Codon optimization of 1, 3-propanediol oxidoreductase expression in escherichia coli and enzymatic properties

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 coli’s 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 beta-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 25ºC 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 Mn2+.

Genetic Engineering Reconstruction of Klebsiella pneumoniae Producing 1,3-propanediol by the Gene yqhD Encoding 1,3-propanediol Oxidoreductase Isoenzyme / 中国生物工程杂志

In the reductive branch, glycerol is first dehydrated to 3-hydroxypropionaldehyde that is then reduced to 1,3-PD under the consumption of reducing power (NADH). If over-expression of the gene dhaB encoding glycerol dehydrase is achieved,the reducing power will be scarce and 3-hydroxypropionaldehyde will be accumulated,which is disadvantage to produce 1,3-propanediol.The structure gene yqhD from E.coli encoding 1,3-propanediol oxidoreductase isoenzyme[under the consumption of reducing power (NADPH)]and the gene dhaB encoding glycerol dehydrase from Klebsiella pneumoniae was amplified using PCR method.The two gene were transferred into expression vector pEtac to construct a novel recombinant Klebsiella pneumoniae (pEtac-dhaB-tac-yqhD).Over-expression of yqhD and dhaB in Klebsiella pneumoniae was achieved with pEtac-dhaB-tac-yqhD.The fermentation result on aerobic conversion showed the increase of 20% of 1,3-propanediol yield by Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD) was obtained compared with Klebsiella pneumoniae.The main by-products,acetic acid and butanediol decreased estrogen receptors 30% obviously.