Gentamicin　C1a　is　an　important　precursor　to　the　synthesis　of　etimicin,　a　potent　antibiotic.　Wild　type　Micromonospora　purpurea　Gb1008　produces　gentamicin　C1a,　besides　four　other　gentamicin　C　components:　C1,　C2,　C2a,　and　C2b.　While　the　previously　reported　engineered　strain　M.　purpurea　GK1101　can　produce　relatively　high　titers　of　C1a　by　blocking　the　genK　pathway,　a　small　amount　of　undesirable　C2b　is　still　being　synthesized　in　cells.　Gene　genL　(orf6255)　is　reported　to　be　responsible　for　converting　C1a　to　C2b　and　C2　to　C1　in　Micromonospora　echinospora　ATCC15835.　In　this　work,　we　identify　the　genL　that　is　also　responsible　for　the　same　methylation　in　Micromonospora　purpurea.　Based　on　M.　purpurea　GK1101,　we　construct　a　new　strain　with　genL　inactivated　and　show　that　no　C2b　is　produced　in　this　strain.　Therefore,　we　successfully　engineer　a　strain　of　M.　purpurea　that　solely　produces　gentamicin　C1a.　This　strain　can　potentially　be　used　in　the　industrial　production　of　C1a　for　the　synthesis　of　etimicin.　©　2019　by　the　authors.　Licensee　MDPI,　Basel,　Switzerland.