Abteilung Mikrobiologie, Institut für Biowissenschaften, Universität Rostock, Albert-Einstein-Str. 3, 18051, Rostock, Germany.
Appl Microbiol Biotechnol. 2012 Nov;96(3):749-61. doi: 10.1007/s00253-012-4112-2. Epub 2012 May 12.
Solventogenic clostridia are characterised by their biphasic fermentative metabolism, and the main final product n-butanol is of particular industrial interest because it can be used as a superior biofuel. During exponential growth, Clostridium acetobutylicum synthesises acetic and butyric acids which are accompanied by the formation of molecular hydrogen and carbon dioxide. During the stationary phase, the solvents acetone, butanol and ethanol are produced. However, the molecular mechanisms of this metabolic switch are largely unknown so far. In this study, in silico, in vitro and in vivo analyses were performed to elucidate the function of the CAC2713-encoded redox-sensing transcriptional repressor Rex and its role in the solventogenic shift of C. acetobutylicum ATCC 824. Electrophoretic mobility shift assays showed that Rex controls the expression of butanol biosynthetic genes as a response to the cellular NADH/NAD(+) ratio. Interestingly, the Rex-negative mutant C. acetobutylicum rex::int(95) produced high amounts of ethanol and butanol, while hydrogen and acetone production were significantly reduced. Both ethanol and butanol (but not acetone) formation started clearly earlier than in the wild type. In addition, the rex mutant showed a de-repression of the bifunctional aldehyde/alcohol dehydrogenase 2 encoded by the adhE2 gene (CAP0035) as demonstrated by increased adhE2 expression as well as high NADH-dependent alcohol dehydrogenase activities. The results presented here clearly indicated that Rex is involved in the redox-dependent solventogenic shift of C. acetobutylicum.
溶剂梭菌的特征是具有两相发酵代谢，主要的最终产物正丁醇因其可用作优质生物燃料而具有特殊的工业意义。在指数生长期，丙酮丁醇梭菌合成乙酸和丁酸，同时伴随着氢气和二氧化碳的形成。在静止期，会产生丙酮、丁醇和乙醇等溶剂。然而，到目前为止，这种代谢转换的分子机制在很大程度上仍不清楚。在这项研究中，通过计算机模拟、体外和体内分析来阐明 CAC2713 编码的氧化还原感应转录阻遏物 Rex 的功能及其在丙酮丁醇梭菌 ATCC 824 溶剂形成中的作用。凝胶电泳迁移率变动分析显示，Rex 作为对细胞 NADH/NAD(+) 比值的响应控制丁醇生物合成基因的表达。有趣的是，Rex 缺失突变体 C. acetobutylicum rex::int(95)产生了大量的乙醇和丁醇，而氢气和丙酮的产量则显著降低。与野生型相比，乙醇和丁醇（但不是丙酮）的形成明显更早开始。此外，如通过增加的 adhE2 表达以及高 NADH 依赖性醇脱氢酶活性所示，rex 突变体还显示出双功能醛/醇脱氢酶 2（由 adhE2 基因编码）的去阻遏，其特征是 adhE2 基因的表达增加。这里呈现的结果清楚地表明，Rex 参与了丙酮丁醇梭菌的氧化还原依赖性溶剂形成转换。
