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Applied and Environmental Microbiology, April 2009, p. 2002-2011, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02315-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression{triangledown}

Ingrid Bakke,1 Laila Berg,1 Trond Erik Vee Aune,1 Trygve Brautaset,2 Håvard Sletta,2 Anne Tøndervik,2 and Svein Valla1*

Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway,1 SINTEF Materials and Chemistry, Department of Biotechnology, SINTEF, Trondheim, Norway2

Received 8 October 2008/ Accepted 26 January 2009

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 β-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 β-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.

* Corresponding author. Mailing address: Department of Biotechnology, Norwegian University of Science and Technology, Sem Saelands vei 6/8, 7491 Trondheim, Norway. Phone: 47 73598694. Fax: 47 73591283. E-mail: svein.valla{at}biotech.ntnu.no

{triangledown} Published ahead of print on 5 February 2009.

Applied and Environmental Microbiology, April 2009, p. 2002-2011, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02315-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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