Taguchi L9 optimization of BsPdaC-CD production in Escherichia coli

The production of sequence-defined partially acetylated chitooligosaccharides (paCOS) is a promising strategy for the valorization of chitin-rich waste into high-value bioactive compounds. The catalytic domain of a deacetylase from Bacillus subtilis (BsPdaC-CD), known to act on N-acetylglucosamine (GlcNAc) residues of chitooligosaccharides, represents a promising biocatalyst for paCOS production. In this work, a fed-batch process to produce the recombinant BsPdaC-CD in Escherichia coli BL21 was developed and optimized using a Taguchi L9 (3⁴) design of experiments. Across nine independent fed-batch processes, BsPdaC-CD titters and space-time yield (STY) varied widely, ranging from 167 to 1197 mg BsPdaC-CD/L and 2.2 to 22.5 mg BsPdaC-CD/(L*h), respectively. Temperature and feed rate were identified as the most influential factors affecting these outcomes. In both cases, the main variability was observed at low feeding rate and temperature, 5 mL/L/h and 22 °C respectively, which showed lower BsPdaC-CD productions. Subsequent experiments at 10 mL/L/h and 30 °C confirmed the robustness of the optimized conditions, consistently yielding 1.2 g/L of BsPdaC-CD within 50 h. These results highlight the utility of Taguchi-based experimental design for rapid identification of critical parameters in recombinant protein bioprocess optimization.