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<p>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 <i>Escherichia coli</i>. In this work, we co-expressed F5H derived from <i>Arabidopsis thaliana</i> (<i>At</i>F5H) and NADPH-dependent cytochrome P450 reductase (CPR) in <i>E. coli</i>, and successfully synthesized <i>ortho</i>-hydroxylated ferulic acid. Simultaneously, by modifying the N-terminal regions of membrane proteins, we increased the concentration of product 5-HFA to 63.6&#xa0;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 <i>E. coli</i>.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月15日

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  • Ping Sun,
  • Yuan Tian,
  • Luyi Wang,
  • Pengcheng Chen,
  • Dan Wu,
  • Pu Zheng

摘要

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.

Graphical Abstract