Tang Yinjie J, Sapra Rajat, Joyner Dominique, Hazen Terry C, Myers Samuel, Reichmuth David, Blanch Harvey, Keasling Jay D
Virtual Institute for Microbial Stress and Survival, Berkeley, California, USA.
Biotechnol Bioeng. 2009 Apr 1;102(5):1377-86. doi: 10.1002/bit.22181.
A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to high ethanol concentrations (10%, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner-Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accurately determined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)(-1) h(-1)) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64 +/- 3 to 25 +/- 2 and from 30 +/- 2 to 19 +/- 2, respectively. The carbon flux under micro-aerobic growth was directed to ethanol, L-lactate (> 99% optical purity), acetate, and formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38 +/- 0.07 mol mol(-1) glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yield by approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.
最近发现的嗜热细菌嗜热栖热放线菌M10EXG能发酵多种戊糖（如木糖）和己糖（如葡萄糖），并能耐受高浓度乙醇（10%，v/v）。我们利用体外酶分析和基于13C的通量分析研究了这种细菌的中心代谢，以深入了解这种极端微生物的生理特性，并探索其代谢在生物乙醇或其他生物工艺应用中的潜力。我们的研究结果表明，嗜热栖热放线菌M10EXG中的葡萄糖代谢通过糖酵解、磷酸戊糖途径和三羧酸循环进行；未检测到Entner-Doudoroff途径和转氢酶活性。回补反应（包括乙醛酸循环、丙酮酸羧化酶和磷酸烯醇式丙酮酸羧激酶）是活跃的，但使用氨基酸标记无法准确测定这些途径的通量。当生长条件从有氧切换到微需氧条件时，通过三羧酸循环和氧化磷酸戊糖途径的通量（基于归一化葡萄糖摄取率100单位（g DCW）（-1）h（-1））分别从64±3降至25±2和从30±2降至19±2。微需氧生长条件下的碳通量流向乙醇、L-乳酸（光学纯度>99%）、乙酸盐和甲酸盐。在完全厌氧条件下，嗜热栖热放线菌M10EXG采用混合酸发酵过程，葡萄糖的最大乙醇产率为0.38±0.07 mol mol（-1）。计算机通量平衡模型表明，嗜热栖热放线菌M10EXG产生的乳酸和乙酸盐使最大乙醇产率降低了约三倍，因此表明这两条途径都应进行改造以实现乙醇产量最大化。
