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

Abstract

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO 2 . This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO 2 to formate. However, feeding the bacterium 13 C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13 C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO 2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO 2 to formate serves as a CO 2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO 2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO 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 2 uptake, and provided physical evidence of a distinct in vivo "rPFOR-PFL shunt" to reduce CO 2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO 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 2 as well.Clostridium thermocellum | pyruvate:ferredoxin oxidoreducase | formate | 13 C-isotopic tracing | one-carbon metabolism T he gram-positive Clostridium thermocellum is a thermophilic and strict anaerobic bacterium. It has gained a great amount of interest due to its cellulolytic abilities. By taking advantage of an extracellular cellulase system called the cellulosome (1), C. thermocellum can depolymerize cellulose into soluble oligosaccharides. The latter are further transported into the cells and fermented through its glycolytic pathway to pyruvate, the precursor to an array of fermentation products (e.g., H 2 , formate, lactate, acetate, ethanol, secreted amino acids) (2, 3). This capability makes C. thermocellum an attractive candidate for consolidated bioprocessing of lignocellulosic biomass, a process configuration that directly converts plant biomass into biofuels and chemicals without separate additions of enzymes (4). To date, there have been numerous works describing the molecular and genetic details of the cellulolytic system and fermentation pathways in C. thermocellum (5-9), whereas other metabolic characteristics of this species have not been adequately understood.Currently, few details regarding inorganic carbon utilization are known for C. thermocellum. However, several investigators routinely add bicarbonate, a dissolved form of CO 2 , into the medium to promote C. thermocellum growth (6, 10-12), implying its ability to use CO 2 . Thi...

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