Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia Coli and enzymatic properties

Wei Li, I. Son Ng, Baishan Fang, Jincong Yu, Guangya Zhang

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The gene dhaT from Klebsiella pneumoniae encoding 1,3-propanediol oxidoreductase (PDOR) was de novo synthesized by splicing overlap extension polymerase chain reaction (SOE-PCR) primarily according to Escherichia colis codon usage, as well as mRNA secondary structure. After optimization, Codon Adaptation Index (CAI) value was improved from 0.75 to 0.83, meanwhile energy of mRNA secondary structure was increased from -400.1 to -86.8 kcal/mol. This synthetic DNA was under control by phage T7 promoter in the expression vector pET-15b and transformed into the E. coli BL21 (DE3) strain. Inducers such as isopropyl (3-D-thiogalactoside (IPTG) and lactose were compared by activity at different inducing time. The activity of PDOR after codon optimized was 385.4 ± 3.6 U/mL, which was almost 5-fold higher than wild type (82.3 ± 1.5 U/ml) under the flask culture at 25 oC for 10 hrs. Then his-tagged enzyme was separated by using Ni-IDA column. The favorite environment for enzyme activity was at 5°C and pH 10.0, PDOR showed a certainly stability in potassium carbonate buffer for 2 hrs at diverse temperatures, enzyme activity was significantly improved by Mn 2+.

Original languageEnglish
Number of pages1
JournalElectronic Journal of Biotechnology
Volume14
Issue number4
DOIs
Publication statusPublished - 2011 Aug 1

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Codon
Escherichia coli
Enzymes
Thiogalactosides
Bacteriophage T7
Escherichia
Messenger RNA
Klebsiella pneumoniae
Lactose
Buffers
Polymerase Chain Reaction
Temperature
DNA
Genes
lactaldehyde reductase
potassium carbonate

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

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title = "Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia Coli and enzymatic properties",
abstract = "The gene dhaT from Klebsiella pneumoniae encoding 1,3-propanediol oxidoreductase (PDOR) was de novo synthesized by splicing overlap extension polymerase chain reaction (SOE-PCR) primarily according to Escherichia colis codon usage, as well as mRNA secondary structure. After optimization, Codon Adaptation Index (CAI) value was improved from 0.75 to 0.83, meanwhile energy of mRNA secondary structure was increased from -400.1 to -86.8 kcal/mol. This synthetic DNA was under control by phage T7 promoter in the expression vector pET-15b and transformed into the E. coli BL21 (DE3) strain. Inducers such as isopropyl (3-D-thiogalactoside (IPTG) and lactose were compared by activity at different inducing time. The activity of PDOR after codon optimized was 385.4 ± 3.6 U/mL, which was almost 5-fold higher than wild type (82.3 ± 1.5 U/ml) under the flask culture at 25 oC for 10 hrs. Then his-tagged enzyme was separated by using Ni-IDA column. The favorite environment for enzyme activity was at 5°C and pH 10.0, PDOR showed a certainly stability in potassium carbonate buffer for 2 hrs at diverse temperatures, enzyme activity was significantly improved by Mn 2+.",
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Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia Coli and enzymatic properties. / Li, Wei; Ng, I. Son; Fang, Baishan; Yu, Jincong; Zhang, Guangya.

In: Electronic Journal of Biotechnology, Vol. 14, No. 4, 01.08.2011.

Research output: Contribution to journalArticle

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