Characterisation of Epicoccum spp. secondary metabolism genes through de novo genome and transcriptome sequencing

The Epicoccum nigrum sensu lato clade of the Didymellaceae family, previously also known as E. purpurascens, consists of a group of very common saprophytic and plant associated fungal species with a worldwide distribution. Over 70 natural products have been identified from Epicoccum nigrum sensu lato. One of the most notable is the antifungal pyronepolyene C-glucoside, epipyrone, which was isolated from an Epicoccum italicum strain (ICMP 19927) from New Zealand. To identify the genes involved in the biosynthesis of epipyrone and other secondary metabolites, the whole genome of Epicoccum italicum strain ICMP 19927 was sequenced using a range of short and long insert shotgun libraries. This was assembled and thoroughly annotated by the funannotate pipeline along with additional specialised packages. Thirty four putative secondary metabolite biosynthetic gene clusters were identified in the genome, including eight clusters containing polyketide synthases (PKS), which were predicted to be required for epipyrone biosynthesis. Protocols for epipyrone production by submerged fermentation of Epicoccum were optimised to compare two conditions that strongly differ by level of epipyrone production. Genome-wide differential expression analysis by mRNA sequencing allowed the identification of the epn gene cluster for epipyrone synthesis, which was strongly upregulated in epipyrone-inducing conditions. The cluster contains six genes, including expected genes for a highly reducing PKS (epnA) and also a glycosyltransferase (epnB), the first time a gene for this class of enzyme has been identified in a fungal biosynthetic cluster with known product. Contiguous mRNA sequence supported that adjacent epnA and epnB genes are expressed as a single polycistronic mRNA, which is very rarely observed in fungi. Various genes known to be involved in nitrogen metabolite repression were found to be differentially expressed simultaneously with epipyrone production, supporting the hypothesis that the epn cluster is regulated via this mechanism in E. italicum. Based on coordinated expression, a range of further known and unknown transcription factor and other genes were identified that are hypothesised to be involved in activation of the epn cluster. Two additional genomes of E. italicum strains and three of closely related E. layuense species were sequenced, assembled and annotated with secondary metabolite gene clusters. The epn cluster was found conserved in all sequenced Epicoccum genomes with more than 96% amino acid identity between polyketide synthase EpnA proteins. Additionally, clusters with identical genic content and high similarity of encoded proteins were surprisingly detected in genomes of two distant Pleosporales species – Clohesyomyces aquaticus and Zopfia rhizophila available from the JGI Mycocosm database. Localisation of the cluster within the genome was species-specific even in closely related Epicoccum species, with some repetitive elements flanking the epn cluster in all species. The availability of annotated reference genomes and comparative genomic data from multiple Epicoccum strains and species, combined with previously reported secondary metabolites and metabolomics datasets will greatly facilitate further studies of the Epicoccum genus. This research has furthered the current state of knowledge of the functional genomics of polyketide clusters in Epicoccum and other Ascomycetes. This will lead to improved understanding of how these clusters function and are regulated in fungi and may lead to biotechnological applications and informed bioprospecting screens.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biological Sciences

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2019

Author dc:creator

• Fokin, Mikhail

Advisors dc:contributor.advisor

• Villas-Boas, SG
• Fleetwood, G
• Weir, BS

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.
• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/48536

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/48536