Extended Biocatalytic Halogenation Cascades Involving a Single‐Polypeptide Regeneration System for Diffusible FADH₂

Abstract

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.

Enzymatic Peptide and Protein Bromination: The BromoTrp Tag

“…Because we and others observed only very minor differences between Thal‐catalyzed chlorination and bromination of l ‐Trp, the observed bromination preference mostly likely results from the binding geometry of this peptide with Thal‐AAC [30,31] …”

“…An intriguing possibility emerged: combining our recently published single‐plasmid coexpression system of halogenase and bifunctional PTDH‐BorF fusion enzyme with the generated Thal‐variant, while also co‐expressing C‐ terminally YNIW‐tagged model proteins, could enable site‐selective protein bromination with just a single purification step from clarified lysates [30] . Given the pronounced bromide preference of the triple mutant, we expected the protein chlorination background to be suppressed by supplementing an excess of bromide to the cultivation medium prior to induction of the T7‐promotor.…”

Enzymatische Peptid‐ und Proteinbromierung: Der BromoTrp Tag

“…Da wir und andere nur sehr geringe Unterschiede zwischen Thal-wt-katalysierter Chlorierung und Bromierung von L-Trp beobachtet haben, resultiert diese Bromierungspräferenz höchst wahrscheinlich von der Bindegeometrie dieses Peptids mit Thal-AAC. [30,31]…”

“…So könnte die selektive Bromierung von Modellproteinen in nur einem Kultivierungs-und Aufreinigungsschritt erfolgen, wenn diese C-terminal mit YNIW getaggt ebenfalls koexprimiert werden. [30] Aufgrund der ausgeprägten Bromid-Präferenz der Dreifachmutante waren wir zuversichtlich, dass der Chlorierungshintergrund durch Zusatz eines Bromid-Überschusses vor Induktion des T7-Promotors unterdrückt werden kann. Als ein erster Test wurde das Modellpeptid GT(GGGGS) 3 YNIW durch FPPS an 2-Chlortritylchlorid-Harz synthetisiert.…”

Tri‐enzyme fusion of tryptophan halogenase achieves a concise strategy for coenzyme self‐sufficiency and the continuous halogenation of L‐tryptophan

“…However, this bienzyme fusion still requires the addition of extra enzymes (GDH or ADH) to reduce the depletion of the costly coenzyme NADH, which is cost‐inefficient and cumbersome for the engineering application of FDHs. Recently, Sewald's group [ 17 ] reported a bifunctional fusion enzyme (PTDH‐BorF) using phosphite as the terminal substrate for flavin regeneration and used this fusion enzyme for co‐expression with FDHs in E. coli and application to L ‐4‐Cl‐kynurenine biosynthesis. We considered whether a new strategy could be adopted to introduce GDH based on FDH/FR‐dual enzyme fusion to form a NAD + ‐NADH‐FAD‐FADH 2 bicoenzyme cycle system with reducing power provided by the cheaper compound glucose and to achieve self‐sufficiency of coenzyme during the bicoenzyme cycle process (Figure 1C).…”