Adaptation of thermophilic enzymes from an ancestral reductive TCA cycle for carbon fixation in plants

The reductive TCA cycle (rTCA) is one of 5 carbon-fixing cycles in prokaryotes other than the Calvin-Benson cycle ubiquitous in plants. Adaptation of these cycles to biofuel crops is one strategy for reducing the greenhouse gas CO2 and simultaneously increasing crop yields. A minimal 5-enzyme synthetic rTCA cycle was designed at NCSU utilizing 2 unique enzymes to carboxylate and reduce 2-oxoglutarate to isocitrate. The enzymes are derived from an evolutionarily primitive configuration of the rTCA cycle that persists in the order Aquificales in hot springs and deep ocean thermal vents. Two strategies are being pursued to engineer mesophilic forms of these enzymes: 1) reducing the temperature of activity of the Aquificales enzymes, and 2) modifying the substrate specificity of homologous mesophilic enzymes. The first enzyme, 2-oxoglutarate carboxylase (OGC), is a member of the biotin-dependent carboxylase family homologous to pyruvate carboxylase. Biotin-dependent carboxylases are multi-subunit complexes. So far, we have identified the temperature-dependent steps of the biotin carboxylase domain of OGC and predicted mutations to reduce the temperature of activity based on the crystal structure. Phylogenetic analysis suggests that OGC and pyruvate carboxylase diverged prior to Aquificales evolution. An ancestral promiscuous enzyme has been proposed to carboxylate both pyruvate and 2-oxoglutarate. Ancestral enzymes are being reconstructed from phylogenetic analyses to switch the substrate specificity of mesophilic pyruvate carboxylase to carboxylate 2-oxoglutarate. The second enzyme, oxalosuccinate reductase (OSR), is homologous with isocitrate dehydrogenase. In fact, isocitrate dehydrogenase has replaced OGC/OSR in most organisms utilizing the rTCA cycle. Isocitrate dehydrogenase favors the oxidative decarboxylase of isocitrate in the TCA cycle, while OGC/OSR favors the reductive cycle. OSR is an ancestral, non-decarboxylating isocitrate dehydrogenase. We propose to engineer a mesophilic OSR by mutating isocitrate dehydrogenase to inhibit decarboxylation, based on phylogenic and structural studies of OSR. Ultimately engineering a mesophilic OGC/OSR will be utilized to increase carbon capture in seed oil crops like Camelina sativa that are carbon limited. Oil from Camelina seeds can be converted to jet fuels.