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 and high salinity. These attributes along with genetic tractability make Synechococcus sp. PCC 7002 an attractive candidate for industrial scale production of specialty and commodi...
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| Format: | Article |
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Frontiers Media S.A.
2022-07-01
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| Series: | Frontiers in Energy Research |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fenrg.2022.896668/full |
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| author | Darrian M. Newman Cara L. Sake Alexander J. Metcalf Fiona K. Davies Melissa Cano Anagha Krishnan Nanette R. Boyle |
| author_facet | Darrian M. Newman Cara L. Sake Alexander J. Metcalf Fiona K. Davies Melissa Cano Anagha Krishnan Nanette R. Boyle |
| author_sort | Darrian M. Newman |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-11T03:30:01Z |
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| institution | Directory Open Access Journal |
| issn | 2296-598X |
| language | English |
| last_indexed | 2024-12-11T03:30:01Z |
| publishDate | 2022-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Energy Research |
| spelling | doaj.art-15ca909aad86446dbbc10b83b746289d2022-12-22T01:22:24ZengFrontiers Media S.A.Frontiers in Energy Research2296-598X2022-07-011010.3389/fenrg.2022.896668896668Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002Darrian M. Newman0Cara L. Sake1Alexander J. Metcalf2Fiona K. Davies3Melissa Cano4Anagha Krishnan5Nanette R. Boyle6Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, CO, United StatesDepartment of Chemical and Biological Engineering, Colorado School of Mines, Golden, CO, United StatesDepartment of Chemical and Biological Engineering, Colorado School of Mines, Golden, CO, United StatesLiving Ink Technologies, Berthoud, CO, United StatesDepartment of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO, United StatesDepartment of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO, United StatesDepartment of Chemical and Biological Engineering, Colorado School of Mines, Golden, CO, United StatesSynechococcus 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.https://www.frontiersin.org/articles/10.3389/fenrg.2022.896668/fullcyanobacteriaterpenoidATP:NADPH ratioPEP carboxylasemalic enzyme |
| spellingShingle | Darrian M. Newman Cara L. Sake Alexander J. Metcalf Fiona K. Davies Melissa Cano Anagha Krishnan Nanette R. Boyle Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002 Frontiers in Energy Research cyanobacteria terpenoid ATP:NADPH ratio PEP carboxylase malic enzyme |
| title | Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002 |
| title_full | Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002 |
| title_fullStr | Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002 |
| title_full_unstemmed | Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002 |
| title_short | Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002 |
| title_sort | characterizing photosynthetic biofuel production isotopically non stationary 13c metabolic flux analysis on limonene producing synechococcus sp pcc 7002 |
| topic | cyanobacteria terpenoid ATP:NADPH ratio PEP carboxylase malic enzyme |
| url | https://www.frontiersin.org/articles/10.3389/fenrg.2022.896668/full |
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