BioIson · Unlocking Fe(II)/αKG-dioxygenase enzymes as sustainable biocatalysts for the synthesis of isonitriles

Isonitriles are valuable organic molecules endowed with unique reactivity features, which find wide applications in a variety of chemical fields, from material sciences to chemical biology, organic, and medicinal chemistry. Current methods to prepare isonitriles suffer from major drawbacks in terms of safety and sustainability, as they rely on the use of large amounts of highly toxic chemicals (e.g., COCl2, POCl3, TMSCN), which make their synthesis hazardous and wasteful, especially for industrial-scale production. Based on the established ability of biocatalytic strategies to enable milder, greener, and more selective chemical processes, the aim of this project is to investigate, characterise, and engineer the protein ScoE from Streptomyces coeruleorubidus and related isonitrile-forming enzymes from the Fe(II)/KG-dioxygenase superfamily with the final goal to turn them into a new class of biocatalysts for the sustainable production of isonitrile compounds. The project will first characterise wild-type ScoE in order to optimise the biotransformation parameters and to evaluate the in vitro catalytic performances of the enzyme and its natural substrate scope (specificity and selectivity). In parallel, representative isonitrile-forming Fe(II)/KG-dioxygenases with potential for different activity and selectivity will be selected from assorted environmental niches and assessed for their substrate flexibility and their ability to biocatalyse the synthesis of isonitrile derivatives in vitro. Such investigations will provide experimental data to drive the identification of best candidates for biocatalyst development. Mutagenesis studies driven by in-silico design will be performed to generate improved Fe(II)/KG-dioxygenase variants (with broader substrate scope) to be exploited as biocatalysts. The hit enzymes will be produced on larger scale and biocatalytic methods for the preparative synthesis of isonitriles will be developed.