Astechrome

Background: With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80 kb in size, breakthrough techniques are needed to characterize this SM wealth. Results: Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery. Conclusion: The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.

Glucose is a widely used carbon source in laboratory practice to culture Aspergillus fumigatus, however, glucose availability is often low in its "natural habitats", including the human body. We used a physiological-transcriptomical approach to reveal differences between A. fumigatus Af293 cultures incubated on glucose, glucose and peptone, peptone (carbon limitation), or without any carbon source (carbon starvation). Autolytic cell wall degradation was upregulated by both carbon starvation and limitation. The importance of autolytic cell wall degradation in the adaptation to carbon stress was also highlighted by approximately 12.4% of the A. fumigatus genomes harboring duplication of genes involved in N-acetyl glucosamine utilization. Glucose withdrawal increased redox imbalance, altered both the transcription of antioxidative enzyme genes and oxidative stress tolerance, and downregulated iron acquisition, but upregulated heme protein genes. Transcriptional activity of the Gliotoxin cluster was low in all experiments, while the Fumagillin cluster showed substantial activity both on glucose and under carbon starvation, and the Hexadehydro-astechrome cluster only on glucose. We concluded that glucose withdrawal substantially modified the physiology of A. fumigatus, including processes that contribute to virulence. This may explain the challenge of predicting the in vivo behavior of A. fumigatus based on data from glucose rich cultures.

Plant pathogenic fungi in the Fusarium genus cause severe damage to crops, resulting in great financial losses and health hazards. Specialized metabolites synthesized by these fungi are known to play key roles in the infection process, and to provide survival advantages inside and outside the host. However, systematic studies of the evolution of specialized metabolite-coding potential across Fusarium have been scarce. Here, we apply a combination of bioinformatic approaches to identify biosynthetic gene clusters (BGCs) across publicly available genomes from Fusarium, to group them into annotated families and to study gain/loss events of BGC families throughout the history of the genus. Comparison with MIBiG reference BGCs allowed assignment of 29 gene cluster families (GCFs) to pathways responsible for the production of known compounds, while for 57 GCFs, the molecular products remain unknown. Comparative analysis of BGC repertoires using ancestral state reconstruction raised several new hypotheses on how BGCs contribute to Fusarium pathogenicity or host specificity, sometimes surprisingly so: for example, a gene cluster for the biosynthesis of hexadehydro-astechrome was identified in the genome of the biocontrol strain Fusarium oxysporum Fo47, while being absent in that of the tomato pathogen F. oxysporum f.sp. lycopersici. Several BGCs were also identified on supernumerary chromosomes; heterologous expression of genes for three terpene synthases encoded on the Fusarium poae supernumerary chromosome and subsequent GC/MS analysis showed that these genes are functional and encode enzymes that each are able to synthesize koraiol; this observed functional redundancy supports the hypothesis that localization of copies of BGCs on supernumerary chromosomes provides freedom for evolutionary innovations to occur, while the original function remains conserved. Altogether, this systematic overview of biosynthetic diversity in Fusarium paves the way for targeted natural product discovery based on automated identification of species-specific pathways as well as for connecting species ecology to the taxonomic distributions of BGCs.