基于α-半乳糖苷酶基因为筛选标记的酿酒酵母诱导型表达载体的构建及应用

Construction of an inducible expression vector based on α-galactosidase gene as a selection marker for Saccharomyces cerevisiae and its application

以α-半乳糖苷酶基因为筛选标记,构建GAL1诱导型启动子介导的酿酒酵母表达载体YGM-α-gal质粒,将枯草芽孢杆菌的β-1,3-1,4-葡聚糖酶基因克隆到此载体中,构建质粒YGMPA-α-gal,转化宿主酵母后,实现β-1,3-1,4-葡聚糖酶在酿酒酵母中的分泌表达.结果表明:在2%半乳糖诱导下,摇瓶发酵24h后分泌表达的β-葡聚糖酶活性达到411.9U爛mL-1,而在培养60h后,发酵液中α-半乳糖苷酶活性可达64.2U爛mL-1.说明α-半乳糖苷酶基因可用作酿酒酵母表达载体转化的有效筛选标记,为食品级酿酒酵母表达系统的构建提供了新选择.

YGM-α-gal,an expression vector for Saccharomyces cerevisiae,was constructed containing α-galactosidase gene as selection marker and an inducible GAL1 promoter.The β-1,3-1,4-glucanase gene cloned from Bacillus subtilis was inserted into the multiple cloning sites of the plasmid YGM-α-gal to generate YGMPA-α-gal,then to transform the host yeast,and β-1,3-1,4-glucanase was efficiently expressed as a secretive protein in S.cerevisiae.The results indicated that the β-1,3-1,4-glucanase activity in the ferment liquid in shake flask was about 411.9 U·mL-1 after adding 2% galactose for 24 h,and the α-galactosidase activity reached 64.2 U·mL-1 after adding 2% galactose for 60 h.So the α-galactosidase gene can be used as an effective selection marker for screening the positive transformants of food-grade yeast expression system.