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)2Cys6–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)2Cys6–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.
Highlights
Filamentous fungi produce various bioactive compounds as secondary metabolites [1,2]
Despite earlier published work suggesting that the Aspergillus nidulans genome has only one terpene cluster [13], our analysis instead revealed multiple terpene synthase genes potentially located in biosynthetic clusters
We searched for genes encoding putative cytochrome P450 monooxygenases, since these enzymes are many times involved in terpenoid biosynthesis [31]
Summary
Filamentous fungi produce various bioactive compounds as secondary metabolites [1,2]. The genes encoding consecutive steps in a biosynthetic pathway of secondary metabolites are often clustered together on the chromosomes [3]. Since secondary metabolites are not crucial for the survival of the organism, their production usually remains silent in normal laboratory conditions [1,6]. The role of secondary metabolites for the producing organism is often unclear. They are most likely used as chemical signals in communication and defense to enhance the survival of the organism in its ecological niche [8]. For many pathogenic fungi, the virulence that has been hypothesized to protect the fungus in an environment with a diverse array of competing organisms [10] is often mediated by secondary metabolites. In many cases the biological importance of secondary metabolites for fungi is elusive, and the conditions triggering the metabolic biosynthesis are unknown [8]
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