Lee Nahum, Lee Sang-Hyuk, Baek Kiheon, Kim Byung-Gee
School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea.
Appl Microbiol Biotechnol. 2015 Oct;99(19):7915-24. doi: 10.1007/s00253-015-6691-1. Epub 2015 Jun 3.
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).
来自阿维链霉菌MA4680的重组酪氨酸酶MelC2（基因标识符：499291317）在大肠杆菌BL21（DE3）中进行了异源表达。通过密码子优化的MelC1载体蛋白（KP198295.1）提高了活性MelC2的表达水平。通过对MelC1的Y91残基进行饱和诱变，发现第91位的芳香族残基如Y、F和W有助于产生正确折叠的MelC2构象。重组MelC2被用作生物催化剂将反式白藜芦醇转化为白皮杉醇。为了通过抑制副产物黑色素的形成来提高产物产量，期望增加MelC2的单加氧作用（k1）与双加氧作用（k2）的比率。这通过蛋白质工程和用葡萄糖脱氢酶（GDH）再生NADH的组合来实现。在距MelC2铜离子8埃半径内的15个残基处进行饱和诱变。共检测了2760个突变体（NNK密码子的概率为99.7%），筛选出了一个突变体I41Y。与野生型MelC2相比，该突变体在酪氨酸上的k1与k2的比率增加了7倍，在白藜芦醇上增加了5倍。结果，在50 mL反应中500 μM白藜芦醇的总产物产量为15.4%（77.4 μM白皮杉醇），比野生型高1.7倍。当I41Y与NADH再生系统结合时，总产物产量为58.0%，增加了8倍（290.2 μM白皮杉醇）。
