大肠杆菌葡萄糖和木糖高效共利用表达机制的研究

Investigating the mechanisms of simultaneous and efficient glucose and xylose co-utilization in Escherichia coli will solve the problem of efficient fermentation of all sugar components of lignocellulosic biomass. In this study, the mechanisms of simultaneous and effiecient glucose and xylose co-utilization will be found on the basis of the engineered E.coli with higher glucose and xylose co-utilization rates.First, the PTS (PEP:carbohydrate phosphotransferase system) will be firstly inactivated to eliminate CCR (carbon catabolite repression) effect in E. coli. In order to improve the utilization rate of glucose and xylose, further efforts will be performed, including 1) combinatorial modulation of essential genes within glucose and xylose metabolic pathways through artificial regulatory parts libraries; 2) Site-directed mutagenesis and directed evolution of XylE transporter to increase xylose transport capability, followed by high-throughput screening; 3) utilization of metabolic evolution to increase glucose and xylose utilization rates. Finally, genome sequencing and transcriptome analysis will be done to investigate the engineered strains. The aims of this project are 1) elucidation of the mechanisms of simultaneous and efficient glucose and xylose co-utilization in Escherichia coli; 2) obtain an efficient E. coli strain which can co-metabolize glucose and xylose efficiently and can be used as a starting strain for production of renewable chemicals from lignocellulosic biomass.

解析大肠杆菌葡萄糖和木糖高效共利用表达机制，将解决木质纤维素水解产物微生物发酵高效利用的问题。本研究将在高效同步利用葡萄糖和木糖的工程菌株基础上揭示葡萄糖和木糖高效共利用表达机制。首先，通过基因敲除使葡萄糖转运系统PTS的失活，解除葡萄糖对木糖吸收利用的阻遏效应。接着，为进一步提高葡萄糖和木糖的吸收利用速率，将通过人工调控元件库对葡萄糖和木糖吸收代谢途径多基因进行协同表达；利用定点突变和定向进化构建木糖转运蛋白XylE突变子库；高通量筛选得到木糖转运性能提高的木糖转运蛋白；采用进化代谢加速自发突变优化细胞性能。最后，对进化代谢突变株进行转录组和重测序分析。通过本项目的研究，将解析大肠杆菌高效葡萄糖和木糖共利用表达机制，并获得高效同步利用葡萄糖和木糖的大肠杆菌工程菌，为利用木质纤维素水解液发酵生产各种可再生化学品提供底盘细胞。

为构建葡萄糖木糖同步高效共利用菌株，木糖转运蛋白xylFGH基因成功敲除，降低消耗ATP的消耗，有效提高细胞生长更多的能量，在葡萄糖木糖共代谢中增强木糖代谢速率。建立了木酮糖激酶双偶联法检测转酮酶TktA的新方法，有效解决了木糖代谢途径中转酮酶酶活检测问题。利用短小芽孢杆菌的木糖苷酶（xynB基因编码）及结构类似物pNPX（硝基酚-木吡喃糖苷），建立了木糖转运蛋白XylE突变96孔板高通量筛选方法。设计了优化木糖代谢途径的新方法，应用与木糖代谢途径基因的调控，得到了木糖代谢速率提高的菌株HQ304。木糖代谢速率的提高有助于葡萄糖和木糖高效共利用，显著提高了共代谢中木糖利用率,使木糖消耗速率提高2倍。构建出同步高效利用葡萄糖和木糖的底盘工程菌，为利用木质纤维素水解液发酵生产各种可再生化学品提供底盘细胞。