Photoautotrophic Growth Rate Enhancement of Synechocystis sp. PCC6803 by Heterologous Production of 2-Oxoglutarate:Ferredoxin Oxidoreductase from Chlorobaculum tepidum

SIMPLE SUMMARY: Synechocystis sp. PCC6803 (Synechocystis) is a photosynthetic organism useful for biotechnological applications. It utilizes light energy and fixes CO(2) to synthesize C3 organic acids for growth and biomass formation. However, carbon fixation is the limiting step for its maximal growth. To enhance the carbon fixation, 2-oxoglutarate:ferredoxin oxidoreductase from Chlorobaculum tepidum (CtOGOR)—a carbon-fixing enzyme in the reductive TCA cycle—was produced in trans in Synechocystis. Overexpression of the CtOGOR gene effectively altered 2-oxoglutarate and glutamate levels and elevated the photoautotrophic growth rate of Synechocystis. ABSTRACT: 2-Oxoglutarate:ferredoxin oxidoreductase from Chlorobaculum tepidum (CtOGOR) is a carbon-fixing enzyme in the reductive TCA cycle that reversibly carboxylates succinyl-CoA to yield 2-oxoglutarate. CtOGOR is a heterotetramer of two large (α = 68 kDa) and two small (β = 38 kDa) subunits. The αβ protomer harbors one thiamine pyrophosphate and two 4Fe-4S clusters. Nonetheless, the enzyme has a considerable oxygen tolerance with a half-life of 143 min at 215 μM dissolved oxygen. Kinetic analyses of the purified recombinant CtOGOR revealed a lower K(m) for succinyl-CoA than for 2-oxoglutarate. Cellular levels of 2-oxoglutarate and glutamate—a product of glutamine oxoglutarate aminotransferase and glutamate dehydrogenase—increased more than twofold in the exponential phase compared with the control strain, leading to an approximately >30% increase in the photoautotrophic growth rate. Thus, CtOGOR was successfully produced in Synechocystis, thereby boosting carboxylation, resulting in enhanced photoautotrophic growth.