LAUSR

Significance Despite sharing the first four reactions, coutilization of the Wood–Ljungdahl pathway (WLP) with the glycine synthase-reductase pathway (GSRP) and reductive glycine pathway (RGP) to fix C1 compounds has remained unknown. In this study, using Clostridium drakei, we elucidated the role of the GSRP and RGP in the presence of the WLP, via a genome-scale metabolic model, RNA-seq, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression. Overall, the data suggested the pathways are functional under autotrophic conditions. Along with the WLP, GSRP and RGP convert CO2 to glycine and then to acetyl-phosphate and serine, which then obtain ATP by producing acetate and operate with limited reducing power. This is a unique coutilization of the pathways under autotrophic conditions in acetogens. Among CO2-fixing metabolic pathways in nature, the linear Wood–Ljungdahl pathway (WLP) in phylogenetically diverse acetate-forming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO2 to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genome-scale metabolic model iSL771 based on the completed genome sequence, transcriptomics, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO2, subsequently converting CO2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.