Identification and molecular genetic analysis of the cichorine gene cluster in Aspergillus nidulans

We recently demonstrated that the phytotoxin cichorine is produced by Aspergillus nidulans. Through a set of targeted deletions, we have found a cluster of seven genes that are required for its biosynthesis. Two of the deletions yielded molecules that give information about the biosynthesis of this metabolite

Differentiating between models of Epothilone binding to microtubules using tubulin mutagenesis, cytotoxicity, and molecular modeling

This is the peer reviewed version of the following article: Entwistle, R. A., Rizk, R. S., Cheng, D. M., Lushington, G. H., Himes, R. H., & Gupta, M. L. (2012). Differentiating between models of Epothilone binding to microtubules using tubulin mutagenesis, cytotoxicity, and molecular modeling. ChemMedChem, 7(9), 1580–1586. http://doi.org/10.1002/cmdc.201200286, which has been published in final form at doi.org/10.1002/cmdc.201200286. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Microtubule stabilizers are powerful anti-mitotic compounds and represent a proven cancer treatment strategy. Several classes of compounds in clinical use or trials, such as the taxanes and epothilones, bind to the same region of β-tubulin. Determining how these molecules interact with tubulin and stabilize microtubules is important both for understanding the mechanism of action and enhancing chemotherapeutic potential, e.g. reducing side effects, increasing solubility, and overcoming resistance. Structural studies using nonpolymerized tubulin or stabilized polymers have produced different models of epothilone binding. Here, we used directed mutagenesis of the binding site on Saccharomyces cerevisiae β-tubulin to analyze interactions between Epothilone B and its biologically relevant substrate, dynamic microtubules. Five engineered amino acid changes contributed to a 125-fold increase in Epothilone B cytotoxicity independent of inherent microtubule stability. The mutagenesis of endogenous β-tubulin was done in otherwise isogenic strains. This facilitated the correlation of amino acid substitutions with altered cytotoxicity using molecular mechanics simulations. The results, which are based on the interaction between Epothilone B and dynamic microtubules, most strongly support the binding mode determined by NMR spectroscopy-based studies. This work establishes a system for discriminating between potential binding modes and among various compounds and/or analogues using a sensitive biological activity-based readout

Inhibition of Tau Aggregation by Three Aspergillus nidulans Secondary Metabolites: 2,ω-Dihydroxyemodin, Asperthecin, and Asperbenzaldehyde

This is the published version. Copyright 2014 George Theime Verlag. All rights reserved.The aggregation of the microtubule-associated protein tau is a significant event in many neurodegenerative diseases including Alzheimerʼs disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activity. We have screened Aspergillus nidulans secondary metabolites containing aromatic ring structures for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol and the previously identified aggregation inhibitor emodin as a positive control. While several compounds showed some activity, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde were potent aggregation inhibitors as determined by both a filter trap assay and electron microscopy. In this study, these three compounds were stronger inhibitors than emodin, which has been shown in a prior study to inhibit the heparin induction of tau aggregation with an IC50 of 1–5 µM. Additionally, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde reduced, but did not block, tau stabilization of microtubules. 2,ω-Dihydroxyemodin and asperthecin have similar structures to previously identified tau aggregation inhibitors, while asperbenzaldehyde represents a new class of compounds with tau aggregation inhibitor activity. Asperbenzaldehyde can be readily modified into compounds with strong lipoxygenase inhibitor activity, suggesting that compounds derived from asperbenzaldehyde could have dual activity. Together, our data demonstrates the potential of 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde as lead compounds for further development as therapeutics to inhibit tau aggregation in Alzheimerʼs disease and neurodegenerative tauopathies

Genome-Based Deletion Analysis Reveals the Prenyl Xanthone Biosynthesis Pathway in Aspergillus nidulans

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja1096682.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

Molecular Genetic Characterization of the Biosynthesis Cluster of a Prenylated Isoindolinone Alkaloid Aspernidine A in Aspergillus nidulans

Aspernidine A is a prenylated isoindolinone alkaloid isolated from the model fungus Aspergillus nidulans. A genome-wide kinase knock out library of A. nidulans was examined and it was found that a mitogen-activated protein kinase gene, mpkA, deletion strain produces aspernidine A. Targeted gene deletions were performed in the kinase deletion background to identify the gene cluster for aspernidine A biosynthesis. Intermediates were isolated from mutant strains which provided information about the aspernidine A biosynthesis pathway

Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids, austinol and dehydroaustinol in Aspergillus nidulans

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja209809t.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

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone

Illuminating the diversity of aromantic polyketide synthases in Aspergillus nidulans

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja3016395.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)

Abstracts from the NIHR INVOLVE Conference 2017

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The paclitaxel site in tubulin probed by site-directed mutagenesis of Saccharomyces cerevisiae β-tubulin

AbstractPreviously, we created a paclitaxel-sensitive strain of Saccharomyces cerevisiae by mutating five amino acid residues in β-tubulin in a strain that has a decreased level of the ABC multidrug transporters. We have used site-directed mutagenesis to examine the relative importance of the five residues in determining sensitivity of this strain to paclitaxel. We found that the change at position 19 from K (brain β-tubulin) to A (yeast β-tubulin) and at position 227 from H (brain β-tubulin) to N (yeast β-tubulin) had no effect on the activity of paclitaxel. On the other hand, the changes V23T, D26G and F270Y, drastically reduced sensitivity of AD1-8-tax to paclitaxel. Molecular modeling and computational studies were used to explain the results