Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast

Abstract

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.

Oxidative Cyclization in Natural Product Biosynthesis

“…Subsequent epoxidation of the residual olefin by AtmM to yield 677 and pyran formation in 678 create the connectivity and architecture found in the prenylated emindole PA ( 679 ) and emindole PB ( 680 ). 480 …”

<i>Aspergillus flavus</i> Secondary Metabolites: More than Just Aflatoxins

“…GGI is then converted to an epoxide by the action of an epoxidase (PaxM/AtmM) and subsequently transformed by an IDT cyclase (PaxB/AtmB) to the first stable intermediate, paspaline 150,151) . In A. flavus, paspaline is then converted to paspalinine by the action of the cytochrome P450 monooxygenases AtmP and AtmQ followed by subsequent conversion to aflatrem by the prenyltransferase AtmD.…”

“…Interestingly, the chemical diversity that is characteristic of IDTs is due in large part to the IDT cyclases (IDTCs). Using a yeast host, Tang et al 151) were able to propose mechanisms by which A. flavus utilizes unique IDTCs to produce both aflatrems and the IDT, aflavinine (Fig. 7).…”

“…They proposed that AtmG/C/M produces a common epoxide intermediate which is then converted to paspaline by the aflatrem cluster IDTC, AtmB, thus leading to subsequent formation of aflatrem. Based on the ability of A. flavus to also produce aflavinine, Tang et al 151) performed a phylogenetic analysis of all atmB homologs in A. flavus to identify candidate IDTCs that may be responsible for formation of aflavinine. When they introduced the standalone (not associated with a typical SM gene cluster) atmB homolog, afB, into a yeast host expressing atmG/C/M, the major product was shown to be aflavinine.…”

Technology development for natural product biosynthesis in Saccharomyces cerevisiae

“…Another glucose repressible promoter, ADH2p, induces strong transcription once glucose is depleted and has been shown to provide higher expression levels than the GAL promoter [13]. The ADH2 promoter has proven highly useful for the heterologous expression of biosynthetic genes and recently enabled the discovery of new fungal indole diterpenes [14]. A library of ADH2-like promoters would prove beneficial for pathway refactoring and characterization of new biosynthetic gene clusters.…”