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Published on: September 10, 2016
Construction of a food-grade multiple-copy integration system for Lactococcus lactis
K Leenhouts1, A Bolhuis, G Venema
1Department of Genetics, University of Groningen, Haren, The Netherlands.
Applied Microbiology and Biotechnology
|June 6, 1998
Summary
A novel food-grade integration system enables stable, multi-copy integration of plasmids into Lactococcus lactis chromosomes. This method simplifies engineering robust bacterial strains for various applications.
Area of Science:
- Microbial genetics and molecular biology
- Food biotechnology
- Bacterial strain engineering
Background:
- Stable integration of genetic material into bacterial chromosomes is crucial for robust strain development.
- Existing methods for Lactococcus lactis strain engineering often lack stability or food-grade compliance.
- Lactococcus lactis is a key organism in food fermentation, making its genetic manipulation highly relevant.
Purpose of the Study:
- To develop a food-grade vector system for stable, multi-copy integration of plasmids into the Lactococcus lactis chromosome.
- To establish a simple and efficient procedure for engineering stable, food-grade Lactococcus lactis strains.
- To assess the stability of integrated plasmid copies under selective and non-selective growth conditions.
Main Methods:
- Development of a food-grade integration vector utilizing pWV01 origin of replication (Ori+) and Pediococcus pentosaceus sucrose genes for selection.
- Utilized specific Lactococcus lactis helper strains (LL108, LL302) for replication and isolation of integration plasmids.
- Employed single-crossover integration into L. lactis MG1363, followed by selection on sucrose-containing medium.
Main Results:
- Achieved stable amplification of approximately 20 plasmid copies per chromosome in Lactococcus lactis MG1363.
- Demonstrated stability of integrated copies under selective conditions, with limited loss (7.5-15 x 10^-2 copies/generation) under non-selective (glucose) conditions.
- Isolated a strain (MG124) retaining 11 integrated copies after 120 generations of non-selective growth.
Conclusions:
- The developed single-crossover integration system provides a simple and effective method for engineering stable, food-grade Lactococcus lactis strains.
- The system allows for stable, multi-copy integration of genes of interest, enhancing potential for industrial applications.
- The high stability of integrated copies, even under non-selective conditions, makes this system highly valuable for food biotechnology.
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