Cytoplasmic expression of the Aspergillus glucoamylase gene integrated into a linear plasmid in Saccharomyces cerevisiae

Journal of Fermentation and Bioengineering Pub Date : 1998-01-01 DOI:10.1016/S0922-338X(98)80133-5

Kazuaki Tomoike , Keisuke Ekino , Mariko Takeshita , Masatoshi Goto , Sadazo Yoshino , Kohsai Fukuda , Norio Gunge , Kensuke Furukawa

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引用次数: 2

Abstract

The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was successfully expressed in cytoplasm of Saccharomyces cerevisiae, using two cytoplasmic linear plasmids, pGKL1 and pGKL2. In order to integrate the glaA gene, two pGKL1-derived plasmids, designated pKTF951 and pKTF952, were constructed. The glaA gene was placed downstream of the ORF2 promoter (UCS2) between ORF1 and ORF3 of pGKL1 to obtain pKTF951. pKTF952 contained an additional ORF5 promoter (UCS5) from pGKL2, connected downstream of the UCS2 in pKTF951. pKTF951 and pKTF952 were respectively transformed to S. cerevisiae carrying the original pGKL1 and pGKL2. Southern analysis revealed that in vivo homologous recombination took between the indigenous pGKL1 and the recombinant pKTF951 or pKTF952 within the common ORF1 and ORF3 regions, which resulted in the replacement of ORF2 by the glaA gene in pGKL1. Transformants carrying the resultant linear recombinant plasmids, pNS951 or pNS952, produced GAI. S. cerevisiae (pNS952) secreted 2.6-fold more GAI than S. cerevisiae (pNS951).

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曲霉糖淀粉酶基因在酿酒酵母细胞质粒中的表达

利用两个细胞质线性质粒pGKL1和pGKL2，成功地在酿酒酵母细胞质中表达了川achi曲霉糖淀粉酶I (GAI)的glaA基因。为了整合glaA基因，构建了两个pgkl1衍生的质粒pKTF951和pKTF952。将gla基因置于ORF2启动子(UCS2)下游，介于pGKL1的ORF1和ORF3之间，获得pKTF951。pKTF952含有一个额外的来自pGKL2的ORF5启动子(UCS5)，连接在pKTF951中UCS2的下游。将pKTF951和pKTF952分别转化为携带原pGKL1和pGKL2的酿酒葡萄球菌。Southern分析显示，在ORF1和ORF3共同区域内，pGKL1与重组基因pKTF951或pKTF952发生了体内同源重组，导致pGKL1中的gla基因取代ORF2。携带线性重组质粒pNS951或pNS952的转化子产生GAI。酿酒葡萄球菌(pNS952)分泌的GAI是酿酒葡萄球菌(pNS951)的2.6倍。

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Journal of Fermentation and Bioengineering

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