Abstract

To overcome protein aggregation and poor secretion during the production of the thermophilic glycogen branching enzyme from Aquifex aeolicus (AaGBE) in Bacillus subtilis, this study developed a two-stage fermentation strategy that decouples cell growth from protein folding. This physiological approach was combined with multilevel engineering, including a protease-deficient host, a signal peptide-independent secretion pathway, a high-copy plasmid with tandem promoters, and N-terminal coding sequence optimization. The integrated strategy effectively alleviated aggregation, achieving an extracellular AaGBE titer of 0.42 g/L and an activity of 1,012 U/mL─representing a 55-fold improvement over the unoptimized strain. The purified AaGBE exhibited a specific activity of 2389 U/mg and retained robust thermostability. These results demonstrate that decoupling cell growth from protein folding provides an effective strategy for the secretion of aggregation-prone industrial enzymes.