The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches.

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

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

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