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

Damien Douchi,Wei Xiong,Bo Wang,Pin-Ching Maness,Jianping Yu,Feiyan Liang,Peter Lindblad,Melissa Cano

doi:10.3389/fmicb.2019.01356

Damien Douchi, Wei Xiong + Show 6 more

Open Access

https://doi.org/10.3389/fmicb.2019.01356

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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 tool to study the extracellular Ci flux and how CBB metabolites regulate Ci uptake and fixation.

Highlights

Photosynthesis has been responsible for decreasing CO2 in the atmosphere from 30–35% to 0.04% over the last 3 billion years or so (Blankenship, 2010) and is the major physicochemical process that generates organic molecules and, as such, supports most of the life on Earth
Under high Ci levels at pH 7–8, bicarbonate transporters and other constituents of the concentrating mechanisms (CCM) are bypassed by the saturating flow of CO2 entering the cell
Air bubbling into the cultures was used to maintain a low but constant Ci supply while avoiding over accumulation of the O2 generated by the photosynthetic electron transport chain (PETC)

Summary

Introduction

Photosynthesis has been responsible for decreasing CO2 in the atmosphere from 30–35% to 0.04% over the last 3 billion years or so (Blankenship, 2010) and is the major physicochemical process that generates organic molecules and, as such, supports most of the life on Earth. MIMS Quantification of Carbon Uptake (RubisCO) appeared 2.5 billion years ago and has evolved to adapt to the decreasing CO2 concentrations and concurrently increasing O2 levels (Whitney et al, 2011). Photosynthetic organisms have evolved various strategies to favor carboxylation over oxygenation by increasing carbon availability for RubisCO. At least five different uptake proteins or complexes are involved in this process, each with different affinities for Ci. Among them are the BicA and SbtA, sodium/bicarbonate symporters, powered by a sodium gradient across the plasma membrane. The involvement of plasma membrane sodium/proton antiporters and ATPase was hypothesized (Kamennaya et al, 2015). Another bicarbonate transporter is the Bct complex, which has its own ATPase activity. The CO2 uptake systems NDH1-3 and NDH1-4 directly convert the CO2 to bicarbonate in the cytoplasm using energy from photosynthetic or respiratory thylakoid electron flow (Battchikova et al, 2011; Artier et al, 2018), locking incoming CO2 which diffuses freely across the membrane and limiting Ci leakages

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