Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Clostridium thermocellum is a thermophilic, obligately anaerobic, gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

热纤梭菌（Clostridium thermocellum）是一种嗜热、专性厌氧的革兰氏阳性菌，是通过整合生物加工（consolidated bioprocessing）将纤维素生物质转化为乙醇的潜在工程微生物。乙醇不耐受是制约工艺经济性的关键评价指标，而乙醇耐受性往往被视为一类复杂且大概率受多基因调控的性状，其分子机制涉及复杂的基因互作网络。本研究对一株乙醇耐受突变株的基因组进行重测序，证实该耐受表型主要源于双功能乙醛辅酶A/乙醇脱氢酶基因（adhE）发生突变；我们通过结构分析提出假说，认为该突变可能改变了酶的辅酶因子特异性，并通过酶活测定验证了这一假说。生化实验结果证实，该突变酶完全丧失了以NADH为辅因子的催化活性，同时获得了以NADPH为辅因子的催化活性，这一变化或可影响突变株体内的电子传递过程。本研究揭示的乙醇耐受表型的简洁遗传基础，可为用于纤维素乙醇生产的工程微生物菌株的理性改造提供重要理论指导。