Construction Of Klebsiella Expression System And Expression Of DhaT And YqhD Genes

1,3-propanediol?1,3-propanediol,1.3-PD?was found in the glycerol fermentation broth of Clostridium pastoris by August Freund.1,3-PD is a very important tricarbon compound,which is widely used in food,chemical industry,cosmetics,medicine and other fields.It is an important chemical raw material.The industrial application of polypropylene terephthalate?PTT?makes 1,3-propanediol have strong application prospects and great commercial value.During the metabolism of 1,3-PD by Klebsiella sp.using glycerol as substrate,1,3-propanediol reductase encoded by dhaT gene is the key rate-limiting enzyme for glycerol to be converted into 1,3-PD.In this study,pET-28a plasmid was modified to construct a new expression vector,and dhaT gene from Klebsiella and yqhD gene from E.coli were enhanced to express 1,3-propanediol reductase in Klebsiella.The aim was to improve the transformation from glycerol to1.3-PD and to study its effect on 1,3-PD production in Klebsiella.In order to increase the production of 1,3-propanediol by microbial fermentation,reduce the cost and improve the competitiveness of microbial fermentation for 1,3-propanediol production,it is necessary to obtain high-yield,high-yield and high conversion strains.In view of the above contents,this study carried out the following research work:?1?pET-28a plasmid promoter was modified to construct new expression vectors recombinant Klebsiella pneumoniae pET-P_tac-dhaT?pET-P_tac-yqhD?pET-P_dhat-dhaT and pET-P_dhat-yqhD for overexpression of dhaT gene and yqhD gene in order to improve the activity of 1,3-propanediol oxidoreductase in Klebsiella.?2?The activity of 1,3-propanediol reductase was 27.4%and 35.3%higher than that of the control strain Klebsiella pneumoniae.Shaking flask fermentation results showed that 1,3-PD of recombinant Klebsiella sp.(pET-P_tac-dhaT and pET-P_tac-yqhD)reached 59.7 g/L,respectively.63.2 g/L,compared with the control strain K.pneumoniae,the yield increased,but the proportion of increase was lower.?3?The fermentation medium and fermentation conditions of recombinant Klebsiella sp.(pET-P_tac-dhaT and pET-P_tac-yqhD)were optimized.The effects of carbon source,carbon source concentration,pH,temperature and feeding mode on the yield of1,3-PD were analyzed.The high-density fermentation of recombinant bacteria was carried out on the optimized medium.The growth and metabolism rate of cells were controlled by adding glycerol and controlling temperature in the later stage.Enzyme activity and 1,3-PD production were increased.The fermentation results showed that the activity of recombinant bacteria was increased.