Genome mining of nonenzymatic ortho-quinone methide-based pseudonatural products from ascidian-derived fungus Diaporthe sp.SYSU-MS4722.

Ortho-quinone methides (o-QMs), generated by oxidative dehydration of clavatol, are highly reactive intermediates in biosynthesis that give rise to a variety of clavatol-containing pseudonatural products (PNPs) in fungi through intra- and intermolecular nonenzymatic cyclization/addition reaction, and some compounds have significant biological activities. Here we report our genome mining efforts on a cryptic clavatol biosynthetic gene cluster (BGC) from an ascidian-derived fungus Diaporthe sp. SYSU-MS4722. The core genes NR-PKS (DiaG), Esterase (DiaF) derived from the fungus Diaporthe sp. SYSU-MS4722 clavatol BGC and the known α-ketoglutarate-dependent nonheme iron enzymes (ClaD) were heterologously expressed in the Aspergillus oryzae NSAR1 (A. oryzae NSAR1). Thirteen new monomeric, dimeric, and trimeric clavatol-based PNPs (7-19), together with three known compounds (20-22) were isolated from the above transformant. Their structures including absolute configurations were elucidated by spectroscopic analysis (UV, IR, HR-ESI-MS, 1D and 2D NMR data), complemented with the X-ray crystallography, the comparison of the experimental and calculated ECD spectra, and gauge-independent atomic orbital (GIAO) NMR calculations. Based on the structural characteristics, their plausible biosynthetic pathways were proposed. Notably, Compounds 8, 9, 14 and 16 exhibited potent anti-fibrotic activity with EC values of 28.9, 10.0, 3.5 and 30.1 μM, respectively.