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).

T cell immunotherapy is emerging as a powerful strategy to treat cancer and may improve outcomes for patients with glioblastoma (GBM). We have developed a chimeric antigen receptor (CAR) T cell immunotherapy targeting IL-13 receptor α2 (IL13Rα2) for the treatment of GBM. Here, we describe the optimization of IL13Rα2-targeted CAR T cells, including the design of a 4-1BB (CD137) co-stimulatory CAR (IL13BBζ) and a manufacturing platform using enriched central memory T cells. Utilizing orthotopic human GBM models with patient-derived tumor sphere lines in NSG mice, we found that IL13BBζ-CAR T cells improved anti-tumor activity and T cell persistence as compared to first-generation IL13ζ-CAR CD8 T cells that had shown evidence for bioactivity in patients. Investigating the impact of corticosteroids, given their frequent use in the clinical management of GBM, we demonstrate that low-dose dexamethasone does not diminish CAR T cell anti-tumor activity in vivo. Furthermore, we found that local intracranial delivery of CAR T cells elicits superior anti-tumor efficacy as compared to intravenous administration, with intraventricular infusions exhibiting possible benefit over intracranial tumor infusions in a multifocal disease model. Overall, these findings help define parameters for the clinical translation of CAR T cell therapy for the treatment of brain tumors.

Xanthones are a class of molecules that bind to a number of drug targets and possess a myriad of biological properties. An understanding of xanthone 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 been found to produce two prenylated xanthones, shamixanthone and emericellin, and we report the discovery of two more, variecoxanthone A and epishamixanthone. Using targeted deletions that we created, we determined that a cluster of 10 genes including a polyketide synthase gene, mdpG, is required for prenyl xanthone biosynthesis. mdpG was shown to be required for the synthesis of the anthraquinone emodin, monodictyphenone, and related compounds, and our data indicate that emodin and monodictyphenone are precursors of prenyl xanthones. Isolation of intermediate compounds from the deletion strains provided valuable clues as to the biosynthetic pathway, but no genes accounting for the prenylations were located within the cluster. To find the genes responsible for prenylation, we identified and deleted seven putative prenyltransferases in the A. nidulans genome. We found that two prenyltransferase genes, distant from the cluster, were necessary for prenyl xanthone synthesis. These genes belong to the fungal indole prenyltransferase family that had previously been shown to be responsible for the prenylation of amino acid derivatives. In addition, another prenyl xanthone biosynthesis gene is proximal to one of the prenyltransferase genes. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans xanthones.

This review studies the impact of whole genome sequencing on Aspergillus secondary metabolite research. There has been a proliferation of many new, intriguing discoveries since sequencing data became widely available. What is more, the genomes disclosed the surprising finding that there are many more secondary metabolite biosynthetic pathways than laboratory research had suggested. Activating these pathways has been met with some success, but many more dormant genes remain to be awakened.

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.

Epipolythiodioxopiperazines (ETPs) are a class of fungal secondary metabolites derived from diketopiperazines. Acetylaranotin belongs to one structural subgroup of ETPs characterized by the presence of a seven-membered 4,5-dihydrooxepine ring. Defining the genes involved in acetylaranotin biosynthesis should provide a means to increase production of these compounds and facilitate the engineering of second-generation molecules. The filamentous fungus Aspergillus terreus produces acetylaranotin and related natural products. Using targeted gene deletions, we have identified a cluster of nine genes including one nonribosomal peptide synthetase gene, ataP, which is required for acetylaranotin biosynthesis. Chemical analysis of the wild type and mutant strains enabled us to isolate seventeen natural products from the acetylaranotin biosynthesis pathway. Nine of the compounds identified in this study are previously not reported natural products. Our data allow us to propose a biosynthetic pathway for acetylaranotin and related natural products.

No abstract