Loss-of-function Aspergillus nidulans CclA, a Bre2 ortholog involved in histone 3 lysine 4 methylation, activated the expression of cryptic secondary metabolite (SM) clusters in A. nidulans. One novel cluster generated monodictyphenone, emodin and emodin derivatives while a second encoded two anti-osteoporosis polyketides, F9775A and F9775B. Modification of the chromatin landscape in fungal SM clusters allows for a simple technological means to express silent fungal secondary metabolite gene clusters.Aspergilli are ubiquitous filamentous fungi whose members include human and plant pathogens and industrial fungi with tremendous medical, agricultural and biotechnological importance. Although demonstrating synteny along large tracks of their sequenced genomes, * Corresponding authors: phone: (323) 442-1670; fax: (323) 442-1390, clayw@usc.edu, phone: (608) 262-9795; fax: (608) (2) clusters 3 . Yet the expression of most SM clusters and their concomitant products remain veiled. Two approaches for activating otherwise silent clusters were recently described. One strategy, utilizing the knowledge that many SM clusters contain a pathway specific transcription factor, fused an inducible promoter to a cluster transcription factor leading to the production of hybrid polyketide-nonribosomal peptide metabolites, the cytotoxic aspyridones A (3) and B (4) 4 . A second approach, based on genomic mining of microarrays generated from mutants of the global regulator of secondary metabolism LaeA 5, 6, 7 , led to the identification of the anti-tumor compound terrequinone A (5) 8 . Efforts to uncover the regulatory role of LaeA revealed that some subtelomeric SM clusters were located in heterochromatic regions of the genome where suppression was relieved by deletion of a key histone deacetylase 9 . The importance of histone modifications in SM clusters was further reflected in the initiation and spread of histone H4 acetylation concurrent with transcriptional activation of the subtelomeric A. parasiticus aflatoxin (6) gene cluster 10 .A consideration of the accruing evidence linking chromatin modifications with SM cluster regulation led us to examine the hypothesis that additional chromatin modifying proteins were important in SM cluster regulation. In particular, we examined a member of the COMPASS (complex associated with Set1) complex for possible regulatory roles in SM silencing. The COMPASS complex is a conserved eukaryotic transcriptional effector both facilitating and repressing chromatin-mediated processes through methylation of lysine 4 of histone 3 (H3K4) 11,12 . While H3K4me2 and H3K4me3 are found predominantly on active loci 12 , the COMPASS complex also regulates homothallic mating silencing, ribosomal DNA silencing, telomere length, and subtelomeric gene expression in yeast [13][14][15] .A critical member of the COMPASS complex is the SPRY domain protein designated Bre2 in Saccharomyces cerevisiae 11 . Analysis of the A. nidulans genome revealed a putative ortholog, here named CclA. Extracts of cclA delet...

Genome sequencing has revealed that fungi have the ability to synthesize many more natural products (NPs) than are currently known, but methods for obtaining suitable expression of NPs have been inadequate. We have developed a successful strategy that bypasses normal regulatory mechanisms. By efficient gene targeting, we have replaced, en masse, the promoters of non-reducing polyketide synthase (NR-PKS) genes, key genes in NP biosynthetic pathways and other genes necessary for NR-PKS product formation or release. This has allowed us to determine the products of eight NR-PKSs of A. nidulans, including seven novel compounds, as well as the NR-PKS genes required for the synthesis of the toxins, alternariol (8) and cichorine (19).

Meroterpenoids are a class of fungal natural products that are produced from polyketide and terpenoid precursors. An understanding of meroterpenoid biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has previously been found to produce two meroterpenoids, austinol and dehydroaustinol. Using targeted deletions that we created, we have determined that, surprisingly, two separate gene clusters are required for meroterpenoid biosynthesis. One is a cluster of four genes including a polyketide synthase gene, ausA. The second is a cluster of ten additional genes including a prenyltransferase gene, ausN, located on a separate chromosome. Chemical analysis of mutant extracts enabled us to isolate 3,5-dimethylorsellinic acid and ten additional meroterpenoids that are either intermediates or shunt products from the biosynthetic pathway. Six of them were identified as novel meroterpenoids in this study. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans meroterpenoids.

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.

Meroterpenoids are natural products produced from polyketide and terpenoid precursors. A gene targeting system for A. terreus NIH2624 was developed and a gene cluster for terretonin biosynthesis was characterized. The intermediates and shunt products were isolated from the mutant strains and a pathway for terretonin biosynthesis is proposed. Analysis of two meroterpenoid pathways corresponding to terretonin in A. terreus and austinol in A. nidulans reveals that they are closely related evolutionarily.