NOVENA · Exploiting an unusual polyketide chain release mechanism for production of novel enacyloxin antibiotic analogues

Exploiting an unusual polyketide chain release mechanism for production of novel enacyloxin antibiotic analogues

Enacyloxin IIa is a polyketide antibiotic with activity against Gram-positive and Gram-negative bacteria that targets ribosomal elongation factor Tu. It has recently been identified as metabolite of Burkholderia ambifaria and shown to have clinically-relevant activity against Acinetobacter baumannii, a problematic panresistant Gram-negative pathogen that is estimated to account for 2-10% of all Gram-negative bacterial infections in intensive care units in Europe and for the death of thousands of European hospital patients every year. Despite its promising biological activity, enacyloxin IIa has not been used in the clinic, presumably due to stability issues. Preliminary experiments have provided evidence for an unusual mechanism of modular polyketide synthase chain release in enacyloxin biosynthesis, involving intermolecular condensation of an acyl carrier protein (ACP)-bound thioester with the C-3 hydroxyl group of (1S, 3R, 4S)-3,4- dihydroxycyclohexane carboxylic acid (DHCCA). This project aimed to elucidate and engineer the biosynthetic pathway to enacyloxin. It also aimed to illuminate the structure-activity relationship of the antibiotic by studying the characteristics of the enacyloxin analogues generated. The specific objectives of the project were: (1) Identify the enzyme responsible for epimerisation of C-1 of the DHCCA moiety (2) Explore the ability of the Bamb_5915 chain release enzyme to catalyse the acylation of diverse DHCCA analogues (3) Create a Burkholderia ambifaria mutant blocked in DHCCA biosynthesis (4) Produce novel enacyloxin IIa derivatives via a mutasynthesis approach involving feeding of DHCCA analogues to a B. ambifaria mutant blocked in DHCCA biosynthesis. The project was highly successful in its aims and fully reached its objectives. The unusual mechanism for polyketide chain release in the biosynthesis of enacyloxin was biochemically characterized and the role of several tailoring enzymes was elucidated. Based on these results, different biosynthetic engineering approaches were developed which afforded over a dozen enacyloxin derivatives, with the potential to yield many more.

Data: CORDIS, © European Union