Directed Evolution and Resolution Mechanism of 1, 3-Propanediol Oxidoreductase from Klebsiella pneumoniae toward Higher Activity by Error-Prone PCR and Bioinformatics

Wei Jiang; Yuan Zhuang; Shizhen Wang; Baishan Fang

doi:10.1371/journal.pone.0141837

Directed Evolution and Resolution Mechanism of 1, 3-Propanediol Oxidoreductase from Klebsiella pneumoniae toward Higher Activity by Error-Prone PCR and Bioinformatics

PLoS One. 2015 Nov 3;10(11):e0141837. doi: 10.1371/journal.pone.0141837. eCollection 2015.

Authors

Wei Jiang¹, Yuan Zhuang², Shizhen Wang¹, Baishan Fang³

Affiliations

¹ 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.
² The Key Laboratory for Industrial Biotechnology of Fujian Higher Education, Hua Qiao University, Xiamen, Fujian, China.
³ 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alcohol Dehydrogenase / chemistry*
Alcohol Dehydrogenase / genetics
Amino Acid Substitution
Bacterial Proteins / chemistry*
Bacterial Proteins / genetics
Computational Biology
Directed Molecular Evolution*
Klebsiella pneumoniae / enzymology*
Klebsiella pneumoniae / genetics
Mutation, Missense*
NAD / chemistry*
NAD / genetics
Polymerase Chain Reaction / methods
Protein Binding

Substances

Bacterial Proteins
NAD
Alcohol Dehydrogenase
1,3-propanediol dehydrogenase

Grants and funding

This work was supported by the National Natural Science Foundation of China (No.41176111, No.41306124), the State Key Program of National Natural Science Foundation of China (No. 21336009), and the Fundamental Research Funds for the Central Universities (No.2013121029).