Modular cascade with engineered HpaB for efficient synthesis of hydroxytyrosol.

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• Hydroxytyrosol is a valuable natural compound with antioxidant and antiviral properties.
• Existing methods for hydroxytyrosol synthesis can be costly or inefficient.
• There is a need for sustainable and scalable production methods.

Purpose of the Study:

• To design and implement a modularized cascade for the bioconversion of l-tyrosine to hydroxytyrosol.
• To engineer robust whole-cell catalysts for efficient intermediate and final product formation.
• To establish a one-pot process for high-yield hydroxytyrosol synthesis.

Main Methods:

• Constructed a four-enzymatic cascade (Module 1) for l-tyrosine to tyrosol conversion.
• Engineered Module 1 into a whole-cell catalyst (BL21 (M1-13)) with optimized enzyme expression.
• Developed Module 2 using a rationally designed 4-hydroxyphenylacetate 3-monooxygenase (HpaBC) variant (HpaB-Mu) for tyrosol hydroxylation.
• Integrated Modules 1 and 2 into a one-pot process using optimized whole-cell catalyst loading.

Main Results:

• Achieved high substrate loading conversion in Module 1 using BL21 (M1-13).
• Engineered HpaB subunit (HpaB-Mu) significantly improved tyrosol hydroxylation in Module 2, creating BL21 (M2-05).
• The one-pot process converted 40 mM l-tyrosine to 35.8 mM hydroxytyrosol with a space-time yield of 1.38 g/L/h.

Conclusions:

• Engineering the HpaB substrate tunnel is an effective strategy to enhance enzyme activity.
• The developed modular cascade and one-pot strategy offer a feasible and economically promising method for hydroxytyrosol synthesis from low-cost substrates.
• This approach has significant potential for industrial application in the nutrition, pharmaceutical, and cosmetic sectors.