Oxepinamide F biosynthesis involves enzymatic d-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration

Liujuan Zheng,Aili Fan,Shu-Ming Li,Haowen Wang

doi:10.1038/s41467-020-18713-0

Abstract

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. Here we report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. The nonribosomal peptide synthase (NRPS) OpaA assembles the quinazolinone core with d-Phe incorporation. The cytochrome P450 enzyme OpaB catalyzes alone the oxepin ring formation. The flavoenzyme OpaC installs subsequently one hydroxyl group at the oxepin ring, accompanied by double bond migration. The epimerase OpaE changes the d-Phe residue back to l-form, which is essential for the final methylation by OpaF.

Highlights

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety
Oxepinamides F, G, and H were isolated from Aspergillus puniceus[1,2], varioloid A and varioxepine A from Paecilomyces variotii[3,4], dihydrocinereain from Aspergillus carneus[5], circumdatin A/B from Aspergillus ochraceus[6], and versicoloid A/B from Aspergillus versicolor[7] (Fig. 1a)
We identify in this study an oxepinamide biosynthetic gene cluster (BGC) in Aspergillus ustus by bioinformatic analysis

Summary

Introduction

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. We report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. Oxepinamides are usually derived from quinazolinones with an anthranilyl (Ant) residue in common They differ from each other by incorporation of two other varying amino acids and additional modifications. Heterologous expression, feeding experiments, and in vitro assays with purified enzymes prove the biosynthetic steps and the 3H-oxepin formation by consecutive ring expansion and regio- and stereospecific hydroxylation. The Dphenylalanyl epimerase OpaE converts the D-Phe residue back to L-form for the last methylation step to form oxepinamide F

Methods

Results

Conclusion