Construction of an alkaline protease overproducer strain based on Bacillus licheniformis 2709 using an integrative approach.

Bacillus licheniformis 2709 is a potential cell factory for the production of alkaline protease AprE, which has important value in industrial application but still lacks sufficient production capacity. To address this problem, we investigated the effects of the secretory viscous materials on the synthesis of AprE, which might seriously affect the industrial fermentation. Furthermore, an iterative chromosomal integration strategy at various chromosomal loci was implemented to achieve stable high-level expression of AprE in B. licheniformis 2709. The host was genetically modified by disrupting the native pgs cluster controlling the biosynthesis of viscous poly-glutamic acid identified in the study by GC/MS, generating a mutant with significantly higher biomass and better bioreactor performance. We further enhanced the expression of alkaline protease by integrating two additional aprE expression cassettes into the genome, generating the integration mutant BL ∆UEP-3 with three aprE expression cassettes, whose AprE enzyme activity in shake flasks reached 25,736 ± 997 U/mL, which was 136% higher than that of the original strain, while the aprE transcription level increased 4.05 times. Thus, an AprE high-yielding strain with excellent fermentation traits was engineered, which was more suitable for bulk-production. Finally, the AprE titer was further increased in a 5-L fermenter, reaching 57,763 ± 1039 U/mL. In summary, genetic modification is an enabling technology for enhancing enzyme production by eliminating the unfavorable characteristics of the host and optimizing the expression of aprE through iterative chromosomal integration. We believe that the protocol developed in this study provides a valuable reference for chromosomal overexpression of proteins or bioactive molecules in other Bacillus species.