Astellifadiene : Structure Determination by NMR Spectroscopy and Crystalline Sponge Method, and Elucidation of its Biosynthesis

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene (1), having a 6-8-6-5-fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope-incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation-initiated second cyclization sequence, 1,2-alkyl migration, and 1,5-hydride shift to generate the novel scaffold of 1. A unique tetracyclic fungal sesterterpene: Genome mining and heterologous expression of a cryptic fungal terpene synthase provided astellifadiene (1) with an unprecedented 6-8-6-5-fused ring system. The structure of 1 was unambiguously determined by the crystalline sponge method coupled with NMR analyses. Further, the biosynthesis of 1 was proposed on the basis of the isotope-incorporation experiments performed both in vivo and in vitro.