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 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.
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.
Hairpin pyrrole-imidazole (Py-Im) polyamides are programmable oligomers that bind the DNA minor groove in a sequence-specific manner with affinities comparable to those of natural DNA-binding proteins. These cell-permeable small molecules have been shown to enter the nuclei of live cells and downregulate endogenous gene expression. We complete here a library of 27 hairpin Py-Im polyamides which bind 7-base-pair sequences of the general form 5′-WWGNNNW-3′ (where W = A or T, N = W, G, or C). Their equilibrium association constants (Ka) range from Ka = 1×108 M−1 to 4×1010 M−1 with good sequence specificity. A table of binding affinities and sequence contexts for this completed 27-member library has been assembled for the benefit of the chemical biology community interested in molecular control of transcription.
molecular recognition; gene regulation; polyamide; small molecule-nucleic acid interaction
The medicinal value associated with complex polyketide and nonribosomal peptide natural products has prompted biosynthetic schemes dependent upon heterologous microbial hosts. Here we report the successful biosynthesis of yersiniabactin (Ybt), a model polyketide-nonribosomal peptide hybrid natural product, using Escherichia coli as a heterologous host. After introducing the biochemical pathway for Ybt into E. coli, biosynthesis was initially monitored qualitatively by mass spectrometry. Next, production of Ybt was quantified in a high-cell-density fermentation environment with titers reaching 67 ± 21 (mean ± standard deviation) mg/liter and a volumetric productivity of 1.1 ± 0.3 mg/liter-h. This success has implications for basic and applied studies on Ybt biosynthesis and also, more generally, for future production of polyketide, nonribosomal peptide, and mixed polyketide-nonribosomal peptide natural products using E. coli.
Induction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood.
In this report, we demonstrate that induction of p53 expression is mediated through NF-κB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-κB and activity of NF-κB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-κB inhibitor, Bay 11-7082 and downregulation of NF-κB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells.
Taken together, NF-κB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress.
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Our previous studies have indicated that expression of Hepatitis B virus pre-S2 large mutant surface antigen (HBV pre-S2Δ) is associated with a significant risk of developing HCC. However, the relationship between HBV pre-S2Δ protein and the resistance of chemotherapeutic drug treatment is still unclear.
Here, we show that the expression of HBV pre-S2Δ mutant surface protein in Huh-7 cell significantly promoted cell growth and colony formation. Furthermore, HBV pre-S2Δ protein increased both mRNA (2.7±0.5-fold vs. vehicle, p = 0.05) and protein (3.2±0.3-fold vs. vehicle, p = 0.01) levels of Bcl-2 in Huh-7 cells. HBV pre-S2Δ protein also enhances Bcl-2 family, Bcl-xL and Mcl-1, expression in Huh-7 cells. Meanwhile, induction of NF-κB p65, ERK, and Akt phosphorylation, and GRP78 expression, an unfolded protein response chaperone, were observed in HBV pre-S2Δ and HBV pre-S-expressing cells. Induction of Bcl-2 expression by HBV pre-S2Δ protein resulted in resistance to 5-fluorouracil treatment in colony formation, caspase-3 assay, and cell apoptosis, and can enhance cell death by co-incubation with Bcl-2 inhibitor. Similarly, transgenic mice showed higher expression of Bcl-2 in liver tissue expressing HBV pre-S2Δ large surface protein in vivo.
Our result demonstrates that HBV pre-S2Δ increased Bcl-2 expression which plays an important role in resistance to 5-fluorouracil-caused cell death. Therefore, these data provide an important chemotherapeutic strategy in HBV pre-S2Δ-associated tumor.
Aspergillus species have the ability to produce a wide range of secondary metabolites including polyketides that are generated by multi-domain polyketide synthases (PKSs). Recent biochemical studies using dissected single or multiple domains from PKSs have provided deep insight into how these PKSs control the structural outcome. Moreover, the recent genome sequencing of several species has greatly facilitated the understanding of the biosynthetic pathways for these secondary metabolites. In this review, we will highlight the current knowledge regarding polyketide biosynthesis in Aspergillus based on the domain architecture of non-reducing, highly reducing, and partially reducing PKSs, and PKS-non-ribosomal peptide synthetases.
Secondary metabolites; Fungi; Polyketide; Nonribosomal peptides