利用高温菌进行纤维素乙醇发酵具有诸多潜在优势。当前提高厌氧菌乙醇产率的主要代谢工程策略是敲除乳酸脱氢酶(LDH)和磷酸乙酰转移酶（PTA）基因，阻断与乙醇代谢途径竞争碳源和还原力的乳酸和乙酸生成途径。但由于ldh和pta在高温厌氧菌中的生理功能及代谢调控机制尚不清楚，导致该策略的改进缺乏理论指导，工程菌的乙醇产量仍然很低。本项目以嗜热厌氧杆菌Rx1为出发菌株，单独或连续敲除ldh和pta基因，在关键酶的活性、基因表达和代谢通量水平上研究ldh和pta基因对糖酵解和乙醇发酵途径的代谢调控作用，再通过分析突变株细胞内NADH/NAD+、ATP/ADP比率与代谢调控作用的关系，进而阐明ldh和pta基因的代谢调控机理。本研究将有助于揭示代谢旁路的阻断对高温厌氧菌中心碳代谢产生的影响及其机理、阐明限制工程菌株乙醇产量的原因。可望为高温厌氧菌的代谢工程改造提供新的靶点和理论依据。

Ethanol fermentation by thermophilic anaerobic cellulolytic bacteria has many potential advantages. Blocking the byproducts metabolic pathway by gene knockout method is a main strategy for enhancing the ethanol yeild of anaerobic bacteria. However, the physiological function and metabolic regulation of relative genes remain unknown. Thus, we are lacking theoretical guidance to improve such strategy, and the alcohol production of engineering bacteria is still at a very low level. In this project, a newly isolated Thermoanerobacterium sp. Rx1 was employed as the original bacteria, with its lactate dehydrogenase gene (pta)and phosphate acetyl transferase gene (ldh) either individually or continuously knocked out. By this means, the metabolic pathway of the byproducts, namely, lactate and acetate, was blocked. The effects of target genes on growth were revealed by analyzing the carbon utilization rate, glycolysis and growth rate of the mutant strain. Next, the effects of gene knockout on the activity of key enzymes, their expression levels, the distribution of products and metabolic flux were then tested. We also analysis its relationship with intracellular oxidation-reduction levels (NADH/NAD). Therefore, the metabolism regulating effects and the mechanism for genes ldh and pta in thermophilic anaerobic bacteria were demonstrated. This project revealed the metabolic regulation mechanism of ldh and pta genes on the level of gene expression, protein and metabolism, and provided important theoretical basis for metabolic engineering modification of thermophilic anaerobic bacteria.