From heterotrophic monoculture to light-driven coculture: A sustainable platform for PHA biosynthesis and insights into interspecies metabolic interactions.

Microbial consortia combining phototrophic and heterotrophic partners offer a sustainable and cost-effective strategy for producing polyhydroxyalkanoates, which have attracted considerable attention as promising alternatives to petroleum-derived plastics. Here, a synthetic cyanobacteria-heterotroph consortium composed of Synechococcus elongatus and Priestia aryabhattai was employed to produce polyhydroxybutyrate (PHB) directly from CO2. Transcriptomic analysis of the heterotrophic partner revealed the sucrose-to-PHB biosynthesis pathway and key gene cluster. Optimal coculture conditions were established to enhance biomass accumulation and PHB production, reaching a maximum titer of 75.1 mg/L and productivity of 33.6 mg/L/day. Moreover, physical separation experiments using transwell inserts and dialysis bags demonstrated that direct cellular contact was not necessarily required for cooperative growth. Under these conditions, PHB production by the consortium was further increased to 102 mg/L. Nevertheless, genome-scale metabolic modeling, targeted metabolite supplementation assays, and untargeted extracellular metabolomics revealed extensive cross-feeding of amino acids, dipeptides, carbohydrates, and cofactors, which are essential nutrients for coculture stability and metabolic coordination. This study not only establishes a sustainable platform for PHB biosynthesis but also provides insights into interspecies metabolic interactions, guiding the rational design of synthetic microbial consortia for biomanufacturing applications.