Present addresses: Department of Poultry Science, Texas A&M University, 101 Kleberg Center, 2472 TAMU, College Station, TX 77843, USA; ‡Department of Food Science and Human Nutrition, University of Illinois, Urbana/Champaign, 905 S. Goodwin Avenue, 439 Bevier Hall, MC-182, Urbana, IL 61801, USA; §Danisco USA Inc., 3329 Agriculture Drive, Madison, WI 53716, USA; ¶Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina, 312B Isaac Taylor Hall, Campus Box 7547, Chapel Hill, NC 27514, USA.
Construction of vectors for inducible and constitutive gene expression in Lactobacillus
Article first published online: 1 SEP 2010
© 2010 The Authors. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd
Special Issue: Lactic Acid Bacteria. Editors: Michiel Kleerebezem and Willem M. de Vos.
Volume 4, Issue 3, pages 357–367, May 2011
How to Cite
Duong, T., Miller, M. J., Barrangou, R., Azcarate-Peril, M. A. and Klaenhammer, T. R. (2011), Construction of vectors for inducible and constitutive gene expression in Lactobacillus. Microbial Biotechnology, 4: 357–367. doi: 10.1111/j.1751-7915.2010.00200.x
- Issue published online: 25 APR 2011
- Article first published online: 1 SEP 2010
- Received 10 May, 2010; accepted 6 July, 2010.
Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.