Construction Of Genetic Engineering Strain Using 1,3-propanediol Dehydrogenase Gene From Clostridium Butyricum And Biosynthesis Of 1,3-propanediol By Whole Cell

1,3-Propanediol（1,3-PD）is one of the most important platform chemicals which is widely used for a solvent and antifreeze,food additives in the food,medicine,cosmetic and chemical industry.Synthesis of 1,3-PD can be done by both chemically and biologically,while the latter has received much attention as it is economically competitive and eco-friendly.Clostridium butyricum could be the most attractive microbial due to its usage as probiotic and containing a co-factor B₁₂independent glycerol dehydratase（GDHt）.1,3-propanediol dehydrogenase（PDOR）is a key enzyme in the biosynthetic pathway when microbial metabolism metabolizes glycerol into 1,3-PD.Therefore,a recombinant strain with PDOR from isolated C.butyricum was used for co-biotransformation of glycerol to 1.3-PD coupled with C.butyricum by whole cell in this study.The main results were as follows:（1）After physiological,and biochemical analysis,two strains designated as YJH-09 and YJH-12 were isolated through preliminary screening.Futhermore,the 16S rDNA sequences were required through TA cloning.According to the results of physiological,biochemical and 16S rDNA sequence analysis,the YJH-09 and YJH-12strains were both identified as C.butyricum.（2）The dhaT gene encoding PDOR was amplified from the isolated strain C.butyricum.DNA sequencing analysis showed that the dhaT is consisted of 1158 bp nucleotides.The recombinant plasmid pET-dhaT was constructed by using vector pET-30a,and recombinant protein was successfully expressed in BL21 strain by inducing with IPTG.The PDOR was then purified by nickel-nickel affinity chromatography and characterized.The characteristic band size was recorded to around 46 kDa after SDS-PAGE analysis.The optimum temperature,pH and specific activity for the reduction reaction of PDOR were found to be 37℃,9.0 and 25.7 U/mg,respectively when propionaldehyde was used as the substrate.The K_m values for propionaldehyde and NADH were 0.36 mM,0.019M,while the V_max values were found to be 34.59 U/mg and 21.47 U/mg,respectively.When 1,3-PD was used as substrate for PDOR,the optimum temperature,pH and specific activity for oxidation reaction was found to be30℃,11.0 and 7.9 U/mg,respectively.The K_m values for 1,3-PD and NAD⁺were recorded as 2.78 mM and 0.107 mM,respectively.The V_max values were 9.68 U/mg for1,3-PD and 7.36 U/mg for NAD⁺.In addition,PDOR showed its catalytic ability to some aldehydes,ketones,and alcohols,among which the highest reducing activity was shown to propionaldehyde,while the maximum oxidizing activity was recorded for 1,3-PD.（3）Finally,co-biotransformation of glycerol was carried out using the whole cells under the optimum biosynthesis conditions including the substrate of glycerol concentration of 50g/L and the mass ratio of C.butyricum YJH-09 to BL21-pET-dhaT,1:1.The fermentation medium was also supplemented with the exogenous coenzyme NADH at a concentration of 0.5 mM.The co-biotransformation resulted a maximum1,3-PD concentration of 25.88g/L,with the yield of 0.54g/g,which was more than a doubled if compared to the 1,3-PD produced by the C.butyricum YJH-09 alone.