Saint-Amans S, Girbal L, Andrade J, Ahrens K, Soucaille P
Centre de Bioingénierie Gilbert Durand, UMR-CNRS 5504, Laboratoire Associé INRA, Institut National des Sciences Appliquées, 31077 Toulouse, France.
J Bacteriol. 2001 Mar;183(5):1748-54. doi: 10.1128/JB.183.5.1748-1754.2001.
The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H(+) ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.
在pH 6.5的条件下，于磷酸盐限制的恒化器培养中，通过使用葡萄糖和甘油的混合物改变底物的整体还原度，对丁酸梭菌的代谢进行了调控。仅以葡萄糖为底物生长的培养物仅产生酸（乙酸盐、丁酸盐和乳酸盐）以及高水平的氢气。相比之下，当甘油被代谢时，1,3 - 丙二醇成为主要产物，酸形成的比速率降低，并且观察到低水平的氢气。甘油的消耗与以下物质的诱导有关：（i）一种将甘油导入中心代谢的甘油脱氢酶和二羟基丙酮激酶，以及（ii）一种对氧敏感的甘油脱水酶和一种参与丙二醇形成的NAD依赖性1,3 - 丙二醇脱氢酶。电子流从氢气形成转向NADH形成与体外氢化酶活性的急剧下降以及乙酰辅酶A（CoA）/游离CoA比率的下降相关，该比率使得NADH - 铁氧化还原蛋白氧化还原酶双向酶能够发挥作用，从而在这种培养物中还原NAD。乙酸盐和丁酸盐形成的减少并非由磷酸转酰酶、乙酸盐激酶和丁酸盐激酶浓度的变化所解释，而是由体内底物浓度的变化所导致，这通过与仅以葡萄糖生长的培养物相比，在以葡萄糖和甘油生长的培养物中乙酰 - CoA/游离CoA和丁酰 - CoA/游离CoA比率的急剧下降以及ATP/ADP比率的急剧增加得以体现。正如先前对丙酮丁醇梭菌所报道的那样（L. Girbal、I. Vasconcelos和P. Soucaille，《细菌学杂志》176:6146 - 6147，1994），当氢化酶的体内活性降低时（在葡萄糖 - 甘油混合物上生长的培养物），丁酸梭菌的跨膜pH值会反转（内部更酸性）。对于这两种培养物，H(+) - ATP酶的化学计量显示保持恒定，等于每消耗一分子ATP输出3个质子。
