Isolation of a versatile Serratia marcescens mutant as a host and molecular cloning of the aspartase gene.
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.
Takagi
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Kisumi
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D000642:Ammonia-Lyases / Q000235:genetics*; D000667:Ampicillin / Q000494:pharmacology; D001220:Aspartate Ammonia-Lyase / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D003851:Deoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007612:Kanamycin / Q000494:pharmacology; D009154:Mutation; D010957:Plasmids; D012706:Serratia marcescens / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D007612:Kanamycin; D000667:Ampicillin; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes; D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase