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

Area of Science:

• Biocatalysis
• Protein Engineering
• Enzyme Technology

Background:

• Vanadium-dependent haloperoxidases (VHPOs) are valuable biocatalysts for halogenation reactions.
• Poor soluble recombinant expression limits the practical application of VHPOs.
• Protein fusion offers a strategy to enhance enzyme expression and function.

Purpose of the Study:

• To improve soluble expression of vanadium chloroperoxidase (CiVCPO) using protein fusion.
• To enable simultaneous in situ hydrogen peroxide (H2O2) generation via formate oxidase (AoFOx).
• To investigate the impact of fusion design on enzyme activity and stability.

Main Methods:

• Constructing a panel of AoFOx-CiVCPO fusion proteins with varied orientations, linker lengths, and architectures.
• Assessing haloperoxidase and formate oxidase activities in crude cell lysates.
• Evaluating the catalytic performance of fusion constructs in model bromination and lactonisation reactions.
• Investigating enzyme stability and deactivation mechanisms through time-course experiments and H2O2 spiking.

Main Results:

• Fusion constructs significantly increased soluble CiVCPO expression and haloperoxidase activity (up to ~9-fold).
• AoFOx activity was reduced in fusion constructs (36%-75%) compared to individual expression.
• A representative fusion construct catalyzed formate-driven bromination and oxidative bromolactonisation in crude extracts.
• Enzyme activity declined over time, with evidence suggesting AoFOx deactivation by hypobromite.

Conclusions:

• Protein fusion is an effective strategy to enhance soluble expression and activity of VHPOs.
• Simultaneous H2O2 generation via AoFOx can be achieved, but requires optimization to maintain oxidase activity.
• Hypobromite-mediated deactivation of the AoFOx domain is a key factor limiting the robustness of these fusion biocatalysts.