We constructed a bifunctional fusion protein for FADH₂ regeneration and successfully coexpressed it with different flavin-dependent halogenases, as well as a dioxygenase that converts 6-chlorotryptophan to 4-Cl-Kynurenine. The figure is a still life of a laboratory bench, with a single oversized E. coli bacterium hovering over an Erlenmeyer flask. Cell disruption is depicted in visual analogy to cracking an egg with the lysate appearing like egg yolk. The lysate in the flask contains all necessary enzymes for the biocatalytic cascade described in the paper rendered as ribbon structures with coloring consistent with the paper. Shown as permanent marker notes on the bench surface are a key reaction scheme as well as a “ToDo-list” that checks off some important goals of the research work. More information can be found in the Research article by N. Montua, N. Sewald.

A bifunctional fusion enzyme with phosphite dehydrogenase and flavin reductase activities has been constructed and characterised. Co-expression of this single polypeptide regeneration system with tryptophan halogenases and carrier-free immobilisation in combiCLEAs facilitates preparative-scale synthesis of halotryptophan from a single cultivation. Extension of the catalytic cascade with a specific dioxygenase enables single cultivation one-pot synthesis of l-4-Cl-kynurenine on a preparative scale.

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 FADH₂, halide salts, and O₂. 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.