Conversion of CO2 into valuable products: Engineering the PirC-PGAM switch in cyanobacteria to direct carbon flux into desired products

BackgroundIn response to rising CO2 emissions driving global warming, there is an urgent need for a transition toward a sustainable bioeconomy. Photo-biotechnological processes based on oxygenic photosynthesis hold high potential for achieving CO2 neutrality and in this regard, cyanobacteria have emerged as promising biocatalysts. Rational metabolic engineering of cyanobacteria depends on a thorough understanding of native regulatory mechanisms governing primary metabolism, which can limit metabolic flux through specific pathways and, consequently, the formation of target products. Recent insights have identified a key regulatory node at the 2,3-bisphosphogylcerate-independent phosphoglycerate mutase (PGAM) reaction, where the metabolic flux from newly fixed carbon is redirected from the Calvin-Benson-Bassham (CBB) cycle towards lower glycolysis. This metabolic valve is controlled by the small inhibitor protein PirC, whose binding to PGAM is determined by the central signal transduction protein PII. ResultsIn this study, we exploit the PirC-PGAM interaction as a novel target for regulatory metabolic engineering in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Chassis strains with engineered control of PGAM, defined as PGAM-ON or PGAM-OFF states, were generated using two complementary approaches: tuning pgam gene expression and modulating PirC abundance to regulate PGAM activity. The effectiveness of this regulatory engineering strategy was demonstrated by redirecting carbon flux toward two representative, naturally occurring products: sucrose, produced via gluconeogenesis fueled by the Calvin-Benson-Bassham (CBB) cycle, and succinate, an intermediate of the tricarboxylic acid (TCA) cycle. Narrowing the PGAM valve resulted in a threefold increase in sucrose accumulation. In contrast, opening the PGAM valve by relieving PGAM inhibition through pirC deletion or separate pgam overexpression resulted in up to an 18-fold increase in succinate excretion. Furthermore, similar genetic configurations were applied to enhance production of a heterologous compound, isoprene, derived from pyruvate. ConclusionsThis study establishes the PGAM valve as a tunable control point for the rational re-direction of carbon flux in Synechocystis and highlights small regulatory proteins as powerful targets for metabolic engineering. Together, these findings provide proof of concept for an advanced level of molecular engineering in cyanobacteria and to fully harness their biocatalytic potential in future photosynthesis-driven biotechnological applications.