Fabri João Henrique T M, Pech-Canul Angel, Ziegler Samantha J, Burgin Tucker Emme, Richardson Isaiah D, Maloney Marybeth I, Bomble Yannick J, Lynd Lee R, Olson Daniel G
Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas (UNICAMP), Campinas, State of São Paulo, Brazil.
Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA.
J Bacteriol. 2025 May 22;207(5):e0001525. doi: 10.1128/jb.00015-25. Epub 2025 Apr 30.
is a thermophilic anaerobic bacterium that natively ferments a variety of hemicellulose substrates to organic acids and alcohols. It has recently been engineered to produce ethanol at high yield and titer; however, it uses a unique metabolic pathway for ethanol production that is poorly characterized. One of the distinctive aspects of this pathway is the presence of acetyl-CoA as an intermediate metabolite. In this organism, acetyl-CoA is converted to ethanol by a bifunctional AdhE enzyme. This enzyme has been a frequent target for mutations, and in many cases, the function of these mutations was unknown. Using a combination of genetic modifications, enzyme assays, and computational analysis, we have developed a better understanding of how mutations in AdhE affect ethanol production in the engineered homoethanologen strain. We identify a set of approximately interchangeable AdhE mutations (G544D, T597K, T597I, and T605I), whose function is to disrupt the activity of the alcohol dehydrogenase (ADH) domain of AdhE. This reduces NADH-linked ADH activity, which dramatically increases ethanol tolerance and changes the overall stoichiometry of acetaldehyde to ethanol conversion. Furthermore, our improved understanding of the function of these AdhE mutations calls into question a proposed feature of AdhE enzymes known as substrate channeling-direct transfer of acetaldehyde between the two domains of the AdhE enzyme. This improved the understanding of the role of AdhE mutations in and provides deeper insights into the function of the unique ethanol production pathway in this organism.
Many anaerobic bacteria maintain redox equilibrium by producing reduced organic compounds such as ethanol. The final two steps of ethanol production are mediated by a bifunctional enzyme, AdhE, and this enzyme is a frequent target of mutations in strains engineered for increased ethanol production. Paradoxically, these mutations increase ethanol production by eliminating the activity of one domain of the AdhE enzyme (the ADH domain). This provides additional support for a redox-imbalance theory of alcohol tolerance, which challenges the prevailing hypothesis that alcohol tolerance is associated with cell membrane effects.
是一种嗜热厌氧菌，天然情况下可将多种半纤维素底物发酵为有机酸和醇类。最近，它经过改造后能够高产且高滴度地生产乙醇；然而，它利用一种独特的代谢途径来生产乙醇，而该途径的特征了解甚少。这条途径的一个显著特点是存在乙酰辅酶A作为中间代谢物。在这种生物体中，乙酰辅酶A通过一种双功能的AdhE酶转化为乙醇。这种酶一直是突变的常见靶点，在许多情况下，这些突变的功能尚不清楚。通过结合基因改造、酶活性测定和计算分析，我们对AdhE中的突变如何影响工程化的同型乙醇生产菌株中的乙醇生产有了更好的理解。我们鉴定出一组大致可互换的AdhE突变（G544D、T597K、T597I和T605I），其功能是破坏AdhE的醇脱氢酶（ADH）结构域的活性。这降低了与NADH相关的ADH活性，从而显著提高了乙醇耐受性，并改变了乙醛向乙醇转化的整体化学计量。此外，我们对这些AdhE突变功能的深入理解对AdhE酶的一个被称为底物通道化（即乙醛在AdhE酶的两个结构域之间直接转移）的假定特征提出了质疑。这增进了对AdhE突变在[此处原文缺失相关内容]中的作用的理解，并为该生物体中独特的乙醇生产途径的功能提供了更深入的见解。
许多厌氧菌通过产生诸如乙醇等还原有机化合物来维持氧化还原平衡。乙醇生产的最后两步由双功能酶AdhE介导，并且这种酶是为提高乙醇产量而改造的菌株中突变的常见靶点。矛盾的是，这些突变通过消除AdhE酶的一个结构域（ADH结构域）的活性来增加乙醇产量。这为酒精耐受性的氧化还原失衡理论提供了额外支持，并对酒精耐受性与细胞膜效应相关的主流假设提出了挑战。
