Abstract

SUMMARY Phycocyanin (PC) is a key light-harvesting pigment protein in the phycobilisome of cyanobacteria and rhodophyta, and is currently the only natural blue pigment successfully commercialized. However, its application is limited by complex biosynthetic and degradative regulation, which constrain efficient CO 2 -based green production. Here, using Synechocystis sp. PCC 6803 as a photosynthetic chassis, the phycocyanobilin (PCB) precursor network was then systematically reconstructed to overcome key biosynthetic bottlenecks. For the first time in cyanobacteria, the C4 synthesis pathway of 5-aminolevulinic acid (5-ALA) was introduced together with enhancement of the native 5-ALA supply route. Combined with ferrochelatase (Ppfc)-mediated redirection of metabolic flux, this strategy expanded and stabilized the tetrapyrrole metabolic pool, resulting in a synergistic enhancement of PCB biosynthesis. In parallel, modulation of apophycocyanin expression was incorporated to better coordinate chromophore supply with phycobiliprotein assembly. In addition, genetic suppression of phycobilisome degradation under high light conditions effectively prolonged the functional half-life of PC. The multimodule engineered strain achieved a PC content of 124.62 mg g −1 under gas-bubbling cultivation, corresponding to a 66.92% increase over the wild-type, accompanied by improved photosystem II (PSII) photochemical efficiency, enhanced electron transport, and elevated photoprotective pigment levels. These results demonstrate that efficient PC biosynthesis emerges from coordinated, system-level engineering of the photosynthetic network rather than amplification of a single metabolic node. This work establishes a scalable paradigm for CO 2 -driven green manufacturing of pigment proteins and the construction of robust photosynthetic cell factories.