title: Directed Evolution and Resolution Mechanism of 1, 3-Propanediol Oxidoreductase from Klebsiella pneumoniae toward Higher Activity by Error-Prone PCR and Bioinformatics.
abstract: 1, 3-propanediol oxidoreductase (PDOR) is a key enzyme in glycerol bioconversion to 1,3-propanediol (1, 3-PD) which is a valuable chemical and one of the six new petrochemical products. We used error-prone PCR and activity screening to identify mutants of Klebsiella pneumoniae (K. pneumoniae) PDOR with improved activity. The activity of one of the identified mutants, PDOR'-24, which includes a single mutation, A199S, was 48 U/mg, 4.9 times that of the wild-type enzyme. Molecular docking was performed to analyze the identified mutants; and amino acids S103, H271, N366, D106, N262 and D364 were predicted to bond with NADH. The origins of the improved activity of PDOR'-24, as well as three other mutants were analyzed by simulating the interaction mechanism of the mutants with the substrate and coenzyme, respectively. This research provides useful information about the use of safranine O plate screening for the directed evolution of oxidoreductases, identifies interesting sites for improving PDOR activity, and demonstrates the utility of using molecular docking to analyze the interaction mechanism of the mutants with the substrate and coenzyme, respectively.
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, 361005, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, Fujian, 361005, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: The Key Laboratory for Industrial Biotechnology of Fujian Higher Education, Hua Qiao University, Xiamen, Fujian, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhuang
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, 361005, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, Fujian, 361005, China.
authors.forename: Shizhen
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, 361005, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, Fujian, 361005, China; The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian, 361005, China.
authors.forename: Baishan
authors.initials: B
authors.lastname: Fang
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D019295:Computational Biology; D019020:Directed Molecular Evolution; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D020125:Mutation, Missense; D009243:NAD / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D009243:NAD; D000426:Alcohol Dehydrogenase; C052370:1,3-propanediol dehydrogenase