Franziska Hufnagel (Leipzig / DE), Nathalie Sofie Becker (Tübingen / DE), Karl Forchhammer (Tübingen / DE), Stephan Klähn (Leipzig / DE)
Introduction
Among prokaryotes oxygenic photosynthesis is a unique feature in cyanobacteria. In the context of establishing a CO2-neutral, sustainable bioeconomy, they receive growing interest as biocatalysts in photo-biotechnological applications. To rationally engineer cyanobacteria and to direct metabolic fluxes towards chemical products, it is important to consider native molecular processes that control metabolism. Recently, the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM) has been shown to serve as a central control point determining the carbon flux from the Calvin-Benson-Bassham cycle (CBB) to lower glycolysis. In particular, PGAM activity is controlled by the small protein PirC that also binds to the central regulatory protein PII. Upon PirC-binding, PGAM is inactivated leading to glycogen accumulation within the cell.1
We aim to target the PirC-PGAM switch in the model organism Synechocystis sp. PCC 6803 to gain control over photosynthetically driven biocatalytic processes. In particular, flux through PGAM shall be blocked to increase product yields that derive from gluconeogenic routes.
Material and Methods
Three different methods to turn off the PGAM activity were developed all of which are controlled by inducible promoters. First, an approach to overexpress pirC is used. Second, the expression of pgam is downregulated. Finally, overexpression of pirC and downregulation of pgam are combined. Once all strains are constructed, Northern Blots are performed to check changes in transcription levels of both pirC and pgam after the induction of the promoters. Besides that, the glycogen concentration in each strain is measured using an enzymatic assay to determine which approach works best to inactivate PGAM.
Results
A rise in glycogen amount can be seen once the activity of PGAM is turned down in pirC-overexpression strains. In addition, we demonstrate that our approach also has implications on other product pathways, e. g. for sucrose, that derive from the CBB.
1 T. Orthwein, J. Scholl, P. Spät, S. Lucius, M. Koch, B. Macek, M. Hagemann, K. Forchhammer. PNAS, 2021, 118(6): e2019988118