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

蓝细菌在光自养和异养条件下生长。光合自养利用光能将无机碳和氮同化为有机物。异养利用先前同化的有机物质进行能量生产和生长所需的生物合成过程。因此，自养和异养以相反的方向使用部分代谢途径。为了避免浪费的循环，蓝细菌调节代谢并通过经由蛋白质体（羧化体）的代谢反应的螯合、通过代谢控制（例如通过使用替代的氧化还原辅因子，如NAD或NADP）或通过代谢通道在异养和自养模式之间切换。SCyCode研究小组在上一个资助期调查了由光暗转换或碳或氮供应改变引起的异养/自养开关。SCyCode合作伙伴确定了后续深入分析的监管方面，在黑暗中RubisCO不完全失活的具体证据和RpaA调节剂对该现象的贡献，或磷酸葡萄糖变位酶（PGM）和葡萄糖-6-磷酸脱氢酶（G6 PDH）的酶促步骤之间的氧化还原调节的葡萄糖-6-磷酸（Glc 6 P）底物通道向Entner-Doudoroff（艾德）的指示和氧化戊糖磷酸（oxidative pentose phosphate，PETP）途径。Kopka小组为SCyCode研究小组的更新提案提供代谢表型和代谢通量分析能力。详细地说，Kopka小组与Wilde和Hagemann小组合作，将应用13 CO2和18 O2标记实验来揭示集胞藻野生型和ΔrpaA突变体细胞在黑暗条件下与光照条件下的RubisCO活性。我们将与Forchhammer小组合作，使用纯化的PGM、G6 PDH和天然或突变的支架和调节蛋白OpcA，开发Glc 6P的体外和体内代谢通道测定。此外，Kopka小组将通过与Hess和Spät/ Maček合作分析蛋白质-代谢物复合物，特别关注在异养/自养转换条件下RubisCO和RpaA的相互作用，为进一步建立代谢组学技术做出贡献。最后，Kopka小组将使用先前生成和提出的通量组数据集来通过计算机探索RubisCO和共同监测的磷酸烯醇式丙酮酸羧化酶反应的13 C-位置同位素异构体分析的选择。该子项目将有助于整合SCyCode的多种研究方法，并将与合作伙伴互动，以实现代谢水平的新功能见解，这些见解只能通过合作伙伴的分子方法的独特组合来获得。

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.