Metabolic fluxes during lipase production by Bacillus subtilis CICC 20034 in synthetic medium were studied using metabolic flux analysis (MFA). The MFA showed that lipase production was dependent on, and coupled to the tributyrin uptake rate, formation of biomass, lactate, ATP, as well as amino acids from the aspartate and glutamate family. Using tributyrin as the sole carbon source, an adaptive evolution strategy was applied to increase the tributyrin uptake rate. B. subtilis SPZ1 was obtained from CICC 20034 by adaptive evolution over 1000 generations of growth-based selection. The tributyrin consumption rate of strain SPZ1 reached 0.89 g/(L·h) which was 1.9-fold higher than that of the original strain. The MFA indicated that the 212% increase of tributyrin uptake flux contributed to the 556% increase of lipase flux. Consequently, the lipase activity (0.65 U/mL) of strain SPZ1 was 1.9-fold higher than that of the original strain. This was the highest lipase activity obtained by fermentation in synthetic medium reported for Bacillus strains. In complex culture medium, lipase activity of SPZ1 reached 3.3 U/mL.
Keywords: Adaptive evolution; Bioprocessing; Fermentation; Lipase; Metabolic flux analysis; Synthetic medium.