利用不同启动子控制木糖代谢关键酶基因构建可共代谢葡萄糖木糖重组酿酒酵母

摘要/Abstract

摘要： 利用不同强度的启动子调控木糖代谢关键酶活性，构建稳定代谢葡萄糖和木糖产乙醇的重组酿酒酵母。以本实验室专利菌株Saccharomyces cerevisiae Y5为宿主菌，将树干毕赤酵母Pichia stipitis CBS6054的木糖还原酶基因XYL1和木糖醇脱氢酶基因XYL2置于磷酸甘油酸激酶基因启动子（PGKp）控制下，酿酒酵母Y5内源的木酮糖激酶基因XKS1分别由己糖激酶基因启动子（HXK2p）及其内源启动子（XKS1p）控制。这3个基因连同各自表达元件导入宿主细胞中，打通其木糖上游代谢途径。酶活测定结果显示，HXK2p对木酮糖激酶表现出更强的启动效率。重组菌Y5-X3-1中木糖还原酶/木糖醇脱氢酶/木酮糖激酶（XR/XDH/XK）的酶活比值为1∶5∶4，其木糖消耗量是宿主菌的5倍，最高乙醇产量为24.35 g/L，达到理论值的73%。结果表明，通过调节XYL1、XYL2及XKS1启动子的强度，调控其表达水平，进而改变3种酶的活性水平，对于提高重组酿酒酵母利用木糖发酵产乙醇有明显效果。

关键词: 重组酿酒酵母, 木糖, 乙醇, 共发酵, 启动子

Abstract: In order to obtain a recombinant yeast strain which can co-ferment both xylose and glucose, the key genes of enzymes in the xylose metabolism were cloned and transformed into Saccharomyces. cerevisiae Y5. The XYL1 and XYL2 genes from Pichia stipitis CBS6054 were placed under the PGKp, which is a constitutive strong promoter；endogenesis XKS1 gene from S.cerevisiae Y5 was controlled by HXK2p and the native promoter XKS1p, respectively. We measured the activity of XR、XDH and XK and evaluated the xylose-fermenting abilities of the engineered strains. The highest xylulokinase activity was observed in the strain Y5-X3-1 whose XKS1 was controlled by HXK2p. The activity of enzyme ratio was 1∶5∶4 between XR, XDH and XK. Furthermore, the xylose consumption of S.cerevisiae Y5-X3-1 was 5 times as the host strain during co-fermentation, and the highest ethanol concentration was 24.35 g/L, which correspond to 73% of the theoretical value.

金怡, 王震, 莫春玲, 杨秀山, 田沈. 利用不同启动子控制木糖代谢关键酶基因构建可共代谢葡萄糖木糖重组酿酒酵母[J]. 生物技术通报, 2014, 0(2): 153-160.

Jin Yi, Wang Zhen, Mo Chunling, Yang XiuShan, Tian Shen. Construction of Recombinant Saccharomyces cerevisiae Strains Through Expressing the Key Genes in the Xylose Metabolism Under the Control of Different Promoters for Co-fermentation Glucose and Xylose[J]. Biotechnology Bulletin, 2014, 0(2): 153-160.

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