Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

Abstract

The genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans by deleting the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). The csnE/CSN5 gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs. Since its discovery by Fleming in the 1920s, fungal penicillin has saved the lives of millions. Currently, the World Health Organization forecasts that the dramatic increase in antimicrobial resistance all over the world might lead to a disaster and proclaims a need for novel drugs (22). Certain fungi, plants, and bacteria produce various potent secondary metabolites (SMs) that span a wide field of structurally and chemically diverse natural products. With almost 1.5 million species (33), the fungal kingdom is a major reservoir for bioactive natural products as beneficial antibiotics and antitumor drugs but also as deleterious mycotoxins and food contaminants (28,38). Although many fungal SMs have been described and tested, their complete potential is by far not exploited.In recent years, different approaches were applied to find novel bioactive SMs either in new species or in already established model organisms. New geographical spots exhibiting extreme conditions were explored in order to find new species producing as-yet-unknown natural products (37). An alternative approach is the exploration of the full genomic potential of already known species by genomic mining (13,14,30,76). Genomic sequencing revealed that there are many more genes for the biosynthesis of SMs than the metabolites already identified. These genes are often clustered, but most of them are rarely expressed under laboratory conditions (35), making the identification of their chemical products challenging. Two major strategies were applied to activate hidden genes: (i) changing the environment or (ii) genetic engineering (19,35,56). (i) The OSMAC (one strain, many compounds) approach activates silent gene clusters by cultivating microorganisms under different conditions (10, 75). Alternatively, physical contact with an opponent results in the uncovering of hidden clusters by activating defense mechanisms (58). (ii) Genetic engineering is focused primarily on expressing complete gene clusters in heterologous hosts (53, 77) or on altering the cellular transcription or protein synthesis machinery. Thus, SM synthesi...

Discovery of McrA, a master regulator of Aspergillus secondary metabolism

“…S6B. Two products have previously been reported for the dba cluster (Ahuja et al ., ; Gerke et al ., ) and felinone A is a third product, not previously known to be produced by A. nidulans .…”

“…. Using a DAD to detect compounds containing chromophores, LO1362 produced approximately 8 detectable compounds under these conditions including sterigmatocystin ( 1 ), 2,4‐dihydroxy‐3‐methyl‐6‐(2‐oxopropyl)benzaldehyde (from the AN7903, dba cluster (Ahuja et al ., ; Gerke et al ., )) ( 34 ), microperfuranone ( 35 ), and four unknowns ( 19 and 31 – 33 ). Only one peak was visible in the alcA (p)AN8694 strains and this peak was identified as lumichrome, which we believe to be metabolized from the riboflavin added to the culture as a nutritional supplement.…”

A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici

“…Secondary metabolites play very important roles in filamentous fungi, but they are often either cryptic or silent in the laboratory conditions and may present complex, difficult to analyze structures, e.g., fungal melanin (Gerke et al ., ; Shao et al ., ; Nosanchuk et al ., ; Rutledge and Challis, ). Thus, it is hard to characterize their structures and to study the function of these metabolites, many of which are emerging as critical for fungal development.…”

Identification of Metabolic Pathways Influenced by the G-Protein Coupled Receptors GprB and GprD in Aspergillus nidulans

“…Most of the genes related to MFS transporters belong to the class of multidrug resistant (MDR) transporters [32]. In the ΔgprD strain, a gene encoding for transport of secondary metabolites, dbaD (AN7898; [33]) was strongly up-regulated (Table S1). Several genes encoding amino acid transporters including AN8659, AN9300 and AN9174 were strongly down-regulated in the ΔgprD strain, compared to wild-type, by 3-, 4-, and 6-fold, respectively.…”