title: Identification and characterization of a novel diterpene gene cluster in Aspergillus nidulans.
abstract: Fungal secondary metabolites are a rich source of medically useful compounds due to their pharmaceutical and toxic properties. Sequencing of fungal genomes has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in normal laboratory conditions. Therefore, to discover new metabolites, it is important to find ways to induce the expression of genes in these otherwise silent biosynthetic clusters. We discovered a novel secondary metabolite in Aspergillus nidulans by predicting a biosynthetic gene cluster with genomic mining. A Zn(II)(2)Cys(6)-type transcription factor, PbcR, was identified, and its role as a pathway-specific activator for the predicted gene cluster was demonstrated. Overexpression of pbcR upregulated the transcription of seven genes in the identified cluster and led to the production of a diterpene compound, which was characterized with GC/MS as ent-pimara-8(14),15-diene. A change in morphology was also observed in the strains overexpressing pbcR. The activation of a cryptic gene cluster by overexpression of its putative Zn(II)(2)Cys(6)-type transcription factor led to discovery of a novel secondary metabolite in Aspergillus nidulans. Quantitative real-time PCR and DNA array analysis allowed us to predict the borders of the biosynthetic gene cluster. Furthermore, we identified a novel fungal pimaradiene cyclase gene as well as genes encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase and a geranylgeranyl pyrophosphate (GGPP) synthase. None of these genes have been previously implicated in the biosynthesis of terpenes in Aspergillus nidulans. These results identify the first Aspergillus nidulans diterpene gene cluster and suggest a biosynthetic pathway for ent-pimara-8(14),15-diene.
authors.affiliation: Metabolic Engineering, VTT Technical Research Centre of Finland, Espoo, Finland. kirsi.bromann@vtt.fi
authors.forename: Kirsi
authors.initials: K
authors.lastname: Bromann
authors.forename: Mervi
authors.initials: M
authors.lastname: Toivari
authors.forename: Kaarina
authors.initials: K
authors.lastname: Viljanen
authors.forename: Anu
authors.initials: A
authors.lastname: Vuoristo
authors.forename: Laura
authors.initials: L
authors.lastname: Ruohonen
authors.forename: Tiina
authors.initials: T
authors.lastname: Nakari-Setälä
mesh_terms: D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D053898:Biosynthetic Pathways; D004224:Diterpenes / Q000378:metabolism*; D051231:Farnesyltranstransferase / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D016681:Genome, Fungal; D005810:Multigene Family; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D059467:Transcriptome
chemical_list: D000214:Acyl Coenzyme A; D004224:Diterpenes; D005656:Fungal Proteins; D014157:Transcription Factors; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; D051231:Farnesyltranstransferase; C024938:HMG-CoA reductase phosphatase; D010749:Phosphoprotein Phosphatases