benzofurans and asperfuranone

Fungal natural products (NPs) comprise a vast number of bioactive molecules with diverse activities, and among them are many important drugs. However, the yields of fungal NPs from native producers are usually low, and total synthesis of structurally complex NPs is challenging. As such, downstream derivatization and optimization of lead fungal NPs can be impeded by the high cost of obtaining sufficient starting material. In recent years, reconstitution of NP biosynthetic pathways in heterologous hosts has become an attractive alternative approach to produce complex NPs. Here, we present an efficient, cloning-free strategy for the cluster refactoring and total biosynthesis of fungal NPs in

Topics: Aspergillus nidulans; Aurovertins; Benzofurans; Biological Products; Biosynthetic Pathways; Diterpenes; Genes, Fungal; Homologous Recombination; Ketones; Multigene Family; Plasmids; Pleuromutilins; Polycyclic Compounds; Regulon

Three undescribed 3(2H)-furanone derivatives, asperfuranones A-C (1-3), along with one known compound (4) were isolated from the Aspergillus sp. strain obtained from the intestines of centipede. Their structures were determined by NMR and MS spectroscopic analyses, and the absolute configurations were established by the Snatzke's sector rules, modified Mosher's method and electronic circular dichroism (ECD) calculation. Meanwhile, the application of the sector rules led to the reassignment of the absolute configurations of 4 and other seventeen previously reported analogues (5-21).

Topics: Animals; Aspergillus; Benzofurans; Circular Dichroism; Mice; Molecular Structure; RAW 264.7 Cells

A facile genetic methodology in the filamentous fungus Aspergillus nidulans allowed exchange of the starter unit ACP transacylase (SAT) domain in the nonreduced polyketide synthase (NR-PKS) AfoE of the asperfuranone pathway with the SAT domains from 10 other NR-PKSs. The newly created hybrid with the NR-PKS AN3386 is able to accept a longer starter unit in place of the native substrate to create a novel aromatic polyketide in vivo.

Topics: Acyltransferases; Aspergillus nidulans; Benzofurans; Molecular Structure; Polyketide Synthases; Protein Structure, Tertiary

Fungal secondary metabolites (SMs) are an important source of medically valuable compounds. Genome projects have revealed that fungi have many SM biosynthetic gene clusters that are not normally expressed. To access these potentially valuable, cryptic clusters, we have developed a heterologous expression system in Aspergillus nidulans . We have developed an efficient system for amplifying genes from a target fungus, placing them under control of a regulatable promoter, transferring them into A. nidulans , and expressing them. We have validated this system by expressing nonreducing polyketide synthases of Aspergillus terreus and additional genes required for compound production and release. We have obtained compound production and release from six of these nonreducing polyketide synthases and have identified the products. To demonstrate that the procedure allows transfer and expression of entire secondary metabolite biosynthetic pathways, we have expressed all the genes of a silent A. terreus cluster and demonstrate that it produces asperfuranone. Further, by expressing the genes of this pathway in various combinations, we have clarified the asperfuranone biosynthetic pathway. We have also developed procedures for deleting entire A. nidulans SM clusters. This allows us to remove clusters that might interfere with analyses of heterologously expressed genes and to eliminate unwanted toxins.

Topics: Aspergillus nidulans; Benzofurans; Molecular Conformation; Polyketide Synthases

An StcA-AfoE hybrid polyketide synthase (PKS), generated by 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.

Topics: Aspergillus nidulans; Benzofurans; Biological Products; Polyketide Synthases; Protein Engineering; Protein Structure, Tertiary

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.

Topics: Antineoplastic Agents; Apoptosis; Aspergillus nidulans; Benzofurans; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Humans; Lung Neoplasms

Filamentous fungi produce numerous natural products that constitute a consistent source of potential drug leads, yet it seems that the majority of natural products are overlooked since most biosynthesis gene clusters are silent under standard cultivation conditions. Screening secondary metabolite genes of the model fungus Aspergillus nidulans, we noted a silent gene cluster on chromosome II comprising two nonribosomal peptide synthetase (NRPS) genes, inpA and inpB, flanked by a regulatory gene that we named scpR for secondary metabolism cross-pathway regulator. The induced expression of the scpR gene using the promoter of the alcohol dehydrogenase AlcA led to the transcriptional activation of both the endogenous scpR gene and the NRPS genes. Surprisingly, metabolic profiling of the supernatant of mycelia overexpressing scpR revealed the production of the polyketide asperfuranone. Through transcriptome analysis we found that another silent secondary metabolite gene cluster located on chromosome VIII coding for asperfuranone biosynthesis was specifically induced. Quantitative reverse transcription-PCR proved the transcription not only of the corresponding polyketide synthase (PKS) biosynthesis genes, afoE and afoG, but also of their activator, afoA, under alcAp-scpR-inducing conditions. To exclude the possibility that the product of the inp cluster induced the asperfuranone gene cluster, a strain carrying a deletion of the NRPS gene inpB and, in addition, the alcAp-scpR overexpression cassette was generated. In this strain, under inducing conditions, transcripts of the biosynthesis genes of both the NRPS-containing gene cluster inp and the asperfuranone gene cluster except gene inpB were detected. Moreover, the existence of the polyketide product asperfuranone indicates that the transcription factor ScpR controls the expression of the asperfuranone biosynthesis gene cluster. This expression as well as the biosynthesis of asperfuranone was abolished after the deletion of the asperfuranone activator gene afoA, indicating that ScpR binds to the afoA promoter. To the best of our knowledge, this is the first report of regulatory cross talk between two biosynthesis gene clusters located on different chromosomes.

Topics: Aspergillus nidulans; Benzofurans; Biosynthetic Pathways; Gene Expression Profiling; Genes, Fungal; Macrolides; Metabolome; Multigene Family; Peptide Synthases

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.

Topics: Aspergillus nidulans; Benzofurans; Fungal Proteins; Furans; Genes, Fungal; Genome, Fungal; Multigene Family; Polyketide Synthases