# Enhancing Caffeic Acid Production in Escherichia coli Through Heterologous Enzyme Combinations and Semi-Rational Design

## TL;DR

This study improves the production of caffeic acid in E. coli by combining different enzymes and using a semi-rational design approach.

## Contribution

A semi-rational design of HpaB and optimized fermentation significantly increased caffeic acid yield in E. coli.

## Key findings

- The highest caffeic acid yield in shake-flask culture was 75.88 mg/L using HpaB from E. coli.
- The mutant S210G/Y117A increased caffeic acid titer by 1.68-fold through iterative saturation mutagenesis.
- Caffeic acid titer reached 2335.48 mg/L in a 5 L fermenter after process optimization.

## Abstract

Background/Objectives: Caffeic acid is a hydroxycinnamic acid that has a wide range of applications in the medical field. The synthesis of caffeic acid using microbial fermentation technology is an environmentally friendly method. Methods: By engaging various enzymes, specifically 4-hydroxyphenylacetate 3-monooxygenase (HpaB), sourced from diverse bacterial strains, we successfully engineered a functional version of this enzyme within Escherichia coli, enabling the production of caffeic acid. In addition to the two common tyrosine ammonia lyases (TAL) and HpaC, different combinations of HpaB demonstrated varying abilities in converting the substrate L-tyrosine into the desired product, caffeic acid. Results: Under shake-flask culture conditions, the highest yield of caffeic acid was achieved with an enzyme mixture containing HpaB from Escherichia coli, reaching 75.88 mg/L. Enhancing the activity of the rate-limiting enzyme through engineering could potentially increase caffeic acid titer. This study aims to conduct a semi-rational design of HpaB through structure-based approaches to screen for mutants that can enhance the production of caffeic acid. Initially, the predicted three-dimensional structure of HpaB was generated using AlphaFold2, and subsequent analysis was conducted to pinpoint the critical mutation sites within the substrate-binding pocket. Five key amino acid residues (R113, Y117, H155, S210 and Y461) located in the vicinity of the flavin adenine dinucleotide binding domain in HpaB from Escherichia coli could be instrumental in modulating enzyme activity. Subsequently, the mutant S210G/Y117A was obtained by iterative saturation mutagenesis, which increased the titer of caffeic acid by 1.68-fold. The caffeic acid titer was further improved to 2335.48 mg/L in a 5 L fermenter. The findings show that the yield of caffeic acid was significantly enhanced through the integration of semi-rational design and fermentation process optimization.