Cyclopropane fatty acid synthase from Oenococcus oeni: expression in Lactococcus lactis subsp. cremoris and...

Area of Science:

• Biochemistry
• Microbiology
• Enology

Background:

• Cyclopropane fatty acids (CFAs) enhance bacterial resistance to environmental stresses like acidity and dryness.
• Cyclopropane fatty acid synthases (CFA synthases) are key enzymes in CFA biosynthesis.
• Oenococcus oeni is an important enological bacterium, and its stress tolerance mechanisms are of significant interest.

Purpose of the Study:

• To perform the first biochemical characterization of a lactic acid bacterium CFA synthase from Oenococcus oeni.
• To investigate the kinetic properties and optimal conditions for O. oeni CFA synthase activity.
• To elucidate the role of O. oeni CFA synthase in the acid tolerance of this bacterium.

Main Methods:

• Overexpression of Oenococcus oeni CFA synthase in E. coli for enzyme purification.
• Biochemical assays to determine optimal pH and temperature for cyclopropanation activity.
• Kinetic analysis to determine the K(m) value for S-adenosyl-L-methionine (AdoMet).

Main Results:

• The optimal cyclopropanation activity for O. oeni CFA synthase was observed at pH 5.6 and 35.8 °C.
• A high K(m) value for AdoMet (2.26 mM) indicated low affinity towards unsaturated phospholipids from L. lactis subsp. cremoris.
• These kinetic properties help explain the partial complementation of a L. lactis subsp. cremoris cfa mutant by the O. oeni cfa gene.

Conclusions:

• The biochemical characterization provides insights into the substrate specificity of O. oeni CFA synthase.
• The enzyme's properties suggest a significant role in the acid tolerance mechanisms of Oenococcus oeni.
• This study lays the groundwork for understanding CFA biosynthesis and its contribution to bacterial resilience in acidic environments.