Cloning Of Adenosylcobalamin Synthase Gene Cobs And Expression In Klebsiella Pneumoniae For 1,3-PD Production

As a chemical raw material,1,3-propanediol has many applications in polymer substances, food lubricants, medicines and many other areas. Now,1,3-propanediol has been received attention as a monomer for synthesizing a new polymer(PTT). The current focus of research is on the conversion of 1,3-propanediol by microbial fermentation using a variety of bacteria, in particular, Klebsiella pneumoniae. The conversion of 1,3-propanediol is carried out by two enzymatic steps. Glycerol dehydratase is a key enzyme in this process, which requires the presence of coenzyme B12 to carry out catalysis effectively. Therefore, bioconvertion of 1,3-propanediol needs to add extra coenzyme B12, however, VB12 is expensive, so it increases the post-industrial costs. The study on the use of genetically engineering to reduce the added coenzyme B12 has not yet been reported in our country.Although there are many researches about the pathway of 1,3-propanediol, but the studies of coenzyme B12 in the 1,3-propanediol metabolism have not been reported. Therefore, the Adenosylcobalamin synthase gene cobs was amplified from E. coli k-12 by using PCR technology in this study. And the promoter tac was used in our laboratory to construct recombinant plasmid pEtac-cobs which contained target gene cobs. And then pEtac-cobs was inserted by tac-dhaB-tac-yqhD from recombinant plasmid pEtac-dhaB-tac-yqhD which harbored glycerol dehydratase gene dhaB and 1,3-propanediol redox isoenzyme gene yqhD. The recombinant plasmid was expressed in K. pneumoniae. The glycerol dehydratase activities of recombinant bacteria and parent strain were determined, the results were 0.93 U/mg and 0.85 U/mg respectively. Compared with the parent strain, glycerol dehydratase activity from recombinant bacteria increased 9.4%. The results of preliminary fermentation showed that recombinant bacteria required less additional VB12 than parent strain from 0.015 g/L to 0.006 g/L, and the production of 1,3-propanedio reached a considerable level.The fermentation medium for recombinant K. pneumoniae producing 1,3-propanediol was preliminary optimized by single factor experiment. Different nitrogen sources and different concentration of yeastextract, glycerol, glucose, MgSO4·7H2O and ATP were investigated respectively. The optimum medium for recombinant K. pneumoniae was as follows:yeast extract 7 g/L, glycerol 60 g/L, MgSO4·7H2O 2.5 g/L, glucose 10 g/L, ATP 0.06 g/L. Effects on 1,3-propanediol production of recombinant K. pneumoniae by inoculum and agitation speed were also studied. The optimum inoculum was 8%, and the optimum agitation speed of fermentor was 100 r/min for 30 hours after 150 r/min for 15 hours.Based on the single factor experiment results, the effect of four factors, glycerol, MgSO4·7H2O, glucose, and VB12 on the results of fermentation was investigated by orthogonal design. The results shows that the yield of 1,3-propanediol could get to the highest level while the concentration of glycerol, MgSO4·7H2O, glucose, and VB12 were:60 g/L,3 g/L,10 g/L and 0.006 g/L respectively. Under these conditions, the highest yield can reach 32 g/L. This shows that the recombinant bacteria outputed more VB12. On one hand, it provided coenzyme B12 for the glycerol dehydratase's effective catalytic reaction, on the other hand, it played a positive role in promoting the glycerol dehydratase's resurrection, thereby increased the production of 1,3-propanediol. The recombinant bacteria significantly reduced the amount of VB12, which provides a new approach to reduce the 1,3-propanediol production costs.