CO sub( 2)-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO sub( 2). This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO sub( 2) to formate. However, feeding the bacterium super( 13)C-bicarbonate and cellobiose followed by NMR analysis showed the production of super( 13)C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO sub( 2) to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO sub( 2) to formate serves as a CO sub( 2) entry point into the reductive one-carbon (C1) metabolism, and internalizes CO sub( 2) via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO sub( 2) 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 CO sub( 2) uptake, and provided physical evidence of a distinct in vivo "rPFOR-PFL shunt" to reduce CO sub( 2) to formate while circumventing the lack of Fdh. Such a pathway precedes CO sub( 2) 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 CO sub( 2) as well.