Modular cascade with engineered HpaB for efficient synthesis of hydroxytyrosol.

Hydroxytyrosol, a naturally occurring chemical with antioxidant and antiviral properties, is widely used in the nutrition, pharmaceutical, and cosmetic industries. In the present study, a modularized cascade composed of Modules 1 and 2 was designed and implemented to convert l-tyrosine to hydroxytyrosol. Module 1 was a four-enzymatic cascade for converting l-tyrosine to tyrosol. Engineering Module 1 by fine-tuning the expression of the desired enzymes resulted in a robust whole-cell catalyst, BL21 (M1-13), which converted l-tyrosine to tyrosol at high substrate loading. Module 2 involved a 4-hydroxyphenylacetate 3-monooxygenase (HpaBC)-catalyzed reaction to hydroxylate tyrosol to form hydroxytyrosol. The rational design of the HpaB subunit led to a positive variant, HpaB-Mu (T292S/R474A), which was subsequently applied to Module 2 for tyrosol hydroxylation, yielding a robust whole-cell catalyst, BL21 (M2-05). The two designed modules were merged for one-pot conversion of l-tyrosine to hydroxytyrosol by adjusting the ratio and total amount of whole-cell catalyst loading, capable of converting 40 mM of l-tyrosine to 35.8 mM of hydroxytyrosol with a high space-time yield (1.38 g/L/h). The current study proved that engineering HpaB at the substrate tunnel was a feasible way to boost its activity and proposed an effective method for synthesizing hydroxytyrosol from low-cost substrates, which has great economic potential.