PUB - Publikationen an der Universität Bielefeld

Flavin dependent halogenases have attracted increasing interest for aryl halogenation at unactivated C‐H positions because they are characterized by high regioselectivity, while requiring only FADH2, halide salts, and O2. Their use in combiCLEAs together with an NADH‐dependent flavin reductase and a NADH‐regeneration system for the preparative halogenation of tryptophan and indole derivatives has been previously described. However, multiple cultivations and protein purification steps are necessary for their production. We present a bifunctional regeneration enzyme for the two‐step catalytic flavin regeneration using phosphite as an inexpensive sacrificial substrate. This fusion protein proved amenable to coexpression with various flavin dependent Trp‐halogenases and enabled the carrier‐free immobilization as combined cross‐linked enzyme aggregates (combiCLEAs) for the preparative synthesis of halotryptophans. Scalability of this system was demonstrated by fed‐batch fermentation in benchtop bioreactors at 2.5 L scale. Furthermore, the inclusion of a 6‐halotryptophan specific dioxygenase into the coexpression strain further converts the halogenation product to the kynurenine derivative. This reaction cascade enables the one‐pot synthesis of l‐4‐Cl‐kynurenine and its brominated analogue on preparative scale from a single cultivation for protein production.