Prototype of an intertwined secondary-metabolite supercluster

Abstract

The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the control of the master regulator of secondary metabolism, LaeA, contains, in its entirety, the genetic machinery for three natural products (fumitremorgin, fumagillin, and pseurotin), where genes for fumagillin and pseurotin are physically intertwined in a single supercluster. Deletions of 29 adjoining genes revealed that fumagillin and pseurotin are coregulated by the supercluster-embedded regulatory gene with biosynthetic genes belonging to one of the two metabolic pathways in a noncontiguous manner. Comparative genomics indicates the fumagillin/pseurotin supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This blended design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution.

Keywords: FapR; Zn(II)2Cys6 transcription factor; biosynthesis; cluster evolution; gene regulation.

Fig. 1.

Subtelomeric region of A. fumigatus chromosome VIII controlled by LaeA and FapR. (A) Schematic representation of A. fumigatus Af2931 genes AfuA_8g00100-AfuA_8g00780. Log₂ expression data from microarray experiments comparing A. fumigatus wild-type to ΔlaeA gene expression, as performed by Perrin et al. (24). Fumitremorgin, fumagillin, and pseurotin cluster genes are color-coded based on literature data (see Results). (B) Schematic representation of A. fumigatus A1163 genes AfuB_086210-AfuB_085980. Black indicates genes not regulated by FapR; green indicates genes involved in fumagillin biosynthesis that are FapR-regulated; red indicates genes involved in pseurotin biosynthesis that are FapR-regulated; yellow indicates genes involved in neither fumagillin nor pseurotin biosynthesis but are FapR-regulated; blue indicates gene encoding FapR.

Fig. 2.

Northern expression analysis of genes of the fumagillin and pseurotin supercluster and adjacent border genes. Northern expression analysis of indicated genes comparing the A. fumigatus wild type (CEA17), the ΔlaeA mutant, and the ΔfapR mutant. Ribosomal RNA was visualized by ethidium bromide staining as loading control. The indicated strains were grown in liquid GMM at 25 °C and 250 rpm for 96 h under ambient light conditions.

Fig. 3.

Comparison of the fumagillin/pseurotin supercluster region in A. fumigatus, A. clavatus, N. fischeri, and M. anisopliae. The genes involved in fumagillin biosynthesis are represented as green arrows. (“A” through “G” in green arrows indicate fmaA to -G.) The genes involved in pseurotin biosynthesis are represented as red arrows. (“A” through “G” in red arrows indicate psoA to -G.) Dark blue arrow depicts the genes encoding FapR. The yellow arrows represent genes with homologs in N. fischeri and/or M. anisopliae in the immediate supercluster region that do not participate in fumagillin or pseurotin biosynthesis; ψ marks pseudogenes that show no bidirectional best hit in the immediate homologous supercluster region of either N. fischeri or M. anisopliae. White arrows represent genes in F. fischeri, M. anisopliae, and A. clavatus with no bidirectional best hit to the immediate homologous supercluster region of each other or A. fumigatus. Connecting gray rectangles indicate homologies among the species. Blue arrows with “R” indicate fapR (fumagillin and pseurotin regulator); orange arrows with “I” indicate fpaI; orange arrows with “II” indicate fpaII.

Fig. 4.

Proposed biosynthetic pathways of fumagillin and pseurotin and regulation mechanism of the participating genes by LaeA and FapR. Green arrows represent genes involved in fumagillin biosynthesis. Red arrows represent genes involved in pseurotin biosynthesis. White arrows represent genes that are regulated by FapR and LaeA but not involved in either fumagillin or pseurotin production. Orange arrows represent supercluster border genes regulated by LaeA but not FapR and not involved in fumagillin or pseurotin biosynthesis. The dark blue arrow represents the gene encoding FapR. ACP, acyl-carrier protein domain; FmaA, terpene cyclase involved in β-trans-bergamotene production; FmaG, P450 monooxygenase involved in oxidative rearrangement of β-trans-bergamotene; FmaD, O-methyltransferase involved in methylation of C4 hydroxyl group; FmaB and -C, PKS and accessory enzyme involved in pentaene chain production and connection to fumagillol, respectively; FmaF, phytanoyl-CoA oxidase involved in oxidation of fumagillin-aldehyde; FPP, farnesyl-pyrophosphate; PCP, peptidyl-carrier protein domain; PsoA, PKS/NRPS hybrid catalyzing formation of the pseurotin scaffold; PsoF, O-methyltransferase involved in methylation of the C8 hydroxyl group of azaspirene; PsoE, GST participating in oxidation of the diene chain. Question marks indicate speculative enzyme assignments.