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.

The Biogenetic Origin of the Biologically Active Naematolin of Hypholoma Species Involves an Unusual Sesquiterpene Synthase

“…Construction of a flexible genetic engineering platform enables yield and chemical structure optimisation of compound of interest and potential discovery of novel bioactive molecules. We have recently had success in analysis of other basidiomycete-derived terpenes such as the diterpene pleuromutilin from Clitopilus passeckerianus [8,9] where we have not only linked the genetic pathway to the chemical synthesis, but have also used expression in a heterologous host to allow pathway manipulation and analysis. Pleuromutilin derivatives are now reaching the clinical market as a new class of antibiotics [8,9].…”

Strategies for Engineering Natural Product Biosynthesis in Fungi

“…In addition, the ability to overproduce valuable NPs enables access to the natural scaffold that can be further modified semisynthetically to optimize biological activity. For example, several pleuromutilin derivatives are under clinical development as systemic antibiotics [37], and nowheterologous expression of selected late stage biosynthetic enzymes in A. oryzae provides a platform for the bioconversion of modified precursors into semisynthetic pleuromutilin derivatives with improved antibiotic activity [38]. Figure 2.…”

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production

“…Fungal Biology and Biotechnology *Correspondence: umemura-m@aist.go.jp 1 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8566, Japan Full list of author information is available at the end of the article Page 2 of 9 Umemura et al Fungal Biol Biotechnol (2020) 7:3 auxotrophic strain (argB − , niaD − , sC − and adeA − ) [12], is used to produce fungal secondary metabolites by simultaneously introducing two to nine genes for biosynthesis of such compounds as pleuromutilin [15], paxilline [16], terretonin [17], helvolic acid [18], menisporopsin A [19] and asperipin-2a [20]. A variety of basidiomycete terpenes have been successfully produced in A. oryzae by heterologous expression of their respective biosynthetic genes using the genome-editing system [21].…”