中文摘要：

【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;从木酮糖激酶的转录水平、酶活水平、胞内的ATP浓度及木糖代谢等性状,对各菌株进行评价。【结果】转录及酶活测定结果显示,与天然状态相比,所选择的启动子对木酮糖激酶均表现出更强的启动效率。菌株体内表达木酮糖激酶活性水平由高至低的顺序为其基因XKS1在启动子PGK1p、TEF1p、HXK2p和XKS1p控制下。随着木酮糖激酶的活性的提高,胞内的ATP水平下降,而转化木糖生成乙醇的能力上升。最高乙醇产率为0.35 g/g消耗的总糖,此时副产物木糖醇产率最低,为0.18 g/g消耗的木糖。【结论】通过在染色体上置换启动子,提高了木酮糖激酶的表达水平。在一定范围内,木酮糖激酶的高活性有利于木糖向乙醇的转化。

英文摘要：

[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.