不同启动子控制下木酮糖激酶的差异表达及其对酿酒酵母木糖代谢的影响

Effect of controlled overexpression of xylulokinase by different promoters on xylose metabolism in Saccharomyces cerevisiae

摘要:

摘要:【目的】以不同强度的启动子控制表达木酮糖激酶基因，并研究其引起的不同木酮糖激酶活性水平对木糖利用酿酒酵母( Saccharomyces cerevisiae) 代谢流向的影响。【方法】以酿酒酵母CEN． PK 113-5D 为出发菌株，选择酿酒酵母内源启动子TEF1p，PGK1p 和HXK2p，利用Cre-loxP 无标记同源重组系统，置换染色体上木酮糖激酶基因XKS1 的启动子(XKS1p) 序列;并通过附加体质粒引入木糖代谢上游途径，构建不同水平表达木酮糖激酶的木糖利用工程菌株;从木酮糖激酶的转

关键词:关键词:酿酒酵母，启动子，木酮糖激酶，木糖，乙醇

Abstract:

Abstract: ［Objective］To investigate xylose metabolism in the Saccharomyces cerevisiae stains overexpressing the xylulokinase gene XKS1 at different levels by replacing the promoter in the chromosome.［Methods］ Based on S．cerevisiae CEN．PK 113-5D，we constructed xylose-metabolizing strains where the promoter of xylulokinase gene XKS1 was replaced by TEF1 promoter，PGK1 promoter and HXK2 promoter on the chromosome． We quantitated the transcriptional level of XKS1 gene ( accumulated mRNA) and measured the activity of xylulokinase in each stains． Furthermore，we also determined the intracellular level of ATP and evaluated the xylose-fermenting abilities of the engineered strains．［Results］ The engineered strains exhibited higher expression of xylulokinase than the parental strain at both transcription and enzyme activity levels． The highest xylulokinase activity was observed in the strain whose XKS1 was controlled by PGK1p，and was decreasingly followed by the strains whose XKS1 was controlled by TEF1p，HXK2p and native promoter．The expression level of xylulokinase negatively correlated with intracellular level of ATP and positively correlated with ability of ethanol production from xylose． The highest ethanol yield was 0. 35 g / g consumed sugars while the lowest xylitol yield，which was 0. 18 g / g consumed xylose，was observed． ［Conclusion］ By promoter replacement，xylulokinase was overexpressed at different levels． In this work，higher expressional level of xylulokinase improved the conversion of xylose to ethanol．

Key words:Keywords: Saccharomyces cerevisiae，promoter，xylulokinase，xylose，ethanol