Fumagillin 1 is a meroterpenoid from Aspergillus fumigatus that is known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The genetic and molecular basis for biosynthesis of 1 had been an enigma despite the availability of the A. fumigatus genome sequence. Here, we reported the identification and verification of the fma gene cluster, followed by characterization of the polyketide synthase and acyltransferase involved in biosynthesis of the dioic acid portion of 1. More significantly, we uncovered the elusive β-trans-bergamotene synthase in A. fumigatus as a membrane-bound terpene cyclase.

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.

We develop a novel synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and encoded metabolites.

The communesins are a prominent class of indole alkaloids isolated from Penicillium species. Due to their daunting structural framework and potential as pharmaceuticals, communesins have inspired numerous total synthesis efforts. However, the genetic and biochemical basis of communesin biosynthesis has remained unexplored. Here, we report the identification and characterization of the communesin (cns) biosynthetic gene cluster from Penicillium expansum. We confirmed communesin is biosynthesized from the coupling of tryptamine and aurantioclavine, two building blocks derived from L-tryptophan. The post-modification steps were mapped by targeted-gene deletion experiments and structural elucidation of intermediates and new analogues. Our studies set the stage for biochemical characterization of the communesin biosynthesis towards understanding how nature generates remarkable structural complexity from simple precursors.

Fumagillin (1), a meroterpenoid from Aspergillus fumigatus, is known for its antiangiogenic activity due to binding to human methionine aminopeptidase 2. 1 has a highly oxygenated structure containing a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene β-trans-bergamotene. The chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted-gene deletion experiments. Here, we report the identification and characterization of three oxygenases from the fumagillin biosynthetic pathway, including a multifunctional cytochrome P450 monooxygenase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates.