具有α-淀粉酶分泌活性以及低双乙酰产量的啤酒酵母工程菌的构建  

Construction of a New Brewing Yeast Strain with Secretive α-amylase Activity and Reduced Diacetyl Production

作　　者：张峰[1] 王肇悦[1] 刘楠[1] 何秀萍[1] 张博润[1] 

机构地区：[1]中国科学院微生物研究所

出　　处：《生物工程学报》2008年第5期837-843,共7页Chinese Journal of Biotechnology

摘　　要：扣囊复膜酵母(Saccharomycopsis fibuligera)因具有较强的α-淀粉酶以及葡聚糖酶活性,使其在以淀粉为唯一碳源的培养基上能够良好的生长。从其基因组中克隆了α-淀粉酶的编码区,构建了由酵母磷酸甘油酸激酶基因(PGK1)启动子、酿酒酵母α-因子信号序列以及扣囊复膜酵母α-淀粉酶基因编码序列组成的基因表达盒。将该表达盒插入到质粒pPLZ-2的ILV2基因序列内部,使其两翼具有ILV2基因的同源区。将该表达盒通过同源重组的方式整合到啤酒酵母工业菌株YSF-5的α-乙酰乳酸合成酶(AHAS)基因ILV2内部。在以淀粉为唯一碳源的培养基上进行转化子的筛选。通过多对引物PCR、α-淀粉酶活性以及AHAS活性分析对转化子进行鉴定,得到一株具有α-淀粉酶分泌表达活性、较低AHAS活性,并且发酵液中双乙酰产量也相对较低的啤酒酵母工程菌。该菌株在非选择压力条件下连续培养50代后仍然保持其遗传稳定性。还对pH、温度以及金属离子对该转化菌株的α-淀粉酶活性的影响进行了研究。由于所构建的菌株不含有非酵母来源的DNA,所以生物安全性相对较高,对酵母育种以及啤酒生产工业都具有较为重要的意义。Saccharomycopsisfibuligera possesses high α-amylase and glucoamylase activities that enable it to utilize raw starch as a carbon source. A expression cassette containing the promoter sequence of 3-phosphogylycerate kinase gene （PGKlp）, the a factor signal sequence from Saccharomyces cerevisiae and the α-amylase coding sequence of S. fibuligera was constructed. The α-amylase expression cassette was inserted in the ILV2 locus of industrial brewer＇s yeast strain YSF-5 encoding α-acetolactate synthase （AHAS） by homologous recombination. The transformed yeast strain was selected on the media with starch as the sole carbon source and verified by PCR. The transformant exhibited secretive α-amylase activity, low AHAS activity and reduced diacetyl production. Effects of temperature, pH, and metal ions on the activity of the α-amylase expressed by the transformant were examined. The fermentation performance of host strain YSF-5 and the transformant was also examined.

关 键 词：啤酒酵母工程菌 Α-淀粉酶 双乙酰 α信号肽 

分 类 号：TS262.5[轻工技术与工程—发酵工程]