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

Jiang W, Zhuang Y, Wang S, Fang B - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Identification of evolved mutants using the high throughput safranin O plates screening method.An example of an active and an inactive clone are shown.
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pone.0141837.g001: Identification of evolved mutants using the high throughput safranin O plates screening method.An example of an active and an inactive clone are shown.

Mentions: The plasmids carrying the mutant genes of the dhaT, were transformed into BL21 DE3 and the resulting strains were grown on the medium-ampicillin-safranine O screening plates for screening of mutants with increased activity. The plates were incubated under 37°C for about 12 h before further use. While PDOR oxidizes 1,3-PD to generate 3-HPA, NADH is produced which could increase the electric potential through the respiratory chain, produce strong proton flow, strengthen the E. coli cell’s intake of safranine O, and make the safranine O aggregate in E. coli cells [19]. Through the safranine O screening, the mutants with desired PDOR activity would show homogeneous peach-like pattern, while inactive cloning has a transparent ring around the colonies (Fig 1).


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

Jiang W, Zhuang Y, Wang S, Fang B - PLoS ONE (2015)

Identification of evolved mutants using the high throughput safranin O plates screening method.An example of an active and an inactive clone are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631369&req=5

pone.0141837.g001: Identification of evolved mutants using the high throughput safranin O plates screening method.An example of an active and an inactive clone are shown.
Mentions: The plasmids carrying the mutant genes of the dhaT, were transformed into BL21 DE3 and the resulting strains were grown on the medium-ampicillin-safranine O screening plates for screening of mutants with increased activity. The plates were incubated under 37°C for about 12 h before further use. While PDOR oxidizes 1,3-PD to generate 3-HPA, NADH is produced which could increase the electric potential through the respiratory chain, produce strong proton flow, strengthen the E. coli cell’s intake of safranine O, and make the safranine O aggregate in E. coli cells [19]. Through the safranine O screening, the mutants with desired PDOR activity would show homogeneous peach-like pattern, while inactive cloning has a transparent ring around the colonies (Fig 1).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus