Tyrosinase is a binuclear copper-containing metalloprotein that leads the fast and regio- selective o- hydroxylation of monophenols to o- diphenols. However, the subsequent second oxidation to produce o- quinones, i. e., melanin precursors, from the o- diphenols has restricted its use to the production of functional o- diphenol derivatives. Herein, we present a combined strategy for the effective inhibition of melanin formation in tyrosinase reaction, which allows the use of tyrosinase as a monophenol monooxygenase. The o-diphenolic products were protected from being oxidized in the tyrosinase reaction by borate ions and L-ascorbic acid ( LAA). Borate- o- diphenol complexes were favorable formed at high pH and consequentially protected the o-diphenolic products from the catecholase activity of tyrosinase. LAA not only directly reduced the byproduct, o- quinones, into o- diphenols but also assisted the completion of the tyrosinase reaction cycle by removing a hydroxyl group attached to the copper metal cluster at the active site of the met-form tyrosinase. The regio- selective o- hydroxylation of 7,4'- dihydroxyisoflavone ( daidzein) to produce 7,30,4'- trihydroxyisoflavone ( 3'- ODI) was successfully carried out by whole E. coli cell biotransformation with heterologously expressed tyrosinase from Bacillus megaterium. The yield of this o- hydroxylation of 5mM daidzein in one- pot 400 mL reaction was ca. 100% in 90 min and the productivity was 16.3 mg 3'- ODI L-1 h-1 DCWmg-1, which is considerably higher than that of other monooxygenases. The method effectively abolished melanin synthesis, so that the o- diphenolic product remained stable without enzyme inactivation. Other monophenolic phytochemicals such as resveratrol and genistein could be subjected to the same strategy. After 1 h, 1mMof genistein and resveratrol were both converted to orobol and piceatannol, respectively, with ca. 95% conversion yield. These results support the strong potential of tyrosinase as a monooxygenase for regio- selective o- hydroxylation of various monophenolic compounds. Biotechnol. Bioeng. 2016; 113: 735- 743. (c) 2015 Wiley Periodicals, Inc.