Construction and Application of a Directed Evolution-Based Multi-Enzyme Cascade Catalytic System for Semi-Synthesis of Chiral β-Hydroxy Acids

Yuxiang Li

doi:10.54254/2755-2721/2025.24774

Applied and Computational EngineeringOpen access

Construction and Application of a Directed Evolution-Based Multi-Enzyme Cascade Catalytic System for Semi-Synthesis of Chiral β-Hydroxy Acids

Research Article

Open Access

Construction and Application of a Directed Evolution-Based Multi-Enzyme Cascade Catalytic System for Semi-Synthesis of Chiral β-Hydroxy Acids

Yuxiang Li ^1*

¹ National University of Singapore

^*Corresponding author: rara481846778@gmail.com

Published on 4 July 2025

ACE Vol.171

ISSN (Print): 2755-273X

ISSN (Online): 2755-2721

ISBN (Print): 978-1-80590-219-5

ISBN (Online): 978-1-80590-220-1

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Abstract

In this study, a directed evolution-driven multi-enzyme cascade system was constructed to achieve efficient semi-synthesis from β-keto acids to chiral (R)-β-hydroxy acids. Through three rounds of directed enzyme modification: ketone reductase (KRED), epoxide hydrolase (EH), and carboxylic acid ligase (CAL) were subjected to random mutagenesis, high-throughput screening, and recombination, respectively, to obtain the mutants KRED-E23, EH-M12, and CAL-F45. Under the same reaction conditions, the catalytic efficiency was increased by 3 to 5 times. In the one-pot reaction at pH 7.5 and 25℃ of this optimized system, the conversion rate of 2-oxo-4-methylvaleric acid to the target product reached 95.2 ± 1.1% within 4 hours, and the enantiomer excess value was 99.4 ± 0.2%. When scaled up to 1 liter, it maintained a high conversion rate (93.7%) and chiral purity (98.9%), obtaining 9.37 grams of product, with a spatio-temporal yield of 2.34 grams · liter ⁻¹ · hour ⁻¹. This achievement proves that the combination of directional evolution and cascade design can efficiently prepare high-value chiral blocks under mild conditions. The modularization and robustness of the system constitute a new platform for the industrial production of complex three-dimensional molecules.

Keywords:

Directed evolution, Multi-enzyme cascade, Ketoreductase, Chiral β-hydroxy acid, Biocatalysis

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References

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References

[2]. Bornscheuer, U. T. (2020). Power of biocatalysis for organic synthesis. ACS Central Science, 6(12), 2046–2057. pubs.acs.org

[3]. Savile, C. K., Faul, M. M., & Turner, N. J. (2021). Biocatalysis. Nature Reviews Methods Primers, 1, Article 44. nature.com

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Cite this article

Li,Y. (2025). Construction and Application of a Directed Evolution-Based Multi-Enzyme Cascade Catalytic System for Semi-Synthesis of Chiral β-Hydroxy Acids. Applied and Computational Engineering,171,53-57.

Data availability

The datasets used and/or analyzed during the current study will be available from the authors upon reasonable request.

About volume

Volume title: Proceedings of the 3rd International Conference on Functional Materials and Civil Engineering

ISBN: 978-1-80590-219-5(Print) / 978-1-80590-220-1(Online)

Editor: Anil Fernando

Conference website: https://2025.conffmce.org/

Conference date: 24 October 2025

Series: Applied and Computational Engineering

Volume number: Vol.171

ISSN: 2755-2721(Print) / 2755-273X(Online)

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