Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
BMC Microbiol. 2012 Sep 21;12:214. doi: 10.1186/1471-2180-12-214.
Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.
Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.
Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.
产热梭菌能够直接将纤维素生物质转化为氢气、乙醇以及二氧化碳、乙酸盐、甲酸盐和乳酸盐。因此，它是通过整合生物加工生产生物燃料的理想模型。优化终产物产率和浓度对于使生物燃料生产具有经济可行性至关重要。相对蛋白质表达谱可能为代谢工程提供目标，而了解在碳限制、pH 值和生长阶段下蛋白质表达和代谢的变化可能有助于优化反应器。我们对细胞外培养物进行了分批式纤维二糖生长的产热梭菌细胞的 2D-HPLC-MS/MS Shotgun 分析，以确定参与碳水化合物利用、能量守恒和终产物合成的核心代谢蛋白的相对蛋白质表达谱。使用 iTRAQ（相对和绝对定量的同重同位素标记）基于蛋白质定量来确定核心代谢蛋白对生长阶段的响应变化。
相对丰度谱揭示了具有催化平行途径能力的假定酶的不同水平。除了醛脱氢酶、依赖于铁氧还蛋白的 Ech 型[NiFe]-氢化酶和 RNF 型 NADH：铁氧还蛋白氧化还原酶外，大多数参与丙酮酸分解代谢和终产物合成的蛋白质都以高丰度检测到。使用 4 重 2D-HPLC-MS/MS，在从指数期到静止期的过渡过程中，检测到的 144 种核心代谢蛋白中有 24%的表达发生了中等变化。值得注意的是，在静止期，参与丙酮酸合成的蛋白质减少，而参与糖原代谢、丙酮酸分解代谢和终产物合成的蛋白质增加。几种可能直接决定终产物合成模式的蛋白质，包括丙酮酸：铁氧还蛋白氧化还原酶、醇脱氢酶和一种假定的分叉氢化酶，在从指数期到静止期的过渡过程中表现出不同的表达。
相对表达谱表明哪些蛋白质可能用于碳水化合物利用和终产物合成，并表明 H2 的合成是通过分叉氢化酶进行的，而乙醇的合成主要由双功能醛/醇脱氢酶催化。核心代谢蛋白对生长阶段的响应差异可能决定碳和电子流向能量储存化合物和终产物。结合相对蛋白质表达水平及其对生理条件的响应变化的知识可能有助于有针对性的代谢工程策略和发酵条件的优化，以提高生物燃料的生产。
