Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803

Iijima, Hiroko; Watanabe, Atsuko; Sukigara, Haruna; Iwazumi, Kaori; Shirai, Toshiaki; Kondo, Akihiko; Osanai, Takashi

doi:10.1016/j.ymben.2021.03.007

Cited by 26 publications

(34 citation statements)

References 42 publications

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“…In the bifurcated structure of the cyanobacterial TCA cycle, the reductive portion supports higher flux than the oxidative portion, which is suppressed in light S4. conditions (Zhang and Bryant, 2011;Xiong et al, 2017); this is typically paired with low flux through TCA intermediates like CIT and AKG and more flux for supplying OAA and MAL directly from PEP (Iijima et al, 2021), which is additionally supplemented by FUM through purine synthesis (Knoop et al, 2010). As expected, we see low fluxes in Figure 2 for the oxidative TCA cycle, driven by the production of biomass precursors (i.e., AKG).…”

Section: Metabolic Flux Analysis Reveals Increased Flux Through Phosp...supporting

confidence: 64%

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

et al. 2022

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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.

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Section: Metabolic Flux Analysis Reveals Increased Flux Through Phosp...supporting

confidence: 64%

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

et al. 2022

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“…The reduction of fumarate to succinate through SDH complex was addressed by deletion of SDH subunits. One of the two homologous iron-sulfur proteins, encoded by sll0823 , has been proposed to be responsible for the fumarate reduction [12], as only the other similar subunit, encoded by sll1625 , seems to be required for the oxidation of succinate to fumarate [3].…”

Section: Discussionmentioning

confidence: 99%

“…In dark conditions and the presence of oxygen, the membrane associated enzyme complex succinate dehydrogenase (SDH) contributes to the reduction of electron transport chain, with concomitant oxidation of succinate to form fumarate [16] and thereby the oxidative branch of TCA cycle is most active. On the other hand, the ability of Synechocystis to excrete succinate during dark anoxic fermentation via the reductive branch of the TCA cycle has been explored in several reports [8, 9, 12, 17]. In this pathway, succinate would presumably be formed via reduction of oxaloacetate to form malate and fumarate and finally succinate, by the enzymes malate dehydrogenase, fumarate hydratase and SDH respectively.…”

Section: Introductionmentioning

confidence: 99%

“…Fumarate is used as the electron acceptor under anaerobic conditions, whereas under aerobic conditions oxygen is used as electron acceptor. Since the genes encoding the NAD cofactor synthesis pathway is present in Synechocystis it is possible that secretion of succinate during anaerobic fermentation can be explained by increased fumarate concentration [12] and Laspo activity.…”

Section: Introductionmentioning

confidence: 99%

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

Kukil

Hawkes

Blikstad

et al. 2022

Preprint

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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 cyanobacteriumSynechocystisPCC 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 inSynechocystiscould 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.

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“…The three-step pathway forms part of the reductive TCA cycle. In the bifurcated structure of the cyanobacterial TCA cycle, the reductive portion supports higher flux than the oxidative portion, which is suppressed in light conditions (Zhang and Bryant, 2011;Xiong et al, 2017), typically with low flux through TCA intermediates like CIT and AKG, and more flux supplying OAA and MAL directly from PEP (Iijima et al, 2021), additionally supplemented by FUM through purine synthesis (Knoop et al, 2010). As expected, we see low fluxes in Figure 2 for the oxidative TCA cycle, with flux driven by the production of biomass precursors (ie.…”

Section: Metabolic Flux Analysis Reveals Increased Flux Through Phosp...mentioning

confidence: 99%

Characterizing Photosynthetic Biofuel Production: Isotopically non-stationary¹³C metabolic flux analysis (INST-¹³CMFA) on limonene producingSynechococcussp. PCC 7002

Newman

Sake

Metcalf

et al. 2022

Preprint

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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.

show abstract

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803

Cited by 26 publications

References 42 publications

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

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

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

Characterizing Photosynthetic Biofuel Production: Isotopically non-stationary¹³C metabolic flux analysis (INST-¹³CMFA) on limonene producingSynechococcussp. PCC 7002

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Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803

Cited by 26 publications

References 42 publications

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

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

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

Characterizing Photosynthetic Biofuel Production: Isotopically non-stationary13C metabolic flux analysis (INST-13CMFA) on limonene producingSynechococcussp. PCC 7002

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Characterizing Photosynthetic Biofuel Production: Isotopically non-stationary¹³C metabolic flux analysis (INST-¹³CMFA) on limonene producingSynechococcussp. PCC 7002