[Objective] To obtain an engineering strain producing G418 single component by directional reconstruction of Micromonospora purpurea G1008 producing gentamicins. [Methods] Using the temperature-sensitive plasmid pKC1139 as vector, the recombinant plasmid pQB303 was constructed and introduced into the M. purpurea G1008 by conjugation. The genQ*-disruption mutant was screened out by replica plating and PCR analysis. Use MS and TLC for analysis of metabolites. [Results] A genQ*-disruption strain, named as M. purpurea GQ175, was obtained to produce G418 single component. The level of antibiotic production was 828 mg/L, similar to the level of the parent strain. [Conclusion] The engineering strain GQ175 produced G418 single component, which had great value for industrial development. Collectively, these results demonstrated that the C-6′ dehydrogenase gene for gentamicin biosynthesis is genQ* and there is no other isoenzyme genes in M. purpurea G1008.