Conversion of CO2 into valuable products: Regulatory engineering of cyanobacteria to direct carbon flux into desired reactions

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Today, it is widely accepted that global warming is caused by anthropogenic CO2 emissions due to the combustion of fossil fuels. Accordingly, climate change and its consequences are of main public interest, which led to a rethinking and the political commitment for the establishment of a CO2-neutral bio-economy in the next decades. This also requires sustainable, e.g. biotechnological processes based on organic carbon (C) delivered by photosynthetic, i.e. light-driven CO2 fixation. As cyanobacteria are the only prokaryotes performing oxygenic photosynthesis, they receive growing interest as biocatalysts in photo-biotechnological applications nowadays. To rationally engineer cyanobacteria by channeling metabolic fluxes to obtain the maximum yield of a desired chemical product, it is also important to consider native molecular processes that control primary metabolism. Although we are only at the beginning of a full understanding of the regulation of cyanobacterial metabolism, recent research has opened the window into a more comprehensive view on fundamental control principles. For instance, we recently discovered a key control step of central C metabolism. The metabolic flux of newly fixed C is majorly controlled at the phosphoglycerate-mutase (PGAM) reaction, which converts the first CO2 fixation product 3-phosphoglycerate (3-PGA) to 2-phosphoglycerate (2-PGA). This reaction acts as a metabolic valve as C is taken out from the Calvin-Benson cycle and re-directed towards lower glycolysis, i.e. into the reaction sequence from glyceraldehyde 3-phosphate (G3P) to pyruvate. As revealed recently, the catalysis provided by PGAM is controlled by the interaction with the small protein PirC, which itself is under control of the central PII signal transduction protein. As another level of metabolic engineering we aim to target the PirC-PGAM switch as key hub of regulatory engineering. In the frame of the project, we will construct chassis strains of the cyanobacterial model strain Synechocystis sp. PCC 6803 with engineered PGAM valves by using three complementary approaches: tuning PGAM gene expression, tuning PGAM activity by modulating PirC abundance and eliminating the control of PII over PirC. In addition, we will combine our approach with “classical” metabolic engineering strategies, e.g. the introduction and expression of genes encoding (heterologous) pathways, feeding or otherwise supporting reactions or the deletion of competing reactions. The significance of regulatory engineering to direct metabolic flux towards the anticipated product will be demonstrated by two representative chemicals derived either from the Calvin Benson cycle (sucrose) or the TCA cycle (succinate). Our study will provide a proof of concept for the next level of molecular engineering of cyanobacteria. Accordingly, it will be crucial to fully exploit their biocatalytic potential, i.e. for the future design of photosynthesis-driven biotechnological applications.