Bioactive expression of eukaryotic cytochrome P450 ferulate-5-hydroxylase in Escherichia coli for sustainable synthesis of antioxidant 5-hydroxyferulic acid

Abstract

The eukaryotic cytochrome P450 ferulate-5-hydroxylase (F5H), a membrane-bound protein, plays a critical role in lignin synthesis involved in the biosynthesis of 5-Hydroxyferulic acid (5-HFA) from ferulic acid, with 5-HFA offering enhanced antioxidant properties. However, there is challenging to engineer recombinants for 5-HFA synthesis by expressing F5H in Escherichia coli. In this work, we co-expressed F5H derived from Arabidopsis thaliana (AtF5H) and NADPH-dependent cytochrome P450 reductase (CPR) in E. coli, and successfully synthesized ortho-hydroxylated ferulic acid. Simultaneously, by modifying the N-terminal regions of membrane proteins, we increased the concentration of product 5-HFA to 63.6 mg/L. Ferulic acid, utilized as the substrate, was extracted from discarded agricultural by-products, demonstrating a sustainable approach to valorizing agricultural waste. This work also advances the application of plant-derived membrane-bound proteins for the production of plant secondary metabolites in E. coli.

Keywords

Ferulate-5-Hydroxylase / Biotransformation / 5-Hydroxyferulic acid / Ferulic acid / N-Terminal modification

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