The D-galactose dehydrogenase gene from Pseudomonas fluorescens: characterization of mutations leading to increased expression in Escherichia coli.
The gene encoding D-galactose dehydrogenase (gld; E.C. 1.1.1.48) from Pseudomonas fluorescens is poorly expressed when cloned into Escherichia coli. Mutagenesis of the wild-type construct leads to a strong expression of gld in the heterologous host. To investigate the mutational events directing the increase in expression we constructed a gld-lacZ translational fusion which facilitated the isolation of mutants by colony screening. From several independent mutants three point mutations could be identified. They were distinguished by the sequence position of their respective single base-pair substitutions in the 5'-untranslated region of the gld gene and the degree of enhancement of enzyme activity of the gene product. The influence of these mutations on gld gene expression was analysed by S1 protection analysis which revealed that their effect was at the level of transcription.
Department of Genetics, Boehringer Mannheim GmbH, Penzberg, Federal Republic of Germany.
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Fiedler
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Buckel
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005691:Galactose Dehydrogenases / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid
D005691:Galactose Dehydrogenases; C047959:galactose dehydrogenase