Function-driven, microfluidics-based discovery of novel microbial dehalogenases – dehalofluidX
The microbial biosphere represents the largest reservoir of catalysts for future industrial application. Yet it remains largely unexplored, with also the majority of genes identified in metagenomes still of unknown function. Using dehalogenation as a key major function with high-potential, versatile biocatalytic activity, dehalofluidX aims to address the major bottleneck faced by metagenomics today: the increasing recognition that genuinely novel biocatalysts with desired properties and unrelated sequences to already known enzymes, will only become accessible through novel approaches and pipelines.
DehalofluidX is a tightly focussed 36-month innovative collaborative research project associating 3 partners from 3 research units in France, with complementary areas of expertise in microbial dehalogenation, microfluidics and omics technologies, and in particular microbial proteomics. The innovative concept of the high-throughput microfluidics-driven workflow is to use fluorescence quenching by halide ions in water-in-oil droplets to detect dehalogenation activity. This readout will be applicable to all halogenated compounds for which discovery of a novel dehalogenase catalyst is desirable. DehalofluidX does not require selective growth of dehalogenating organisms, and will target a much larger diversity of dehalogenating microbes and biocatalysts of the microbial biosphere than that accessed so far.
DehalofluidX is organised in 3 successive well-defined, interconnected experimental tasks: (i) Droplet-based activity screening (validation of the microfluidics screening pipeline; isolating dehalogenating bacteria from environmental samples); (ii) Processing of cells with dehalogenase activity (processing of cultivable, poorly cultivable or non-cultivable cells with dehalogenase activity; phylogenetic and proteomic characterization, PCR screening for known dehalogenases); and (iii) Characterisation of novel catalysts (identification of genomic regions encoding dehalogenase activity; enzymatic characterisation of newly identified dehalogenases).
Through its goal of discovering novel enzymes from the environmental microbiome for subsequent application and/or optimisation by biotech companies, dehalofluidX perfectly fits axis 3 of ANR Défi 3, as it may yield both new basic knowledge and technological know-how indispensable for the discovery of new biocatalysts. DehalofluidX also fits well with the National Strategy of Research ("green factory", Orientation 12 of the SNR), and has a direct link with the “Investments for the Future” program through partner DBR, member of LabEx NetRNA.
The project PI has access to both pristine and contaminated environments as a source of new dehalogenating microbes and enzymes. Strong consortium expertise in all aspects of the proposed work strongly reduces potential challenges to success. Key requirements of droplet-based microfluidic high-throughput screening (droplet stability, maintenance of phenotype-genotype linkage, and signal sensitivity) have already been validated in preliminary experiments of halide-dependent fluorescence quenching in the presence of halogenated substrates. A thorough risk assessment was also performed on all stages of the dehalofluidX workflow, and potential issues and experimental fall-back solutions were identified and described.
DehalofluidX will have major scientific impact, through delivery of novel biocatalysts for sustainable production of chemicals and bioremediation, and proof-of-concept of a novel activity-driven microfluidics high-throughput screening pipeline, expected to become generally applicable to other types of catalysts and R&D topics in the future. Technological impact will also be significant due to the novelty of the proposed activity-based approach for enzyme discovery. From an economic viewpoint, newly identified catalysts will be patented and licensed, as handled by SATT Conectus, the technology transfer instrument at Université de Strasbourg.
Monsieur Stéphane Vuilleumier (Génétique moléculaire, génomique et microbiologie)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
GMGM Génétique moléculaire, génomique et microbiologie
Architecture et Réactivité de l'ARN
CEA/DRF/JOLIOT/SPI/Li2D Laboratoire Innovations technologiques pour la Détection et le Diagnostic
Help of the ANR 443,806 euros
Beginning and duration of the scientific project: November 2017 - 36 Months