Identification of Two Aflatrem Biosynthesis Gene Loci in Aspergillus flavus and Metabolic Engineering of Penicillium paxilli To Elucidate Their Function

Abstract

Aflatrem is a potent tremorgenic toxin produced by the soil fungus Aspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search of Aspergillus complete genome sequence data identified putative aflatrem gene clusters in the genomes of A. flavus and Aspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first, ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes, atmG, atmC, and atmM, and the second, ATM2, is telomere distal on chromosome 7 and contains five genes, atmD, atmQ, atmB, atmA, and atmP. Reverse transcriptase PCR in A. flavus demonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer of atmP and atmQ into Penicillium paxilli paxP and paxQ deletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis in A. flavus based on these reconstitution experiments in P. paxilli and identification of putative intermediates in wild-type cultures of A. flavus.

Secondary metabolite arsenal of an opportunistic pathogenic fungus

“…This uncharacterized BGC is likely to produce a polyketide-terpene hybrid, possibly with some similarity to the Aspergillus flavus toxin aflatrem [83], and is found in N. fischeri.…”

Geranylation of Cyclic Dipeptides by the Dimethylallyl Transferase AnaPT Resulting in a Shift of Prenylation Position on the Indole Ring

“…[2,3] However, indole derivatives with larger prenyl moieties have also been found in nature, for example, indole sesquiterpenoid xiamycin and derivatives from Streptomyces, [5,6] and indole diterpenoids paxilline and aflatrem from Penicillium and Aspergillus species. [7][8][9] In many cases, the prenylated derivatives differ clearly from their precursors in their biological and pharmacological activities. [10][11][12] Prenyltransferases from plants, bacteria and fungi are responsible for the transfer reactions of the prenyl moieties, and belong to different superfamilies/groups, based on their primary amino acid sequences and biochemical properties.…”

A fungal prenyltransferase catalyzes the regular di-prenylation at positions 20 and 21 of paxilline

“…23) Moreover, the prenylation of indoles/indole-3-glycerol phosphates by PaxC and stepwise regular di-prenylation at positions 21 and 22 of paxilline by PaxD was clearly confirmed by in vitro experiments with purified recombinant enzymes. 23,24) We have also reported on the function of atmD, which is located in the aflatrem biosynthetic gene cluster in Aspergillus flavus 25) and encodes an enzyme with 32% amino acid identity to PaxD. It catalyzed the reverse mono-prenylation of paxilline at position 20 and position 21.…”