Overproduction Of Gentamicin B By The Discovery And Overexpression Of Its Synthetic Genes GenR And GenS

As the second-generation aminoglycoside antibiotic,isepamicin possesses a high level of stability to aminoglycoside modifying enzymes.In pharmaceutical industry,isepamicin is mainly manufactured from gentamicin B,which is produced by M.echinospora as a minor component of the gentamicin complex.Improvement of gentamicin B production through metabolic engineering is therefore important to satisfy the increasing demand for isepamicin.We hypothesized that gentamicin B was generated from gentamicin JI-20 A via deamination of the C2’ amino group based on structural similarity.Using kan J and kan K as the gene probes,we identified the putative deamination-related genes,genR and genS,through genome mining of the gentamicin B producing strain M.echinospora CCTCC M 2018898.Interestingly,genR and genS constitute a gene cassette located approximately 28.7 kb away from the gentamicin gene cluster.It was confirmed that genR and genS are responsible for the last steps in gentamicin B biosynthesis by knockout and complementation experiments in vivo.Based on this finding,we successfully constructed a highyielding strain by overexpressing genR and genS with the high-intensity promoter,increasing the production of gentamicin B to 798 mg/L,which was 64% higher than the original strain.We believe this work constitutes the missing part of the known gentamicin biosynthetic pathway and provides optional components and technology for further metabolic engineering to obtain high-yielding strains of gentamicin B to meet the needs of industrial production of isepamicin.