Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recent ly sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonri bosomal peptide synthetase. Expression of all or parts of the peptide synth etase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether C epA, a three-module 3,158-residue peptide synthetase expected to assemble t he first three residues of the heptapeptide precursor, could be heterologou sly expressed in E. coil and converted to active, hole form by posttranslat ional priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-315 8), All three constructs were expressed in soluble form. We find that the C epA1-575 fragment, containing adenylation and peptidyl carrier protein doma ins (A1-PCP1), specifically adenylates L-leucine and D-leucine in a 6:1 rat io, and it can be converted to hole form by the phosphopantetheinyltransfer ase Sfp; also, we find that holo-CepA1-575 can he covalently aminoacylated with L-leucine on the peptidyl carrier protein 1 domain, However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with L-leucine or other expected amino acid substrat es. suggesting severe folding problems in the multidomain proteins.