| Kanamycin A and kanamycin B are the secondary metabolites of wild-type S.Kanamyceticus ATCC12853.Both of them belong to 2-DOS aminoglycoside antibiotics,which are of broad spectrum and significant bactericidal activity againsting Gram-negative bacteria,so that they got a wide range of clinical use.Recent years,with the continuous application of these antibiotics,many bacteria have been resistant to this kind of antibiotics,what's more,the strong side effects also limit their further clinical use.In order to solve these problems,on one hand,people bent themselves to modifing the functional group to reduce its side effects;on the other hand,they recured to semi-synthetic methods to develop more new aminoglycoside antibiotics.In which,the most typical case is using kanamycin B as raw materials to synthesize the Dibekacin and Arbekacin.This part of work has already been mature in our research group.However,as the secondary metabolite of S.Kanamyceticus,kanamycin B production is less than 10%of the total yield,which is far from being satisfied as an industrial raw material for semi-synthesis of Arbekacin,and makes these drugs expensive.Nowadays,by analyzing the biosynthesis information of Kanamycin complex,the key enzyme genes in the gene clusters of biosynthesis pathway was revealed,which has provided a way for gene transformation to realize the enrichment of kanamycin B and reduce the production cost of Arbekacin.In this work,with the study of the kanamycin biosynthetic gene clusters,it was clear that kanF and kanJ genes were in charge of the low yield of kanamycin B.The kanF gene encodes a glycosyltransferase that can simultaneously select two sugar donors ligating into 2-DOS,which is resulting in the upstream branch of the kanamycin parallel pathway.While the kanJ gene encodes the enzyme to catalyze the first step of conversion to kanamycin A from Kanamycin B.Based on the method of genetic engineering,two genes were transformed by the biology engineering methods to construct the engineering strain rich in kanamycin B.For the upstream kanF gene,the neo8 expression plasmid was first constructed and the heterologous expression result in S.Kanamycin shows that compared with the wild-type strain,the engineering strain got a 2.1-fold increase in kanamycin B production,and the proportion of kanamycin A:B in the fermentation broth reduced from the original 18.2%to 5.7%which provided the further possibilities for the enrichment of kanamycin B.On the basis of this,the neo8-kanF recombinant plasmid was successfully constructed,and the method of split joint of large gene fragments to the original plasmid was explored.The transconjugation system of pKC1139 was also established.In addition,a PermE*+neo8 expression system was established to validate the role of the erythromycin promoter PermE*in S.kanamyceticus,laying the foundation for the subsequent expression of the kanamycin synthesis gene cluster.For the downstream kanJ gene,the kanJ-knockout strain was successfully constructed and the yield of kanamycin B was 661.0 mg·L-1,which was more than 200 times higher than that of the original strain.Further more,it changed the distribution of fermentation products,so that kanamycin B fermentation broth as the main product of the yield for 10.5 times to kanamycin A. |
PDF Full Text Request