嗜热芽孢杆菌木糖厌氧发酵制备乙醇新代谢途径的研究

An effective ethanol production from xylose is one of the critical techniques in biotechnological production of ethanol using all components of cellulosic material. There had been not new breakthrough for many years. The existing yeast and engineering bacteria presented a wide variety of problems, such as the difficult control in industrical production and the low yield. Recently we found the xylose metabolism pathway of Bacillus thermophilus was single and the convertion yield from sugar to acid was higher. In this study, the upstream pathway of near-perfect xylose anaerobic metabolism was utilized and an new metabolic pathway was developed. Using gene knock-out technique, the assignment of metabolic flux was changed, the lactic acid production by Bacillus thermophilus was cut off for the ethanol production from xylose under anaerobic conditions. This research not only could reveal xylose anaerobic metabolism mechanism of Bacillus thermophilus, but also makes a practical support and theoretial basis for the metalbolic engineering of biochemical production using cellulosic material by Bacillus thermophilus. Additionally, a new method was proposed for the pathway reconstruction of ethanol efficient production form xylose by this study.

木糖高效生产乙醇是植物纤维原料全组分利用的难点之一，多年来一直未能实现有效突破。现有天然酵母及工程菌进行木糖发酵乙醇存在工业生产调控难度大、乙醇产率低等多方面问题，前期工作发现用于乳酸发酵的嗜热芽孢杆菌木糖厌氧代谢具有途径单一，糖酸转化率高的优势。本项目拟借助嗜热芽孢杆菌近乎完美的木糖厌氧代谢上游途径，采用基因敲除技术进行代谢途径改造，截断或抑制乳酸生成途径，改变代谢通量分配，打通乙醇合成途径，最终实现厌氧条件下木糖生产乙醇。项目的研究成果不仅有利于深入揭示嗜热芽孢杆菌木糖厌氧代谢机理，还可以为嗜热芽孢杆菌利用植物纤维原料合成生物化学品的代谢调控奠定理论基础和实践支撑，并为木糖高效生产乙醇的途径改造提供新思路。

本项目以能高效利用木糖的嗜热芽孢杆菌为研究对象，通过等离子体诱变育种技术进一步提高了嗜热芽孢杆菌木糖厌氧发酵制备乳酸性能；对嗜热芽孢杆菌NL01进行全基因组测序获得GC含量为46.23%的144个Contig，基因组全长为3,505,081 bp；考察了厌氧条件下嗜热芽孢杆菌葡萄糖和木糖转录谱差异，对差异表达的793个基因进一步筛选分析获得12个高丰度差异表达的基因作为该菌株高效木糖代谢途径中的关键基因进行荧光定量PCR验证，并初步探索了嗜热芽孢杆菌遗传操作平台和基因敲除体系。项目还基于大肠杆菌体系采用基因敲除技术进行代谢途径改造，截断或抑制代谢旁路途径：丙酮酸甲酸裂解酶（pflB）、D-乳酸脱氢酶（ldhA），乙酸激酶（adhE）、磷酸转乙酰酶（pta）和富马酸还原酶（frdA），导入嗜热芽孢杆菌木糖厌氧代谢关键基因木糖异构酶、木酮糖激酶和乳酸脱氢酶，重构了厌氧条件下木糖代谢途径，实现厌氧条件下木糖生产乙醇和乳酸，大幅度提高了宿主菌木糖转化效率。项目的研究成果不仅有利于深入揭示嗜热芽孢杆菌木糖厌氧代谢机理，还可以为利用植物纤维原料合成生物化学品的代谢调控奠定理论基础和实践支撑，并为木糖高效生产乙醇的途径改造提供启发。