Optimization of a biotransformation process to produce 4-(2,3,5,6-tetramethylpyrazine-1)-4′-demethylepipodophyllotoxin

Abstract

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 alternata S-f6 was systematically optimized. 28°C of culture temperature and 120rpm of rotary shaker speed were suitable for the accumulation of 4-TMP-DMEP. The production (i.e., 11.1±1.4mg/L) of 4-TMP-DMEP was remarkably improved by using an initial yeast extract concentration of 2.5g/L. 2.0g/L of Span 80 was beneficial for the 4-TMP-DMEP production (i.e., 25.0±1.5mg/L). Furthermore, the 4-TMP-DMEP production was remarkably improved by one pulse feeding of 50mg/L of DMEP on day 6 and two pulse feedings of 40mg/L of TMP on days 8 and 14 when its residual level was below 50mg/L and 10mg/L, respectively. The 4-TMP-DMEP production of 45.1±1.6mg/L was obtained in the fed-batch biotransformation process, which was enhanced by 726% and 256%, comparing to that (i.e., 5.4±0.4mg/L and 0.9mg/L/day) obtained in the batch biotransformation before optimization.