L-Aspartate oxidase provides new insights into fumarate reduction in anaerobic darkness in Synechocystis sp. PCC6803

Cyanobacteria are promising microbial hosts for production of various industrially relevant compounds, such as succinate, a central metabolite of the tricarboxylic acid cycle (TCA). Cyanobacteria have been engineered to produce succinate during photoautotrophic growth, and are also able to secrete it during anoxic fermentation conditions. It has been assumed that under anoxic darkness, succinate can be formed by reduction of fumarate catalyzed by the succinate dehydrogenase complex (SDH), however, no characterization of SDH regarding this activity has been performed. In this study, we address this issue by generating strains of the unicellular cyanobacterium Synechocystis PCC 6803 (Synechocystis) deficient in one or several subunits of SDH, and investigating succinate accumulation in these strains during dark anaerobic fermentation. The results showed higher succinate accumulation in SDH deletion strains than in the wild type, indicating a succinate dehydrogenase activity of SDH rather than fumarate reduction under these conditions. We further explored the possibility of another potential route for succinate formation from fumarate via L-aspartate oxidase (Laspo). The gene encoding Laspo in Synechocystis could not be inactivated, indicating an essential function for this enzyme. Using purified SynLaspo, we could demonstrate in vitro that in addition to L-aspartate oxidation the enzyme exhibits an L-aspartate-fumarate oxidoreductase activity. We therefore suggest that reduction of fumarate to succinate during anoxic darkness can be a byproduct of the Laspo reaction, which is the first step in biosynthesis of NAD cofactors. This work contributes to the understanding of cyanobacterial TCA cycle for future engineering and sustainable production of dicarboxylic acids.