Biosynthesis of Indole Diterpene Lolitrems: Radical‐Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton

Abstract

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome‐editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical‐induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue.

Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

“…CRISPR-KI has been mostly used to integrate fragments that span few kilobases, and although recently it has been used for the integration of larger BGC fragments in Aspergilli, the efficiency of targeted integration is not reported. 20,21 In contrast, recombinase-mediated integration does not generate dsDNA breaks and is orthogonal to the host machinery. This makes recombinase-based systems a convenient and reliable alternative for the single-step integration of large DNA constructs, where the efficiency of HR declines.…”

Branching and converging pathways in fungal natural product biosynthesis

“…The P450 LtmQ, which is homologous to PaxQ and AtmQ, performs hydroxylation to provide terpendole E (131) [105,106]. Compound 131 is subsequently transformed into terpendole C (132) by four enzymes, and finally, 130 is synthesized by the prenyltransferase LtmE and the P450 LtmJ from 132 [107,108]. In Chaunopycnis alba, an uncharacterized dehydrogenase oxidizes 132 to yield terpendole K (133) [106].…”

Heterologous production of fungal natural products: Reconstitution of biosynthetic gene clusters in model host <i>Aspergillus oryzae</i>

“…Tandem transformation utilizing two plasmids with the same selectable marker is efficient, requiring the use of fewer vectors and allowing a rapid introduction of biosynthetic genes to obtain TFs. By applying this simple method, we completely elucidated the detailed biosynthetic pathway of fungal indole diterpenes such as shearinine, 34) lolitrem, 35) and especially penitrem 36) whose biosynthesis requires 17 genes, one of the largest number of genes in the fungal metabolite biosynthesis (Fig. 5).…”