Gentamicin　and　sisomicin　are　two　different　aminoglycoside　antibiotics.　The　comparison　of　their　chemical　structure　and　biosynthetic　gene　clusters,　coupled　with　bioinformatic　analysis,　suggested　that　the　gntK　gene　would　be　associated　with　methylation.　The　gntK　gene　fragment　in　M.　purpurea　G1008　was　inactivated　by　genetic　engineering　and　its　mutant　strain　M.　purpurea　GK1101　(Delta　gntK)　was　screened.　The　metabolites　of　G1008　and　GK1101　was　analyzed　by　HPLC-MS,　which　revealed　that　GK1101　no　longer　produced　gentamicin　C-1　or　C-2,　while　mainly　synthesizing　gentamicin　C-1a,　and　the　production　of　C-1a　increased　significantly.　This　indicated　that　the　metabolic　flow　for　the　gentamicin　C-1　and　C-2　biosynthesis　was　blocked　by　disrupting　the　gntK　gene,　which　substantiated　that　the　gntK　gene　encoded　the　enzyme　that　catalyzes　the　methylation　of　purpurosamine　C-6＇.　The　mutant　GK1101　has　good　prospects　for　industrial　application.　In　addition,　our　study　provides　information　that　can　be　used　to　clarify　the　function　of　a　single　gene　and　simplify　the　targeted　genetic　breeding　of　important　pharmaceutical　microorganisms.