Extended Biocatalytic Halogenation Cascades Involving a Single‐Polypeptide Regeneration System for Diffusible FADH<sub>2</sub>
Nicolai Montua, Norbert Sewald
Abstract
Abstract Flavin‐dependent halogenases have attracted increasing interest for aryl halogenation at unactivated C−H positions because they are characterised by high regioselectivity, while requiring only FADH 2 , halide salts, and O 2 . Their use in combined crosslinked enzyme aggregates (combiCLEAs) together with an NADH‐dependent flavin reductase and an 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 two‐step catalytic flavin regeneration using phosphite as an inexpensive sacrificial substrate. This fusion protein proved amenable to co‐expression with various flavin‐dependent Trp‐halogenases and enables carrier‐free immobilisation as combiCLEAs from a single cultivation for protein production and the preparative synthesis of halotryptophan. The scalability of this system was demonstrated by fed‐batch fermentation in bench‐top bioreactors on a 2.5 L scale. Furthermore, the inclusion of a 6‐halotryptophan‐specific dioxygenase into the co‐expression 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 a preparative scale.