Direct carbon monoxide fixation via the bacterial and archaeal Wood–Ljungdahl pathways

The Wood–Ljungdahl (WL) pathway, which is widely distributed in both archaea and bacteria, is an ancient carbon fixation pathway from CO 2 . CO 2 fixation proceeds via two branches of pathway: progressive reduction to the methyl group in the methyl branch and one-step reduction to CO in the carbonyl branch. In the final step of the pathway, the methyl group, CO, and CoA are combined into a carbon monoxide dehydrogenase (CODH)/acetyl-CoA synthase (ACS) complex to form acetyl-CoA. Here, we show direct CO fixation to the carbonyl group of acetyl-CoA in both archaeal and bacterial WL pathways under hydrogenogenic growth conditions using 13 C tracer-based metabolomics. A combination of metabolomics and proteomics suggested that the hydrogenotrophically grown Thermodesulfatator indicus and Archaeoglobus sp. strain MCR cells, directly fixed CO using free-form ACS in the carbonyl branch with relatively low CO availability. In contrast, carboxydotrophically grown Archaeoglobus cells utilize the CODH/ACS complex for CO 2 fixation rather than CO fixation. Direct CO fixation by free-form ACS is more advantageous for conserving reduced ferredoxin compared with the thermodynamically challenged CO 2 reduction by CODH. These findings provide further insight into the origin and evolution of the most ancient inorganic carbon fixation pathway and geochemical cycles on early Earth.