An engineered dual-functional L-DOPA decarboxylase enables a minimized hydroxytyrosol cascade

Shiming Tang; Zhilin Ouyang; Ying Huo; Daocheng Liao; Zhiteng Zhang; Ying Lin; Suiping Zheng

doi:10.1016/j.ijbiomac.2024.138176

An engineered dual-functional L-DOPA decarboxylase enables a minimized hydroxytyrosol cascade

Int J Biol Macromol. 2024 Nov 28:138176. doi: 10.1016/j.ijbiomac.2024.138176. Online ahead of print.

Authors

Shiming Tang¹, Zhilin Ouyang¹, Ying Huo¹, Daocheng Liao¹, Zhiteng Zhang¹, Ying Lin¹, Suiping Zheng²

Affiliations

¹ Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, PR China.
² Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, PR China. Electronic address: spzheng@scut.edu.cn.

PMID: 39615727
DOI: 10.1016/j.ijbiomac.2024.138176

Abstract

Hydroxytyrosol has been proven beneficial to human health. However, the process involving the conversion of L-DOPA to 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) in hydroxytyrosol biosynthesis typically required the simultaneous use of decarboxylase and oxidative deaminase. In addition, phenylacetaldehyde reductase from Solanum lycopersicum (SlPAR) in hydroxytyrosol biosynthesis exhibits poor thermal stability. In this study, we unexpectedly discovered that L-DOPA decarboxylase from Pseudomonas putida (PpDODC) exhibits weak dual-functional activity for both decarboxylation and oxidative deamination of L-DOPA. Through a dual-function reshaping strategy, the best dual-functional mutant PpDODC/Y79F/Y324F achieved an enzyme activity of 0.95 U/mg and exhibited a 256.8-fold increase in activity compared to the wild-type. Through rational design of SlPAR, the optimal mutant SlPAR/G52D/V188I/N234W/Q286P (SlPAR-M4) maintained stability after 12 h' treatment at 40 °C. Based on these mutants, we established a simplified cascade to synthesize hydroxytyrosol from L-DOPA, achieving a hydroxytyrosol yield of 31.4 mM from 32 mM L-DOPA in a 5-hour reaction. This process achieved the highest molar conversion rate (98.2 %) currently reported for the synthesis of hydroxytyrosol from L-DOPA. This study provides a novel solution for hydroxytyrosol synthesis from a new perspective, and the mutants hold promise for widespread application in biotransformation studies of hydroxytyrosol and other structurally-similar compounds.

Keywords: Dual-functional activity; Hydroxytyrosol; L-DOPA decarboxylase.