Characterization of the Aspergillus nidulans monodictyphenone gene cluster.

Deletion of cclA, a component of the COMPASS complex of Aspergillus nidulans, results in the production of monodictyphenone and emodin derivatives. Through a set of targeted deletions in a cclA deletion strain, we have identified the genes required for monodictyphenone and emodin analog biosynthesis. Identification of an intermediate, endocrocin, from an mdpHDelta strain suggests that mdpH might encode a decarboxylase. Furthermore, by replacing the promoter of mdpA (a putative aflJ homolog) and mdpE (a putative aflR homolog) with the inducible alcA promoter, we have confirmed that MdpA functions as a coactivator. We propose a biosynthetic pathway for monodictyphenone and emodin derivatives based on bioinformatic analysis and characterization of biosynthetic intermediates.

Molecular genetic analysis of the orsellinic acid/F9775 gene cluster of Aspergillus nidulans.

F-9775A and F-9775B are cathepsin K inhibitors that arise from a chromatin remodelling deletant strain of Aspergillus nidulans. A polyketide synthase gene has been determined to be responsible for their formation and for the simpler, archetypical polyketide orsellinic acid. We have discovered simple culture conditions that result in the production of the three compounds, and this facilitates analysis of the genes responsible for their synthesis. We have now analysed the F9775/orsellinic acid gene cluster using a set of targeted deletions. We find that the polyketide synthase alone is required for orsellinic acid biosynthesis and only two additional genes in the cluster are required for F9775 A and B synthesis. Our deletions also yielded the bioactive metabolites gerfelin and diorcinol.

Chromatin-level regulation of biosynthetic gene clusters.

Loss-of-function Aspergillus nidulans CclA, a Bre2 ortholog involved in histone H3 lysine 4 methylation, activated the expression of cryptic secondary metabolite clusters in A. nidulans. One new cluster generated monodictyphenone, emodin and emodin derivatives, whereas a second encoded two anti-osteoporosis polyketides, F9775A and F9775B. Modification of the chromatin landscape in fungal secondary metabolite clusters allows for a simple technological means to express silent fungal secondary metabolite gene clusters.

A gene cluster containing two fungal polyketide synthases encodes the biosynthetic pathway for a polyketide, asperfuranone, in Aspergillus nidulans.

The genome sequencing of Aspergillus species including A. nidulans reveals that the products of many of the secondary metabolism pathways in these fungi have not been elucidated. Our examination of the 27 polyketide synthases (PKS) in A. nidulans revealed that one highly reduced PKS (HR-PKS, AN1034.3) and one nonreduced PKS (NR-PKS, AN1036.3) are located next to each other in the genome. Since no known A. nidulans secondary metabolites could be produced by two PKS enzymes, we hypothesized that this cryptic gene cluster produces an unknown natural product. Indeed after numerous attempts we found that the products from this cluster could not be detected under normal laboratory culture conditions in wild type strains. Closer examination of the gene cluster revealed a gene with high homology to a citrinin biosynthesis transcriptional activator (CtnR, 32% identity/47% similarity), a fungal transcription activator located next to the two PKSs. We replaced the promoter of the transcription activator with the inducible alcA promoter, which enabled the production of a novel polyketide that we have named asperfuranone. A series of gene deletions has allowed us to confirm that the two PKSs together with five additional genes comprise the asperfuranone biosynthetic pathway and leads us to propose a biosynthetic pathway for asperfuranone. Our results confirm and substantiate the potential to discover novel compounds even from a well-studied fungus by using a genomic mining approach.

Identification and characterization of the asperthecin gene cluster of Aspergillus nidulans.

The sequencing of Aspergillus genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of sumO, the gene that encodes the small ubiquitin-like protein SUMO in A. nidulans, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the sumO deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.

Molecular genetic mining of the Aspergillus secondary metabolome: discovery of the emericellamide biosynthetic pathway.

The recently sequenced genomes of several Aspergillus species have revealed that these organisms have the potential to produce a surprisingly large range of natural products, many of which are currently unknown. We have found that A. nidulans produces emericellamide A, an antibiotic compound of mixed origins with polyketide and amino acid building blocks. Additionally, we describe the discovery of four previously unidentified, related compounds that we designate emericellamide C-F. Using recently developed gene targeting techniques, we have identified the genes involved in emericellamide biosynthesis. The emericellamide gene cluster contains one polyketide synthase and one nonribosomal peptide synthetase. From the sequences of the genes, we are able to deduce a biosynthetic pathway for the emericellamides. The identification of this biosynthetic pathway opens the door to engineering novel analogs of this structurally complex metabolite.