A C₃₅ carotenoid biosynthetic pathway

Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C₃₀ carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C₃₅ backbone. The products of condensation of farnesyldiphosphate and GDP, C₃₅ structures comprise 40 to 60% of total carotenoid accumulated. Carotene desaturases and carotene cyclases from C₄₀ or C₃₀ pathways accepted and converted the C₃₅ substrate, thus creating a C₃₅ carotenoid biosynthetic pathway in E. coli. Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described. This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.