Andrimid producers encode an acetyl-CoA carboxyltransferase subunit resistant to the action of the antibiotic

摘要

Andrimid is a hybrid nonribosomal peptide-polyketide antibiotic that blocks the carboxyl-transfer reaction of bacterial acetyl-CoA carboxylase (ACC) and thereby inhibits fatty acid biosynthesis with submicromolar potency. The andrimid biosynthetic gene cluster frorn Pantoea agglomerans encodes an admT gene with homology to the acetyl-CoA carboxyltransferase (CT) β-subunit gene accD. Escherichia coli cells overexpressing admT showed resistance to andrimid. Co-overproduction of AdmT with E. coli CT α-subunit AccA allowed for the in vitro reconstitution of an active heterol-ogous tetrameric CT A_2T_2 complex. A subsequent andrimid-inhibition assay revealed an IC_(50) of 500 nM for this hybrid A_2T_2 in contrast to that of 12 nM for E. coli CT A_2D_2. These results validated that AdmT is an AccD homolog that confers resistance in the andrimid producer. Mutagenesis studies guided by the x-ray crystal structure of the E. coli A_2D_2 complex disclosed a single amino acid mutation of AdmT (L203M) responsible for 5-fold andrimid sensitivity (IC_(50) = 100 nM). Complementarity, the E. coli AccD mutant M203L became 5-fold more resistant in the CT assays. This observation allowed for bioinformatic identification of several Vibrio cholerae strains in which accD genes encode the Met← → Leu switches, and their occurrences correlate predictively with sensitivities to andrimid in vivo.