Reprogramming metalloenzyme reactivity for abiological catalysis – Abiozyme

As part of the transition to a sustainable bioeconomy, it is expected that the chemical industry will increasingly rely on biotechnologies such as biocatalysis and synthetic biology for the production of compounds such as pharmaceuticals, agrochemicals, flavors and fragrances. However, to reach their full potential, it is necessary to design new enzymes catalyzing abiological reactions that chemists can do. Catalysis with transition metals has considerably contributed to the development of new reactions, in particular by means of reactive intermediates of metal-carbon or metal-heteroatom type. For instance, cyclopropyl or aziridyl functional groups are synthetically accessible from metallocarbene or metallonitrene species which do not find their counterparts in biological processes. Engineering this type of reactivity in artificial metalloenzymes will allow the development of new biocatalysts that can be easily integrated in innovative biocatalytic cascades or in artificial metabolic pathways in vivo (metabolic engineering) for the bioproduction of complex synthetic molecules.

Within this project, we will explore the high potential of copper-dependent lytic polysaccharide monooxygenases (LPMOs) as unique scaffold for the engineering of abiological reactivity. Indeed, LPMOs harbor a solvent exposed active site with a rare histidine brace motif which ensures a high stability and reactivity of the metal center. This motif corresponds to an N-terminal histidine residue coordinating a metal ion by both its side chain and its N-terminal free amine. More specifically, we aim at demonstrating that LPMOs can be reprogrammed into innovative biocatalysts performing metal-catalyzed abiological transformations. Our approach will consist in: i) diversifying the structural topology of their unique active site (by site-directed mutagenesis, including non-canonical amino acids) and explore their metal promiscuity; ii) characterizing the variants harboring new-metal coordinating motifs; iii) screening these new metalloenzymes for alternative abiological reactivity (with a focus on carbene and nitrene transfer reactions); iv) improving the catalytic efficiency (total turnover number) and scope of the reactions (substrate scope, steric and electronic effects, stereoselectivity) of these new biocatalysts by directed evolution, and v) using these biocatalysts for the synthesis of molecules of interest.