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 recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase 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 CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in E. coli and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates l-leucine and d-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be 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 substrates, suggesting severe folding problems in the multidomain proteins.
Trauger
J W
JW
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Walsh
C T
CT
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007930:Leucine / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D014640:Vancomycin / Q000031:analogs & derivatives* / Q000096:biosynthesis
D000900:Anti-Bacterial Agents; D017931:DNA Primers; C112142:chloroeremomycin; D014640:Vancomycin; D010453:Peptide Synthases; D007930:Leucine