title: Heterologous expression of tyrosinase (MelC2) from Streptomyces avermitilis MA4680 in E. coli and its application for ortho-hydroxylation of resveratrol to produce piceatannol.
abstract: Recombinant tyrosinase from Streptomyces avermitilis MA4680, MelC2 (gi:499291317), was heterologously expressed in Escherichia coli BL21 (DE3). The expression level of active MelC2 was increased by the codon-optimized MelC1 caddie protein (KP198295.1). By performing saturation mutagenesis of the Y91 residue of MelC1, it was found that aromatic residues such as Y, F, and W at the 91st position help produce a correctly folded conformation of MelC2. The recombinant MelC2 was utilized as a biocatalyst to convert trans-resveratrol into piceatannol. In order to improve the product yield through suppression of the formation of melanin, a by-product, an increase in the ratio of monooxygenation (k 1) to dioxygenation (k 2) of MelC2 is desirable. This was achieved by a combination of protein engineering and regeneration of NADH with glucose dehydrogenase (GDH). Saturation mutagenesis was performed at 15 residues within 8-Å radius from copper ions of MelC2. A total of 2760 mutants were examined (99.7 % probability for NNK codon) and I41Y, a mutant, was screened. The ratio of k 1 to k 2 of the mutant increased sevenfold on tyrosine and fivefold on resveratrol, when compared to wild-type MelC2. As a result, the overall product yield from 500 μM resveratrol in 50-mL reaction was 15.4 % (77.4 μM piceatannol), 1.7 times higher than wild type. When I41Y was incorporated with the NADH regeneration system, the total product yield was 58.0 %, an eightfold increase (290.2 μM of piceatannol).
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea.
authors.forename: Nahum
authors.initials: N
authors.lastname: Lee
authors.forename: Sang-Hyuk
authors.initials: SH
authors.lastname: Lee
authors.forename: Kiheon
authors.initials: K
authors.lastname: Baek
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006900:Hydroxylation; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism; D000077185:Resveratrol; D013267:Stilbenes / Q000737:chemistry / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D013267:Stilbenes; C041525:3,3',4,5'-tetrahydroxystilbene; D014442:Monophenol Monooxygenase; D000077185:Resveratrol