Abstract
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 Klebsiella pneumoniae and PaHpaC from Pseudomonas aeruginosa (or SeHpaC from Salmonella enterica) were obtained for the de novo synthesis of l-DOPA in E. coli, 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 P. aeruginosa and SeHpaC from S. enterica, and PlHpaB from Photorhabdus luminescens and KpHpaC from K. pneumoniae, respectively. Next, PaHpaB and PlHpaB were further engineered to improve their catalytic efficiency by the semi-rational method. PaHpaB(A211W) and PlHpaB(S210G) were obtained. The titer of caffeic acid was further increased to 1281.25 mg/L without l-DOPA accumulation using the PaHpaB(A211W)-UTR-SeHpaC hybrid. The production of hydroxytyrosol was further enhanced to 1681.42 mg/L using the combination of PlHpaB(S210G)-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 Escherichia coli, 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.