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Expression of the Staphylococcus hyicus lipase in Lactococcus lactis
S Drouault1, G Corthier, S D Ehrlich
1Unité de Génétique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy en Josas Cedex, France.
Applied and Environmental Microbiology
|February 2, 2000
Summary
High expression of Staphylococcus hyicus lipase in Lactococcus lactis is toxic and leads to cellular accumulation. Lipase degradation and export issues were observed, suggesting subspecies-specific proteases and potential secretion inefficiencies.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Extracellular enzymes like Staphylococcus hyicus lipase are valuable biocatalysts.
- Understanding heterologous protein expression and secretion in microbial hosts is crucial for biotechnology.
Purpose of the Study:
- To investigate the expression, localization, and degradation of Staphylococcus hyicus lipase in Lactococcus lactis and Bacillus subtilis.
- To identify factors affecting the secretion and stability of the lipase in these bacterial hosts.
Main Methods:
- Gene expression under inducible promoters (PnisA) in Lactococcus lactis and Bacillus subtilis.
- Protein localization analysis using cell fractionation and Western blotting.
- Assessment of lipase degradation using proteases and analysis of subspecies-specific differences.
Main Results:
- High-level expression of S. hyicus lipase in L. lactis was toxic and resulted in up to 30% of cellular protein, with 80% cell-associated.
- Lipase was found associated with the cytoplasmic membrane (unprocessed) and cell wall (partially degraded).
- Lipase degradation patterns differed between L. lactis subsp. cremoris and L. lactis subsp. lactis, indicating distinct extracellular proteolytic systems.
Conclusions:
- The leader peptide is not responsible for incorrect lipase export; secretion may be inefficient.
- Subtleties in lipase synthesis kinetics or the need for accessory export systems might be critical for proper folding and secretion.
- Extracellular proteases in L. lactis subspecies differentially degrade the lipase, highlighting subspecies-specific differences in proteolytic activity.