Data_Sheet_1_Developing a Cell-Free Extract Reaction (CFER) System in Clostridium thermocellum to Identify Metabolic Limitations to Ethanol Production.zip

The cellulolytic bacterium Clostridium thermocellum is a promising candidate for lignocellulosic biofuel production; however, ethanol titer needs to be improved for commercialization. To understand the factors limiting ethanol titer in C. thermocellum, we developed a cell-free extract reaction (CFER) system. We demonstrated that 15 mM cellobiose could be converted, in vitro, to 25 mM ethanol and that this reaction functions both at thermophilic (55°C) and mesophilic (37°C) temperatures. Although the yield was similar to that produced by whole cells, the rate was much slower (~0.5 vs. 12 mM/h). In order to reliably quantify metabolites, rapid CFER quenching is necessary. Among the methods tested, filtration with a 3-kDa molecular weight cutoff filter proved to be the most effective. Metabolomic analysis revealed high levels of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) in the CFER, identifying potential rate-limiting enzymes downstream of F6P. NADH was also found to accumulate in the CFER, suggesting that NADH recycling is rate-limiting. We used two complementary strategies to identify enzymes that limit metabolic flux, including feeding different substrates and supplementing with exogenous enzymes. In the enzyme addition experiment, the largest improvement was observed with the addition of yeast alcohol dehydrogenase (ADH), indicating a limitation at that reaction. The development of a CFER system for C. thermocellum, combined with detailed measurements of intermediate metabolites, allowed us to directly observe the metabolism of this organism, and suggested several potential metabolic engineering interventions for increasing ethanol titer. This demonstrates a technique that may be of general use for metabolic engineering in non-model organisms.

纤维素分解细菌热纤梭菌（Clostridium thermocellum）是木质纤维素生物燃料生产的极具潜力的候选菌株，但其乙醇滴度仍需进一步提升以满足商业化应用需求。为解析限制热纤梭菌乙醇滴度的关键因素，本研究构建了无细胞提取物反应（cell-free extract reaction, CFER）体系。研究证实，15 mM纤维二糖可在体外转化为25 mM乙醇，且该反应可在嗜热（55℃）与中温（37℃）条件下正常进行。尽管该反应的产物得率与完整细胞体系相近，但反应速率却显著更低（约0.5 mM/h vs. 12 mM/h）。为实现代谢物的准确定量，需对CFER体系进行快速淬灭处理。在所测试的多种方法中，采用3 kDa分子量截留滤膜进行过滤的效果最为优异。代谢组学分析显示，CFER体系中葡萄糖-6-磷酸（glucose-6-phosphate, G6P）与果糖-6-磷酸（fructose-6-phosphate, F6P）水平显著升高，由此明确了F6P下游潜在的限速酶靶点。研究还发现还原型烟酰胺腺嘌呤二核苷酸（NADH）在CFER体系中发生积累，提示NADH循环再生过程为代谢通量的限速步骤。本研究采用两种互补策略以筛选限制代谢通量的关键酶，包括饲喂不同底物与添加外源酶。在外源酶添加实验中，添加酵母乙醇脱氢酶（alcohol dehydrogenase, ADH）后乙醇产量提升最为显著，证明该反应步骤存在代谢瓶颈。热纤梭菌CFER体系的构建，结合中间代谢物的精准定量分析，使我们能够直接观测该菌株的代谢过程，并为提升乙醇滴度提出了多项潜在的代谢工程改造靶点。本研究建立的技术方法，有望在非模式生物的代谢工程研究中得到广泛应用。