Linking PII regulation with central carbon control in Synechocystis PCC 6803
Final Report Abstract
Cyanobacteria, as the pioneers of oxygenic photosynthesis, have started over 2 billions of years ago, to enrich the earth’s atmosphere with oxygen, which paved the way for the evolution of life as we know it today. During their evolution, cyanobacteria have tuned the regulatory mechanisms to perfection, which enabled them to adapt and acclimate to the ever-changing environmental conditions. One of the mist prominent examples of such regulatory systems is the interaction network of the nitrogen-controlling PII signalling protein, which was studied in depth in the non-diazotrophic model organism Synechocystis PCC6803. In this organism, PII regulates numerous metabolic processes, which are required to maintain a proper carbon- and nitrogen homeostasis. Thereby, PII interacts with various target proteins (the “PII interactors”) to tune their activity. In this way, PII coordinates the carbon-nitrogen balance and the storage of excess nitrogen in form of arginine and the polymer cyanophycin. Moreover, PII controls indirectly the global nitrogen transcription factor NtcA via interaction with the NtcA co-activator PipX. In this project, we identified and characterized a novel PII-interactor, the PirC protein. PirC turned out to be a central switch of the flow of newly fixed CO2. PirC acts as inhibitor of the enzyme co-factor independent 2,3-bisphosphoglycerat mutase (PGAM). PGAM catalyzes the conversion of the first product of CO2 fixation, 3-phosphoglycerate into 2-phosphoglycerate, which guides newly fixed carbon towards anabolic reactions of amino acid synthesis and fatty acid synthesis. By inhibiting PGAM, carbon flow is mainly re-directed towards the carbon-storage polymer glycogen. Under natural conditions, nitrogen limitation leads to inhibition of PGAM by interaction with PirC. By contrast, a PirC deficient mutant accumulates less glycogen but increased levels of polyhydroxybutyrate (PHB) during nitrogen limitation. To analyse in more detail the mechanism of PirC-mediated PGAM inhibition, biochemical structure-function analysis and enzymatic characterization was carried out. We could show that the interaction of PII with PirC alleviates the inhibitory effect of PirC on PGAM. The interaction of PII with PirC depends on the leve of the metabooic status-reporter metabolites ATP, ADP and 2-oxoglutarate. Increased levels of 2-oxoglutarate (the indicator of the state of nitrogen assimilation via the GS-GOGAT pathway) lead to dissociation of the PII-PirC complex. We identified specific structural elements in the cyanobacterial PGAM enzymes that are required to transmit the inhibitory effect of PirC on PGAM activity. A strain with a C- terminal truncated PGAM exhibits constitutively high PGAM activities, leading to increased PHB levels. Overall, this work laid the foundation for the construction of Synechocystis strains that can be used as superior chassis for the synthesis of sustainable production of bioplastic PHB and other valuable goods from CO2.
Publications
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Kinetic Analysis of a Protein-protein Complex to Determine its Dissociation Constant (KD) and the Effective Concentration (EC50) of an Interplaying Effector Molecule Using Bio-layer Interferometry. BIO-PROTOCOL, 11(17).
Orthwein, Tim; Huergo, Luciano; Forchhammer, Karl & Selim, Khaled
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The novel P II -interactor PirC identifies phosphoglycerate mutase as key control point of carbon storage metabolism in cyanobacteria. Proceedings of the National Academy of Sciences, 118(6).
Orthwein, Tim; Scholl, Jörg; Spät, Philipp; Lucius, Stefan; Koch, Moritz; Macek, Boris; Hagemann, Martin & Forchhammer, Karl
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New views on PII signaling: from nitrogen sensing to global metabolic control. Trends in Microbiology, 30(8), 722-735.
Forchhammer, Karl; Selim, Khaled A. & Huergo, Luciano F.
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Structural elements of cyanobacterial co-factor-independent phosphoglycerate mutase that mediate regulation by PirC. openRxiv.
Orthwein, Tim; Alford, Janette Theresa; Becker, Nathalie Sofie; Fink, Phillipp & Forchhammer, Karl
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Unravelling the Function of Sll0944 in the Regulation of Carbon and Nitrogen Metabolism in Synechocystis sp. PCC 6803. Dissertation der Universität Tübingen
Orthwein, T.
