Discovery and engineering of colchicine alkaloid biosynthesis

Few complete pathways are established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture.(1) A lead example is colchicine, an FDA-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species.(2-5) Here we use a combination of transcriptomics, metabolic logic, and pathway reconstitution to elucidate a near complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome, or genetic tools in the native host. We have uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine.(6) Notably, in doing so we have identified a non-canonical cytochrome P450 that catalyzes the remarkable ring expansion reaction required to produce the distinct carbon scaffold of colchicine. We further utilize the newly identified genes to engineer a biosynthetic pathway (16 enzymes total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This work establishes a metabolic route to tropolone-containing colchicine alkaloids and provides new insights into the unique chemistry plants use to generate complex, bioactive metabolites from simple amino acids.