skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria

Abstract

Photosynthesis uses solar energy to drive inorganic carbon (Ci) uptake, fixation, and biomass formation. In cyanobacteria, Ci uptake is assisted by carbon concentrating mechanisms (CCM), and CO 2 fixation is catalyzed by RubisCO in the Calvin-Benson-Bassham (CBB) cycle. Understanding the regulation that governs CCM and CBB cycle activities in natural and engineered strains requires methods and parameters that quantify these activities. Here, we used membrane-inlet mass spectrometry (MIMS) to simultaneously quantify Ci concentrating and fixation processes in the cyanobacterium Synechocystis 6803. By comparing cultures acclimated to ambient air conditions to cultures transitioning to high Ci conditions, we show that acclimation to high Ci involves a concurrent decline of Ci uptake and fixation parameters. By varying light input, we show that both CCM and CBB reactions become energy limited under low light conditions. A strain over-expressing the gene for the CBB cycle enzyme fructose-bisphosphate aldolase showed higher CCM and carbon fixation capabilities, suggesting a regulatory link between CBB metabolites and CCM capacity. While the engineering of an ethanol production pathway had no effect on CCM or carbon fixation parameters, additional fructose-bisphosphate aldolase gene over-expression enhanced both activities while simultaneously increasing ethanol productivity. These observations show that MIMS can be a usefulmore » tool to study the extracellular Ci flux and how CBB metabolites regulate Ci uptake and fixation.« less

Authors:
 [1];  [2];  [1];  [1];  [1];  [1];  [2];  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. Uppsala Univ., Uppsala (Sweden)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1544524
Report Number(s):
NREL/JA-2700-72738
Journal ID: ISSN 1664-302X
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Frontiers in Microbiology
Additional Journal Information:
Journal Volume: 10; Journal ID: ISSN 1664-302X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; CCU; MIMS; carbon uptake rate; cyanobacteria; FbaA

Citation Formats

Douchi, Damien, Liang, Feiyan, Cano, Melissa, Xiong, Wei, Wang, Bo, Maness, Pin -Ching, Lindblad, Peter, and Yu, Jianping. Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria. United States: N. p., 2019. Web. doi:10.3389/fmicb.2019.01356.
Douchi, Damien, Liang, Feiyan, Cano, Melissa, Xiong, Wei, Wang, Bo, Maness, Pin -Ching, Lindblad, Peter, & Yu, Jianping. Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria. United States. doi:10.3389/fmicb.2019.01356.
Douchi, Damien, Liang, Feiyan, Cano, Melissa, Xiong, Wei, Wang, Bo, Maness, Pin -Ching, Lindblad, Peter, and Yu, Jianping. Tue . "Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria". United States. doi:10.3389/fmicb.2019.01356. https://www.osti.gov/servlets/purl/1544524.
@article{osti_1544524,
title = {Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria},
author = {Douchi, Damien and Liang, Feiyan and Cano, Melissa and Xiong, Wei and Wang, Bo and Maness, Pin -Ching and Lindblad, Peter and Yu, Jianping},
abstractNote = {Photosynthesis uses solar energy to drive inorganic carbon (Ci) uptake, fixation, and biomass formation. In cyanobacteria, Ci uptake is assisted by carbon concentrating mechanisms (CCM), and CO2 fixation is catalyzed by RubisCO in the Calvin-Benson-Bassham (CBB) cycle. Understanding the regulation that governs CCM and CBB cycle activities in natural and engineered strains requires methods and parameters that quantify these activities. Here, we used membrane-inlet mass spectrometry (MIMS) to simultaneously quantify Ci concentrating and fixation processes in the cyanobacterium Synechocystis 6803. By comparing cultures acclimated to ambient air conditions to cultures transitioning to high Ci conditions, we show that acclimation to high Ci involves a concurrent decline of Ci uptake and fixation parameters. By varying light input, we show that both CCM and CBB reactions become energy limited under low light conditions. A strain over-expressing the gene for the CBB cycle enzyme fructose-bisphosphate aldolase showed higher CCM and carbon fixation capabilities, suggesting a regulatory link between CBB metabolites and CCM capacity. While the engineering of an ethanol production pathway had no effect on CCM or carbon fixation parameters, additional fructose-bisphosphate aldolase gene over-expression enhanced both activities while simultaneously increasing ethanol productivity. These observations show that MIMS can be a useful tool to study the extracellular Ci flux and how CBB metabolites regulate Ci uptake and fixation.},
doi = {10.3389/fmicb.2019.01356},
journal = {Frontiers in Microbiology},
number = ,
volume = 10,
place = {United States},
year = {2019},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Figures / Tables:

FIGURE 1 FIGURE 1: A schematic representation of the relationship between photosynthetically harvested energy by the photosynthetic electron transfer chain (PETC), inorganic carbon (Ci) uptake and fixation, and the anabolic metabolism. Orange arrows indicate chemical energy fluxes. Black arrows indicate carbon flux.

Save / Share:

Works referenced in this record:

13C flux analysis of cyanobacterial metabolism
journal, October 2014

  • Adebiyi, Adeola O.; Jazmin, Lara J.; Young, Jamey D.
  • Photosynthesis Research, Vol. 126, Issue 1
  • DOI: 10.1007/s11120-014-0045-1

Synthetic DNA system for structure-function studies of the high affinity CO2 uptake NDH-13 protein complex in cyanobacteria
journal, October 2018

  • Artier, Juliana; Holland, Steven C.; Miller, Neil T.
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1859, Issue 10
  • DOI: 10.1016/j.bbabio.2018.06.015

Cyanobacterial NDH-1 complexes: Novel insights and remaining puzzles
journal, August 2011

  • Battchikova, Natalia; Eisenhut, Marion; Aro, Eva-Mari
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1807, Issue 8
  • DOI: 10.1016/j.bbabio.2010.10.017

Early Evolution of Photosynthesis
journal, October 2010


Regulation of CO2 Concentrating Mechanism in Cyanobacteria
journal, January 2015

  • Burnap, Robert; Hagemann, Martin; Kaplan, Aaron
  • Life, Vol. 5, Issue 1
  • DOI: 10.3390/life5010348

Genome-wide analysis of the fructose 1,6-bisphosphate aldolase (FBA) gene family and functional characterization of FBA7 in tomato
journal, November 2016


The kinetics of the reaction CO2 + H2O → H+ + HCO3− as one of the rate limiting steps for the dissolution of calcite in the system H2OCO2CaCO3
journal, September 1996


Cellular inorganic carbon fluxes in Trichodesmium: a combined approach using measurements and modelling
journal, November 2014

  • Eichner, Meri; Thoms, Silke; Kranz, Sven A.
  • Journal of Experimental Botany, Vol. 66, Issue 3
  • DOI: 10.1093/jxb/eru427

Simultaneous Transport of CO 2 and HCO 3 by the Cyanobacterium Synechococcus UTEX 625
journal, July 1988

  • Espie, George S.; Miller, Anthony G.; Birch, Douglas G.
  • Plant Physiology, Vol. 87, Issue 3
  • DOI: 10.1104/pp.87.3.551

Engineering the methylerythritol phosphate pathway in cyanobacteria for photosynthetic isoprene production from CO 2
journal, January 2016

  • Gao, Xiang; Gao, Fang; Liu, Deng
  • Energy & Environmental Science, Vol. 9, Issue 4
  • DOI: 10.1039/C5EE03102H

A mass spectrometer inlet system for sampling gases dissolved in liquid phases
journal, April 1963


Impacts of genetically engineered alterations in carbon sink pathways on photosynthetic performance
journal, December 2016


Installing extra bicarbonate transporters in the cyanobacterium Synechocystis sp. PCC6803 enhances biomass production
journal, May 2015


CO 2 CONCENTRATING MECHANISMS IN PHOTOSYNTHETIC MICROORGANISMS
journal, June 1999


Combined Effects of CO 2 and Light on the N 2 -Fixing Cyanobacterium Trichodesmium IMS101: Physiological Responses
journal, July 2010

  • Kranz, Sven A.; Levitan, Orly; Richter, Klaus-Uwe
  • Plant Physiology, Vol. 154, Issue 1
  • DOI: 10.1104/pp.110.159145

Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio
journal, March 2018


Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803
journal, November 2016


Synechocystis PCC 6803 overexpressing RuBisCO grow faster with increased photosynthesis
journal, June 2017


Combinatory strategy for characterizing and understanding the ethanol synthesis pathway in cyanobacteria cell factories
journal, November 2015


Active Transport of CO 2 by the Cyanobacterium Synechococcus UTEX 625 : Measurement by Mass Spectrometry
journal, March 1988

  • Miller, Anthony G.; Espie, George S.; Canvin, David T.
  • Plant Physiology, Vol. 86, Issue 3
  • DOI: 10.1104/pp.86.3.677

Genome Engineering of the 2,3-Butanediol Biosynthetic Pathway for Tight Regulation in Cyanobacteria
journal, May 2015


A modular system for the measurement of CO 2 and O 2 gas flux and photosynthetic electron transport in microalgae : Algal photosynthetic efficiency system
journal, December 2012

  • Oakley, Clinton A.; Hopkinson, Brian M.; Schmidt, Gregory W.
  • Limnology and Oceanography: Methods, Vol. 10, Issue 12
  • DOI: 10.4319/lom.2012.10.968

CyAbrB2 Contributes to the Transcriptional Regulation of Low CO 2 Acclimation in Synechocystis sp. PCC 6803
journal, September 2016

  • Orf, Isabel; Schwarz, Doreen; Kaplan, Aaron
  • Plant and Cell Physiology, Vol. 57, Issue 10
  • DOI: 10.1093/pcp/pcw146

The functioning of the CO 2 concentrating mechanism in several cyanobacterial strains: a review of general physiological characteristics, genes, proteins, and recent advances
journal, June 1998

  • Price, G. Dean; Sültemeyer, Dieter; Klughammer, Barbara
  • Canadian Journal of Botany, Vol. 76, Issue 6
  • DOI: 10.1139/b98-081

Photoreduction of O 2 Primes and Replaces CO 2 Assimilation
journal, September 1976


Consistent Sets of Spectrophotometric Chlorophyll Equations for Acetone, Methanol and Ethanol Solvents
journal, June 2006


Active uptake of CO2 during photosynthesis in the green alga Eremosphaera viridis is mediated by a CO2-ATPase
journal, January 1992

  • Rotatore, Caterina; Lew, RogerR.; Colman, Brian
  • Planta, Vol. 188, Issue 4
  • DOI: 10.1007/BF00197046

Catalytic exchange of 18 O from 13 C 18 O-labelled CO 2 by wild-type cells and ecaA , ecaB , and ccaA mutants of the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803
journal, June 1998

  • So, Anthony KC; Van Spall, Harriette GC; Coleman, John R.
  • Canadian Journal of Botany, Vol. 76, Issue 6
  • DOI: 10.1139/b98-063

A thylakoid‐located carbonic anhydrase regulates CO 2 uptake in the cyanobacterium Synechocystis sp. PCC 6803
journal, December 2018

  • Sun, Nan; Han, Xunling; Xu, Min
  • New Phytologist, Vol. 222, Issue 1
  • DOI: 10.1111/nph.15575

Passive Entry of CO 2 and Its Energy-dependent Intracellular Conversion to HCO in Cyanobacteria Are Driven by a Photosystem I-generated ΔμH +
journal, April 2001

  • Tchernov, Dan; Helman, Yael; Keren, Nir
  • Journal of Biological Chemistry, Vol. 276, Issue 26
  • DOI: 10.1074/jbc.M101973200

Increased fructose 1,6-bisphosphate aldolase in plastids enhances growth and photosynthesis of tobacco plants
journal, February 2012

  • Uematsu, Kimio; Suzuki, Nobuaki; Iwamae, Tomoko
  • Journal of Experimental Botany, Vol. 63, Issue 8
  • DOI: 10.1093/jxb/ers004

Alterations in Global Patterns of Gene Expression in Synechocystis sp. PCC 6803 in Response to Inorganic Carbon Limitation and the Inactivation of ndhR , a LysR Family Regulator
journal, November 2003

  • Wang, Hong-Liang; Postier, Bradley L.; Burnap, Robert L.
  • Journal of Biological Chemistry, Vol. 279, Issue 7
  • DOI: 10.1074/jbc.M311336200

Advancing Our Understanding and Capacity to Engineer Nature’s CO 2 -Sequestering Enzyme, Rubisco
journal, October 2010

  • Whitney, Spencer M.; Houtz, Robert L.; Alonso, Hernan
  • Plant Physiology, Vol. 155, Issue 1
  • DOI: 10.1104/pp.110.164814

Sensing of Inorganic Carbon Limitation in Synechococcus PCC7942 Is Correlated with the Size of the Internal Inorganic Carbon Pool and Involves Oxygen
journal, November 2005

  • Woodger, Fiona J.; Badger, Murray R.; Price, G. Dean
  • Plant Physiology, Vol. 139, Issue 4
  • DOI: 10.1104/pp.105.069146

The plasticity of cyanobacterial carbon metabolism
journal, December 2017


Genetic engineering of the Calvin cycle toward enhanced photosynthetic CO2 fixation in microalgae
journal, October 2017


    Works referencing / citing this record:

    Consistent Sets of Spectrophotometric Chlorophyll Equations for Acetone, Methanol and Ethanol Solvents
    journal, June 2006


    13C flux analysis of cyanobacterial metabolism
    journal, October 2014

    • Adebiyi, Adeola O.; Jazmin, Lara J.; Young, Jamey D.
    • Photosynthesis Research, Vol. 126, Issue 1
    • DOI: 10.1007/s11120-014-0045-1

    A mass spectrometer inlet system for sampling gases dissolved in liquid phases
    journal, April 1963


    The kinetics of the reaction CO2 + H2O → H+ + HCO3− as one of the rate limiting steps for the dissolution of calcite in the system H2OCO2CaCO3
    journal, September 1996


    Impacts of genetically engineered alterations in carbon sink pathways on photosynthetic performance
    journal, December 2016


    Cyanobacterial NDH-1 complexes: Novel insights and remaining puzzles
    journal, August 2011

    • Battchikova, Natalia; Eisenhut, Marion; Aro, Eva-Mari
    • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1807, Issue 8
    • DOI: 10.1016/j.bbabio.2010.10.017

    Synthetic DNA system for structure-function studies of the high affinity CO2 uptake NDH-13 protein complex in cyanobacteria
    journal, October 2018

    • Artier, Juliana; Holland, Steven C.; Miller, Neil T.
    • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1859, Issue 10
    • DOI: 10.1016/j.bbabio.2018.06.015

    The plasticity of cyanobacterial carbon metabolism
    journal, December 2017


    Synechocystis PCC 6803 overexpressing RuBisCO grow faster with increased photosynthesis
    journal, June 2017


    Genome-wide analysis of the fructose 1,6-bisphosphate aldolase (FBA) gene family and functional characterization of FBA7 in tomato
    journal, November 2016


    Installing extra bicarbonate transporters in the cyanobacterium Synechocystis sp. PCC6803 enhances biomass production
    journal, May 2015


    Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803
    journal, November 2016


    Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio
    journal, March 2018


    Genome Engineering of the 2,3-Butanediol Biosynthetic Pathway for Tight Regulation in Cyanobacteria
    journal, May 2015


    The plasticity of cyanobacterial metabolism supports direct CO2 conversion to ethylene
    journal, April 2015


    Increased fructose 1,6-bisphosphate aldolase in plastids enhances growth and photosynthesis of tobacco plants
    journal, February 2012

    • Uematsu, Kimio; Suzuki, Nobuaki; Iwamae, Tomoko
    • Journal of Experimental Botany, Vol. 63, Issue 8
    • DOI: 10.1093/jxb/ers004

    Cellular inorganic carbon fluxes in Trichodesmium: a combined approach using measurements and modelling
    journal, November 2014

    • Eichner, Meri; Thoms, Silke; Kranz, Sven A.
    • Journal of Experimental Botany, Vol. 66, Issue 3
    • DOI: 10.1093/jxb/eru427

    CyAbrB2 Contributes to the Transcriptional Regulation of Low CO 2 Acclimation in Synechocystis sp. PCC 6803
    journal, September 2016

    • Orf, Isabel; Schwarz, Doreen; Kaplan, Aaron
    • Plant and Cell Physiology, Vol. 57, Issue 10
    • DOI: 10.1093/pcp/pcw146

    Sensing of Inorganic Carbon Limitation in Synechococcus PCC7942 Is Correlated with the Size of the Internal Inorganic Carbon Pool and Involves Oxygen
    journal, November 2005

    • Woodger, Fiona J.; Badger, Murray R.; Price, G. Dean
    • Plant Physiology, Vol. 139, Issue 4
    • DOI: 10.1104/pp.105.069146

    Combined Effects of CO 2 and Light on the N 2 -Fixing Cyanobacterium Trichodesmium IMS101: Physiological Responses
    journal, July 2010

    • Kranz, Sven A.; Levitan, Orly; Richter, Klaus-Uwe
    • Plant Physiology, Vol. 154, Issue 1
    • DOI: 10.1104/pp.110.159145

    Early Evolution of Photosynthesis
    journal, October 2010


    Advancing Our Understanding and Capacity to Engineer Nature’s CO 2 -Sequestering Enzyme, Rubisco
    journal, October 2010

    • Whitney, Spencer M.; Houtz, Robert L.; Alonso, Hernan
    • Plant Physiology, Vol. 155, Issue 1
    • DOI: 10.1104/pp.110.164814

    Photoreduction of O 2 Primes and Replaces CO 2 Assimilation
    journal, September 1976


    Active Transport of CO 2 by the Cyanobacterium Synechococcus UTEX 625 : Measurement by Mass Spectrometry
    journal, March 1988

    • Miller, Anthony G.; Espie, George S.; Canvin, David T.
    • Plant Physiology, Vol. 86, Issue 3
    • DOI: 10.1104/pp.86.3.677

    Simultaneous Transport of CO 2 and HCO 3 by the Cyanobacterium Synechococcus UTEX 625
    journal, July 1988

    • Espie, George S.; Miller, Anthony G.; Birch, Douglas G.
    • Plant Physiology, Vol. 87, Issue 3
    • DOI: 10.1104/pp.87.3.551

    A thylakoid‐located carbonic anhydrase regulates CO 2 uptake in the cyanobacterium Synechocystis sp. PCC 6803
    journal, December 2018

    • Sun, Nan; Han, Xunling; Xu, Min
    • New Phytologist, Vol. 222, Issue 1
    • DOI: 10.1111/nph.15575

    Catalytic exchange of 18 O from 13 C 18 O-labelled CO 2 by wild-type cells and ecaA , ecaB , and ccaA mutants of the cyanobacteria Synechococcus PCC7942 and Synechocystis PCC6803
    journal, June 1998

    • So, Anthony KC; Van Spall, Harriette GC; Coleman, John R.
    • Canadian Journal of Botany, Vol. 76, Issue 6
    • DOI: 10.1139/b98-063

    The functioning of the CO 2 concentrating mechanism in several cyanobacterial strains: a review of general physiological characteristics, genes, proteins, and recent advances
    journal, June 1998

    • Price, G. Dean; Sültemeyer, Dieter; Klughammer, Barbara
    • Canadian Journal of Botany, Vol. 76, Issue 6
    • DOI: 10.1139/b98-081

    CO 2 CONCENTRATING MECHANISMS IN PHOTOSYNTHETIC MICROORGANISMS
    journal, June 1999


    Combinatory strategy for characterizing and understanding the ethanol synthesis pathway in cyanobacteria cell factories
    journal, November 2015


    Genetic engineering of the Calvin cycle toward enhanced photosynthetic CO2 fixation in microalgae
    journal, October 2017


    Regulation of CO2 Concentrating Mechanism in Cyanobacteria
    journal, January 2015

    • Burnap, Robert; Hagemann, Martin; Kaplan, Aaron
    • Life, Vol. 5, Issue 1
    • DOI: 10.3390/life5010348

    A modular system for the measurement of CO 2 and O 2 gas flux and photosynthetic electron transport in microalgae : Algal photosynthetic efficiency system
    journal, December 2012

    • Oakley, Clinton A.; Hopkinson, Brian M.; Schmidt, Gregory W.
    • Limnology and Oceanography: Methods, Vol. 10, Issue 12
    • DOI: 10.4319/lom.2012.10.968

      Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.