Rapamycin is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications. Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have rationally designed approaches to generate a rapamycin high producer strain of S. hygroscopicus HD-04-S. These have included alleviation of glucose repression, improved tolerance towards lysine and shikimic acid, and auxotrophy of tryptophan and phenylalanine through the application of stepwise UV mutagenesis. The resultant strain produced rapamycin at 450 mg/L in the shake flask scale. These fermentations were further scaled up in 120 and 20,000 L fermentors, respectively, at the pilot plant. Selected fermentation factors including agitation speed, pH, and on-line supplementation were systematically evaluated. A fed-batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. The final titer of rapamycin was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor. This work highlights a high rapamycin producing strain derived by mutagenesis and subsequent screening, fermentation optimization of which has now made it feasible to produce rapamycin on an industrial scale by fermentation. The strategies developed here should also be applicable to titer improvement of other important microbial natural products on an industrial scale. Biotechnol. Bioeng. 2010; 107: 506-515. (C) 2010 Wiley Periodicals, Inc.