Project Title: Protein engineering (rational and evolutive) and biocatalysis.

Supervisor: Prof. Ulrich Schaneberg, RWTH Aachen, Germany

Objectives: (1) To understand on molecular level how heme-dependent monooxygenases hydroxylate N-, O-, and S-heterocyclic compounds; (2) To discover new functional recombinant eukaryotic heme-dependent monooxygenases; (3) To improve the catalytic performance of heme-dependent monooxygenases for heterocyclic compounds and to explore the synthetic potential of novel heme-dependent monooxygenases; (4) To produce selected heme-dependent monooxygenases and variants in 10 L scale and to kinetically characterise them for valuable drug metabolites; (5) To explore standardized storage conditions and developing in 96-well plates kits of heme-dependent monooxygenases (incl. variants) for rapid screening of conversion of N-, O-, and S-heterocyclic compounds.

Methodology: (1) Investigation of three heme-dependent monooxygenases for the conversion of heterocyclic compounds. Through modelling and random mutagenesis positions will be identified and mutated to elucidate on the molecular level structure-function relationships (ESR4, RWTH); (2) Novel heterocycle hydroxylating heme-dependent monooxygenases. Novel enzymes will be identified by in silico and functional screening of plant and fungal transcriptomes (ESR5, TUG); (3) Random mutagenesis libraries for identification of beneficial positions and production of heme-dependent monoxygeneases for formulation as lyophilisates in 96 well plates to develop novel metabolite test kits for the hydroxylation of hetercyclic compounds which are key intermediates for active pharmaceutical intermediates (API) synthesis (ESR6, SSB).

Expected Results: (1) Identification of amino acid positions that govern selectivity of three heme-dependent monooxygenases; (2) Three simultaneous saturation mutagenesis libraries per monooxygenase.