Construction of an expression system for engineering of the lantibiotic Pep5.
AUTOR(ES)
Bierbaum, G
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=201989Documentos Relacionados
- Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5.
- Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin
- Molecular Analysis of Expression of the Lantibiotic Pep5 Immunity Phenotype
- Producer immunity towards the lantibiotic Pep5: identification of the immunity gene pepI and localization and functional analysis of its gene product.
- Molecular cloning, sequencing, and expression of the crr gene: the structural gene for IIIGlc of the bacterial PEP:glucose phosphotransferase system.
