Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002

Synechococcus sp. PCC 7002 is a unicellular cyanobacterium capable of fast growth and tolerance to high light intensity salinity. These attributes along with genetic tractability make an attractive candidate for industrial scale production specialty commodity chemicals. LS (Davies et al., Front Bioeng Biotechnol, 2014, 2, 21–11) produces limonene, energy dense diesel jet fuel drop-in additive, at titer 4 mg/L over 4-day incubation period. In this study, we use the state-of-the-art whole-cell characterization tool, isotopically non-stationary 13 C metabolic flux analysis (INST- CMFA) determine intracellular fluxes through pathways central metabolism limonene producing strain wild type 7002. We find similar distribution in Calvin-Benson-Bassham cycle, photorespiration, oxidative pentose phosphate pathway, tricarboxylic acid cycle. The key difference between strains observed pyruvate. displays significantly higher amphibolic phosphoenolpyruvate carboxylase malic enzyme synthesize pyruvate, while uses pyruvate kinase single step. Our findings suggest that mechanism recover physiologically optimal ratio ATP NADPH. upregulation pathway may act restore physiological ATP:NADPH has been disturbed by biosynthesis. This study demonstrates value INST- CMFA as tool cyanobacterial engineering provides new avenues research improving .