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

蓝藻在光自养和异养条件下都能生长。光自养利用光能将无机碳和氮同化为有机物。异养利用以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，自养和异养利用了部分相反方向的代谢途径。为了避免浪费的循环，蓝藻通过蛋白质体（羧酸体）隔离代谢反应，通过代谢控制，例如通过使用替代氧化还原辅助因子，如NAD或NADP，或通过代谢通道来调节代谢并在异养和自养模式之间切换。SCyCode研究小组在之前的资助期内研究了由光暗转换或碳或氮供应改变引起的异养/自养开关。SCyCode合作伙伴确定了随后深入分析的调控方面，特别是在黑暗中RubisCO不完全失活的证据，以及RpaA调节剂对这种现象的贡献，或者是氧化还原调节的葡萄糖-6-磷酸（Glc6P）底物通道在磷酸葡萄糖糖化酶（PGM）和葡萄糖-6-磷酸脱氢酶（G6PDH）通往enner - doudoroff （ED）和氧化磷酸五糖（OPP）途径之间的指示。Kopka小组为SCyCode研究组的更新建议贡献了代谢表型和代谢通量分析的能力。Kopka小组与Wilde和Hagemann小组合作，将采用13CO2-和18O2-标记实验来揭示聚囊藻野生型和ΔrpaA突变细胞在黑暗条件下与光照条件下的RubisCO活性。我们将与Forchhammer集团合作，使用纯化的PGM、G6PDH和天然或突变的支架和调节蛋白OpcA，开发Glc6P的体外和体内代谢通道测定。此外，Kopka团队将与Hess和Spät/ ma<e:1> ek合作，进一步建立代谢组学支持技术，分析蛋白质-代谢物复合物，特别关注异养/自养开关条件下RubisCO和RpaA的相互作用。最后，Kopka小组将使用先前生成的和建议的通量组数据集来探索硅片，13c -位置同位素分析RubisCO的选择，并共同监测磷酸烯醇丙酮酸羧化酶反应。该子项目将有助于整合SCyCode的各种研究方法，并将与合作伙伴进行互动，以获得代谢水平上的新功能见解，这些见解只能通过合作伙伴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.