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The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathway is therefore important towards understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered. Here using Saccharomyces cerevisiae as a heterologous host and a phylogenetically-guided enzyme mining approach, we combinatorially assembled IDT biosynthetic pathways using IDTCs homologs identified from different fungal hosts. We identified the genetically standalone IDTCs involved in the cyclization of aflavine and anominine, and produced new IDTs not previously isolated. The cyclization mechanisms of the new IDTCs were proposed based on the yeast reconstitution results. Our studies demonstrate heterologous pathway assembly is a useful tool in the reconstitution of unclustered biosynthetic pathways.

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Six new indole alkaloids including five new glyantrypine derivatives (1, 2a, 2b, 3, 4) and a new pyrazinoquinazoline derivative (5), together with eight known alkaloids (6-13), were isolated from the culture of the mangrove-derived fungus Cladosporium sp. PJX-41. Their structures were elucidated primarily by spectroscopic and physical data. The absolute configurations of compounds 1-9 were established on the basis of CD, NOESY data, and single-crystal X-ray diffraction analysis. Compounds 2b, 5, 7-9, and 11 exhibited significant activities against influenza virus A (H1N1), with IC50 values of 82-89 μM.

The pericyclases are a growing superfamily of enzymes that catalyze pericyclic reactions. We report a pericyclase IccD catalyzing an inverse-electron demand Diels-Alder (IEDDA) reaction with a rate acceleration of 3 × 10 5 fold in the biosynthesis of fungal natural product ilicicolin H. We demonstrate IccD is highly periselective towards the IEDDA cycloaddition over a competing normal electron demand Diels-Alder (NEDDA) reaction from an ambimodal transition state. A predicted flavoenzyme IccE was identified to epimerize the IEDDA product 8-epi-ilicicolin H to ilicicolin H, a step that is critical for the observed antifungal activity of ilicicolin H. Our results reveal the ilicicolin H biosynthetic pathway and add to the collection of pericyclic reactions that are catalyzed by pericyclases.