<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Nikkomycins are a group of peptidyl nucleoside antibiotics produced by <jats:italic>Streptomyces ansochromogenes</jats:italic>. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by <jats:italic>S. ansochromogenes</jats:italic>. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials. </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>To increase the yields of nikkomycins, an additional copy of nikkomycin biosynthetic gene cluster (35 kb) was introduced into nikkomycin producing strain, <jats:italic>S. ansochromogenes</jats:italic> 7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIK)was introduced into <jats:italic>S. ansochromogenes</jats:italic> by conjugal transfer. Introduction of pNIK led to enhanced production of nikkomycins (880 mg L<jats:sup>-1</jats:sup>, 4 -fold nikkomycin X and 210 mg L<jats:sup>-1</jats:sup>, 1.8-fold nikkomycin Z) in the resulting exconjugants comparing with the parent strain (220 mg L<jats:sup>-1</jats:sup> nikkomycin X and 120 mg L<jats:sup>-1</jats:sup> nikkomycin Z). The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure. </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>A high nikkomycins producing strain (1100 mg L<jats:sup>-1</jats:sup> nikkomycins) was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of <jats:italic>S. ansochromogenes</jats:italic>. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites. </jats:sec>