The Research And Exploration Of Building Kanamycin B Overproducing Strain

Kanamycin is an important aminoglycoside antibiotic with wide antimicrobial spectrum and strong antibacterial ability. The kanamycin B, one variant of kanamycin, is the raw materials of arbekacin, which is the latest aminoglycoside antibiotic of strong antibacterial ability against the aminoglycoside -resistant pathogens and has low toxicity. Arbekacin is good at killing drug-resistant bacteria and has a wide prospect of market. But there are no manufacturers able to produce the arbekacin independently in the domestic, as a result the price of arbekacin is quite high. The synthetic route of arbekacin from kanamycin B has been completed in our laboratory, But the kanamycin B is expensive because of the low fermentation yield. we have made efforts to screen the strain of producing more kanamycin B, and it would be important and meaningful if we can apply this high-producing strain into industrial productions.First, we mutanted the S.kanamyceticus through the protoplast mutagenesis method to screen the mutant strain which is resistant to its own metabolite, and we have gained a mutant strain.The yield of kanamycin B could reach up to 80.3 mg·L-1, which is increased by 46.53% than the wide-typle strain.Secondly, we attempted to modify the biosythesis gene of S.kanamyceticus by gene engineering method to obtain the kanamycin B over-producing strain. We optimized the Conjugative transfer system of S.kanamyceticus and E.coli successfully and constructed a series of recombinant plasmid. The neo8 has been integrated into the genome of S.kanamyceticus, and the production of kanamycin B is 124 mg·L-1, which is increased by 126% than the original strain and increased by 54.4% than the mutant strain through the protoplast mutagenesis method. On the basis of these study, we constructed a recombiant plasmid with PermE to study the function of PermE in the S.kanamyceticus, and we constructed disrupt plasmid of kanJ and kanF to prevent the biosynthesis of kanamycin A in the S.kanamyceticus. Through these study, we have explored a PCR reaction system of high GC target sequence.