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

Journal Article · 24 June 2019 · Frontiers in Microbiology

DOI: https://doi.org/10.3389/fmicb.2019.01356 · OSTI ID:1544524

Douchi, Damien ^[1]; Liang, Feiyan ^[2]; Cano, Melissa ^[1]; Xiong, Wei ^[1]; Wang, Bo ^[1]; Maness, Pin -Ching ^[1]; Lindblad, Peter ^[2]; Yu, Jianping ^[1]

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1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
2. Uppsala Univ., Uppsala (Sweden)

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.

• Research Organization: National Renewable Energy Laboratory (NREL), Golden, CO (United States)
• Sponsoring Organization: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
• Grant/Contract Number: AC36-08GO28308
• OSTI ID: 1544524
• Report Number(s): NREL/JA--2700-72738
• Journal Information: Frontiers in Microbiology, Journal Name: Frontiers in Microbiology Vol. 10; ISSN 1664-302X
• Publisher: Frontiers Research FoundationCopyright Statement
• Country of Publication: United States
• Language: English