A perplexing aspect of fungal secondary metabolite gene clusters is that most clusters remain ‘silent’ under common laboratory growth conditions where activation is obtained through gene manipulation or encounters with environmental signals. Few proteins have been found involved in repression of silent clusters. Through multicopy suppressor mutagenesis, we have identified a novel cluster suppressor in Aspergillus nidulans, MvlA (modulator of veA
loss). Genetic assessment of MvlA mutants revealed the role of both itself and VeA (but not the VeA partner LaeA) in the suppression of the cryptic ors gene cluster producing orsellinic acid and its F9775 derivatives. Loss of veA upregulates F9775A and F9775B production and this increase is reduced 4~5 fold when an overexpression mvlA (OE::mvlA) allele is introduced into the ΔveA background. Previous studies have implicated a positive role for GcnE (H3K9 acetyltransferase of the SAGA/ADA complex) in ors cluster expression and here we find expression of gcnE is upregulated in ΔveA and suppressed by OE::mvlA in the ΔveA background. H3K9 acetylation levels of ors cluster genes correlated with gcnE expression and F9775 production in ΔveA and OE::mvlAΔveA strains. Finally, deletion of gcnE in the ΔveA background abolishes ors cluster activation and F9775 production. Together, this work supports a role for VeA and MvlA in modifying SAGA/ADA complex activity.
LaeA; Velvet Complex; F9775A; Urc4; suppressor; CclA; SAGA/ADA
Filamentous fungi have long been recognized to be a rich source of secondary metabolites with potential medicinal applications. The recent genomic sequencing of several Aspergillus species has revealed that many secondary metabolite gene clusters are apparently silent under standard laboratory conditions. Several successful approaches have been utilized to upregulate these genes and unearth the corresponding natural products. A straightforward, reliable method to purify and characterize new metabolites therefore should be useful. Details are provided herein on the cultivation of Aspergillus nidulans and the LC/MS analysis of the metabolic profile. Following is an explanation of silica gel chromatography, HPLC, and preparative TLC. Finally, the NMR characterization of previously unknown A. nidulans metabolites is detailed.
Secondary metabolites; natural products; polyketides; purification methods; Aspergillus nidulans
Norsolorinic acid, isolated from the Aspergillus nidulans, was investigated for its anti-proliferative activity in human breast adenocarcinoma MCF-7 cells. To identity the anticancer mechanism of norsolorinic acid, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor, and Fas ligand. The results showed that norsolorinic acid induced apoptosis of MCF-7 cells without mediation of p53 and p21/WAF1. We suggest that Fas/Fas ligand apoptotic system is the main pathway of norsolorinic acid-mediated apoptosis of MCF-7 cells. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the anti-proliferative activity of norsolorinic acid in MCF-7 cells.
norsolorinic acid; breast cancer; p53; Fas/APO-1; Fas ligand; apoptosis
Mining for novel natural compounds is of eminent importance owing to the continuous need for new pharmaceuticals. Filamentous fungi are historically known to harbor the genetic capacity for an arsenal of natural compounds, both beneficial and detrimental to humans. The majority of these metabolites are still cryptic or silent under standard laboratory culture conditions. Mining for these cryptic natural products can be an excellent source for identifying new compound classes. Capitalizing on the current knowledge on how secondary metabolite gene clusters are regulated has allowed the research community to unlock many hidden fungal treasures, as described in this chapter.
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).
The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new “function-omic” avenues downstream of the metabolomics, identification, and purification phases.
Several fungal pathogens produce bioactive small molecules, commonly known as secondary metabolites (SMs) that contribute towards disease development in susceptible hosts. Genome assessment of human pathogenic Aspergillus species indicates these fungi have the capabilities of producing hundreds of SMs, most of which are currently not characterized for their effect on human health and the immune system. This lack of knowledge is directly correlated to the difficulties of obtaining assayable quantities of pure metabolites. To overcome this roadblock in assessing the potential impact of SMs on the immune system, our laboratories have developed a two-tiered cost-effective, high-throughput program utilizing microfluidic platforms and a novel zebrafish model to identify SMs inhibiting neutrophil chemotaxis. Using minimal and physiologically relevant amounts of SMs, this systematic approach has identified the A. fumigatus spore SM, endocrocin, as a potent chemotaxis inhibitor. Interestingly, the production of endocrocin is temperature dependent and virulence studies with the endocrocin null mutant implicates the temperature at which the fungus forms spores as a factor in disease development.
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.
Sclerotiorin, an azaphilone polyketide, is a bioactive natural product known to inhibit 15-lipoxygenase and many other biological targets. To readily access sclerotiorin and analogs, we developed a 2–3 step semisynthetic route to produce a variety of azaphilones starting from an advanced, putative azaphilone intermediate (5) over-produced by an engineered strain of Aspergillus nidulans. The inhibitory activities of the semisynthetic azaphilones against 15-lipoxygenase were evaluated with several compounds displaying low micromolar potency.
Regulation of secondary metabolite (SM) gene clusters in Aspergillus nidulans has been shown to occur through cluster specific transcription factors or through global regulators of chromatin structure such as histone methyltransferases, histone deacetylases, or the putative methyltransferase LaeA. A multi-copy suppressor screen for genes capable of returning SM production to the SM deficient ΔlaeA mutant resulted in identification of the essential histone acetyltransferase EsaA, able to complement an esa1 deletion in Saccharomyces cereviseae. Here we report that EsaA plays a novel role in SM cluster activation through histone 4 lysine 12 (H4K12) acetylation in four examined SM gene clusters (sterigmatocystin, penicillin, terrequinone, and orsellinic acid), in contrast to no increase in H4K12 acetylation of the housekeeping tubA promoter. This augmented SM cluster acetylation requires LaeA for full effect and correlates with both increased transcript levels and metabolite production relative to wild type. H4K12 levels may thus represent a unique indicator of relative production potential, notably of SMs.
A StcA-AfoE hybrid PKS, generated from swapping the AfoE (asperfuranone biosynthesis) SAT domain with the StcA (sterigmatocystin biosynthesis) SAT domian, produced a major new metabolite with the same chain length as the native AfoE product. Structure elucidation allowed us to propose a likely pathway and feeding studies supported the hypothesis that the chain length of PKS metabolites may be under precise control of KS and PT domains.
The genome sequencing of the fungus Aspergillus niger uncovered a large cache of genes encoding enzymes thought to be involved in the production of secondary metabolites yet to be identified. Identification and structural characterization of many of these predicted secondary metabolites are hampered by their low concentration relative to the known A. niger metabolites such as the naphtho-γ-pyrone family of polyketides. We deleted a nonreducing PKS gene in A. niger strain ATCC 11414, a daughter strain of A. niger ATCC strain 1015 whose genome was sequenced by the DOE Joint Genome Institute. This PKS encoding gene we name albA is a predicted ortholog of alb1 from Aspergillus fumigatus which is responsible for production of the naphtho-γ-pyrone precursor for the 1,8-dihydroxynaphthalene (DHN) melanin/spore pigment. Our results show that the A. nigeralbA PKS is responsible for both the production of the spore pigment precursor and a family of naphtho-γ-pyrones commonly found in significant quantity in A. niger culture extracts. The generation of an A. niger strain devoid of naphtho-γ-pyrones will greatly facilitate the elucidation of cryptic biosynthetic pathways in this organism.
Secondary Metabolism; Aspergillus niger; Natural Products; Genomics; Naphtho-γ-pyrone; Polyketides
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.
Secondary metabolites from microorganisms have a broad spectrum of applications, particularly in therapeutics. The growing number of sequenced microbial genomes has revealed a remarkably large number of natural product biosynthetic clusters for which the products are still unknown. These cryptic clusters are potentially a treasure house of medically useful compounds. The recent development of new methodologies has made it possible to begin unlock this treasure house, to discover new natural products and determine their biosynthesis pathways. This review will highlight some of the most recent strategies to activate silent biosynthetic gene clusters and to elucidate of their corresponding products and pathways.
Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans.
Secondary metabolite (SM) production by fungi is hypothesized to provide some fitness attribute for the producing organisms. However, most SM clusters are “silent” when fungi are grown in traditional laboratory settings, and it is difficult to ascertain any function or activity of these SM cluster products. Recently, the creation of a chromatin remodeling mutant in Aspergillus nidulans induced activation of several cryptic SM gene clusters. Systematic testing of nine purified metabolites from this mutant identified an emodin derivate with efficacy against both human fungal pathogens (inhibiting both spore germination and hyphal growth) and several bacteria. The ability of catalase to diminish this antimicrobial activity implicates reactive oxygen species generation, specifically, the generation of hydrogen peroxide, as the mechanism of emodin hydroxyl activity.
Asperfuranone, a novel compound of genomic mining in Aspergillus nidulans, was investigated for its anti-proliferative activity in human non-small cell lung cancer A549 cells. To identity the anti-cancer mechanism of asperfuranone, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21 Waf1/Cip1, Fas/APO-1 receptor and Fas ligand. Enzyme-linked immunosorbent assay showed that the G0/G1 phase arrest might be due to p53-dependent induction of p21 Waf1/Cip1. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by asperfuranone. Our study reports here for the first time that the induction of p53 and the activity of Fas/Fas ligand apoptotic system may participate in the anti-proliferative activity of asperfuranone in A549 cells.
Recent published sequencing of fungal genomes has revealed that these microorganisms have a surprisingly large number of secondary metabolite pathways that can serve as potential sources for new and useful natural products. Most of the secondary metabolites and their biosynthesis pathways are currently unknown, possibly because they are produced in very small amounts and are thus difficult to detect or are produced only under specific conditions. Elucidating these fungal metabolites will require new molecular genetic tools, better understanding of the regulation of secondary metabolism, and state of the art analytical methods. This review describes recent strategies to mine the cryptic natural products and their biosynthetic pathways in fungi.
natural products biosynthesis; genomic mining; polyketide synthase; nonribosomal peptide synthetase
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.
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 mdpHΔ 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.
Understanding the molecular details associated with aberrant high mobility group A2 (HMGA2) gene expression is key to establishing the mechanism(s) underlying its oncogenic potential and impact on the development of therapeutic strategies. Here, we report the involvement of HMGA2 in impairing DNA-dependent protein kinase (DNA-PK) during the non-homologous end joining (NHEJ) process. We demonstrated that HMGA2-expressing cells displayed deficiency in overall and precise DNA end-joining repair and accumulated more endogenous DNA damage. Proper and timely activation of DNA-PK, consisting of Ku70, Ku80 and DNA-PKcs subunits, is essential for the repair of DNA double strand breaks (DSBs) generated endogenously or by exposure to genotoxins. In cells overexpressing HMGA2, accumulation of histone 2A variant X phosphorylation at Ser-139 (γ-H2AX) was associated with hyper-phosphorylation of DNA-PKcs at Thr-2609 and Ser-2056 before and after the induction of DSBs. Also, the steady-state complex of Ku and DNA ends was altered by HMGA2. Microirradiation and real-time imaging in living cells revealed that HMGA2 delayed the release of DNA-PKcs from DSB sites, similar to observations found in DNA-PKcs mutants. Moreover, HMGA2 alone was sufficient to induce chromosomal aberrations, a hallmark of deficiency in NHEJ-mediated DNA repair. In summary, a novel role for HMGA2 to interfere with NHEJ processes was uncovered, implicating HMGA2 in the promotion of genome instability and tumorigenesis.
HMGA2; Ku70/80; DNA-PKcs; NHEJ; genome instability
Natural products display impressive activities against a wide range of targets, including viruses, microbes and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily-scalable production of valuable yet hard-to-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. The quinomycin family of nonribosomal peptides, including echinomycin, trtiostin A and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown the production of echinomycin and trtiostin A in E. coli using our convenient and modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products in E. coli. Here we report an identification of a new gene cluster responsible for the biosynthesis of SW-163s that involves previously unknown biosynthesis of (+)-(1S, 2S)-norcoronamic acid and generation of aliphatic side chains of various sizes via iterative methylation of an unactivated carbon center. Substituting an echinomycin biosynthetic gene with a gene from the newly identified SW-163 biosynthetic gene cluster, we were able to rationally re-engineer the plasmid-based echinomycin biosynthetic pathway for the production of a novel bioactive compound in E. coli.
depsipeptide; hybrid molecule; nonribosomal peptide synthetase; engineered biosynthesis; E. coli
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.
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 non-reduced 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.
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.
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.