Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain
Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain
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DOI：
10.1002/bit.22181
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发表时间：
2009-04-01
影响因子：
3.8
通讯作者：
Keasling, Jay D.
中科院分区：
文献类型：
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作者：
Tang, Yinjie J.;Sapra, Rajat;Keasling, Jay D.
一种新近发现的嗜热菌，热葡萄糖苷地杆菌M10EXG，能发酵一系列C5(如木糖)和C6糖(如葡萄糖)，并能耐受高浓度的乙醇(10%，v/v)。我们利用体外酶分析和基于C-13的通量分析对该细菌的中心代谢进行了研究，以深入了解该极端细菌的生理特性，并探索其在生物乙醇或其他生物过程中的代谢应用。我们的发现表明，高温葡萄糖苷杆菌M10EXG中的葡萄糖代谢通过糖酵解、戊糖磷酸途径和TCA循环进行，未检测到Entner-Doudoroff途径和转氢酶活性。逆转录反应(包括乙醛分流、丙酮酸羧化酶和磷酸烯醇式丙酮酸羧酸激酶)是活跃的，但通过这些途径的通量不能用氨基酸标记准确地测定。当生长条件从好氧条件切换到微好氧条件时，通过三氯乙酸循环和磷酸戊糖途径的通量(以此为基础)分别从30+/-2减少到25+/-2和从30+/-2减少到19+/-2。微好氧生长条件下的碳通量指向乙醇、L乳酸盐(光学纯度99%)、乙酸盐和甲酸盐。在全厌氧条件下，热葡萄糖苷杆菌M10EXG采用混合酸发酵工艺，最高酒精产量为0.38+/-0.07摩尔摩尔(-1)葡萄糖。在硅通量平衡模型中显示，热葡萄糖苷菌M10EXG的乳酸和乙酸酯的产生使最大乙醇产量减少了大约三倍，因此表明这两条途径都应该被修改，以最大限度地提高乙醇产量。
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 C-13-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 he accurately determined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on it normalized glucose uptake rate of 100 units (gDCW) (1)h (1)) through the TCA cycle and oxidative pentose phosphate pathway were reduced front 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.