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

Area of Science:

• Biocatalysis and enzyme engineering
• Metabolic engineering
• Green chemistry

Background:

• Hydroxytyrosol, a beneficial human health compound, has complex biosynthesis pathways.
• Existing methods for hydroxytyrosol synthesis require multiple enzymes and face challenges with enzyme stability.
• The conversion of L-DOPA to 3,4-DHPAA typically needs both decarboxylase and oxidative deaminase activities.

Purpose of the Study:

• To develop a simplified and efficient biocatalytic cascade for hydroxytyrosol synthesis from L-DOPA.
• To engineer a dual-functional enzyme with both decarboxylase and oxidative deaminase activity.
• To enhance the thermal stability of phenylacetaldehyde reductase for improved process viability.

Main Methods:

• Engineered L-DOPA decarboxylase from Pseudomonas putida (PpDODC) for dual-function activity via a reshaping strategy.
• Utilized rational design to create a thermostable mutant of phenylacetaldehyde reductase from Solanum lycopersicum (SlPAR-M4).
• Established a two-enzyme cascade system using the engineered PpDODC and SlPAR-M4 mutants for L-DOPA conversion.

Main Results:

• Developed a dual-functional PpDODC mutant (PpDODC/Y79F/Y324F) with a 256.8-fold increase in activity.
• Created a stabilized SlPAR mutant (SlPAR-M4) that maintained activity after 12 hours at 40°C.
• Achieved a 98.2% molar conversion rate of L-DOPA to hydroxytyrosol in a 5-hour reaction, yielding 31.4 mM.

Conclusions:

• A novel, simplified biocatalytic cascade for hydroxytyrosol synthesis was successfully established.
• Engineered enzymes offer a promising solution for efficient and stable hydroxytyrosol production.
• The developed mutants have potential for broad applications in biotransformation of similar compounds.