Construction of a Nonnatural C₆₀ Carotenoid Biosynthetic Pathway

Longer-chain carotenoids have interesting physiological and electronic/photonic properties due to their extensive polyene structures. Establishing nonnatural biosynthetic pathways for longer-chain carotenoids in engineerable microorganisms will provide a platform to diversify and explore the potential of these molecules. We have previously reported the biosynthesis of nonnatural C₅₀ carotenoids by engineering a C₃₀-carotenoid backbone synthase (CrtM) from Staphylococcus aureus. In the present work, we conducted a series of experiments to engineer C₆₀ carotenoid pathways. Stepwise introduction of cavity-expanding mutations together with stabilizing mutations progressively shifted the product size specificity of CrtM toward efficient synthases for C₆₀ carotenoids. By coexpressing these CrtM variants with hexaprenyl diphosphate synthase, we observed that C₆₀-phytoene accumulated together with a small amount of C₆₅-phytoene, which is the largest carotenoid biosynthesized to date. Although these carotenoids failed to serve as a substrate for carotene desaturases, the C₂₅-half of the C₅₅-phytoene was accepted by the variant of phytoene desaturase CrtI, leading to accumulation of the largest carotenoid-based pigments. Continuing effort should further expand the scope of carotenoids, which are promising components for various biological (light-harvesting, antioxidant, and communicating) and nonbiological (photovoltaic, photonic, and field-effect transistor) systems.