Adaptive_Evolution_2026

Thermoanaerobacterium saccharolyticum, an anaerobic and thermophilic bacterium capable of metabolizing sugar monomers and soluble oligomers into ethanol, has been proposed for use in consolidated bioprocessing in coculture with compatible cellulolytic bacteria such asClostridium thermocellum. Although the mixed acid fermentation of both of these strains has been engineered to produce ethanol as the major fermentation product, the maximum titer produced thus far byC. thermocellum, about 3% (w/v) ethanol, is half that produced byT. saccharolyticum. There is thus motivation to understand the mechanistic basis of the robust ethanol pathway inT. saccharolyticumso that key features can be recapitulated inC. thermocellumand other organisms.Previously, we characterized theindividualrole of the main genes responsible for electron transfer in the ethanol production ofT. saccharolyticum. However, the consequences of thecombinedloss of function of all these genes have not been investigated, nor has the way in which fermentative metabolism adapts to such constraints. In this work, we combined knockouts of ferredoxin nicotinamide oxidoreductase (fnor) genes (nfnAandnfnB) and hydrogenase genes (hydAandhfsD) and studied their effects on fermentation and grow. We showed that these genetic modifications together impair growth and decrease electron transfer from reduced ferredoxin, thereby redirecting flux from the pyruvate ferredoxin oxidoreductase enzyme to the pyruvate formate lyase enzyme. We also performed adaptive evolution of these mutants to rescue their growth, and most notably, we observed a single nucleotide variation in the alcohol dehydrogenaseadhAgene. Through molecular dynamics simulations and enzymatic assays, we determined that this point mutation causes a structural change that impairs the AdhA specificity for the NADPH cofactor and increases NADH-linked activity to restore redox balance. These findings consolidate our understanding of the functioning of electron transfer pathways in this organism.

解糖热厌氧杆菌(Thermoanaerobacterium saccharolyticum)是一类厌氧嗜热细菌，可将单糖与可溶性寡糖代谢为乙醇，其被提议与热纤梭菌(Clostridium thermocellum)等相容性纤维素分解细菌进行共培养，用于整合生物加工工艺。尽管已对这两种菌株的混合酸发酵进行工程改造，使其以乙醇作为主要发酵产物，但目前热纤梭菌(C. thermocellum)所能达到的最大乙醇滴度约为3%（w/v），仅为解糖热厌氧杆菌(T. saccharolyticum)产量的一半。因此，我们有必要阐明解糖热厌氧杆菌中高效乙醇合成途径的分子机制，以便将其关键特征复刻到热纤梭菌及其他生物中。此前，我们已对解糖热厌氧杆菌乙醇生产过程中负责电子传递的主要基因的个体功能进行了表征，但尚未探究这些基因全部功能丧失的联合效应，也未阐明发酵代谢如何适应此类遗传限制。本研究中，我们对铁氧还蛋白烟酰胺氧化还原酶(ferredoxin nicotinamide oxidoreductase, fnor)基因nfnA与nfnB以及氢化酶基因hydA与hfsD进行了联合敲除，并研究了其对发酵与生长的影响。我们发现，这些遗传修饰共同削弱了菌株生长，并降低了还原型铁氧还蛋白的电子传递效率，从而将丙酮酸铁氧还蛋白氧化还原酶的代谢流重定向至丙酮酸甲酸裂解酶途径。我们还对这些突变株进行了适应性进化以挽救其生长，最显著的是，我们在乙醇脱氢酶(alcohol dehydrogenase, adhA)基因中观察到了单核苷酸变异。通过分子动力学模拟与酶学实验，我们确定该点突变会引发蛋白质结构改变，削弱AdhA对NADPH辅酶的特异性，并增强其与NADH相关的酶活，从而恢复氧化还原平衡。这些发现进一步加深了我们对该物种电子传递途径功能机制的理解。