Identification of the cytochrome P450 monooxygenase that bridges the clavine and ergoline alkaloid pathways

Clavines and D-lysergic acid-derived alkaloid amides and alkaloid peptides ore two different families of compounds that have the indole-derived tetracyclic metergoline ring system in common. Previous work has shown that D-lysergic acid is biosynthetically derived from clavine alkaloids. Recent cloning and analysis of the ergot alkaloid biosynthesis gene cluster from the D-lysergic acid peptide (ergopeptines)-producing Claviceps purpurea, has shown that it most probably contains all genes necessary for D-lysergic acid synthesis as well as those that encode the assembly Of D-lysergic acid peptides, such as ergotamine. To address the role of the oxygenase genes of alkaloid-gene clusters, the only cyto-chrome P450 monooxygenase gene of this cluster was inactivated by disruption. The resultant mutant accumulated agroclavine, elymoclavine, and chonoclavine in substantial amounts but not ergopeptines. Feeding the mutant with D-lysergic acid restored ergopeptine synthesis; this suggests a block in the conversion of elymoclavine to D-lysergic acid. The gene was designated cloA (for encoding a clavine oxidose, CLOA). Retransformotion of the mutant with the intact cloA gene also restored ergopeptine synthesis. These data show that CLOA catalyses the conversion of clavines to D-lysergic acid, it acts as a critical enzyme in the ergot alkaloid gene cluster, and bridges the biosynthesis of the two different families of alkaloids.