Engineering Fluoroacetate Dehalogenase by Growth-Based Selections on Non-Natural Organofluorides

Area of Science:

• Biocatalysis and enzyme engineering
• Environmental biotechnology
• Organic chemistry

Background:

• Organofluorides, including per- and polyfluorinated alkyl substances (PFAS), are persistent environmental pollutants due to the stability of the carbon-fluorine (C─F) bond.
• Nature possesses limited mechanisms to break C─F bonds, with fluoroacetate dehalogenases (FAcDs) being an exception, known for efficient fluoroacetate hydrolysis.
• Developing biocatalysts to degrade non-natural organofluorides is crucial for environmental remediation.

Purpose of the Study:

• To engineer fluoroacetate dehalogenases (FAcDs) for enhanced activity and broader substrate specificity towards various organofluorides.
• To establish a scalable and robust selection platform for directed evolution of FAcDs.
• To develop sustainable biocatalytic solutions for the degradation of environmental organofluoride pollutants.

Main Methods:

• A growth-based selection strategy was developed to enrich FAcD variants capable of cleaving C─F bonds in organofluorides.
• Engineered FAcD libraries were subjected to selection using various organofluorides as sole carbon sources for bacterial growth.
• Activity and substrate profiles of evolved FAcD variants were characterized for fluoroacetate, 2-fluoropropionate, and 2,2-difluoroacetate.

Main Results:

• A large-scale engineering campaign successfully generated FAcD variants with improved catalytic activities and altered substrate specificities.
• The selection platform demonstrated the ability to enrich FAcDs that can metabolize synthetic organofluorides.
• An inhibition pathway affecting the conversion of gem-difluoride compounds by FAcDs was identified.

Conclusions:

• The study presents the first large-scale engineering of FAcDs using a novel, operationally simple selection platform.
• This platform enables the adaptation of FAcDs for the sustainable degradation of environmentally relevant organofluorides.
• The engineered FAcDs hold promise for bioremediation strategies targeting persistent fluorinated pollutants.