Add time:09/02/2019         Source:sciencedirect.com

In pharmaceutical industry, isepamicin is mainly manufactured from Gentamicin (cas 1403-66-3) B, which is produced by Micromonospora 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-20A via deamination of the C2’ amino group. Using kanJ and kanK 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. Gene knockout of genR and genS almost abolished the production of gentamicin B in the mutant strain, suggesting that these two genes, which are responsible for the last steps in gentamicin B biosynthesis, constitute the missing part of the known gentamicin biosynthetic pathway. Based on these finding, we successfully constructed a gentamicin B high-yielding strain (798 mg/L), in which an overexpression cassette of genR and genS was introduced. Our work fills the missing piece to solve the puzzle of gentamicin B biosynthesis and may inspire future metabolic engineering efforts to generate gentamycin B high-yielding strains that could eventually satisfy the need for industrial manufacturing of isepamicin.

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