Reconstitution of biosynthetic machinery for the synthesis of the highly elaborated indole diterpene penitrem.

Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation-initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two-step P450-catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products.

Rapid reconstitution of biosynthetic machinery for fungal metabolites in Aspergillus oryzae: total biosynthesis of aflatrem.

Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.

Reconstitution of biosynthetic machinery for indole-diterpene paxilline in Aspergillus oryzae.

Indole-diterpenes represented by paxilline share a common pentacyclic core skeleton derived from indole and geranylgeranyl diphosphate. To shed light on the detailed biosynthetic mechanism of the paspaline-type hexacyclic skeleton, we examined the reconstitution of paxilline biosynthetic machinery in Aspergillus oryzae NSAR1. Stepwise introduction of the six pax genes enabled us to isolate all biosynthetic intermediates and to synthesize paxilline. In vitro and in vivo studies on the key enzymes, prenyltransferase PaxC and cyclase PaxB, allowed us to elucidate actual substrates of these enzymes. Using the isolated and the synthesized epoxide substrates, the highly intriguing stepwide epoxidation/cyclization mechanism for the construction of core structure has been confirmed. In addition, we also demonstrated "tandem transformation" to simultaneously introduce two genes using a single vector (paxG/paxB, pAdeA; paxP/paxQ, pUNA). This may provide further option for the reconstitution strategy to synthesize more complex fungal metabolites.