Random mutagenesis of the PM promoter as a powerful strategy for improvement of recombinant-gene expression.
The inducible Pm-xylS promoter system has proven useful for production of recombinant proteins in several gram-negative species and in high-cell-density cultivations of Escherichia coli. In this study we subjected a 24-bp region of Pm (including the -10 element) to random mutagenesis, leading to large mutant libraries in E. coli. Low-frequency-occurring Pm mutants displaying strongly increased promoter activity (up-mutants) could be efficiently identified by using beta-lactamase as a reporter. The up-mutants typically carried multiple point mutations positioned throughout the mutagenized region, combined with deletions around the transcription start site. Mutants displaying up to about a 14-fold increase in beta-lactamase expression (relative to wild-type Pm) were identified without loss of the inducible phenotype. The mutants also strongly stimulated the expression of two other reporter genes, luc (encoding firefly luciferase) and celB (encoding phosphoglucomutase), and were found to significantly improve (twofold) a previously optimized process for high-level recombinant production of the medically important granulocyte-macrophage colony-stimulating factor in E. coli under high-cell-density conditions. These results demonstrate the potential of using random mutagenesis of promoters to improve protein expression at industrial levels and indicate that targeted modifications of individual functional elements are not sufficient to obtain optimized promoter sequences.
Bakke
Ingrid
I
Department of Biotechnology, Norwegian University of Science and Technology, Sem Saelands vei 6/8, 7491 Trondheim, Norway.
Berg
Laila
L
Aune
Trond Erik Vee
TE
Brautaset
Trygve
T
Sletta
Håvard
H
Tøndervik
Anne
A
Valla
Svein
S
D001483:Base Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010733:Phosphoglucomutase / Q000096:biosynthesis / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion; D015854:Up-Regulation; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D008156:Luciferases; D001618:beta-Lactamases; D010733:Phosphoglucomutase