CO2-Fixing One-Carbon Metabolism in a Cellulose-Degrading Bacterium Clostridium thermocellum

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO₂. This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO₂ to formate. However, feeding the bacterium ¹³C-bicarbonate and cellobiose followed by NMR analysis showed the production of ¹³C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO₂ to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO₂ to formate serves as a CO₂ entry point into the reductive one-carbon (C1) metabolism, and internalizes CO₂ via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO₂ using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO₂ uptake, and provided physical evidence of a distinct in vivo "rPFOR-PFL shunt" to reduce CO₂ to formate while circumventing the lack of Fdh. Such a pathway precedes CO₂ fixation via the reductive C1 metabolic pathway in C. thermocellum. These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO₂ as well.