Construction of an expression system for engineering of the lantibiotic Pep5.
Pep5 is a lanthionine-containing antimicrobial peptide which is produced by Staphylococcus epidermidis 5. Its structural gene, pepA, is located on the 20-kb plasmid pED503. A 6.2-kb fragment of pED503 containing pepA, the immunity gene pepI, and 5.4 kb of downstream sequence was able to direct biosynthesis of biologically active Pep5 in a nonproducing variant of the producer strain which is devoid of pED503. In addition to producing wild-type Pep5 with a molecular mass of 3,488 Da, the clone produced a peptide with an eightfold-lower bactericidal activity and a mass of 3,506 Da, indicative of incomplete dehydration of one hydroxyamino acid. For construction of the expression system, this 6.2-kb fragment was cut into a 1.39-kb fragment containing pepA and pepI and a 4.8-kb fragment covering the remaining downstream region. This 4.8-kb fragment was directly cloned into an Escherichia coli-Staphylococcus shuttle vector, yielding a new plasmid (pGB9) into which mutated pepA genes generated on the 1.39-kb fragment can be reinserted to yield a functional Pep5 biosynthesis gene cluster. To test the expression system, two mutants were constructed. Lys-18-Pro Pep5 was produced in its dehydrated form and a partially hydrated form in amounts comparable to those of the wild-type peptide. In contrast, only small amounts of Phe-23-Asp Pep5 were excreted, indicating that some residues in the propeptide part of the prelantibiotic may be crucial for certain steps in the biosynthetic pathway of lantibiotics.
Bierbaum
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Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, Germany.
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D000595:Amino Acid Sequence; D000626:Aminopeptidases; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D001430:Bacteriocins; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010455:Peptides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013212:Staphylococcus epidermidis / Q000235:genetics*
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C054222:lantibiotic Pep5; D000626:Aminopeptidases; C022450:prolyl aminopeptidase