ABSTRACT Tacrolimus is a polyketide macrolide produced by Streptomyces species which is widely used as anti-fibrotic agent and potent immunosuppressant. In this article dual mutagenesis approach using mutagens (NTG+EMS+UV) was used to develop a mutant strain of Streptomyces tacrolimicus (ATCC 55098) for higher tacrolimus production and this strain showed higher tacrolimus production at 82.5 mg/l. Interestingly; addition of L-Lysine (0.2 g/l) into the production medium further enhanced the tacrolimus production to ~102 mg/l at 7-L fed-batch bioreactor. To the best of our knowledge this is the first report mentioning efficient strain development for higher production of tacrolimus using dual mutagenesis. The obtained data presents an impressive model for higher production of tacrolimus and enhanced our understanding regarding improvement in production capacity of tacrolimus in Streptomyces tacrolimicus.