Abstract
Photoautotrophic microorganisms are increasingly explored for the conversion of atmospheric carbon dioxide into biomass and valuable products. The Calvin-Benson-Bassham (CBB) cycle is the primary metabolic pathway for net CO2 fixation within oxygenic photosynthetic organisms. The cyanobacteria, Synechocystis sp. PCC 6803, is a model organism for the study of photosynthesis and a platform for many metabolic engineering efforts. The CBB cycle is regulated by complex mechanisms including enzymatic abundance, intracellular metabolite concentrations, energetic cofactors and post-translational enzymatic modifications that depend on the external conditions such as the intensity and quality of light. However, the extent to which each of these mechanisms play a role under different light intensities remains unclear. In this work, we conducted non-targeted proteomics in tandem with isotopically non-stationary metabolic flux analysis (INST-MFA) at four different light intensities to determine the extent to which fluxes within the CBB cycle are controlled by enzymatic abundance. The correlation between specific enzyme abundances and their corresponding reaction fluxes is examined, revealing several enzymes with uncorrelated enzyme abundance and their corresponding flux, suggesting flux regulation by mechanisms other than enzyme abundance. Additionally, the kinetics of 13C labeling of CBB cycle intermediates and estimated inactive pool sizes varied significantly as a function of light intensity suggesting the presence of metabolite channeling, an additional method of flux regulation. These results highlight the importance of the diverse methods of regulation of CBB enzyme activity as a function of light intensity, and highlights the importance of considering these effects in future kinetic models.
Highlights
The Calvin-Benson-Bassham (CBB) cycle is the primary metabolic pathway through which inorganic carbon is fixed by photosynthetic organisms (Bassham et al, 1954)
While the abundances of many CBB cycle enzymes are well correlated with light levels, other enzymes such as both PRK isozymes, FBP/SBPase, and GAPDH2 are not, suggesting the importance of additional forms of regulation
Our 13C labeling data taken across multiple light levels suggest that metabolite channeling is present but plays an important role in modulating specific reaction fluxes, as revealed by the presence of inactive metabolite pools
Summary
The Calvin-Benson-Bassham (CBB) cycle is the primary metabolic pathway through which inorganic carbon is fixed by photosynthetic organisms (Bassham et al, 1954). The rate limiting steps and factors controlling the pathway are only partially captured by existing models (Arnold and Nikoloski, 2011; Jablonsky et al, 2011; Mills et al, 2020). Varying enzymatic abundance is one way in which metabolic processes are regulated. Cyanobacteria are interesting model organisms for their fast growth and their ability to be genetically transformed (Ducat et al, 2011). Cyanobacteria can serve as a way to transform atmospheric CO2 into useful end products (Carroll et al, 2018), such as astaxanthin (Diao et al, 2020), ethylene (Ungerer et al, 2012), ethanol (Dexter and Fu, 2009), or omega-3 fatty acids (Santos-Merino et al, 2018)
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