Effects of spoIIE and rsfA Knockout on Spore Formation, Cell Growth, 2,3-Butanediol Synthesis and Heterologous Protein Expression in Bacillus licheniformis

Sporulation represents a complex metabolic reprogramming process in bacteria. In this study, we used CRISPR-Cpf1 to knock out spoIIE and rsfA in Bacillus licheniformis. The ΔspoIIE strain completely lost sporulataion capacity, while ΔrsfA showed a 25% reduction. Although viable cell counts decreased by 80.7% and 45.7%, respectively, glucose consumption and 2,3-butanediol synthesis remained unchanged, and acetoin synthesis increased by 19% in ΔspoIIE. Per-cell metabolic rates were significantly enhanced: glucose uptake increased 2.7–3.4-fold, acetoin synthesis 2.3–4.2-fold, 2,3-butanediol synthesis 1.7-fold, and heterologous protein expression 10–15-fold. These findings demonstrate that blocking sporulation liberates metabolic resources and enhances the specific productivity of vegetative cells, providing a strategy for engineering high-performance B. licheniformis cell factories.