Isolation of a versatile Serratia marcescens mutant as a host and molecular cloning of the aspartase gene

Abstract

An extracellular nuclease-deficient, antibiotic-sensitive, and restrictionless mutant was isolated from the wild-type strain of Serratia marcescens Sr41 by four rounds of mutagenesis. The mutant was transformed efficiently with plasmid DNAs prepared from Escherichia coli and S. marcescens, and was used as a host for the cloning of the aspartase gene (aspA+) of S. marcescens. Cells carrying the cloned aspA+ gene on a multicopy plasmid produced ca. 39-fold more aspartase than did control cells, and the level of enzyme overproduction was in proportion to the copy number of the aspA+ recombinant plasmid. Aspartase was identified as a polypeptide of molecular weight 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.