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

Abstract

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 ( Ci VCPO) and enable in situ H 2 O 2 generation via formate oxidase from Aspergillus oryzae ( Ao FOx). A panel of Ao FOx– Ci VCPO 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 Ci VCPO), whereas Ao FOx activity decreased (approximately 36%–75%) compared to the individually expressed oxidase. A representative construct ( Ci VCPO–10 aa flexible linker– Ao FOx) 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. H 2 O 2 - spiking partially restored activity, and sustained turnover was observed in a hypohalite - free sulfoxidation model reaction, implicating hypobromite - mediated deactivation of the Ao FOx domain as a principal robustness - limiting factor