P73: Functional Studies of [FeFe]-Hydrogenase in Clostridium thermocellum

Clostridium thermocellum is a thermophilic and ethanogenic bacterium that can degrade cellulosic biomass using a complex extracellular cellulolytic organelle, cellulosome. C. thermocellum metabolizes cellulose to cellodextrin and cellobiose, which are then taken up by the cells. During cellulose fermentation, it produces hydrogen gas with concomitant release of typical carbon byproducts. Central to biological H₂ metabolism is hydrogenase enzyme. Hydrogenases catalyze the oxidation or evolution of H₂ according to the equation: 2H⁺ + 2e^- <-> H₂. C. thermocellum contains three putative [FeFe]-hydrogenases (CtHydA1, CtHydA2 and CtHydA3) and one putative hexameric [NiFe]-hydrogenase, yet little is known about the physiological functions of these hydrogenases. To elucidate the function of the hydrogenase, we chose the CtHydA3 due to its novelty and simplicity of the structure. Four plasmids were constructed containing CtHydA3 (C- or N-terminus 6X His-tag), a putative ferredoxin-like protein (Ct_3004), and three [FeFe]-hydrogenase maturation proteins (CtHydE, CtHydF, and CtHydG) and were co-transformed into E. coli strain Rosetta (DE3) and BL21 (DE3) for heterologous expression. Protein immunoblots confirmed the expression of the His-tagged CtHydA3 only in E. coli Rosetta (DE3), but not in E. coli BL21 (DE3) likely due to differences in codon usage between C. thermocellum and E. coli. This is further corroborated by a two-fold increase in in vitro hydrogenase activity over a high background from native E. coli hydrogenases. Affinity purification of both C- and N-terminal His-tagged hydrogenase was accomplished, albeit with low yield. Work is underway to improve heterologous expression for the characterization of the CthydA3 hydrogenase.