The putative tunicamycin biosynthetic gene cluster was identified in Streptomyces clavuligerus with genome mining approach and this genetic information was used to isolate the tunicamycin biosynthetic gene set from S. chartreusis. Gene inactivation of $tun1_{Sch}$ abolished tunicamycin accumulation in S. chartreusis, confirming that the tunicamycin biosynthetic gene cluster was cloned.