Studies On Cloning, Sequence Analysis And Construction Of Expression Vector Of Genes Encoding Glycerol Dehydratase From Klebsiella Pneumoniae

1,3-Propanediol is an important substance in chemical industries. It has a lotof good character and wide application future. It has been paid more and moreattention for its utilization rebirth resource and little pollution to the environmentin the bioconversion of 1,3-PD. The glycerol dehydratase (GDHt) is the firstenzyme involved in the process of bioconversion of 1,3-PD and catalyzes therate-limiting step in the anaerobic conversion of glycerol to 1,3-PD, it has themost important effect to the conversion speed of 1,3-PD. So the research ofglycerol dehydratase has important actual meaning to home further developmentand utilization of 1,3-PD. The aim of our study is to initiate the reseach by therecombinant microorganism in the bioconversion of 1,3-PD and improve theyield of 1,3-PD by the search of microbial fermentation production of 1,3-PD byKlebsiella pneumoniae and the study of genes encoding glycerol dehydratase.In this experiment, the microbial fermentation production of 1,3-PD by K.pneumoniae was studied and gene cloning, sequence analysis and construction ofexpression vector of GDHt were first described in our country. Firstly, theoptimum fermentation culture medium and conditions of K. pneumoniae weredetermined. Based on what mentioned above, the genes encoding GDHt were clonedby using PCR method. The sequence analysis and construction of expression vectorfrom pMAL-c2X and pGEX-4T-1 vector in E.coli wereaccomplished respectively.The major results are as the following:The optimum fermentation culture and conditions in the microbialfermentation production of 1,3-PD by K. pneumoniae were determined. Effects ofC,N,P and Fe2+ was studied. The optimum culture medium is: glycerol 60g/L,NH4Cl 1.8g/L,KH2PO4 1.6g/L,Fe2+ 0.03‰;Through orthogonal experiment,theoptimum culture conditions are:pH 7.0,temperature 37℃,culture time 40h,inoculum size 8%;Under the optimum conditions: the maximal 1,3-PD yield was38.62g/L.The genes encoding glycerol dehydratase of K. pneumoniae were clonedby using PCR method respectively. The PCR products were sequenced directlyand cloned to pMD18-T and identified by sequencing respectively. The sequenceresult of the amplified DNA fragments shows: Large gene fragment was 1668base pairs encoding 555 amino acid residues, it included an initiation codon(ATG)and a stop condon(TGA), the G+C content was 60.54%;The middle gene...