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

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. •It was discovered that the wild-type PpDODC exhibits weak dual-function activity, capable of both decarboxylation and deamination of L-DOPA.•The dual-functional mutant PpDODC/Y79F/Y324F achieved an enzyme activity of 0.95 U/mg, exhibiting a 256.8-fold increase in activity compared to the wild-type.•The rational design of SlPAR resulted in the highly thermally stable mutant SlPAR-M4 (SlPAR/G52D/V188I/N234W/Q286P).•31.4 mM of hydroxytyrosol was produced from 32 mM L-DOPA in 5 hours, with a molar conversion rate and productivity of 98.2% and 0.97 g/L/h, respectively.