An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus

Abstract

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.

FIG. 1.

(A) Diagrammatic representation of homologous recombination at dmaW. In the wild-type (wt) diagram, F and R are annealing sites for primers dmaW forward and dmaW reverse, respectively, which amplify an internal portion of dmaW. The gene knockout construct pDMAT1, containing the internal fragment of dmaW, was linearized at a unique PpuMI site internal to dmaW prior to transformation. The diagram labeled ko represents the dmaW locus with three copies of pDMAT1 integrated in tandem, disrupting dmaW. UF, annealing site for a primer derived from universal primer sequences in the vector; 3S, annealing site for a primer flanking the 3′ border of the site of integration; UR, annealing site for a primer derived from reverse primer sequences in the vector; 5S, annealing site for a primer flanking the 5′ border of the site of integration. (B) PCR from primers UR and 5S amplified the expected 852-bp band from a strain carrying the disrupted dmaW allele but not from the wild-type isolate. (C) PCR from primers UF and 3S amplified the 1.2-kb product only from the strain with a disrupted dmaW gene. Although additional amplicons can be predicted from duplicate annealing sites for primers UF and UR, which result from the multiple integrations of pDMAT1, these theoretical amplicons are much too large to be amplified with the PCR conditions and reagents used in this study. The relative mobilities of relevant fragments (in kilobases) of BstEII-digested bacteriophage lambda are indicated on the left in panels B and C.

FIG. 2.

Southern blot of SalI-digested DNA from nontransformed A. fumigatus (wt) and three dmaW knockout mutants probed with a dmaW fragment derived from primers dmaW forward and dmaW reverse (see Fig. 1A). The relative intensities of hybridization reflect minor differences in loading of the samples. The relative mobilities of relevant fragments (in kilobases) of BstEII-digested bacteriophage lambda are indicated on the left.

FIG. 3.

High-performance liquid chromatography traces of nontransformed A. fumigatus (wt), a dmaW knockout strain (dmaW ko6), and strain dmaW ko6 complemented by ectopic integration of a DNA fragment containing the gene (dmaW ct). The insert shows overlaid traces for nontransformed A. fumigatus (solid line; wt), strain dmaW ko6 (dotted line; ko), and the complemented strain (dashed line; ct) at a higher sensitivity to demonstrate the presence or absence of fumigaclavine B. B, fumigaclavine B; F, festuclavine; A, fumigaclavine A; C, fumigaclavine C.

FIG. 4.

Map of the genes clustered with dmaW of A. fumigatus compared to the cluster surrounding dmaW of C. purpurea (redrawn from data in reference 25). The arrows above the chromosomes indicate the positions of genes and the orientations of transcription. Abbreviations: Cp, C. purpurea; ox/red, oxidoreductase; cat, catalase; OAc trans, O-acetyltransferase; P450, cytochrome P450 monooxygenase; lovA P450, molecule similar to cytochrome P450 involved in lovastatin biosynthesis; pda P450, molecule similar to cytochrome P450 involved in pisatin demethylase activity; DH, short-chain alcohol dehydrogenase; alt, alternate; OYE, old yellow enzyme; hydrox, hydroxylase; monoox, monooxygenase; ps, peptide synthetase; FAD, flavin adenine dinucleotide. Genes that are found in the same relative position and orientation in the two clusters are enclosed in a solid boxes. Genes previously proposed to be involved in ergot alkaloid production that are present in both clusters but differ in their relative positions and orientations are enclosed in dashed boxes. Genes for which a function has been demonstrated by gene knockout (5, 16, 29) are indicated with asterisks. The scale applies to both clusters. The A. fumigatus sequences illustrated span from nucleotide 2894958 (left end of diagram) to nucleotide 2926335 (right end of diagram) of assembly 92 (chromosome 2; formerly assembly 72) of the A. fumigatus genome. The identifiers for the A. fumigatus sequences (from left to right) are m19874, m19875, m19876, m19877, m19878, m19880, m19881, m19882, m19883, m19884, m19885, m19886, m19888, and m19891. Data are available from The Institute for Genomic Research (www.tigr.org). The GenBank accession numbers for the C. purpurea sequences are as follows: lysergyl ps2, AJ438610; cytochrome P450-1, no accession number available (described by Correia et al. [5]); catalase, AJ703808; cpox2 (short-chain alcohol dehydrogenase oxidoreductase), CAB39316; cpox1 (flavin adenine dinucleotide oxidoreductase), AJ703809; orfB, AY836772; orfA, AY836771; dmaW, CAC37397; and lysergyl ps1, CAB39315.