Evidence for enzyme catalysed intramolecular [4+2] Diels–Alder cyclization during the biosynthesis of pyrichalasin H

Abstract

Cytochalasans are highly complex fungal metabolites which exhibit diverse biological activities. Little is known of the chemical steps involved in the construction of the tricyclic core, which consists of an octahydro-isoindole skeleton fused to a macrocyclic ring. Here, using a directed gene knockout and complementation strategy, we show that PyiF is implicated as the proposed intramolecular [4+2] Diels-Alderase required for construction of the tricyclic core of pyrichalasin H 1.

Predicting the chemical space of fungal polyketides by phylogeny-based bioinformatics analysis of polyketide synthase-nonribosomal peptide synthetase and its modification enzymes

“…3 and S2 , Table S6 ). Additionally, we found that both type-I and type-II DAases can be further divided into two subclades: clade Iα functionally characterized DAase: LepI 25 ), Iβ (IccD 23 ), IIα (EqxF 31 , Fsa2 32 , Phm7 32 , and Tas3 33 ), and IIβ (CcsF 34 , CHGG_01241 35 , PoxQ 36 , and MycB 37 ). IIβ can be further divided into two groups, which reflect the two alternative functions of synthesizing either a macrocyclic (IIβ-1) or decalin (IIβ-2) ring.…”

Diversely Functionalised Cytochalasins through Mutasynthesis and Semi‐Synthesis

“… [10] We recently reported the discovery and characterisation of the biosynthetic gene cluster (BGC) encoding the biosynthesis of 1 a [11, 12] . Knockout experiments showed that the biosynthesis starts with the O ‐methylation of l ‐tyrosine 2 to give 3 a (Scheme 1), which is the substrate for a hybrid polyketide synthase non‐ribosomal peptide synthetase (PKS‐NRPS, PyiS) that, in association with auxiliary proteins, [13] produces the cytochalasin skeleton 4 a . This undergoes various oxygenation [14] and acylation reactions to give 1 a .…”

Chemical and Genetic Studies on the Formation of Pyrrolones During the Biosynthesis of Cytochalasans

“… Structure of Δ pyiE shunt metabolite 9 isolated in this study and shunt metabolite 10 isolated in the Δ pyiF study [11] …”

“…We then re‐introduced a copy of either pyiE itself or its homolog ORFZ , under the control of the Aspergillus oryzae amyB promoter ( P amyB ), into the KO strain using previously reported methods [21] . In previous work we had shown that P amyB is functional in M. grisea and we therefore expected that biosynthesis of 1 should be restored to WT titres [7, 11] . However, LCMS analysis showed that 1 was not produced in significantly increased titre for either complementation experiment (Figure 1 and Figures S6.1–S6.6).…”

“…Meanwhile, pyiR encodes a transcription‐factor which we have shown to be responsible for increasing the titre of 1 [10] . Recently we showed that PyiF is involved in the proposed DA reaction [11,12] …”