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Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives.

ABSTRACT: The rise in antibiotic resistance is a major threat for human health. Basidiomycete fungi represent an untapped source of underexploited antimicrobials, with pleuromutilin-a diterpene produced by Clitopilus passeckerianus-being the only antibiotic from these fungi leading to commercial derivatives. Here we report genetic characterisation of the steps involved in pleuromutilin biosynthesis, through rational heterologous expression in Aspergillus oryzae coupled with isolation and detailed structural elucidation of the pathway intermediates by spectroscopic methods and comparison with synthetic standards. A. oryzae was further established as a platform for bio-conversion of chemically modified analogues of pleuromutilin intermediates, and was employed to generate a semi-synthetic pleuromutilin derivative with enhanced antibiotic activity. These studies pave the way for future characterisation of biosynthetic pathways of other basidiomycete natural products in ascomycete heterologous hosts, and open up new possibilities of further chemical modification for the growing class of potent pleuromutilin antibiotics.

SUBMITTER: Alberti F

PROVIDER: S-EPMC5705593 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives.

Alberti Fabrizio F Khairudin Khairunisa K Venegas Edith Rodriguez ER Davies Jonathan A JA Hayes Patrick M PM Willis Christine L CL Bailey Andy M AM Foster Gary D GD

Nature communications 20171128 1

The rise in antibiotic resistance is a major threat for human health. Basidiomycete fungi represent an untapped source of underexploited antimicrobials, with pleuromutilin-a diterpene produced by Clitopilus passeckerianus-being the only antibiotic from these fungi leading to commercial derivatives. Here we report genetic characterisation of the steps involved in pleuromutilin biosynthesis, through rational heterologous expression in Aspergillus oryzae coupled with isolation and detailed structur ...[more]

PMID: 29184068

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Project description:BackgroundStreptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of non-glycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in biological membranes and as a tool for the diagnosis of Niemann-Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have proven useful for the discovery of products with improved properties. Here, we describe the late biosynthetic steps for filipin III biosynthesis and strategies for the generation of bioactive filipin III derivatives at high yield.ResultsA region of 13,778 base pairs of DNA from the S. filipinensis genome was isolated, sequenced, and characterized. Nine complete genes and two truncated ORFs were located. Disruption of genes proved that this genomic region is part of the biosynthetic cluster for the 28-membered ring of the polyene macrolide filipin. This set of genes includes two cytochrome P450 monooxygenase encoding genes, filC and filD, which are proposed to catalyse specific hydroxylations of the macrolide ring at C26 and C1' respectively. Gene deletion and complementation experiments provided evidence for their role during filipin III biosynthesis. Filipin III derivatives were accumulated by the recombinant mutants at high yield. These have been characterized by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification thus revealing the post-polyketide steps during polyene biosynthesis. Two alternative routes lead to the formation of filipin III from the initial product of polyketide synthase chain assembly and cyclization filipin I, one trough filipin II, and the other one trough 1'-hydroxyfilipin I, all filipin III intermediates being biologically active. Moreover, minimal inhibitory concentration values against Candida utilis and Saccharomyces cerevisiae were obtained for all filipin derivatives, finding that 1'-hydroxyfilipin and especially filipin II show remarkably enhanced antifungal bioactivity. Complete nuclear magnetic resonance assignments have been obtained for the first time for 1'-hydroxyfilipin I.ConclusionsThis report reveals the existence of two alternative routes for filipin III formation and opens new possibilities for the generation of biologically active filipin derivatives at high yield and with improved properties.

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Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives.

Publications

Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives.

Similar Datasets

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