System reconstruction of Bacillus licheniformis for efficient expression of alkaline protease

Abstract

Industrial enzymes are widely used in diverse applications, but low productivity limits their further widespread utilization. This research aimed to develop high-performance alkaline protease (AprE) expression strains of Bacillus licheniformis through element optimization and modular engineering. Firstly, the aprE gene expression cassette was systematically optimized through element engineering. To minimize host background interference, five large gene fragments were deleted from the genome of B. licheniformis DW2. This expression cassette and genome-reduced strain resulted in 5.77-, 4.84- and 1.31-fold increases in the activities of alkaline protease, nattokinase and chitinase, respectively. Crucially, metabolomics analysis then served as the pivotal discovery tool, revealing that high expression of AprE was constrained by insufficient precursor amino acids and excessive metabolic overflow. Subsequently, the amino acid biosynthesis, energy metabolism, overflow metabolism, and cell membrane/wall modules of the strain were successively modified. The final AprE expression host DM6E10 achieved a remarkable enzyme activity of 34,343 U/mL, with a maximum activity of 107,100 U/mL in a 5-L bioreactor. This study built an efficient cell factory for AprE production and provided insights for the optimization of other protein expression hosts.