Identification of Cytochrome P450s Required for Fumitremorgin Biosynthesis in Aspergillus fumigatus

Abstract

Fumitremorgin C, a diketopiperazine mycotoxin produced by Aspergillus fumigatus, is a potent and specific inhibitor of breast cancer resistance protein (BCRP). Elucidation of the fumitremorgin C biosynthetic pathway provides a strategy for new drug design. A structure-activity relationship study based on metabolites related to the ftm gene cluster revealed that the process most crucial for inhibitory activity against BCRP was cyclization to form fumitremorgin C. To determine the gene involved in the cyclization reaction, targeted gene inactivation was performed with candidate genes in the ftm cluster. Analysis of the gene disruptants allowed us to identify ftmE, one of the cytochrome P450 genes in the cluster, as the gene responsible for the key step in fumitremorgin biosynthesis. Additionally, we demonstrated that the other two cytochrome P450 genes, ftmC and ftmG, were involved in hydroxylation of the indole ring and successive hydroxylation of fumitremorgin C, respectively.

Genome mining and biosynthesis of fumitremorgin-type alkaloids in ascomycetes

“…30 Therefore, we speculate that the mentioned clusters consist of nine genes each as illustrated in Figure 2, which was also identified in A. fumigatus BM939 (Figure 2). 31 However, it cannot be excluded that different strains carry similar, but not identical clusters and produce different end products. The nine genes from the four strains share high homology to each other with sequence identities of 85-99% on the amino acid level.…”

“…Functional proof of the biosynthetic genes and gene cluster of fumitremorgin-type alkaloids was carried out by gene inactivation experiments in the producing strain BM939, 31 by identification of the accumulated secondary metabolites after heterologous expression in Aspergillus strains 33,34 or by biochemical investigation of the recombinant enzymes from Escherichia coli or Saccharomyces cerevisiae. 27,31,32,35 In total, functions of seven genes have been proven experimentally.…”

“…27,31,32,35 In total, functions of seven genes have been proven experimentally. On the basis of the obtained bioinformatic, molecular biological and biochemical results, the following biosynthetic pathway has been postulated ( Figure 3).…”

“…1 The biosynthesis of fumitremorgin-type alkaloids begins by condensation of the two amino acids L-tryptophan and L-proline to brevianamide F, catalyzed by the non-ribosomal peptide synthetase FtmPS (also termed FtmA), which was proven by heterologous overexpression of the NRPS gene ftmPS in Aspergillus and identification of the accumulated product brevianamide F. 33 Brevianamide F is then prenylated by the prenyltransferase FtmPT1/FtmB in the presence of dimethylallyl diphosphate, resulting in the formation of tryprostatin B, as demonstrated by using the recombinant FtmPT1. 27 By gene inactivation in A. fumigatus BM939 and heterologous expression in S. cerevisiae, Kato et al 31 showed that the three cytochrome P450 enzymes, FtmP450-1/FtmC, FtmP450-2/FtmE and FtmP450-3/FtmG, are responsible for the conversion of tryprostatin B (2) to 6-hydroxytryprostatin B, tryprostatin A (3) to fumitremorgin C (8) and fumitremorgin C (8) to 12,13-dihydroxyfumitremorgin C, respectively. Using recombinant enzymes, we have demonstrated that FtmPT2/FtmH catalyzes the prenylation of 12,13-dihydroxyfumitremorgin C in the presence of dimethylallyl diphosphate, resulting in the formation of fumitremorgin B, 35 which is then converted to verruculogen by FtmOx1/FtmF by inserting an endoperoxide bond between the two prenyl moieties.…”

“…The putative methyltransferase FtmMT/FtmD is expected for the conversion of 6-hydroxytryprostatin B to tryprostatin A. 27 Kato et al 31 reported that inactivation of ftmO/ftmI in A. fumigatus BM939 did not abolish the fumitremorgin B accumulation. Using overproduced and purified His 6 -FtmO, we could not provide any evidence on its role in the biosynthesis of fumitremorgin-type alkaloids (unpublished results).…”