(SCyCode): Metabolomics and metabolic flux analyses for probing transitions between heterotrophic and autotrophic growth

• Applicant: Dr. Joachim Kopka
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 397695561

Cyanobacteria grow both, under photo-autotrophic and under heterotrophic conditions. Photo-autotrophy assimilates inorganic carbon and nitrogen into organic matter using light energy. Heterotrophy uses previously assimilated organic matter for energy production and the biosynthetic processes required for growth. Accordingly, autotrophy and heterotrophy use part of the metabolic pathways in opposite directions. To avoid wasteful circles, cyanobacteria regulate metabolism and switch between heterotrophic and autotrophic modes by sequestration of metabolic reactions via protein bodies (carboxysomes), by metabolic control, e.g. via use of alternative redox cofactors, such as NAD or NADP, or by metabolic channeling. The SCyCode research group investigated in the previous funding period heterotrophy/ autotrophy switches that are induced by transition between light and dark or altered carbon- or nitrogen-supply. SCyCode partners identified regulatory aspects for subsequent in-depth analysis, specifically evidence of incomplete RubisCO deactivation in the dark and contributions of the RpaA regulator to this phenomenon or indications of redox-regulated glucose-6-phosphate (Glc6P) substrate channeling between the enzymatic steps of phosphoglucomutase (PGM) and glucose-6-phosphate dehydrogenase (G6PDH) towards the Entner-Doudoroff (ED) and oxidative pentose phosphate (OPP) pathways. The Kopka group contributes competence of metabolic phenotyping and metabolic flux analysis to the renewal proposals of the SCyCode research group. In detail, the Kopka group in cooperation with the Wilde and Hagemann groups will apply 13CO2- and 18O2- labelling experiments to unravel RubisCO activity of Synechocystis wild type and ΔrpaA mutant cells under dark compared to light conditions. We will develop in cooperation with the Forchhammer group in vitro and in vivo metabolic channeling assays for Glc6P using purified PGM, G6PDH and the native or mutated scaffold and regulator protein, OpcA. In addition, the Kopka group will contribute to further establishment of metabolomics enabling technologies by teaming up with the Hess and Spät/ Maček for the analysis of protein-metabolite complexes with a specific focus on RubisCO and RpaA interactions under heterotrophy/autotrophy switch conditions. Finally, The Kopka group will use previously generated and proposed fluxomic data sets to explore in silico, the option of 13C-positional isotopomer analysis for the RubisCO and co-monitored phosphoenolpyruvate carboxylase reactions. The sub-project will be instrumental to the integration of the diverse research approaches of SCyCode and will interact with the cooperating partners towards novel functional insights at metabolic level that can only be gained by the unique combination of the partners´ molecular approaches.

• DFG Programme: Research Units
• Subproject of: FOR 2816: The Autotrophy-Heterotrophy Switch in Cyanobacteria: Coherent Decision-Making at Multiple Regulatory Layers
• International Connection: USA
• Cooperation Partner: Dr. Aleksandra Skirycz