Engineering non-P450 3-hydroxylase for synthesizes catechol-containing compounds in .

Catechols (such as l-DOPA, caffeic acid and hydroxytyrosol, etc.) are a class of phenolic derivatives with ortho-hydroxyl groups which represents various bioactivities including antioxidative, anti-inflammatory, antiviral, and anticancer properties. Non-P450-dependent 3'-hydroxylases HpaBC are the rate-limiting enzymes in catechol biosynthesis. Herein, different HpaB/HpaC combinations were first investigated. The best combinations of KpHpaB from and PaHpaC from (or SeHpaC from ) were obtained for the synthesis of l-DOPA in , resulting in 1838.56 mg/L l-DOPA (or 1822.99 mg/L l-DOPA). The highest production of caffeic acid and hydroxytyrosol were obtained with the enzyme combinations of PaHpaB from and SeHpaC from . and PlHpaB from and KpHpaC from , respectively. Next, PaHpaB and PlHpaB were further engineered to improve their catalytic efficiency by the semi-rational method. PaHpaB and PlHpaB were obtained. The titer of caffeic acid was further increased to 1281.25 mg/L without l-DOPA accumulation using the PaHpaB-UTR-SeHpaC hybrid. The production of hydroxytyrosol was further enhanced to 1681.42 mg/L using the combination of PlHpaB-UTR- KpHpaC. The production of l-DOPA, caffeic acid and hydroxytyrosol was increased using these hybrids of HpaB/HpaC by 4.6-fold, 10.1-fold, and 8.4-fold compared to EcHpaBC from , respectively. This work demonstrates that pairing of HpaB/HpaC and engineering HpaB is an powerful method for improving 3-hydroxylase activity and the production of catechol-containing compounds.