produces bioactive metabolite recognized as lipstatin and its intermediate orlistat. The main focus of this study is to enhance lipstatin production by strain improvement and precursor feeding. In this study, strain improvement to enhance the production of lipstatin was carried out by different doses (50, 100, 150, 200, and 250 Gy) of gamma radiation and precursors (Linoleic acid, Oleic acid, and l-Leucine). Screening showed that the highest yield of lipstatin (4.58 mg/g) was produced by mutant designated as SRN 7. The production of lipstatin (5.011 mg/g) increased significantly when the medium was supplemented with ratio 1:1.5 (linoleic acid + oleic acid). The addition of 1.5% l-Leucine leads to further increment in the production of lipstatin (5.765 mg/g). The addition of 10% soy flour in the culture medium resulted in the maximum production of lipstatin to 5.886 mg/g.