1BrDNA: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。

Abstract:
Plasmid-carrying Saccharomyces cerevisia (W303-1BpYeDP60/G/ADS) and genome-transformed S. cerevisia (W303-1BrDNA: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 W303-1BpYeDP60/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 W303-1BrDNA: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 W303-1BpYeDP60/G/ADS was higher than that of W303-1BrDNA:ADS. Southern blot analysis showed that there is only one copy of ADS gene in the genome of W303-1BrDNA:ADS. It implied that the amorpha-4,11-diene yield can be improved by increasing the ADS gene copies.

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