High-specificity synthesis of novel monomers by remodeled alcohol hydroxylase.
High-specificity synthesis of novel monomers by remodeled alcohol hydroxylase.
复制标题
重构醇羟化酶高特异性合成新型单体
DOI：
10.1186/s12896-016-0291-8
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发表时间：
2016-08-24
影响因子：
3.5
通讯作者：
Liu H
中科院分区：
文献类型：
--
作者：
Zheng Y;Li L;Liu Q;Zhang H;Cao Y;Xian M;Liu H
背景二醇是生产塑料和聚氨酯的重要单体，广泛应用于日常生活中。在其亚末端具有一个羟基的中链二醇能够赋予所合成的材料更大的柔性。但遗憾的是，这种类型的二醇迄今尚未合成。对先进材料的强烈需求促使我们开发了一种生产这些新型二醇的新策略。结果天然P450 BM 3能够将中链醇转化为相应的α，ω1-，α，ω2-和α，ω3-二醇，每种二醇约占总二醇的1/3，但对短链醇几乎没有活性。通过实验室进化P450 BM 3获得了醇羟基化的极大改善的区域特异性。在12个氨基酸残基（J2-F87 A）取代后，1，7-癸烷二醇（ω-3羟基化）占总癸二醇的比例从34.0%增加到86.8%。结构模拟和定点突变表明，血红素末端残基如Ala 78、Phe 87和Arg 255在控制醇羟基化的区域选择性中起关键作用，而底物结合位点口的残基不负责区域选择性。其是用于开发先进材料的有前景的单体。确定了几个控制醇羟基化反应区域选择性的关键氨基酸残基，为如何提高醇羟基化反应的区域选择性提供了一些新的见解。本研究不仅为1，7-癸烷醇的生物合成提供了一个很好的策略，而且为其它有用的二醇的生产提供了一条有前途的途径。
BackgroundDiols are important monomers for the production of plastics and polyurethanes, which are widely used in our daily life. The medium-chain diols with one hydroxyl group at its subterminal end are able to confer more flexibility upon the synthesized materials. But unfortunately, this type of diols has not been synthesized so far. The strong need for advanced materials impelled us to develop a new strategy for the production of these novel diols. In this study, we use the remodeled P450BM3for high-specificity production of 1,7-decanediol.ResultsThe native P450BM3was capable of converting medium-chain alcohols into corresponding α, ω1-, α, ω2- and α, ω3-diols, with each of them accounting for about one third of the total diols, but it exhibited a little or no activity on the short-chain alcohols. Greatly improved regiospecificity of alcohol hydroxylation was obtained by laboratory evolution of P450BM3. After substitution of 12 amino acid residues (J2-F87A), the ratio of 1,7-decanediol (ω-3 hydroxylation) to total decanediols increased to 86.8 % from 34.0 %. Structure modeling and site-directed mutagenesis demonstrated that the heme end residues such as Ala78, Phe87and Arg255play a key role in controlling the regioselectivity of the alcohol hydroxylation, while the residues at the mouth of substrate binding site is not responsible for the regioselectivity.ConclusionsHerein we employ an engineered P450BM3for the first time to enable the high-specificity biosynthesis of 1,7-decanediol, which is a promising monomer for the development of advanced materials. Several key amino acid residues that control the regioselectivity of alcohol hydroxylation were identified, providing some new insights into how to improve the regiospecificity of alcohol hydroxylation. This report not only provides a good strategy for the biosynthesis of 1,7-decanediol, but also gives a promising approach for the production of other useful diols.