Physiological and transcriptomic changes by enfR overexpression in a formate- utilizing archaeon

The rmoc occus onnurineus NA1 exhibits growth by oxidizing carbon monoxide (CO) or formate to generate hydrogen gas (H 2 ). The EnfR protein has been identified as a crucial regulator of carboxydotrophic metabolism in this strain, controlling the expression of the codh-mch-mnh3 gene cluster. This study investigated the regulatory effect of EnfR on formate metabolism. Deletion of the enfR gene negatively affected cell growth on formate, whereas overexpression of enfR significantly enhanced cell growth and H 2 production in pH-stat batch cultures compared to the parental strain. Transcriptomic analysis of the enfR -overexpression strain (EnfR OE) revealed differential expression of 219 genes, including clusters encoding ferredoxin-dependent membrane-bound hydrogenase, 2-ketoacid ferredoxin oxidoreductases, and flagellins. Notably, for the fdh2 gene encoding formate dehydrogenase, which is essential for formate-dependent growth, no significant differences in transcript or protein levels were observed. The collective metabolic outcome of those DEGs likely contributes to the enhanced formate-dependent growth of the EnfR OE strain, partially by modulating the levels of reduced electron carriers. This study provides meaningful insights into understanding formate-dependent growth and H 2 production. Key points • Deletion of the enfR gene significantly impaired growth under formate conditions. • Overexpression of enfR not only accelerated cell growth but also enhanced H 2 production. • Transcriptomic analysis revealed that enfR overexpression promotes growth by regulating cellular redox balance rather than by directly inducing transcription of the fdh2-mfh2-mnh2 cluster.