Redirecting Carbon Flux Toward 2-Ketoglutarate Enables Photoautotrophic 1,4-Butanediol Production from CO2.

Engineered cyanobacteria offer a sustainable platform for the conversion of light and CO2 into value-added chemicals. Unlike heterotrophic systems, cyanobacteria do not require organic carbon sources such as glycerol, glucose, or sucrose. In this study, we engineered Synechococcus elongatus PCC 7942 (hereafter 7942) for the production of 1,4-butanediol (14BDO), a precursor used in the synthesis of pharmaceuticals, biodegradable polymers, and resins, directly from CO2. To identify a suitable biosynthetic route, we evaluated three heterologous 14BDO pathways previously developed in heterotrophic hosts. Among these, the carboxylic acid reductase (Car) pathway supported 14BDO production directly from CO2 without supplementation of an exogenous carbon source, whereas the Weimberg pathway required d-xylose supplementation and the CoA-dependent pathway did not produce detectable 14BDO. We therefore focused on the Car pathway and improved production by rewiring carbon flux through the incomplete tricarboxylic acid pathway, directing intermediates toward 2-ketoglutarate as a key metabolic node. Further engineering, including additional expression of ppc and gltA and deletion of glgC, increased 14BDO production, consistent with enhanced carbon availability for 14BDO biosynthesis. This work establishes a functional route for photoautotrophic 14BDO production from CO2 and demonstrates the importance of carbon flux engineering for improving production of non-native chemicals in cyanobacteria.