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.
Highlights
The rise in antibiotic resistance is a major threat for human health
This suggested that silencing of individual genes from the cluster triggered the downregulation of all the other genes known to be involved in biosynthesis of pleuromutilin
The diterpene antibiotic pleuromutilin has provided leads for the only commercial antibiotic produced from a basidiomycete fungus, from C. passeckerianus and other related species[3], and has led to the generation of semi-synthetic derivatives[7, 8, 10] for human and veterinary use
Summary
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. We report here genetic characterisation of the steps involved in the biosynthesis of 1, through a logical stepwise expression of genes from the pleuromutilin gene cluster in A. oryzae, coupled with isolation and detailed structural elucidation of the pathway intermediates by spectroscopic methods and comparison with synthetic standards. This pathway engineering approach has given insights into pleuromutilin biosynthesis and creation of a panel of A. oryzae strains designed to accumulate each pathway intermediate as a final product. In industrial production of antibiotics, replacing chemical synthesis steps with a OH
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