Secondary metabolism and development in the filamentous fungus <i>Aspergillus nidulans</i> - Activation of silent gene clusters and characterization of the SAM synthetase SasA

Abstract

Antimicrobial resistance is spreading whereas the number of newly discovered antibiotics is declining. The genomes of filamentous fungi comprise numerous putative gene clusters coding for biosynthetic enzymes of structurally diverse secondary metabolites, which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were primarily based on artificially targeting the cellular protein synthesis apparatus. In this work, an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans was applied, by utilizing the deletion mutant of the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in transcriptional activation of a previously silenced biosynthetic gene cluster (dba), comprising an orphaned polyketide synthase (PKS) gene. The direct product of the PKS was isolated and identified as 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA), which to our knowledge had never been described in an Aspergillus species before and which showed antibiotic activity against Micrococcus luteus. Additionally, a second new A. nidulans compound was identified in wild type as 3,3-(2,3-dihydroxypropyl)diindole (DHPDI), which is lost in a strain overexpressing the specific transcription factor gene dbaA of the PKS gene cluster. This supports the idea of interplay between secondary metabolite pathways, resulting in mutually exclusive metabolite production. Genes for CSN are highly conserved and can easily be identified. Therefore, the construction and analysis of csn mutant strains of other fungi can be a highly promising approach to uncover hidden biosynthetic gene clusters. Expression of biosynthetic gene clusters is regulated by heterochromatin formation, in which S-adenosylmethionine- (SAM-) dependent methylations play a crucial role. The biosynthesis of the ubiquitous methyl group donor SAM from methionine and ATP is catalyzed by the conserved SAM synthetase. The filamentous fungus A. nidulans carries a single gene coding for the SAM synthetase SasA. In this work, the A. nidulans SAM synthetase was comprehensively characterized by genetic, cell biological and biochemical analysis. Deletion of its encoding gene sasA is lethal, and overexpression leads to impaired secondary metabolism and development, including very small sterile fruiting bodies and unusually pigmented auxiliary Hülle cells. This suggests defects in coordination of development and secondary metabolite production, which is emphasized by a putative interaction of the predominantly cytoplasmic SasA with histone-2B, reflecting a putative epigenetic link to gene expression.