Synthetic Biology Driven Biosynthesis of Unnatural Tropolone Sesquiterpenoids

Tropolone sesquiterpenoids (TS) are an intriguing family of biologically active fungal meroterpenoids that arise through a unique intermolecular hetero Diels–Alder (hDA) reaction between humulene and tropolones. Here, we report on the combinatorial biosynthesis of a series of unprecedented analogs of the TS pycnidione 1 and xenovulene A 2. In a systematic synthetic biology driven approach, we recombined genes from three TS biosynthetic gene clusters (pycnidione 1, xenovulene A 2 and eupenifeldin 3) in the fungal host Aspergillus oryzae NSAR1. Rational design of the reconstituted pathways granted control over the number of hDA reactions taking place, the chemical nature of the fused polyketide moiety (tropolono‐ vs. monobenzo‐pyranyl) and the degree of hydroxylation. Formation of unexpected monobenzopyranyl sesquiterpenoids was investigated using isotope‐feeding studies to reveal a new and highly unusual oxidative ring contraction rearrangement.