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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 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 toolmore » 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]
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  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), Transportation Office. Bioenergy Technologies Office
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.
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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. https://doi.org/10.3389/fmicb.2019.01356
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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. https://doi.org/10.3389/fmicb.2019.01356. https://www.osti.gov/servlets/purl/1544524.
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@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}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.3389/fmicb.2019.01356
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Cited by: 2 works
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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.
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    Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803
    journal, January 2020

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