Biosynthesis of Amphi-enterobactin Siderophores by <i>Vibrio harveyi</i> BAA-1116: Identification of a Bifunctional Nonribosomal Peptide Synthetase Condensation Domain

The genome of <i>Vibrio harveyi</i> BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (<i>aebA–F</i>) resembling that for entero­bactin, yet entero­bactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative entero­bactin-like biosynthetic gene cluster (<i>aebG</i>). The proximity of this FACL gene to the entero­bactin-like synthetase suggested that <i>V. harveyi</i> may produce amphiphilic entero­bactin-like siderophores. Extraction of the bacterial cell pellet of <i>V. harveyi</i> led to the isolation and structure determination of a suite of eight amphi-entero­bactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxy­benzoyl-l-serine and acyl-l-serine. The FACL knockout mutant, Δ<i>aebG V. harveyi</i>, and the NRPS knockout mutant, Δ<i>aebF V. harveyi</i>, do not produce amphi-entero­bactins. The amphi-entero­bactin biosynthetic machinery was heterologously expressed in <i>Escherichia coli</i> and reconstituted <i>in vitro</i>, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions