Metabolic regulation of carbon allocation from the CBB cycle under different CO2 supply

蓝藻是光自养生物，白天吸收二氧化碳，晚上通过异养途径利用积累的有机碳。此外，Synechocystis在将细胞从高二氧化碳环境转移到低二氧化碳环境后，激活了卡尔文循环中低糖酵解方向的碳流，减少了糖原储备，这表明细胞在光照下的代谢从自养到异养的变化。因此，初级碳代谢的合成代谢和分解代谢途径都发生在非区隔细胞中，需要严格调节以避免无效循环。我们假设在卡尔文循环和糖酵解之间的代谢分支点上的同工酶（如磷酸甘油突变酶，PGAM，或甘油醛-3-磷酸脱氢酶，GapDH）至少是这些变化的部分原因。在第一个项目阶段，我们在特定突变体的帮助下研究了GapDHs和PGAMs在二氧化碳含量不同的合成代谢或分解代谢碳流中的作用。对于低糖酵解方向的碳流，PGAM1主要负责，其活性由调节蛋白PirC开启或关闭，而其他PGAM同工酶往往不参与。进一步实验表明，GapDH1不能利用NADPH2，参与异养代谢。GapDH2参与卡尔文循环，主要受CP12蛋白调控。对磷酸化蛋白质组的研究表明，CP12和一个碳酸氢盐转运体的亚基在低二氧化碳条件下被去磷酸化，而蛋白激酶SpkC的磷酸化则增加。此外，其他蛋白激酶似乎也参与了二氧化碳的适应。最后，对昼夜节律细胞的代谢组学研究表明，RubisCO在夜间也有活性。基于这些结果，在接下来的阶段，我们希望与其他SCyCode小组合作，将研究重点放在以下四个方面。1. CP12，特别是它的差异磷酸化，在调节光中的卡尔文循环活性中起什么作用？2. 在二氧化碳驯化过程中，哪些蛋白激酶或磷酸酶负责蛋白的不同磷酸化？3. RubisCO在聚胞菌暗期的活跃程度是多少？它是否受到二磷酸化糖的调节？4. 现有的数据集最终将与墨卡托研究员一起整合到初级碳代谢的二氧化碳驯化动力学模型中。这些研究也将使优化蓝藻碳代谢在未来的生物技术应用成为可能。

Cyanobacteria are photoautotrophic organisms, which assimilate CO2 during the day and use accumulated organic carbon by heterotrophic pathways during the night. Furthermore, Synechocystis activates the carbon flow from the Calvin cycle in the direction of lower glycolysis and reduces glycogen reserves after shifting cells from high to low CO2 conditions, which indicates a change from autotrophic to heterotrophic metabolism in light. Hence, anabolic and catabolic routes of primary carbon metabolism are both occurring in the non- compartmentalized cell, which need to be tightly regulated to avoid futile cycles. We assume that isoenzymes at metabolic branch points between the Calvin cycle and glycolysis (e.g. phosphoglycerate mutases, PGAM, or glyceraldehyde-3-phosphate dehydrogenases, GapDH) are at least partially responsible for these changes.In the first project phase, we investigated the role of GapDHs and PGAMs with the help of specific mutants in the anabolic or catabolic carbon flow depending on the CO2 content. For the carbon flow in the direction of the lower glycolysis, PGAM1 is mainly responsible, the activity of which is switched on or off by the regulatory protein PirC, while the other PGAM isoenzymes tend not to be involved. Further experiments showed that GapDH1 cannot use NADPH2 and participates in heterotrophic metabolism. The GapDH2 is involved in the Calvin cycle and is mainly regulated by the protein CP12. Investigations of the phosphoproteome showed that CP12 and subunits of a bicarbonate transporter are dephosphorylated under low CO2 conditions, while the phosphorylation of the protein kinase SpkC increases. In addition, other protein kinases seem to be involved in the CO2 acclimation. Finally, metabolome studies on cells in a day / night rhythm indicated that RubisCO is also active at night.Based on these results, we want to focus our research in cooperation with other SCyCode groups on the following four points in the upcoming phase. 1. What role does CP12, in particular its differential phosphorylation, play in regulating the Calvin cycle activity in light? 2. Which protein kinases or phosphatases are responsible for the differential phosphorylation of proteins during the CO2 acclimation? 3. To what extent is RubisCO active in Synechocystis in the dark phase and is it regulated by di-phosphorylated sugars? 4. The existing data sets will finally be integrated into a kinetic model of the CO2 acclimation of the primary carbon metabolism together with a Mercator Fellow. These investigations should also make it possible to optimize the cyanobacterial carbon metabolism specifically for biotechnological applications in the future.