后熟期短的酿酒酵母工程菌构建

Construction of A Strain of Saccharomyces cerevisiae with Less Maturation Time

借助质粒pBluescriptM13-,以淀粉酶基因(AMY)为筛选标记构建乙酰乳酸合成酶基因(ILV2)的重组整合质粒pMGI。用重组质粒pMGI所含ilv2∷AMY嵌合基因片段转化工业酿酒酵母菌Sc11,获得转化子(Sc11-ilv)。Sc-11-ilv不含细菌载体序列和酵母抗药性标记,所表达的乙酰乳酸合成酶活性降低30%;模拟发酵实验表明Sc11-ilv发酵液的双乙酰含量降低70%,Sc11-ilv发酵性能保持不变,遗传稳定性为100%,可以比较安全地用于啤酒生产。

An integrative plasmid pMGI carrying yeast ct-acetolactate synthase gene （ILV2）, that used the cloned amylase gene （AMY） as the selective marker, was constructed from pBluescript M 13^-. The ilv2：：AM expression cassette released from pMGI was transformed into an industrial strain ofSaccharomyces cerevisiae and integrated at the ILV2 gene of the host genome through one-step replacement, gaining transformants （Scll-ilv） free of undesired sequences （bacterial or viral vector sequences and yeast selective marker）. The ct-acetolactate synthase activity of the recombinant strain was lowered by 30%. Fermentation tests confirmed that the diacetyl concentration was reduced by 70% in fermented wort by the recombinant strain, while the brewing performances of the recombinant strain were retained. This type of integration is genetically stable even after 100 generations of cell multiplication under non-selective conditions and can be used in beer production safely.