Non-canonical Biosynthesis of the Brexane-Type Bishomosesquiterpene Chlororaphen through Two Consecutive Methylation Steps in Pseudomonas chlororaphis O6 and Variovorax boronicumulans PHE5-4

A non-canonical biosynthetic pathway furnishing the first natural brexane-type bishomosesquiterpene (chlororaphen, C17H28) was elucidated in the gamma-proteobacterium Pseudomonas chlororaphis O6. A combination of genome mining, pathway cloning, in vitro enzyme assays, and NMR spectroscopy revealed a three-step pathway initiated by C10 methylation of farnesyl pyrophosphate (FPP, C-15) along with cyclization and ring contraction to furnish monocyclic gamma-presodorifen pyrophosphate (gamma-PSPP, C-16). Subsequent C-methylation of gamma-PSPP by a second C-methyltransferase furnishes the monocyclic alpha-prechlororaphen pyrophosphate (alpha-PCPP, C-17), serving as the substrate for the terpene synthase. The same biosynthetic pathway was characterized in the beta-proteobacterium Variovorax boronicumulans PHE5-4, demonstrating that non-canonical homosesquiterpene biosynthesis is more widespread in the bacterial domain than previously anticipated.