A comparative genomics study of 23 Aspergillus species from section Flavi

Abstract

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.

Fig. 1. Phylogeny and genome statistics of section Flavi plus eight other Aspergillus, Penicillium, and Neurospora species.

a Phylogenetic tree constructed using RAxML, MUSCLE, and Gblocks based on 200 monocore genes (a single homolog in each of the species). The red star indicates an uncertain leaf most likely caused by a different gene calling method^–, and the arrow shows where A. sojae should be placed in the phylogenetic tree. The zoom shows the branching in a clade around A. oryzae. b The colors illustrate the clades found within section Flavi and X indicates species sequenced in this study. Earlier sequenced genomes such as A. oryzae and A. fumigatus were assembled using optical mapping and genetic maps. c Seven bubble plots illustrating key genome numbers and sequencing quality parameter. The bubble sizes have been scaled to each panel and are not comparable across panels.

Fig. 2. Core-specific, section-specific, and clade-specific and species-unique genes.

a A dendrogram representing the phylogenetic relationship between the 29 Aspergilli. The black boxes in the nodes represent the homologous protein families shared among the species branching from that node. The white boxes at the tips represent the protein families unique to that individual species. b A barplot showing the number of total (green), core (turquoise), and species-specific (orange) proteins for each species. The dark shading illustrates the number of proteins with a least one functional annotation based on InterPro.

Fig. 3. Location of species unique and secondary metabolite genes in the A. oryzae genome.

The gray bars represent the A. oryzae genome. Above the chromosome the species-specific (turquoise) and secondary metabolite genes (orange) are mapped to the genome, each line represents a gene. The curve shows the percentage of the density calculated from the total number of genes within 30 kbp in steps of 5 kb. Below the genome, the core genes are mapped by the gray dots and the density of the total number of genes is shown by the black graph (with a window of 30 kbp).

Fig. 4. Carbohydrate-active enzymes (CAZymes) in section Flavi.

a The total number of CAZymes in each species distributed on six categories of enzyme activity: auxiliary activities, carbohydrate-binding molecules, carbohydrate esterases, glycoside hydrolases, glycosyltransferases, and polysaccharide lyases. b Boxplot representing the diversity of CAZyme family content and abundance among clade A. flavus (light blue), A. tamarii (yellow), A. nomius (dark blue), A. alliaceus (light turquoise), the rest of the Flavi section (orange), other Aspergilli (dark turquoise), and non-Aspergillus species (gray). For each CAZyme class the total number of CAZymes (top row) and the number of unique CAZyme families (bottom row) are displayed. In the boxplot the midline represents the median, the upper and lower limit of the box represents the third and first quartile, and the whiskers extend up to 1.5 times the interquartile.

Fig. 5. Carbohydrate-active enzymes in section Flavi sorted according to the phylogram of Fig. 1.

a Heatmap representing the growth profiles of 23 Flavi species and 8 additional species on 35 different media. b Comparison of the CAZyme sets related to plant biomass degradation in the genomes of species from Aspergillus section Flavi, and some other fungi. The colors reflect the polysaccharides the enzymes are active toward.

Fig. 6. Dereplication of known compounds and predicted secondary metabolite backbone genes per species.

a A dendrogram representing the phylogenetic relationship between the species. The black boxes in the nodes represent the secondary metabolite gene cluster (SMGC) families shared among the species branching from that node. If there is no black box there are zero clusters shared. The gray boxes at the tips show the number of unique SMGC families only found in one species for the Flavi section. b Matrix indicating the presence and absence of SMGC families coupled to known clusters from the MIBiG database for each species. Overview of the cluster family for aflatoxin can be found in Supplementary Figure 11. c Predicted secondary metabolite genes for each species divided by the backbone enzyme. DMAT: dimethylallyltransferase (prenyl transferases), HYBRID: a backbone gene containing domains from NRPS and PKS backbones, NRPS: non-ribosomal peptide synthetase, NRPS-like: non-ribosomal peptide synthetase like, containing at least two NRPS-specific domains and another domain or one NRPS A domain in combination with NAD binding 4 domain or short-chain dehydrogenase, PKS: polyketide synthase, PKS-like: polyketide synthase like, containing at least two PKS-specific domains and another domain, TC: terpene cyclase.