Engineering the Heterologous Expression of a Thermophilic Glycogen Branching Enzyme in Bacillus subtilis

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.