Evolutionary tuning of a biosynthetic gene cluster drives furanocoumarin accumulation and diversification

Deciphering evolutionary drivers of biosynthetic pathways could enhance bioactive compound production. In Angelica, interspecific variation in furanocoumarins (FCs) accumulation reflects divergent pathway evolution. Here, we conduct comparative genomics between high-FC Angelica sensu stricto (s.s.) and low-FC Angelica sensu lato (s.l.) species. We reveal an FC biosynthetic gene cluster (BGC) comprising core enzymes (p-coumaroyl-CoA 2'-hydroxylases (C2'Hs), prenyltransferases (PTs)) and peripheral O-methyltransferases (OMTs). The ancestral Angelica s.l. clade retains an FC BGC configuration with OMTs on separate chromosomes and PTs performing only C-prenylation. In contrast, Angelica s.s. evolves an FC BGC, where core enzymes and OMTs co-localise on the same chromosome, with C2'H copy number expansion correlating with elevated expression and PTs enabling both C- and O-prenylation, collectively enhancing FC production and structural diversity. These findings elucidate how BGC architecture, gene copy number and functional innovation collectively drive phytochemical innovation, providing a blueprint for engineering medicinal FC biosynthesis.