Deletion of electron-bifurcating [FeFe]-hydrogenase enhances H2-driven CO2 fixation and ethanol production in Clostridium ljungdahlii.

Hydrogenases are central to energy conservation and CO2 fixation in acetogenic bacteria during H2-driven autotrophic growth. The model acetogenic bacterium Clostridium ljungdahlii encodes multiple hydrogenases, with the NADP+- and ferredoxin-dependent electron-bifurcating [FeFe]-hydrogenase HytCBDE1AE2 presumed to be primary. In this study, the hydrogenase catalytic subunit of the HytCBDE1AE2 complex, HytA was knocked out by the CRISPR/Cas9-mediated mutagenesis. Unexpectedly, the ΔhytA mutant exhibited significantly enhanced autotrophic growth on H2/CO2, achieving a ∼ 3-fold higher cell density and approximately 2-fold and 3-fold increases in acetate and ethanol titers, respectively. This was accompanied by elevated intracellular ATP levels and a shifted redox state (decreased NAD+/NADH and increased NADP+/NADPH ratios), indicating altered bioenergetics. Proteomics revealed compensatory upregulation of three single-subunit hydrogenases in ΔhytA, among which, the Hyd encoded by CLJU_RS18325 exhibited the highest expression level. Hyd was predicted to bypass the electron bifurcation, directly reducing ferredoxin with H2. While hyd deletion alone abolished ethanol synthesis without affecting acetate production, the double knockout of hytA and hyd failed to grow on H2/CO2, establishing Hyt and Hyd as the two primary functional hydrogenases under lab cultivation conditions. Improved fed-batch fermentation further demonstrated the biotechnological potential of the ΔhytA strain, which achieved high biomass (OD600 = 7.5) and elevated ethanol titers (22.3 g/L). This work provides new insights into the function of hydrogenases in C. ljungdahlii and presents an engineered chassis with enhanced capacity for CO2-based chemical production.