Discovery of a Metagenome-Derived Enzyme that Produces Branched-Chain Acyl-(Acyl-Carrier-Protein)s from Branched-Chain α-Keto Acids

Abstract

Clones that confer the production of N-acyl amino acids to model cultured bacterial hosts are frequently identified in activity-based screens of soil DNA libraries. The N-acyl amino acids encoded by these clones are synthesized from acyl-(acyl-carrier-protein)s (acyl-ACPs, ACP) and amino acids using a diverse group of bacterial enzymes referred to as N-acyl amino acid synthases (NASs).[1, 2] The acyl-ACP substrates of NAS enzymes are common intermediates in the de novo synthesis of fatty acids by the type II fatty acid synthase system (FAS II).[3] In addition to N-acyl amino acids, FAS II-derived acyl-ACPs also serve as acyl-donors in the production of N-acyl homoserine lactone autoinducers, the cofactor lipoic acid and several lipopeptide antibiotics (Figure 1A, compounds 1–4).[4–9] During a recent screen of soil metagenomic libraries for clones exhibiting antimicrobial activity, we uncovered an NAS-containing clone (clone EC5) that confers the production of long-chain N-acyl-phenylalanines and N-acyl-tryptophans to multiple Gram-negative host species.[10] The NAS-encoding gene found on clone EC5, nasA, is the first gene in a predicted two gene operon, nasAB (GenBank Accession No. {type:entrez-nucleotide,attrs:{text:GQ869383,term_id:260177223,term_text:GQ869383}}GQ869383). The second gene in this operon, nasB, encodes for a putative 1118 residue protein that contains six well-characterized Pfam homology domains (Figure 1B).[11] Based on generic functional predictions for these domains, we hypothesized that this enzyme was likely to be involved in the formation of ACP-linked fatty acids similar to those produced by bacterial FAS II systems. Here we report results from a series of heterologous expression experiments in Burkholderia graminis C4D1M, which show that nasB functions to provide branched-chain acyl-ACPs for nasA. Many bacteria, particularly Gram-negative species, do not natively produce branched-chain fatty acids.[3] NasB and its homologs are likely used by bacteria with straight-chain specific FAS II systems for generating branched-chain acyl-ACP substrates and should be useful as tools for synthesizing these substrates in model heterologous expression systems in which they are not natively produced (e.g. E. coli).