Biosynthesis of pleuromutilin congeners using an Aspergillus oryzae expression platform.

Pleuromutilin is an antibiotic diterpenoid made by Clitopilus passeckerianus and related fungi, and it is the progenitor of a growing class of semi-synthetic antibiotics used in veterinary and human medicine. To harness the biotechnological potential of this natural product class, a full understanding of its biosynthetic pathway is essential. Previously, a linear pathway for pleuromutilin biosynthesis was established. Here we report two shunt pathways involving Pl-sdr and Pl-atf that were identified through the rational heterologous expression of combinations of pleuromutilin biosynthetic genes in Aspergillus oryzae. Three novel pleuromutilin congeners were isolated, and their antimicrobial activity was investigated, alongside that of an additional derivative produced through a semi-synthetic approach. It was observed that the absence of various functional groups - 3 ketone, 11 hydroxyl group or 21 ketone - from the pleuromutilin framework affected the antibacterial activity of pleuromutilin congeners. This study expands our knowledge on the biosynthesis of pleuromutilin and provides avenues for the development of novel pleuromutilin analogues by combining synthetic biology and synthetic chemistry.

Dose-Dependent Effect of Wedelia trilobata Essential Oil (EO) on Lettuce (Lactuca sativa L.) with Multivariate Analysis.

Understanding metabolite changes and underlying metabolic pathways that may be affected in target plants following essential oils (EOs) exposure is of great importance. In this study, a gas chromatography-mass spectrometry (GC/MS) based metabolomics approach was used to determine the metabolite changes in lettuce (Lactuca sativa L.) shoot and root after exposure to different concentrations of W. trilobata EO. Multivariate analyses of principal component analysis (PCA) and orthogonal partial least-discriminant analysis (OPLS-DA) corroborated that shoot and root of lettuce responded differently to W. trilobata EO. In EO-exposed shoot samples, an increase in the levels of malic acid, glutamine, serine, lactose and α-glucopyranose affected important metabolism pathways such as glycolysis, fructose and mannose metabolism and galactose metabolism. The findings suggest that lettuce may be up-regulating these metabolites to increase tolerance against W. trilobata EO. In EO-exposed root samples, changes in fatty acid biosynthesis, elongation, degradation, phenylalanine, tyrosine and tryptophan metabolism were linked to a decrease in lyxose, palmitic acid, octadecanoic acid, aspartic acid, phenylalanine and myo-inositol. These results indicate that W. trilobata EO could cause alterations in fatty acid compositions and lead to inhibition of roots growth. Together, these findings provide insight into the metabolic responses of lettuce upon W. trilobata EO exposure, as well as potential mechanisms of action of W. trilobata EO as bio-herbicides.

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

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.