title: High-specificity synthesis of novel monomers by remodeled alcohol hydroxylase.
abstract: Diols 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 P450BM3 for high-specificity production of 1,7-decanediol.
  
  
  
  The native P450BM3 was 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 Ala(78), Phe(87) and Arg(255) play 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.
  
  
  
  Herein we employ an engineered P450BM3 for 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.
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Yanning
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.|College of Food Science, Sichuan Agricultural University, Yaan, 625014, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.|College of Food Science, Sichuan Agricultural University, Yaan, 625014, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China.
authors.forename: Yujin
authors.initials: Y
authors.lastname: Cao
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China. xianmo@qibebt.ac.cn.
authors.forename: Mo
authors.initials: M
authors.lastname: Xian
authors.affiliation: CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No.189 Songling Road, Laoshan District, Qingdao, 266101, China. liuhz@qibebt.ac.cn.
authors.forename: Huizhou
authors.initials: H
authors.lastname: Liu
mesh_terms: D000438:Alcohols / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D003198:Computer Simulation; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004789:Enzyme Activation; D006018:Glycols / Q000138:chemical synthesis*; D017665:Hydroxyl Radical; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D001426:Bacterial Proteins; D006018:Glycols; D017665:Hydroxyl Radical; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; C000617955:1,2-decanediol
keywords: 1,7-decanediol; Alcohol hydroxylation; Diols; Escherichia coli; P450BM3; Regiospecificity