Discovery and engineering of colchicine alkaloid biosynthesis

Few complete pathways have been established for the biosynthesis of medicinal compounds from Plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal Plants or Plant Cell Culture¹. A lead example is colchicine, a US Food and Drug Administration (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 uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine⁶. Notably, we identified a non-canonical Cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 Enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the Amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine Alkaloids and provides insights into the unique chemistry that Plants use to generate complex, bioactive metabolites from simple Amino acids.