Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

Clostridium thermocellum is a gram-positive, acetogenic, thermophilic, anaerobic bacterium that degrades cellulose and carries out mixed product fermentation, catabolising cellulose to acetate, lactate, and ethanol under various growth conditions, with the concomitant release of H-2 and CO2. Very little is known about the factors that determine metabolic fluxes influencing H-2 synthesis in anaerobic, cellulolytic bacteria like C. thermocellum. We have begun to investigate the relationships between genome content, gene expression, and end-product synthesis in C. thermocellum cultured under different conditions. Using bioinformatics tools and the complete C. thermocellum 27405 genome sequence, we identified genes encoding key enzymes in pyruvate catabolism and H-2-synthesis pathways, and have confirmed transcription of these genes throughout growth on alpha-cellulose by reverse transcriptase polymerase chain reaction. Bioinformatic analyses revealed two putative lactate dehydrogenases, one pyruvate formate lyase, four pyruvate: formate lyase activating enzymes, and at least three putative pyruvate: ferredoxin oxidoreductase (POR) or POR-like enzymes. Our data suggests that hydrogen may be generated through the action of either a Ferredoxin (Fd)-dependent NiFe hydrogenase, often referred to as "Energy-converting Hydrogenases", or via NAD(P)H-dependent Fe-only hydrogenases which would permit H-2 production from NADH generated during the glyceraldehyde3- phosphate dehydrogenase reaction. Furthermore, our findings show the presence of a gene cluster putatively encoding a membrane integral NADH: Fd oxidoreductase, suggesting a possible mechanism in which electrons could be transferred between NADH and ferredoxin. The elucidation of pyruvate catabolism pathways and mechanisms of H-2 synthesis is the first step in developing strategies to increase hydrogen yields from biomass. Our studies have outlined the likely pathways leading to hydrogen synthesis in C. thermocellum strain 27405, but the actual functional roles of these gene products during pyruvate catabolism and in H-2 synthesis remain to be elucidated, and will need to be confirmed using both expression analysis and protein characterization.