Discovery and selection of novel defluorinases for bioremediation – MICROFLUOR

Positioned at the interface of environmental sciences and chemical biology, the 42-month MICROFLUOR project aims at renewing and accelerating the discovery of protein catalysts for the degradation of "forever chemicals" polyfluoroalkyl substances (PFAS). This very large and diverse class of recalcitrant compounds is essentially unknown in nature. Industrially produced PFAS are used for a wide range of applications in all areas of human activities, and now represent ubiquitous contaminants in the environment. It is increasingly being realised that PFAS are highly toxic to both humans and wildlife, so that remediation solutions for these chemicals are in urgent demand. However, degradation of PFAS represents a huge challenge, due to the strength of the carbon-fluorine bond, the most stable covalent bond known. Indeed, very few defluorinating bacterial strains have been reported so far, and enzymes, genes and degradation pathways involved in PFAS degradation remain to be discovered and characterised.
MICROFLUOR will tackle this issue with an innovative RNA-based fluoride-specific biosensor recently developed by the two project partner teams "Adaptations et Interactions Microbiennes dans l'Environnement" and "Digital Biology of RNA" in Strasbourg. This biosensor was designed for fluorescence-based detection of the fluoride released by enzymatic dehalogenation of fluorinated compounds. In MICROFLUOR, this biosensor will be coupled with a state-of-the-art high-throughput microfluidics pipeline for screening of proteins and cells displaying defluorination activity.
The goal of the MICROFLUOR project is to deliver novel and laboratory-evolved enzymes active in defluorination reactions, and thereby to contribute nature-based solutions for remediation of PFAS contamination in the environment. Building on the expertise and collaborative track record of the two partners in microbial dehalogenation metabolism and microfluidics, respectively, MICROFLUOR will develop and use multiple approaches to bioprospecting and experimental evolution in the laboratory.
Work in MCROFLUOR will be organised in three work packages, of process pipeline validation; enzyme discovery in reported defluorinating strains and laboratory enrichment cultures from sites contaminated by PFAS; and directed and random mutagenesis of selected dehalogenases in the laboratory, for development of new protein catalysts with defluorinase activity.
Multiple starting points and approaches (e.g. cells and genes, genomic and metagenomic libraries, and enrichment cultures set up from PFAS-contaminated sites to which partners have secured access) maximize the chances that MICROFLUOR will deliver new catalysts for future operational bioremediation of soils and waters contaminated by fluorinated chemicals. Work will first focus on achieving biodegradation of reference fluorinated compounds, and then extend to obtaining enzymes with defluorinase activity with selected PFAS.
In a longer-term perspective, MICROFLUOR also ambitions to provide "proof-of-concept" that the proposed RNA biosensor-based, microfluidics activity-driven screening strategy can be widely applicable in the development and implementation of tools for bioremediation of other chemical contaminants in the future.