SCyCode - Unraveling the cyanobacterial carbon switch and its regulation using enzyme kinetic analyses and mathematical modeling

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Synechocystis sp. PCC 6803 (hereafter named Synechocystis) possesses a broad metabolic versatility and is able to grow photoautrotrophic, heterotrophic as well as photomixotrophic. In addition to the auto- and heterotrophic diurnal rhythm, transitions from a photoautotrophic to a heterotrophic life style “autotrophy-heterotrophy switch” are also induced in response to inorganic carbon and nitrogen fluctuations.Notably, the Synechocystis primary metabolism is characterized by parallel pathways with partly shared enzyme reactions and multiple isoenzymes. Among the different pathways the Embden-Meyerhof-Parnas (EMP) pathway is the only reversible pathway with function in gluconeogenesis and glycolysis. In previous studies it was demonstrated that in Synechocystis the biochemical control likely dominates over the transcriptional control in order to adjust the central carbon metabolism to response to changed carbon availability. Hence, we hypothesized that these isoenzymes might possess different kinetic and regulatory properties to perform the autotrophy-heterotrophy switch. In the first funding period, we identified the two phosphofructokinase (PFK) and pyruvate kinase (PK) isoenzymes as well as phosphoglycerate kinase (PGK) as novel control points of the cyanobacterial EMP pathway. These studies were performed in close collaboration with Jacky Snoep (Co-PI) and the groups of Martin Hagemann and Kirstin Gutekunst. Our results hint at a complex regulation, which takes place and is governed by the energy charge of the cell, the availability of reduced/oxidized electron carrier (i.e. NAD(P)+ or NAD(P)H + H+) and the redox state of the cell (i.e. thioredoxin/ferredoxin). In addition, the cellular concentrations of key intermediates such as glucose 6-phosphate or 3-phosphoglycerate seems to play a significant role. The aim of this subproject in the second funding period is to provide additional detailed enzyme characterizations combined with detailed reaction kinetic mathematical models (Co-PI Jacky Snoep) in order to characterize these important control points that regulate the carbon flux in response to growth conditions, i.e. the autotrophy-heterotrophy switch. Therefore, we will focus on the antagonistic enzyme couples, and the triosephosphate hub as control points of the EMP pathway. In addition, the flux of glucose 6-phophate from glycogen or glucose into the EMP or alternative catabolic pathways as well as the regulation of the lower EMP shunt will be addressed. These studies will be performed in close collaboration with our SCyCode partners Karl Forchhammer, Sofia Doello, Martin Hagemann and Kirstin Gutekunst.