This work optimized the novel biotransformation process of podophyllotoxin to produce podophyllic acid by Pseudomonas aeruginosa CCTCC AB93066. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/l of yeast extract and 5 g/l of peptone were favorable for podophyllic acid production (i.e. 25.3 +/- 3.7 mg/l), while not beneficial for the cell growth of P. aeruginosa. This indicated that the accumulation of podophyllic acid was not corresponded well to the cell growth of P. aeruginosa. 0 g/l of sucrose was beneficial for podophyllic acid production (i.e. 34.3 +/- 3.9 mg/l), which led to high podophyllotoxin conversion (i.e. 98.2 +/- 0.1%). 1 g/l of NaCl was the best for podophyllic acid production (i.e. 47.6 +/- 4.0 mg/l). Secondly, the production of podophyllic acid was significantly enhanced by fed-batch biotransformation. When each 100 mg/l of podophyllotoxin was added to the biotransformation system after 4, 10 and 25 h of culture, respectively, podophyllic acid concentration reached 99.9 +/- 12.3 mg/l, enhanced by 284% comparing to one-time addition (i.e. 26.0 +/- 2.1 mg/l). The fundamental information obtained in this study provides a simple and efficient way to produce podophyllic acid. (C) 2008 Elsevier Ltd. All rights reserved.