The developed tandem biotransformation process for the directional biosynthesis of a designed compound 4-(2,3,5,6-tetramethylpyrazine-1)-4'-demethylepipodophyllotoxin (4-TMP-DMEP) by Alternaria alternate S-f6 was systematically optimized. 28 degrees C of culture temperature and 120 rpm of rotary shaker speed were suitable for the accumulation of 4-TMP-DMEP. The production (i.e., 11.1 +/- 1.4 mg/L) of 4-TMP-DMEP was remarkably improved by using an initial yeast extract concentration of 2.5 g/L. 2.0 g/L of Span 80 was beneficial for the 4-TMP-DMEP production (i.e., 25.0 +/- 1.5 mg/L). Furthermore, the 4-TMP-DMEP production was remarkably improved by one pulse feeding of 50 mg/L of DMEP on day 6 and two pulse feedings of 40 mg/L of TMP on days 8 and 14 when its residual level was below 50 mg/L and 10 mg/L, respectively. The 4-TMP-DMEP production of 45.1 +/- 1.6 mg/L was obtained in the fed-batch biotransformation process, which was enhanced by 726% and 256%, comparing to that (i.e., 5.4 +/- 0.4 mg/L and 0.9 mg/L/day) obtained in the batch biotransformation before optimization. (C) 2012 Elsevier Ltd. All rights reserved.