The biosynthetic gene cluster encoding the phytotoxin pyrichalasin H 5 was discovered in Magnaporthe grisea NI980, and the late-stage biosynthetic pathway of 5 was fully elucidated using targeted gene inactivations resulting in the isolation of 13 novel cytochalasans. This study reveals that the nonproteinogenic amino acid O-methyltyrosine is the true precursor of 5, and other cryptic cytochalasans and mutasynthesis experiments produce novel halogenated pyrichalasin H analogues.

A key step during the biosynthesis of cytochalasans is a proposed Knoevenagel condensation to form the pyrrolone core, enabling the subsequent 4+2 cycloaddition reaction that results in the characteristic octahydroisoindolone motif of all cytochalasans. In this work, we investigate the role of the highly conserved α,β‐hydrolase enzymes PyiE and ORFZ during the biosynthesis of pyrichalasin H and the ACE1 metabolite, respectively, using gene knockout and complementation techniques. Using synthetic aldehyde models we demonstrate that the Knoevenagel condensation proceeds spontaneously but results in the 1,3‐dihydro‐2H‐pyrrol‐2‐one tautomer, rather than the required 1,5‐dihydro‐2H‐pyrrol‐2‐one tautomer. Taken together our results suggest that the α,β‐hydrolase enzymes are essential for first ring cyclisation, but the precise nature of the intermediates remains to be determined.

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.

Transcription factors from the biosynthesis of fungal metabolites were investigated by ectopic expression: pyiR from the pyrichalasin cluster enhanced titres of pyrichalasin H 1; but BC1 from the ACE1 cluster unexpectedly induced hinnulin A 4.