Discovery of (±)-Penindolenes Reveals an Unusual Indole Ring Cleavage Pathway Catalyzed by P450 Monooxygenase.

(±)-Penindolenes A-D (1-4), the first representatives of indole terpenoids featuring a γ-lactam skeleton, were isolated from the mangrove-derived endophytic fungus Penicillium brocae MA-231. Our bioactivity tests revealed their potent antimicrobial and acetylcholinesterase inhibitory activities. The biosynthetic reactions by the five enzymes PbaABCDE leading to γ-lactam ring formation were identified with heterologous expression and in vitro enzymatic assays.

Recent developments in the engineered biosynthesis of fungal meroterpenoids.

Meroterpenoids are hybrid compounds that are partially derived from terpenoids. This group of natural products displays large structural diversity, and many members exhibit beneficial biological activities. This mini-review highlights recent advances in the engineered biosynthesis of meroterpenoid compounds with C15 and C20 terpenoid moieties, with the reconstruction of fungal meroterpenoid biosynthetic pathways in heterologous expression hosts and the mutagenesis of key enzymes, including terpene cyclases and α-ketoglutarate (αKG)-dependent dioxygenases, that contribute to the structural diversity.

A Case of Convergent Evolution: The Bacterial Sesquiterpene Synthase for 1-epi-Cubenol from Nonomuraea coxensis.

A terpene synthase from Nonomuraea coxensis was identified as (+)-1-epi-cubenol synthase. The enzyme is phylogenetically unrelated to the known enzyme of the same function that is widespread in streptomycetes. Isotopic labelling experiments were performed to unambiguously assign the NMR data and to investigate hydrogen migrations during terpene cyclisations.

Mechanism of the Bifunctional Multiple Product Sesterterpene Synthase AcAS from Aspergillus calidoustus.

The multiproduct chimeric sesterterpene synthase AcAS from Aspergillus calidoustus yielded spirocyclic calidoustene, which exhibits a novel skeleton, besides five known sesterterpenes. The complex cyclisation mechanism to all six compounds was investigated by isotopic labelling experiments in combination with DFT calculations. Chemically synthesised 8-hydroxyfarnesyl diphosphate was converted with isopentenyl diphosphate and AcAS into four oxygenated sesterterpenoids that structurally resemble cytochrome P450 oxidation products of the sesterterpene hydrocarbons.

Biosynthetic Gene Cluster for Asperterpenols A and B and the Cyclization Mechanism of Asperterpenol A Synthase.

The biosynthetic gene cluster for the sesterterpenoids asperterpenols A and B from CBS121601 is described. The cluster contains two genes, for a chimeric sesterterpene synthase and - for a cytochrome P450 monooxygenase. The cyclization mechanism of the asperterpenol A synthase, catalyzing the formation of a unique sesterterpene with a 6/6/8/5 tetracyclic ring system, was studied using isotopically labeled terpene precursors.

On the mechanism of ophiobolin F synthase and the absolute configuration of its product by isotopic labelling experiments.

An ophiobolin F synthase homolog was discovered from Aspergillus calidoustus CBS121601. The cyclisation mechanism of this terpene synthase was investigated by extensive isotopic labelling experiments and the absolute configuration of its product ophiobolin F was elucidated by enantioselective deuteration.

An Unusual Skeletal Rearrangement in the Biosynthesis of the Sesquiterpene Trichobrasilenol from Trichoderma.

The skeletons of some classes of terpenoids are unusual in that they contain a larger number of Me groups (or their biosynthetic equivalents such as olefinic methylene groups, hydroxymethyl groups, aldehydes, or carboxylic acids and their derivatives) than provided by their oligoprenyl diphosphate precursor. This is sometimes the result of an oxidative ring-opening reaction at a terpene-cyclase-derived molecule containing the regular number of Me group equivalents, as observed for picrotoxan sesquiterpenes. In this study a sesquiterpene cyclase from Trichoderma spp.

Multidomain P450 Epoxidase and a Terpene Cyclase from the Ascochlorin Biosynthetic Pathway in Fusarium sp.

Ascochlorin is a medicinally important fungal meroterpenoid. Its biosynthetic pathway in Fusarium sp. was identified, and the stereoselective epoxidation of the farnesyl group by the multidomain, soluble P450 monooxygenase AscE and the subsequent formation of the unique timethylcyclohexanone ring by the membrane-bound cyclase AscF were investigated.

Elucidation and Heterologous Reconstitution of Chrodrimanin B Biosynthesis.

The biosynthetic gene cluster of the fungal meroterpenoid chrodrimanin B (4) was discovered in Penicillium verruculosum TPU1311, and the complete biosynthetic pathway of 4 has been elucidated by heterologous reconstitution of its biosynthesis in Aspergillus oryzae, as well as by in vitro characterizations of selected enzymes. The present study has identified the polyketide synthase that produces 6-hydroxymellein (3) and also provided a biosynthetic platform of chrodrimanins for further bioengineering.

Genome-Based Discovery of an Unprecedented Cyclization Mode in Fungal Sesterterpenoid Biosynthesis.

Sesterterpenoids are a group of terpenoid natural products that are primarily biosynthesized via cyclization of the C25 linear substrate geranylfarnesyl pyrophosphate (GFPP). Although the long carbon chain of GFPP in theory allows for many different cyclization patterns, sesterterpenoids are relatively rare species among terpenoids, suggesting that many intriguing sesterterpenoid scaffolds have been overlooked. Meanwhile, the recent identification of the first sesterterpene synthase has allowed the discovery of new sesterterpenoids by the genome mining approach.

Cytochrome P450 for Citreohybridonol Synthesis: Oxidative Derivatization of the Andrastin Scaffold.

A biosynthetic gene cluster similar to that for andrastin A (1) was discovered in Emericella variecolor NBRC 32302. Ctr-P450, a cytochrome P450 uniquely present in the cluster, was coexpressed with the andrastin A biosynthetic genes, leading to the production of the antifeedant agent citreohybridonol (4), along with four new andrastin derivatives. The results revealed the unusual multifunctionality of Ctr-P450 and indicated that this approach can be applied for further natural product diversification.

An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon.

Molecular Basis for Stellatic Acid Biosynthesis: A Genome Mining Approach for Discovery of Sesterterpene Synthases.

The search for a new sesterterpene synthase in the genome of Emericella variecolor, which reportedly produces diverse sesterterpenoids, is described. One gene product (a chimeric protein with prenyltransferase and terpene cyclase domains) led to the synthesis of a novel tricyclic sesterterpene, stellata-2,6,19-triene (1), from DMAPP and IPP, and the hydrocarbon was further transformed into stellatic acid (2) by cytochrome P450 monooxygenase encoded by the gene adjacent to the sesterterpene synthase gene.