Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to
anthocyanidins attributed to the action of
leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that
anthocyanin-related
glutathione transferases, currently implicated only in
anthocyanin transport, catalyse an essential
dehydration of the
leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate
cyanidin. Building on this knowledge, introduction of
anthocyanin-related
glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased
anthocyanin production. In addition to unravelling the long-elusive
anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants.