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Applied and Environmental Microbiology, May 2002, p. 2397-2403, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2397-2403.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Modulation of Gene Expression Made Easy

Christian Solem and Peter Ruhdal Jensen*

Section of Molecular Microbiology, BioCentrum, Technical University of Denmark, DK-2800 Lyngby, Denmark

Received 1 November 2001/ Accepted 11 February 2002

A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding ß-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected the activities of all three enzymes to the same extent, and enzyme activities ranging from 0.5 to 3.5 times the wild-type level were obtained.

* Corresponding author. Mailing address: Section of Molecular Microbiology, BioCentrum, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark. Phone: 45 45-252510. Fax: 45 45-932809. E-mail: imprj{at}pop.dtu.dk.

Applied and Environmental Microbiology, May 2002, p. 2397-2403, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2397-2403.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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