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Fungal unspecific peroxygenases (UPOs) have gained substantial attention for their versatile oxyfunctionalization chemistry paired with impressive catalytic capabilities. A major drawback, however, remains their sensitivity towards their co‐substrate hydrogen peroxide, necessitating the use of smart in situ hydrogen peroxide generation methods to enable efficient catalysis setups. Herein, we introduce flavin‐containing protein photosensitizers as a new general tool for light‐controlled in situ hydrogen peroxide production. By genetically fusing flavin binding fluorescent proteins and UPOs, we have created two virtually self‐sufficient photo‐enzymes (PhotUPO). Subsequent testing of a versatile substrate panel with the two divergent PhotUPOs revealed two stereoselective conversions. The catalytic performance of the fusion protein was optimized through enzyme and substrate loading variation, enabling up to 24300 turnover numbers (TONs) for the sulfoxidation of methyl phenyl sulfide. The PhotUPO concept was upscaled to a 100 mg substrate preparative scale, enabling the extraction of enantiomerically pure alcohol products. [ABSTRACT FROM AUTHOR]

Catalyst discovery and development requires the screening of large reaction sets necessitating analytic methods with the potential for high‐throughput screening. These techniques often suffer from substrate dependency or the requirement of expert knowledge. Chromatographic techniques (GC/LC) can overcome these limitations but are generally hampered by long analysis time or the need for special equipment. The herein developed multiple injections in a single experimental run (MISER) GC‐MS technique allows a substrate independent 96‐well microtiter plate analysis within 60 min. This method can be applied to any laboratory equipped with a standard GC‐MS. With this concept novel, unspecific peroxygenase (UPO) chimeras, could be identified, consisting of subdomains from three different fungal UPO genes. The GC‐technique was additionally applied to evaluate an YfeX library in an E. coli whole‐cell system for the carbene‐transfer reaction on indole, which revealed the thus far unknown axial heme ligand tryptophan. [ABSTRACT FROM AUTHOR]