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A high-throughput visual screening method for p-hydroxybenzoate hydroxylase to increase phenolic compounds

Zhenya Chen1, Tongtong Chen1, Shengzhu Yu1

  • 1Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Haidian District, Beijing, 100081, China.

Biotechnology for Biofuels and Bioproducts
|May 3, 2022
PubMed
Summary

Researchers developed a novel screening method to create a highly active p-hydroxybenzoate hydroxylase (PobA) mutant. This engineered enzyme enables efficient microbial production of gallic acid (GA) and pyrogallol from simple carbon sources.

Keywords:
BiosynthesisGallic acidHydroxylationPyrogallolScreeningp-Hydroxybenzoate hydroxylase

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Area of Science:

  • Biotechnology
  • Enzyme Engineering
  • Metabolic Engineering

Background:

  • Gallic acid (GA) and pyrogallol are valuable phenolic compounds with diverse biological activities.
  • Microbial biosynthesis offers an eco-friendly approach for producing GA and pyrogallol.
  • Limited hydroxylation activity of p-hydroxybenzoate hydroxylase (PobA) hinders efficient GA and pyrogallol biosynthesis.

Purpose of the Study:

  • To engineer a PobA enzyme with enhanced activity towards 3,4-dihydroxybenzoic acid (3,4-DHBA).
  • To develop an efficient screening method for identifying high-activity PobA mutants.
  • To establish microbial production of GA and pyrogallol using the engineered enzyme.

Main Methods:

  • Random mutagenesis of PobA and a novel visual screening method were employed to identify improved enzyme variants.
  • In vitro enzyme assays and molecular docking simulations were used to characterize the mutant PobA's activity and mechanism.
  • Engineered PobA was incorporated into de novo biosynthetic pathways in E. coli for GA and pyrogallol production.

Main Results:

  • A PobA mutant (Y385F/T294A/V349A) exhibited a 4.92-fold increase in catalytic efficiency (kcat/Km) towards 3,4-DHBA.
  • Expression of the mutant PobA in E. coli yielded 840 mg/L of GA from 3,4-DHBA.
  • The engineered pathway achieved 301 mg/L GA and 129 mg/L pyrogallol production from simple carbon sources.

Conclusions:

  • An efficient screening strategy and a high-activity PobA mutant were successfully developed.
  • De novo biosynthesis pathways utilizing the engineered PobA enabled significant GA and pyrogallol production.
  • The developed PobA mutant shows potential for producing GA/pyrogallol derivatives and the screening method can be applied to other enzymes.