High‐Yield Expression of Arylmalonate Decarboxylase in Escherichia coli Through High‐Cell‐Density Cultivation Strategies

This study develops and optimizes a high-cell-density cultivation (HCDC) of E. coli BL21 (DE3) to improve enzyme production utilizing DASGIP multibioreactor systems. By shifting from traditional shake flask methods to HCDC, we observed a 43-fold increase in biomass production, with a comparable mass-specific activity of the cell-free extract. The HCDC demonstrated a 30-fold increase in the active, soluble expression of arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica (BbAMDase) compared to conventional methodology. The protocol was designed to be readily adapted to various expression approaches and to provide a robust foundation for broader use in recombinant protein production. The HCDC was achieved with a linear glucose feed to promote robust cell growth. We optimized the glycerol feeding strategy during isopropyl β-D-1-thiogalactopyranoside (IPTG) induction to maximize AMDase production. Throughout the induction phase, we monitored both soluble expression yield and enzymatic activity, aiming to establish a process that is not only profitable but also efficient and stable.