Cloning And Expression Of Genes Of 1,3-Propanediol Oxidoreductase And Aldehyde Reductase

1,3-Propanediol has been widely applied for large volume markets,particularly in polymer business.Microbial production of 1,3-propanediol has been considered as a competitor to the traditional petrochemical routes.Recently,microbial production has been taken into account more and more.The formation of 1,3-propanediol is limited by the amount of NADH supplied by the oxidative pathway of glycerol dismutation.It is revealed that relaxation of the coenzyme specificity of 1,3-propanediol oxidoreductase for both NADH and NADPH would increase the production of 1,3-propanediol by metabolic flux analysis.According to computational result,Asp41 was mutated to glycine to relax the coenzyme specificity of 1,3-propanediol oxidoreductase.With NADH and NADPH as coenzymes,the specific activity of the mutated enzyme was 629.38 and 277.35 U/mg,while the specific activity of the wild type enzyme was 706.90 and 20.95 U/mg,respectively.With NADH as coenzyme,the Michaelis constant(K_m) and turnover number(k_cat) of the wild type enzyme were 0.015 mmol/L and 90.64 l/s, respectively.With NADH as coenzyme,K_m and k_cat of the mutated enzyme were 0.48 mmol/L and 411.61 l/s,respectively;while with NADPH as coenzyme,Km and k_cat of the mutated enzyme were 0.14 mmol/L and 187.35 l/s,respectively.Although the K_m values of the mutated enzyme for the two coenzymes(NADH and NADPH) increased,the k_cat with NADH was 3.54-fold higher than that of the wild type enzyme,and the k_cat with NADPH was 1.07-fold higher than that of the wild type enzyme.The yqhD gene from Klebsiella pneumoniae DSM2026,encoding the aldehyde reductase, was cloned and overexpressed in E.coli BL21(DE3).After purified by Q-Sepharose and Sephacryl S-300 chromatography to homogeneity,the specific activity of aldehyde reductase (3.8 U/mg) was measured with 3-hydroxypropionaldehyde as substrate.It was interesting that the reversible reaction activity was not detected with 1,3-propanediol as substrate.The optimal pH and optimal temperature of the enzyme were pH 5.8 and 60℃,respectively.The enzyme had a wide range of substrates,in which butyraldehyde was tested with the highest activity,followed by 3-hydroxypropionaldehyde.The K_m values of the enzyme for NADPH and NADH were 0.07 mmol/L and 1.42 mmol/L,respectively,indicating the flexibility of dependence on coenzymes. On the basis of previous work,the recombinant plasmids pDK6-dhaT,pDK6-dhaT' and pDK6-yqhD were constructed.Then the recombinant plasmids were transformed into Klebsiella pneumoniae DSM2026.During the flask culture,the enhanced production of 1,3-propanediol in the recombinants were measured in comparison with the wild type at an initial glycerol concentration of 50 g/L.