Peng Bingyin, Chen Xiao, Shen Yu, Bao Xiaoming
The State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
Wei Sheng Wu Xue Bao. 2011 Jul;51(7):914-22.
To investigate xylose metabolism in the Saccharomyces cerevisiae stains overexpressing the xylulokinase gene XKS1 at different levels by replacing the promoter in the chromosome.
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.
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 PGKlp, 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.
By promoter replacement, xylulokinase was overexpressed at different levels. In this work, higher expressional level of xylulokinase improved the conversion of xylose to ethanol.
通过替换染色体上的启动子，研究不同水平过表达木酮糖激酶基因XKS1的酿酒酵母菌株中的木糖代谢。
基于酿酒酵母CEN.PK 113-5D，构建了木糖代谢菌株，其中木酮糖激酶基因XKS1的启动子在染色体上被TEF1启动子、PGK1启动子和HXK2启动子取代。我们定量了XKS1基因的转录水平（累积mRNA），并测量了各菌株中木酮糖激酶的活性。此外，我们还测定了细胞内ATP水平，并评估了工程菌株的木糖发酵能力。
在转录和酶活性水平上，工程菌株均表现出比亲本菌株更高的木酮糖激酶表达。在XKS1由PGK1p控制的菌株中观察到最高的木酮糖激酶活性，其次是XKS1由TEF1p、HXK2p和天然启动子控制（活性）依次降低的菌株。木酮糖激酶的表达水平与细胞内ATP水平呈负相关，与木糖产乙醇能力呈正相关。最高乙醇产量为0.35 g/g消耗糖，同时观察到最低木糖醇产量，为0.18 g/g消耗木糖。
通过启动子替换，木酮糖激酶在不同水平上实现了过表达。在本研究中，木酮糖激酶较高的表达水平提高了木糖向乙醇的转化。
