Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species

Abstract

The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus, and A. steynii) have been whole-genome PacBio sequenced to provide genetic references in three Aspergillus sections. A. taichungensis and A. candidus also were sequenced for SM elucidation. Thirteen Aspergillus genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, A. novofumigatus was compared with the pathogenic species A. fumigatus This suggests that A. novofumigatus can produce most of the same allergens, virulence, and pathogenicity factors as A. fumigatus, suggesting that A. novofumigatus could be as pathogenic as A. fumigatus Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris, and A. steynii, respectively, and novofumigatonin, ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus Our study delivers six fungal genomes, showing the large diversity found in the Aspergillus genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of A. novofumigatus pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.

Keywords: Aspergillus; comparative genomics; fumigatus; secondary metabolism.

Fig. 1.

Phylogenetic tree of 15 strains constructed from a composition vector approach of whole-proteome sequences using CVTree3 with a K’mer size of eight (19, 20). The time scale has been scaled to the root, thereby making the branch lengths relative to the distance between the root and the species. This time will therefore depend on what species are included in the set. The four PacBio-sequenced species are marked with orange, and the Fumigati section is marked with blue.

Fig. 2.

(A) Overview of the SM gene clusters predicted in A. novofumigatus and their homologs in the reference species. (B) Overview of the SM gene clusters predicted in A. fumigatus and their homologs in the reference species. (C) A Venn diagram of the A. fumigatus and A. novofumigatus SM gene clusters. (D) The number and different types of SM gene clusters predicted in A. fumigatus and A. novofumigatus. DMATS, dimethylallyl tryptophan synthase; NRPS, nonribosomal peptide synthetase; PKS, polyketide synthase; TC, terpene cyclase.

Fig. 3.

Synteny plots of investigated clusters made using Easyfig tBLASTx. (A) The synteny of the predicted aflatoxin cluster in A. flavus NRRL3357 and the identified candidate aflatoxin cluster in A. ochraceoroseus (scaffold 2, 4,201,774–4,251,209 bp). (B) Synteny plot of the candidate cluster for ochrindol in A. steynii (scaffold 7, 2,783,445–2,824,507 bp) and terrequinone cluster in A. nidulans. The terrequinone cluster consists of a single-module nonribosomal peptide synthetase (tdiA), a prenyltransferase (tdiB), an oxidoreductase (tdiC), an aminotransferase (tdiD), and a gene of unknown function similar to a methyl transferase (tdiE). (C) Synteny plot of the known meroterpenoid cluster of terretonin in A. terreus and the candidate cluster of novofumigatonin in A. novofumigatus (scaffold 14, 103,246–136,450 bp). (D) The chemical structure of chlorflavonin and the candidate cluster for chlorflavonin in A. campestris (scaffold 1, 576,100–603,958 bp). The hydroxylation has been highlighted with blue, the O-methylation has been highlighted in purple, and the PKS backbone has been highlighted in orange. (E) Synteny plot of putative chlorflavonin clusters in A. candidus and A. campestris.