Elevating Haloperoxidase Expression in Escherichia coli through Fusion with a Formate Oxidase

Vanadium-dependent haloperoxidases (VHPOs) are attractive biocatalysts for halofunctionalisation chemistry, but their routine use is frequently constrained by poor soluble recombinant expression. Here, we explore protein fusion as a construct-level strategy to simultaneously improve soluble expression of the vanadium chloroperoxidase from Curvularia inaequalis (CiVCPO) and enable in situ H₂O₂ generation via formate oxidase from Aspergillus oryzae (AoFOx). A panel of AoFOx-CiVCPO fusion designs was generated by varying enzyme orientation, linker length and linker architecture. Notably, fusion constructs displayed markedly increased haloperoxidase activity yields in crude lysates (up to ~9-fold relative to non-fused CiVCPO), whereas AoFOx activity decreased (approximately 36%-75%) compared to the individually expressed oxidase. A representative construct (CiVCPO-10 aa flexible linker-AoFOx) catalysed formate-driven bromination of activated arenes (phenol, thymol) and oxidative bromolactonisation of 4-pentenoic acid in crude extracts, giving product distributions consistent with hypobromite-mediated reactivity. Time-course experiments revealed that product formation was concentrated in the first 2 h and subsequently declined. H₂O₂-spiking partially restored activity, and sustained turnover was observed in a hypohalite-free sulfoxidation model reaction, implicating hypobromite-mediated deactivation of the AoFOx domain as a principal robustness-limiting factor.