Clostridium cellulovorans is among the most promising candidates for consolidated bioprocessing (CBP) of cellulosic biomass to liquid biofuels (ethanol, butanol). C. cellulovorans metabolizes all the main plant polysaccharides and mainly produces butyrate. Since most butyrate and butanol biosynthetic reactions from acetyl-CoA are common, introduction of single heterologous alcohol/aldehyde dehydrogenase can divert the branching-point intermediate (butyryl-CoA) towards butanol production in this strain. However, engineering C. cellulovorans metabolic pathways towards industrial utilization requires better understanding of its metabolism. The present study aimed at improving comprehension of cellulose metabolism in C. cellulovorans by comparing growth kinetics, substrate consumption/product accumulation and whole-cell soluble proteome (data available via ProteomeXchange, identifier PXD015487) with those of the same strain grown on a soluble carbohydrate, glucose, as the main carbon source. Growth substrate-dependent modulations of the central metabolism were detected, including regulation of several glycolytic enzymes, fermentation pathways (e.g. hydrogenase, pyruvate formate lyase, phosphate transacetylase) and nitrogen assimilation (e.g. glutamate dehydrogenase). Overexpression of hydrogenase and increased ethanol production by glucose-grown bacteria suggest a more reduced redox state. Higher energy expenditure seems to occur in cellulose-grown C. cellulovorans (likely related to overexpression and secretion of (hemi-)cellulases), which induces up-regulation of ATP synthetic pathways, e.g. acetate production and ATP synthase. SIGNIFICANCE: C. cellulovorans can metabolize all the main plant polysaccharides (cellulose, hemicelluloses and pectins) and, unlike other well established cellulolytic microorganisms, can produce butyrate. C. cellulovorans is therefore among the most attractive candidates for direct fermentation of lignocellulose to high-value chemicals and, especially, n-butanol, i.e. one of the most promising liquid biofuels for the future. Recent studies aimed at engineering n-butanol production in C. cellulovorans represent milestones towards production of biofuels through one-step fermentation of lignocellulose but also indicated that more detailed understanding of the C. cellulovorans central carbon metabolism is essential to refine metabolic engineering strategies towards improved n-butanol production in this strain. The present study helped identifying key genes associated with specific catabolic reactions and indicated modulations of central carbon metabolism (including redox and energy balance) associated with cellulose consumption. This information will be useful to determine key enzymes and possible metabolic bottlenecks to be addressed towards improved metabolic engineering of this strain.

中文翻译：

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通过比较蛋白质组学方法研究纤维素纤维梭菌的纤维素代谢。

纤维素梭菌是将纤维素生物质整合为液体生物燃料（乙醇，丁醇）的最有前途的候选产品之一。C. cellulovorans代谢所有主要的植物多糖，并主要产生丁酸。由于大多数来自乙酰辅酶A的丁酸酯和丁醇生物合成反应是常见的，因此引入单个异源醇/醛脱氢酶可将分支点中间体（丁酰CoA）转移至该菌株的丁醇生产中。但是，工程化C.cellulovorans代谢途径向工业利用需要对其代谢有更好的了解。本研究旨在通过比较生长动力学来提高对C.cellulovorans中纤维素代谢的理解，底物消耗/产品积累和全细胞可溶性蛋白质组（可通过ProteomeXchange获得的数据，标识号PXD015487），与以可溶性碳水化合物，葡萄糖为主要碳源生长的同一菌株的蛋白质相同。检测到中央代谢的生长底物依赖性调节，包括几种糖酵解酶，发酵途径（例如氢化酶，丙酮酸甲酸裂解酶，磷酸转乙酰酶）和氮同化（例如谷氨酸脱氢酶）的调节。葡萄糖生长细菌过度表达氢化酶和增加乙醇生产，表明氧化还原状态更加降低。纤维素生长的C. cellulovorans中似乎发生了更高的能量消耗（可能与（半）纤维素酶的过度表达和分泌有关），这会诱导ATP合成途径的上调。生成乙酸盐和ATP合酶。意义：C.纤维素纤维素可以代谢所有主要的植物多糖（纤维素，半纤维素和果胶），并且与其他成熟的纤维素分解微生物不同，它可以生成丁酸酯。因此，C.cellulovorans是将木质纤维素直接发酵成高价值化学品，尤其是正丁醇（即未来最有希望的液体生物燃料之一）的最有吸引力的候选对象之一。旨在工程化纤维素丙三醇的正丁醇生产的最新研究代表了通过木质纤维素一步发酵生产生物燃料的里程碑，但同时也表明，对纤维素丙三醇中心碳代谢的更详细的了解对于完善代谢工程策略以改善其发展至关重要。该菌株中正丁醇的生产。本研究有助于鉴定与特定分解代谢反应有关的关键基因，并表明与纤维素消耗有关的中央碳代谢（包括氧化还原和能量平衡）的调节。该信息将有助于确定关键酶和可能的代谢瓶颈，以解决该菌株改善的代谢工程问题。