Construction Of A High Yield Kanamycin B Strain And Studies On Its Regulatory Genes

As one of aminoglycoside-aminocyclitol(AmAc)antibiotics containing 2-deoxystreptamine(DOS),kanamycin has kept broad range antibacterial activities for decades.Although recent clinical application of kanamycin has been limited because of the emergence of resistant pathogenic bacteria,the semisynthetic derivatives of kanamycin,such as amikacin,dibekacin and arbekacin,are widely used to solve the issue of resistance effectively.Our group has successfully designed and optimized the synthesis processes of dibekacin and arbekacin from kanamycin B(KB)with good yields.However,the inadequate supply of KB seriously confines the output and leads to the high price of these crucial antibiotics.Generally,the wild-type S.kanamyceticus produce small amounts of KB compared with kanamycin A(KA)which is the main product.To solve the problem of KB in short supply,it is urgent and significant to construct engineered strain with high-yield ability of KB.Firstly,two different sgRNAs were designed for the kanJ gene,which were respectively ligated to the original plasmid pCRISPR-Cas9.And then the homologous arms of kanJ which were used as templates to achieve homologous repair were inserted into the StuI site through Gibson assembly.After successful construction of the kanJ knockout plasmid,the kanJ knockout plasmid was introduced into S.kanamyceticus by conjugal transfer.The Cas9 protein was activated under the stimulation of thiostreomycin to disrupte the kanJ gene successfully.The kanamycin B yield of ?kanJ strain can reach 537.20 mg/L,which was nearly 10.7 times higher than that of the wild-type strain.Kanamycin B became the only product in fermentation of the AkanJ strain.Secondly,the function of regulating genes kanG and kanL in the metabolic pathway of kanamycin was discussed.The kanG and kanL multi-expression plasmids were integrated into the genome of S.kanamyceticus by conjugal transfer.Finally,kanG and kanL multiple expression strains were obtained through resistance selection.The kanamycin A yield of kanG multi-expression strain was 387.00 mg/L,and the yield of kanamycin B was 46.66 mg/L.The kanamycin A yield of kanL multi-expression strain was 408.07 mg/L,and the yield of kanamycin B was 51.62 mg/L.