Oxidative Ring Contraction by a Multifunctional Dioxygenase Generates the Core Cycloocatadiene in the Biosynthesis of Fungal Dimeric Anhydride Zopfiellin

Abstract

To elucidate the biosynthesis of a fungicidal dimeric anhydride zopfiellin, the putative biosynthetic gene cluster was identified. We conducted heterologous expression of candidate genes for the synthesis of maleic anhydride and its dimerization and identified the two isomeric dimers with 9-membered rings as products. Notably, α-ketoglutarate-dependent dioxygenase ZopK oxidized one of the dimers, giving the 8-membered ring of zopfiellin. The mechanism of oxidative rearrangement is proposed by analyzing the incorporation of 13C-labeled precursors.

Uncovering biosynthetic relationships between antifungal nonadrides and octadrides

“…ZopK, which is equivalent to ZopL9 of the D. curvata BGC reported here, was shown to catalyse the oxidative ring contraction in the zopellin pathway to produce deoxyzopellin 24. 25 This proceeds via the intermediate 19 (which we now know to be 5-hydroxy-deoxyscytalidin) and is consistent with our identication, via feeding studies, of 19 as an intermediate in the zopellin pathway. In vitro assays using puried recombinant ZopL9 gave results which are entirely consistent with the ndings of Shiina et al 25 In the presence of a-ketoglutarate, ZopL9 was capable of catalysing highly efficient hydroxylation of deoxyscytalidin 17 to give 5-hydroxy-deoxyscytalidin 19 (Fig.…”

“…Additional genes identied in both clusters encode an enoyl-CoA hydratase, an isochorismatase, a small hypothetical protein (zopL8 and scyR11), a GAL4 type transcription factor and two MFS (major facilitator superfamily) transporters. The enoyl-CoA hydratase encoded by the zopellin cluster (zopR2) was not identied in the work by Oikawa and colleagues, 25 but RNAseq data conrmed its annotation.…”

“…Recent work by Shiina et al 25 on the zopellin pathway of Z. curvata, using heterologous expression as well as in vitro assays, has begun to elucidate the later stages of zopellin biosynthesis. ZopK, which is equivalent to ZopL9 of the D. curvata BGC reported here, was shown to catalyse the oxidative ring contraction in the zopellin pathway to produce deoxyzopellin 24.…”

“…2). Our biosynthetic investigations involved a different approach from those previously reported 25 and used a combination of gene disruptions in Diffractella curvata and Scytalidium album, feeding studies to mutant strains and full structure elucidation of known and novel compounds. These studies establish the biosynthetic relationships between zopellin 15, scytalidin 16, and deoxyscytalidin 17.…”

“…These studies showed that a ring contraction occurs from a nonadride to an octadride. 25 Herein we describe our recent studies which conrm the absolute and relative congurations of the nonadride scytalidin 16 and octadride zopellin 15 and reveal that they share…”