scholarly journals Characterization of the P450 Monooxygenase NysL, Responsible for C-10 Hydroxylation during Biosynthesis of the Polyene Macrolide Antibiotic Nystatin in Streptomyces noursei

2006 ◽  
Vol 72 (4) ◽  
pp. 2514-2519 ◽  
Author(s):  
Olga Volokhan ◽  
Håvard Sletta ◽  
Trond E. Ellingsen ◽  
Sergey B. Zotchev
Keyword(s):  
Polyketide Synthase ◽  
Kinetic Studies ◽  
Biosynthetic Gene Cluster ◽  
Gene Replacement ◽  
Gene Encoding ◽  
P450 Monooxygenase ◽  
Polyene Macrolide Antibiotic ◽  
Streptomyces Noursei

ABSTRACT The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of Km and k cat and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.

scholarly journals Analysis of the Mycosamine Biosynthesis and Attachment Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455

2007 ◽  
Vol 73 (22) ◽  
pp. 7400-7407 ◽  
Author(s):  
Aina Nedal ◽  
Håvard Sletta ◽  
Trygve Brautaset ◽  
Sven E. F. Borgos ◽  
Olga N. Sekurova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Gene Cluster ◽  
Macrolide Antibiotic ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Polyene Macrolide Antibiotic ◽  
Streptomyces Noursei ◽  
Dehydratase Activity

ABSTRACT The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the β-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches

2007 ◽  
Vol 27 (1-3) ◽  
pp. 151-163 ◽  
Author(s):  
M. C. Lara ◽  
M. L. Valentino ◽  
J. Torres-Torronteras ◽  
M. Hirano ◽  
R. Martí
Keyword(s):  
Molecular Genetic ◽  
In Vivo Studies ◽  
Gene Encoding ◽  
Mitochondrial Neurogastrointestinal Encephalomyopathy ◽  
Mtdna Replication ◽  
Biochemical Features

Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE.

scholarly journals Differential Expression of the Aspergillus fumigatus pksP Gene Detected In Vitro and In Vivo with Green Fluorescent Protein

2001 ◽  
Vol 69 (10) ◽  
pp. 6411-6418 ◽  
Author(s):  
Kim Langfelder ◽  
Bruno Philippe ◽  
Bernhard Jahn ◽  
Jean-Paul Latgé ◽  
Axel A. Brakhage
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Fumigatus ◽  
Polyketide Synthase ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Gene Encoding ◽  
Immunocompromised Mice ◽  
Green Fluorescent

ABSTRACT Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To be able to analyze the expression of putative virulence-associated genes of A. fumigatus, the use of the enhanced green fluorescent protein (EGFP) as a reporter was established. Two 5′ sequences, containing the putative promoters of thepyrG gene, encoding orotidine-5′-phosphate decarboxylase, and the pksP gene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence ofA. fumigatus, were fused with the egfpgene. The PpksP-egfp construct was integrated via homologous recombination into the genomicpksP locus. EGFP production was analyzed by fluorescence spectrometry, Western blot analysis, and fluorescence microscopy. Differential gene expression in A. fumigatus was observed. Fluorescence derived from the PYRG-EGFP fusion protein was detected during all developmental stages of the fungus, i.e., during germination, during vegetative growth, in conidiophores, and weakly in conidia. In addition, it was also detected in germinating conidia when isolated from the lungs of immunocompromised mice. By contrast, PKSP-EGFP-derived fluorescence was not found in hyphae or stalks of conidiophores but was found in phialides and conidia in vitro when the fungus was grown under standard conditions, indicating a developmentally controlled expression of the gene. Interestingly,pksP-egfp expression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice. This finding indicates that thepksP gene can also be expressed in hyphae under certain conditions and, furthermore, that the pksP gene might also contribute to invasive growth of the fungus.

scholarly journals A High-Throughput Method for Enzyme Kinetic Studies

2003 ◽  
Vol 8 (5) ◽  
pp. 544-554 ◽  
Author(s):  
Lakshmi D. Saraswat ◽  
Kimberley A. Caserta ◽  
Kathy Laws ◽  
Darren Wei ◽  
Simon S. Jones ◽  
...  
Keyword(s):  
Drug Metabolism ◽  
High Throughput ◽  
Kinetic Studies ◽  
Metabolic Stability ◽  
Metabolic Parameter ◽  
High Throughput Method ◽  
Multiple Samples ◽  
Parameter Values

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 °C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery. ( Journal of Biomolecular Screening 2003:544-554)

scholarly journals Transcriptome Analysis Identifies a Gene Cluster for the Biosynthesis of Biruloquinone, a Rare Phenanthraquinone, in a Lichen-Forming Fungus Cladonia macilenta

2021 ◽  
Vol 7 (5) ◽  
pp. 398
Author(s):  
Won-Yong Kim ◽  
Min-Hye Jeong ◽  
Sung-Hwan Yun ◽  
Jae-Seoun Hur
Keyword(s):  
Gene Cluster ◽  
Transcriptome Analysis ◽  
Polyketide Synthase ◽  
Expression Patterns ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Whole Transcriptome Analysis ◽  
High Degree ◽  
Whole Transcriptome

Lichens are prolific producers of natural products of polyketide origin. We previously described a culture of lichen-forming fungus (LFF) Cladonia macilenta that produces biruloquinone, a purple pigment that is a phenanthraquinone rarely found in nature. However, there was no genetic information on the biosynthesis of biruloquinone. To identify a biosynthetic gene cluster for biruloquinone, we mined polyketide synthase (PKS) genes from the genome sequence of a LFF isolated from thalli of C. macilenta. The 38 PKS in C. macilenta are highly diverse, many of which form phylogenetic clades with PKS previously characterized in non-lichenized fungi. We compared transcriptional profiles of the 38 PKS genes in two chemotypic variants, one producing biruloquinone and the other producing no appreciable metabolite in vitro. We identified a PKS gene (hereafter PKS21) that was highly upregulated in the LFF that produces biruloquinone. The boundaries of a putative biruloquinone gene cluster were demarcated by co-expression patterns of six clustered genes, including the PKS21. Biruloquinone gene clusters exhibited a high degree of synteny between related species. In this study we identified a novel PKS family responsible for the biosynthesis of biruloquinone through whole-transcriptome analysis.

scholarly journals New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

2011 ◽  
Vol 77 (18) ◽  
pp. 6636-6643 ◽  
Author(s):  
Trygve Brautaset ◽  
Håvard Sletta ◽  
Kristin F. Degnes ◽  
Olga N. Sekurova ◽  
Ingrid Bakke ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Fungal Infections ◽  
Therapeutic Index ◽  
Hydroxyl Group ◽  
Hydroxyl Groups ◽  
Marginal Effect ◽  
P450 Monooxygenase ◽  
Polyene Macrolides ◽  
Biosynthetic Engineering

ABSTRACTPolyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected toin vitroassays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

scholarly journals δ-Aminolaevulinic acid synthesis is required for virulence of the wheat pathogen Stagonospora nodorum

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1533-1538 ◽  
Author(s):  
Peter S. Solomon ◽  
Cordula I. Jörgens ◽  
Richard P. Oliver
Keyword(s):  
Single Gene ◽  
Acid Synthesis ◽  
Gene Replacement ◽  
Stagonospora Nodorum ◽  
Homologous Gene ◽  
In Planta ◽  
Gene Encoding ◽  
Aminolaevulinic Acid ◽  
Key Enzyme

δ-Aminolaevulinic acid (ALA) is synthesized in fungi by ALA synthase, a key enzyme in the synthesis of haem. The requirement for ALA synthase in Stagonospora nodorum to cause disease in wheat was investigated. The single gene encoding ALA synthase (Als1) was cloned and characterized. Expression analysis determined that Als1 transcription was up-regulated during germination and also towards the latter stages of the infection. The Als1 gene was further characterized by homologous gene replacement. The inactivation of Als1 resulted in strains producing severely stunted germ tubes leading quickly to death. The strains could be recovered by supplementation with 33 μM ALA. Pathogenicity assays revealed the als1 strains were essentially non-pathogenic, inferring a key role for the synthesis of ALA during in planta growth. Supplementing the strains with ALA restored growth in vitro and also pathogenicity for up to 5 days after inoculation. Further examination by inoculating the als1 strains onto wounded leaves found that pathogenicity was only partially restored, suggesting that host-derived in planta levels of ALA are not sufficient to support growth. This study has identified a key role for fungal ALA synthesis during infection and revealed its potential as an antifungal target.

scholarly journals An Unconventional Melanin Biosynthetic Pathway in Ustilago maydis

2020 ◽  
Author(s):  
Esmeralda Z. Reyes-Fernández ◽  
Yi-Ming Shi ◽  
Peter Grün ◽  
Helge B. Bode ◽  
Michael Bölker
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Model Organism ◽  
Ustilago Maydis ◽  
Biosynthetic Gene Cluster ◽  
Polyketide Synthases ◽  
Phytopathogenic Fungus ◽  
Deletion Mutants ◽  
P450 Monooxygenase ◽  
Corn Smut

Ustilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well established model organism for the study of plant-microbe interactions, its potential to produce specialized metabolites, which might contribute to this interaction, has not been studied in detail. By analyzing the U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4 and pks5), a cytochrome P450 monooxygenase (cyp4) and a protein with similarity to versicolorin B synthase (vbs1). Metabolic profiles of deletion mutants in this gene cluster suggested that Pks3 and Pks4 act in concert as heterodimer to generate orsellinic acid (OA) which is reduced to the corresponding aldehyde by Pks5. The OA-aldehyde can then react with triacetic acid lactone (TAL) also derived from Pks3/Pks4 heterodimers to form larger molecules including novel coumarin derivatives. Our findings suggest that U. maydis synthesizes a novel type of melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or L-3,4-dihydroxyphenylalanine (L-DOPA). Along with these observations, this work also provides an insight into the mechanisms of polyketide synthases in this filamentous fungus. IMPORTANCE The fungus Ustilago maydis represents one of the major threats for maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in an unconventional way, requiring more than one polyketide synthase for producing melanin precursors, gives a different perspective on the complexity of these multimodular enzymes and their evolution in the fungal kingdom.

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