Fluorinases, the only enzymes known to catalyze the transfer of fluorine to an organic molecule, are essential catalysts for the biological synthesis of valuable organofluorines. However, the few fluorinases identified so far have low turnover rates that hamper biotechnological applications. Here, we isolated and characterized putative fluorinases retrieved from systematic in silico mining and identified a nonconventional archaeal enzyme from Methanosaeta sp. that mediates the fastest SN2 fluorination rate reported to date. Furthermore, we demonstrate enhanced production of fluoronucleotides in vivo in a bacterial host engineered with this archaeal fluorinase, paving the way toward synthetic metabolism for efficient biohalogenation.

International Clinical Research Centre St Anne's University Hospital 656 91 Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University 601 77 Brno Czech Republic

The Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark 2800 Kongens Lyngby Denmark

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Machine Learning-Guided Protein Engineering