De novo coassembly of functional chlorophyll- and carotenoid-protein complexes in <i>Saccharomyces cerevisiae</i>.

Abstract

Chlorophylls are essential for photosynthesis, yet their heterologous production is a major challenge in synthetic biology due to pathway complexity and the diversion of resources away from essential cofactor biosynthesis. Here, we reconstruct the complete chlorophyll biosynthetic pathway in <i>Saccharomyces cerevisiae</i>. By integrating 13 heterologous genes and deleting 3 endogenous genes, we engineered a yeast chassis capable of producing chlorophyll a at titers of 26 micrograms per gram of dry cell weight. This platform was further expanded with a ketocarotenoid biosynthetic module to produce echinenone and canthaxanthin, enabling the in vivo coassembly of two distinct pigment-protein complexes: the plant-derived water-soluble chlorophyll protein and the cyanobacterial orange carotenoid protein (Syn_OCP). Spectroscopic and biochemical analyses confirm that these yeast-assembled complexes retain native spectral and functional signatures, including Syn_OCP photoactivity, demonstrating high-fidelity pigment binding. Our work establishes a versatile eukaryotic platform for producing photosynthetic complexes, which could help open avenues for studying their assembly outside plants and for engineering light-driven metabolism in nonphotosynthetic eukaryotes.