Optimization of Isoamylase Production in Escherichia coli Under Different Nutrient Limitation Conditions

Isoamylase (IA), a starch debranching enzyme that cleaves α-1,6-d-glycosidic bonds in amylose, is used in various industries. However, its low expression in Escherichia coli BL21 (DE3) limits its application, necessitating fermentation optimization. Limiting carbon (C) and other nutrients can optimize bacterial growth for better IA expression. Herein, using E. coli expressing IA-mCherry as the research object, we explored whether low concentrations of the nutrients nitrogen (N), phosphorus (P), magnesium (Mg), and sulfur (S) have a limiting effect on bacterial growth. We subsequently investigated differences in expression under restricted nutrient conditions and performed batch fermentation experiments under various conditions to determine optimal nutrient limitation conditions. Low concentrations of N and P had a limiting effect on bacterial growth. IA expression in the P-limited group was increased by ∼10% but limiting N had no influence. Combinations of C and P restriction during fed-batch fermentation showed that the total yield was 73.73% higher under C excess and P-limited conditions than under other combinations, while corresponding expression per unit biomass was 24.42% higher. The results revealed that P restriction plays a key role in increasing IA production in E. coli.