Unspecific peroxygenases (UPOs) recently arose as dream biocatalysts for oxyfunctionalisation reactions. They enable the direct transformation of oxygen from hydrogen peroxide to a broad substrate scope capable of reaching impressive total turnover numbers (TONs) beyond 200,000. There is a vast diversity of UPO sequences available within the fungal kingdom with more than 4,300 unique annotated sequences. However, the widespread application and subsequent protein engineering of UPOs are hampered by their challenging heterologous production in fast-growing host organisms. To address these challenges, Alcalde and coworkers as well as our group, developed yeast-based heterologous production setups. These systems gave access to novel UPOs as well as the opportunity for protein engineering. Amongst the recently discovered enzymes, a UPO derived from the thermophilic fungus Myceliophthora thermophila (MthUPO) achieved the thus far highest UPO shake flask production yields. In contrast to the predominantly studied AaeUPO derived from Agrocybe aegerita, the MthUPO revealed the oxidation of naphthalene to 1,4-naphthoquinone as one of two main products. These 1,4-naphthoquinone core structures are intensely studied moieties with application in the industry as an intermediate for the anthraquinone synthesis and are widely distributed in the plant and animal kingdom. In nature, naphthoquinones occur in the human body as the blood clotting precursor vitamin K3, in plants as carcinogenesis plumbagin analogues or in insects as chemical defence. We hypothesised that MthUPO's ability to perform efficient aromatic as well as benzylic oxyfunctionalisations could provide a starting point to engineer chemoselective UPO variants for either specificity. Using the colourimetric assay 5-nitro-1,3-benzodioxole (NBD), around 5,300 transformants were screened to obtain improved variants exhibiting complementary regio-and chemoselectivities as well as improved enantioselectivities. Computational modelling described the molecular basis behind the observed catalytic and selectivity improvements. We commenced our studies by generating a homology model for the MthUPO structure based on the solved crystal structure of the UPO from Marasmius rotula (MroUPO, PDB: 5FUJ, 29% identity and 41 % similarity, see SI for details). The NBD substrate was docked into a representative structure of the most populated cluster, which aided in identifying substrate-binding residues (Fig. ). Nine positions (L56, F59, L60, L86, F154, T155, S159, A161, L206, see Fig. ) were randomised by the Golden Mutagenesis technique. The mutant library was transformed in Saccharomyces cerevisiae producing the corresponding variants. We screened the library using the colourimetric NBD assay in combination with the recently established split-GFP analysis in yeast. The combination of these two assays enabled the distinction between substrate conversion and secretion of the respective enzyme. Variations at position L56 substantially influenced the production of the enzyme. Only 52 % of the transformants displayed a split-GFP signal (Tab. S3). Site saturation at position L60 yielded improved variants with superior TONs for NBD conversion. The variants L60M (1.2-fold relative to the wildtype), L60Q (1.3-fold) and L60F (2.7-fold) showed the most noticeable improvements (Tab. 1 and S4). Variant L60F also exhibited a 1.4-fold improved catalytic efficiency (kcat/Km, Tab. 2). The variant library of position F154, which is located at the entrance channel, turned out to be a pivotal position for the NBD conversion. Even though 81 % of the variants were secreted according to the split-GFP signal, only the rediscovered wildtype enzymes displayed activity (Fig. ).