Enhancing Bacillus cereus antibacterial ability through improved cofactor supply.

Bacillus cereus 0-9 is a biocontrol microorganism that antagonizes Gram-positive bacteria and pathogenic fungi, such as Staphylococcus aureus and Gaeumannomyces graminis, through the secretion of antimicrobial peptides. However, its low antibacterial activity limits its biocontrol application. In this study, a significant enhancement in antibacterial activity against S. aureus was achieved by overexpressing glucose dehydrogenase from Bacillus subtilis (BsGDH) in B. cereus 0-9, expanding the activity from 6.98 to 11.59 U/mL, representing a 66% improvement. To further improve its biocontrol capability, we aimed to improve the catalytic efficiency of BsGDH by screening 11 low-conserved residues in the protein's second-shell via conservation analysis and molecular docking. Following three rounds of saturation mutagenesis, the specific enzyme activity and K_cat/K_m value of the variant N97F/N192S/E198G reached to 289.74 U/mg and 4.95 µM⁻¹·min⁻¹, representing 5.66 and 11.38 times greater than that of the wild-type BsGDH, respectively. Molecular docking suggested that residues Gly94, Gly14, and Ile191 form a triangular region enhancing substrate affinity and enzymatic activity. Furthermore, the Root Mean Square Fluctuation analysis from molecular dynamics showed significant conformational changes in five regions of the mutants (α2 helix, α3 helix, α5 helix + β4 sheet, α8 helix + β5 sheet, and α13-14 helix), increasing the flexibility of the active pocket. Ultimately, the antibacterial activity of B. cereus 0-9 expressing N97F/N192S/E198G reached 22.79 U/mL, 2.26 times higher than that of B. cereus 0-9. This study offers a promising candidate for enhancing NAD(P)⁺ metabolic cycling and antimicrobial peptide synthesis in cells for industrial applications.