Genus-scale Analysis of Gene Cluster Evolution in Fungi

The Aspergillus genus is highly diverse and contains more than 300 species. Several of these have high impact on society, beneficial as well as harmful, including the opportunistic pathogen A. fumigatus, the food spoiler A. flavus, the citric acid and enzyme producer A. niger, and the food fermentor A. oryzae.

Aspergillus species are known to produce a high number of secondary metabolites and have been shown to have an even higher genomic potential based on the number of predicted secondary metabolite gene clusters in the genomes. Some secondary metabolites have bioactivities which are useful medically such as antibiotics, immunosupressants, and cholesterol-lowering agents.

The Aspergillus whole genus sequencing project (of which this project is a part of) aims to produce a genome sequence for a representative strain of each species within the genus. In this thesis, we investigate both the opportunities and challenges in exploring this genus using the vast data created from the genome sequencing project.

We focus on secondary metabolism addressing the challenge of how we can identify beneficial bioactive clusters using the diversity and self-resistance mechanisms. Moreover, we investigate the diversity and similarities of species spanning several sections and across the Flavi section. As part of the Aspergillus sequencing project, this thesis work will publish 25 genomes.

We have investigated the genome characteristics and used the diversity of the genomes to create novel insights and hence knowledge-based hypothesis. To characterize and utilize a bioactive compound industrially, it is important to know which genes are responsible for the biosynthesis. There are many ways of linking gene clusters to compounds and here we present an overview of these by divide them into strategies.

Moreover, we have demonstrated the use of some of the strategies based on comparative genomics and retrobiosynthesis to identify putative clusters for, among others, the bioactive compounds novofumigatonin and chlorflavonin, of which the novofumigatonin cluster has been experimentally verified subsequently [1].

Investigating the Flavi section, we have identified common traits such as large genomes and a high number of predicted biosynthetic gene clusters, but also differences such as variations in gene cluster families. Examination of the phylogeny have led to questions regarding the evolution in the A. flavus clade and domestication of A. oryzae and A. sojae.

The carbohydrate-active enzyme (CAZy) potential is large within the Flavi section with a maximum of 663 CAZymes found in A. parasiticus. With the large number of predicted secondary metabolite gene clusters it is a challenge to select the most promising clusters and prioritize the experimental efforts in the quest of novel bioactive compounds.

In order to overcome this challenge we have developed a pipeline, FRIGG (Fungal ResIstance Gene-directed Genome mining), using genome sequences to identify putative bioactive gene clusters based on duplicated self-resistance genes. This pipeline thereby provides a means of selecting which clusters to investigate and dramatically shortens the experimental process.

Applying the pipeline to 51 Aspergillus and Penicillium species, we have identified a total of 72 protein families with putative resistance genes found in clusters including the verified resistance gene for fellutamide B. We selected a cluster for experimental investigation and preliminary results indicate it could be producing N-acetyl-glutamine which have shown bioactivity as a psycostimulant and in a complex with aluminium as an antiulcer agent and [2, 3, 4].

In summary, this work has not only contributed to to the Aspergillus community with new genome sequences and insights from comparative genomics analysis, but also with strategies to link gene clusters and compounds and a pipeline identifying putative resistance genes and bioactive clusters. In the future, this pipeline can be used as a guide in the quest for novel bioactive compounds, which are desperately needed.

The exploration of the newly sequenced genomes has only just started with our generated insights and hypothesis and it will continue to move the field forward gaining many more insights in the time ahead.