Regioselective Hydroxylation of <i>trans</i>-Resveratrol <i>via</i> Inhibition of Tyrosinase from <i>Streptomyces avermitilis</i> MA4680

Secreted tyrosinase from melanin-forming <i>Streptomyces avermitilis</i> MA4680 was involved in both ortho-hydroxylation and further oxidation of <i>trans</i>-resveratrol, leading to the formation of melanin. This finding was confirmed by constructing deletion mutants of <i>melC</i>_<i>2</i> and <i>melD</i>_<i>2</i> encoding extracellular and intracellular tyrosinase, respectively; the <i>melC2</i> deletion mutant did not produce piceatannol as well as melanin, whereas the <i>melD2</i> deletion mutant oxidized resveratrol and synthesized melanin with the same yields, suggesting that MelC2 is responsible for ortho-hydroxylation of resveratrol. Extracellular tyrosinase (MelC2) efficiently converted <i>trans</i>-resveratrol into piceatannol in the presence of either tyrosinase inhibitors or reducing agents such as catechol, NADH, and ascorbic acid. Reducing agents slow down the dioxygenase reaction of tyrosinase. In the presence of catechol, the regio-specific hydroxylation of <i>trans</i>-resveratrol was successfully performed by whole cell biotransformation, and further oxidation of <i>trans</i>-resveratrol was efficiently blocked. The yield of this ortho-hydroxylation of <i>trans</i>-resveratrol was dependent upon inhibitor concentration. Using 1.8 mg of wild-type <i>Streptomyces avermitilis</i> cells, the conversion yield of 100 μM <i>trans</i>-resveratrol to piceatannol was 78% in 3 h in the presence of 1 mM catechol, indicating 14 μM piceatannol h^–1 DCW mg^–1 specific productivity, which was a 14-fold increase in conversion yield compared to that without catechol, which is a remarkably higher reaction rate than that of P450 bioconversion. This method could be generally applied to biocatalysis of various dioxygenases

Bioconjugation of l-3,4-Dihydroxyphenylalanine Containing Protein with a Polysaccharide

We describe the simple bioconjugation strategy in combination of periodate chemistry and unnatural amino acid incorporation. The residue specific incorporation of 3,4-dihydroxy-l-phenylalanine can alter the properties of protein to conjugate into the polymers. The homogeneously modified protein will yield quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for widespread use to prepare protein conjugates and synthetic biology applications