Tailored high-cell-density fed-batch cultivation and induction strategies for optimized production of a low-immunogenic L-asparaginase mutant in Escherichia coli

An integrated upstream process was developed for the high-level production of a low-immunogenic L-asparaginase mutant (EcaII P40S/S206C) in Escherichia coli, combining high-cell-density fed-batch cultivation with controlled induction and post-induction nutrient feeding. Biomass accumulation was achieved by exponential substrate feeding, followed by pH-stat-based nutrient supply during the expression phase. Induction protocols were optimized using biomass-specific IPTG dosing (µmol IPTG/g_cell), applied either as a pulse or via exponential feeding. This combined strategy resulted in a maximum volumetric enzyme activity of 134,516 U/L and a specific productivity of 3387 U/g_cell, corresponding to 91-fold and 24-fold increases, respectively, compared with standard batch cultivation. Overall, these results indicate that the coordinated control of feeding strategy and induction conditions substantially improves both volumetric and cell-specific productivity of recombinant L-asparaginase and provides a robust upstream framework for the efficient manufacture of improved enzyme variants.