Thermoanaerobacter ethanolicus can produce acetate, lactate, hydrogen and ethanol from sugars resulting from plant carbohydrate polymer degradation at temperatures above 65°C. T. ethanolicus is a promising candidate for thermophilic ethanol fermentations due to both the pentose and hexose utilization. Although an ethanol balance model in T. ethanolicus has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in ethanol formation have been carried out. To address this issue, we developed a thermostable Cas9 based system for genome editing of T. ethanolicus. As a proof of principle, three genes including thymidine kinase gene (tdk), acetaldehyde-alcohol dehydrogenase gene (adhE) and redox sensing protein gene (rsp) were chosen as editing targets, and these genes were edited successfully. As a genetic tool, we tested the gene knock-out and a small DNA fragment knock-in. After optimization of the transformation strategies, 77% genome editing efficiency was observed. Furthermore, our results in vivo revealed that redox sensing protein (RSP) play a more improtant role to regulate energy metabolism, including hydrogen production and ethanol formation. The genetic system provides us an effective strategy to identify genes involved in biosynthesis and energy metabolism. IMPORTANCE Interest in thermophilic microorganisms as emerging metabolic engineering platforms to produce biofuels and chemicals has surged. Thermophilic microbes for biofuels have been attracted great attention, due to their tolerance of high temperature and wide range of substrate utilization. Based on the biochemical experiments of previous investigation, the ethanol formation was controlled via transcriptional regulation and influenced by the relevant properties of specific enzymes in T. ethanolicus. Thus, there is an urgent need to understand the physiological function of these key enzymes, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Here we developed a thermostable Cas9-based engineering tool for gene editing in T. ethanolicus. Thermostable Cas9 based genome-editing tool may further be applied to metabolically engineer T. ethanolicus to produce biofuels. This genetic system is an important expansion to the genetic tool box of anaerobic thermophile T. ethanolicus strain.

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