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 γ‐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, C15) along with cyclization and ring contraction to furnish monocyclic γ‐presodorifen pyrophosphate (γ‐PSPP, C16). Subsequent C‐methylation of γ‐PSPP by a second C‐methyltransferase furnishes the monocyclic α‐prechlororaphen pyrophosphate (α‐PCPP, C17), serving as the substrate for the terpene synthase. The same biosynthetic pathway was characterized in the β‐proteobacterium Variovorax boronicumulans PHE5‐4, demonstrating that non‐canonical homosesquiterpene biosynthesis is more widespread in the bacterial domain than previously anticipated. In the biosynthetic pathway of the first natural brexane‐type compound, chlororaphen, farnesyl pyrophosphate (FPP) is methylated and cyclized by a non‐canonical FPP methyltransferase (FPP‐MT), furnishing γ‐presodorifen pyrophosphate (γ‐PSPP). Methylation by the γ‐PSPP‐MT yields α‐prechlororaphen pyrophosphate (α‐PCPP), which is converted into the bishomosesquiterpene chlororaphen by the chlororaphen synthase (ChloS).