The direct and selective hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a major challenge in organic chemistry. The monooxygenase P450 BM3 from Bacillus megaterium is known to catalyze the CH-activating functionalization of benzene with atmospheric dioxygen. The main objective of this thesis project has been the engineering of P450 BM3 toward the aromatic hydroxylation of pseudocumene to produce trimethylhydroquinone (TMHQ). The aromatic TMHQ is a key building block in vitamin E synthesis. The capability of P450 BM3 to catalyze the direct aromatic hydroxylation of pseudocumene to TMHQ was validated and a first biocatalytic route to TMHQ in a one-pot reaction was established. P450 BM3 variants were investigated for their side product formation and selectivity. TMHQ concentrations up to 0.19 g L- 1 were obtained with P450 BM3 M3 (R47S, Y51W, A330F, I401M). This P450 BM3 variant was subsequently used as a starting variant in protein engineering campaigns. Identification of P450 BM3 variants with improved TMHQ formation in large mutant libraries required an effective screening strategy. Therefore, a hydroquinone (HQ) specific screening assay based on the interaction of 4-nitrophenylacetonitrile (NpCN) with HQ under alkaline conditions was established. In the 96-well plate format, a low detection limit (5 μM), broad linear detection range (5 to 250 μM) and low standard deviation (10-14%) was obtained. The NpCN assay was successfully applied for screening of random mutagenesis and semi-rational designed libraries. The P450 BM3 variant with the highest TMHQ formation obtained in protein engineering campaigns was P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P). P450 BM3 AW2 had a 70-fold improved TMHQ formation when compared to the wildtype (WT) and a 1.8-fold improved TMHQ formation compared to P450 BM3 M3. Until now, the industrial application of P450s is restricted to whole-cell systems which bring along several benefits e.g., cofactor regeneration and process stability. Whole-cell catalysts co-expressing P450 BM3 variants and alcohol dehydrogenases (ADHs) in an E. coli host were evaluated to produce cyclooctanone. Furthermore, solvent-resistant Pseudomonas putida strains were explored as hosts for whole-cell conversions as well as the co-expression of FhuAΔ1-160 for improved substrate uptake was studied
