The redox-sensing protein Rex modulates ethanol production in Thermoanaerobacterium saccharolyticum

Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred.解糖热厌氧杆菌（Thermoanaerobacterium saccharolyticum）是一种嗜热厌氧菌，经基因工程改造后可高效合成乙醇，在乙醇滴度达70 g/L时，理论得率可达约90%。本研究报道了在野生型解糖热厌氧杆菌中敲除氧化还原感应转录调控因子Rex（redox-sensing transcriptional regulator Rex）后所产生的生理变化：单独敲除rex基因可使乙醇理论得率提升一倍（从40%增至91%），但所得重组菌株的生长速率仅为野生型菌株的约三分之一。敲除rex基因还可上调乙醇脱氢酶基因adhE与adhA的转录表达水平。经过数次连续传代培养后，菌株的平均乙醇得率从91%降至55%，而生长速率则有所回升。我们通过全基因组重测序（whole-genome resequencing）对生长速率加快的Δrex缺失株进行次生突变位点鉴定，结果在多个菌株的adhE基因中均发现了次生突变。此外，这类菌株的NADH依赖型乙醇脱氢酶活性大幅降低。我们开展了基因互补实验，将rex基因重新导入Δrex缺失株：在未携带adhE突变的Δrex菌株中，重新导入rex可使乙醇得率降至野生型水平以下；而在携带adhE突变的Δrex菌株中，乙醇得率未发生明显变化。