Dissection and rational engineering of the biosynthetic pathway to enacyloxin, a promising anti-Gram-negative antibiotic

Enacyloxin IIa is a polyketide antibiotic with potent activity against Gram-positive and Gram-negative bacteria that targets ribosomal elongation factor Tu [1 – 5]. It has been identified as a metabolite of Burkholderia ambifaria AMMD and shown to have clinically-relevant activity against Acinetobacter baumannii, a problematic multidrug-resistant Gram-negative pathogen.⁶ Despite its promising biological activity, enacyloxin IIa is unlikely to find direct clinical application, given the densely-packed array of similar and potentially labile functional groups in the antibiotic (see figure below). Enacyloxin biosynthesis has recently been mapped to an 80 kb gene cluster in the sequenced genome of B. ambifaria AMMD and a pathway for its biosynthesis has been proposed, involving assembly of the 27-carbon acyl chain by a modular polyketide synthase (PKS) [6]. The polyketide chain undergoes various modifications by on-/post-PKS tailoring enzymes, including halogenation, hydroxylation, carbamoylation and oxidation. Detailed structural and biochemical analyses have provided insights into an unusual mechanism of chain release in enacyloxin biosynthesis, showing that the enzymatic machinery is able to accommodate (1R,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid (DHCCA) as well as a diverse set of analogues. Using a variety of rational engineering approaches, including mutasynthesis, gene deletion and gene replacement strategies, we are currently exploiting this biosynthetic knowledge for the production of novel analogues with improved pharmacological properties.

Fig. 1: A Structure of enacyloxin IIa. B X-ray crystal structure of enacyloxin IIa bound to an E. coli EF-Tu complex (rendered from PDB entry 2KVN) [5].

Keywords: Synthetic biology, enzymology, polyketide biosynthesis, Burkholderia, antibiotic.

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