| 具有a-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 |
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| 引用本文： | 张峰,王肇悦,刘楠,何秀萍,张博润.具有a-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[J].生物工程学报,2008,24(5):837-843. |
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| 作者姓名： | 张峰 王肇悦 刘楠 何秀萍 张博润 |
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| 作者单位： | 1. 中国科学院微生物研究所,北京,100101;中国科学院研究生院,北京,100039
2. 中国科学院微生物研究所,北京,100101 |
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| 摘 要： | 扣囊复膜酵母(Saccharomycopsis fibuligera)因具有较强的a-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a-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a-因子信号序列以及扣囊复膜酵母a-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a-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、a-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a-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a-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。
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| 关 键 词： | 啤酒酵母工程菌 a-淀粉酶 双乙酰 a信号肽 |
| 收稿时间： | 2007/9/26 0:00:00 |
| 修稿时间： | 2007年9月26日 |
Construction of a New Brewing Yeast Strain with Secretive a-amylase Activity and Reduced Diacetyl Production |
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| Feng Zhang,Zhaoyue Wang,Nan Liu,Xiuping He and Borun Zhang.Construction of a New Brewing Yeast Strain with Secretive a-amylase Activity and Reduced Diacetyl Production[J].Chinese Journal of Biotechnology,2008,24(5):837-843. |
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| Authors: | Feng Zhang Zhaoyue Wang Nan Liu Xiuping He and Borun Zhang |
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| Institution: | The Laboratory of Molecular Genetics and Breeding of Yeast, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; Graduate School of Chinese Academy of Sciences, Beijing 100039, China;The Laboratory of Molecular Genetics and Breeding of Yeast, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;The Laboratory of Molecular Genetics and Breeding of Yeast, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;The Laboratory of Molecular Genetics and Breeding of Yeast, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;The Laboratory of Molecular Genetics and Breeding of Yeast, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China |
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| Abstract: | Saccharomycopsis fibuligera possesses high alpha-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 (PGK1p), the alpha factor signal sequence from Saccharomyces cerevisiae and the alpha-amylase coding sequence of S. fibuligera was constructed. The alpha-amylase expression cassette was inserted in the ILV2 locus of industrial brewer's yeast strain YSF-5 encoding alpha-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 alpha-amylase activity, low AHAS activity and reduced diacetyl production. Effects of temperature, pH, and metal ions on the activity of the alpha-amylase expressed by the transformant were examined. The fermentation performance of host strain YSF-5 and the transformant was also examined. |
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