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

Abstract

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.

Berberine bridge enzyme-like oxidase-catalysed double bond isomerization acts as the pathway switch in cytochalasin synthesis

“…44 – 49 , the elucidation process for compound 3 is described in SI). Although we obtained shunt compound 3 rather than the expected product 4 from strain AN - aspoEH , possibly due to the rapid tautomerization of 4 to 3 in vivo 29 , the production of 3 fully demonstrates that (1) the working programs of both the hrPKS module (for polyketide chain extension) and NRPS module (for polyketide chain transfer and amino acid selection) of AspoE are correct; and (2) under our culture conditions, no enzymes from A. nidulans can catalyse the reduction of putative key aldehyde intermediate 5 to yield alcohol product 5′ (Fig. 3a ), which is usually observed during the reconstitution of other CYT pathways (Fig.…”

“…2b, vi ). Recently, using synthesized mimic substrates, Zhang et al proposed that the hydrolase-catalysed reaction might occur prior to the Diels-Alderase-catalysed reaction during pyrichalasin H biosynthesis 29 . Formation of a hydrolase-bound intermediate (via covalent binding to retain the correct tautomer form of the substrate) is crucial for the subsequent Diels-Alder reaction.…”

Secondary metabolite biosynthetic diversity in the fungal family Hypoxylaceae and Xylaria hypoxylon

“…To establish the typical octahydroisoindolone core, the activities of a hybrid PKS-NRPS, trans -enoyl reductase, α,β-hydrolase and a Diels-Alderase are required ( Skellam 2017 , Hantke et al. 2020 , Zhang et al. 2021 ).…”

Total Mycosynthesis: Rational Bioconstruction and Bioengineering of Fungal Natural Products

“…18 Tyrosine 18a is first O-methylated (PyiA), and O-methyltyrosine 18b is fused to an octaketide and probably released as the aldehyde 19 by a PKS-NRPS (PyiS) and trans-ER (PyiC), similar to those involved in the construction of pretenellin A 2. 19 Knoevenagel (PyiE) 20 and Diels-Alder (PyiF) 21 reactions then form the distinctive isoindolone core 20 and four tailoring steps reduce the 21-carbonyl (PyiH), acetylate the resulting secondary alcohol (PyiB), and hydroxylate at C-18 (PyiD) and C-7 (PyiG). The fungus Magnaporthe grisea produces pyrichalasin H at around 60 mg•L -1 , although overexpression of the pathway-specific regulator pyiR can increase this titre three-fold.…”