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

Information for choice: what people need, prefer and use - Executive summary for the National Institute for Health Research Service Delivery and Organisation programme

First paragraph: Choice is at the heart of all Government health policies but is meaningless without information. Information is pivotal to people's experience of choice and self-management. To make optimal choices with confidence and to build on their existing self-management strategies people need the right information, at the right time, with the right support to use it. We already know that people want information but not necessarily for making choices and that people facing complex treatment choices often prefer decisions to be made on their behalf by a well-informed and trusted health professional. SDO 08/1710/153 was commissioned to understand the types of information that people take account of when making choices, the format of information that they prefer, and whether preferences vary systematically according to socio-economic status, ethnicity, gender and age

Information for choice: what people need, prefer and use - Report for the National Institute for Health Research Service Delivery and Organisation programme

First paragraph: Choice is at the heart of all Government health policies (1-5). The SDO's scoping review of the evidence on patient choice in the NHS, commissioned in 2004, suggested that: people want information but not necessarily for making choices; that people facing complex treatment choices often prefer decisions to be made on their behalf by a well-informed and trusted health professional; that wanting the option of choosing a distant hospital for non urgent care is limited to those situations where there is a long wait for a local hospital and there is a history of poor quality care; that wealthy and better educated people are likely to benefit most from choice; and that there is little evidence that giving people more choice will, in itself, improve quality of care (6). It is recognised that information is pivotal to people's experience of choice and self-management; to make optimal choices with confidence and to build on their existing self-management strategies people need the right information, at the right time with right support to use it (7). Lord Darzi's Next Stage Review (8) made it clear that the English NHS was to be focused as: "an NHS that gives patients and the public more information and choice, works in partnership and has quality of care at its heart" (page 7) (our emphasis). SDO 08/1710/153 was commissioned in 2005. The brief called for research to understand the types of information that people take account of when making choices, the format of information that they prefer, and whether preferences vary systematically according to socio-economic status, ethnicity, gender and age. In responding to this brief we focused on two key types of information: 'general facts' and 'personal experience' information. By 'general facts' we mean research-based information about health care interventions and the risks and outcomes associated with them; medical knowledge that reflects consensus based on what has been observed among many patients/people; and other information that is widely accepted to be both reasonably reliable and fairly broadly applicable (e.g. statements of legal requirement or policy). By 'personal experience' information we mean information about the experiences of particular individuals, as communicated by themselves or others

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

An Efficient System for Heterologous Expression of Secondary Metabolite Genes 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/ja401945a.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 non-reducing 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 NR-PKSs 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

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