Identification and engineering of the aprE regulatory region and relevant regulatory proteins in Bacillus licheniformis 2709.

Bacillus licheniformis 2709 is the main industrial producer of alkaline protease (AprE), but its biosynthesis is strictly controlled by a highly sophisticated transcriptional network. In this study, the UP elements of aprE located 74-98, 98-119 and 140-340 bp upstream of the transcriptional start site (TSS) were identified, which presented obvious effects on the transcription of aprE. To further analyze the transcriptional mechanism, the specific proteins binding to the approximately 500-bp DNA sequences were subsequently captured by reverse-chromatin immunoprecipitation (reverse-ChIP) and DNA pull-down (DPD) assays, which captured the transcriptional factors CggR, FruR, and YhcZ. The study demonstrated that CggR, FruR and YhcZ had no significant effect on cell growth and aprE expression. Then, aprE expression was significantly enhanced by deleting a potential negative regulatory factor binding site in the genome. The AprE enzyme activity in shake flasks of the genomic mutant BL ∆1 was 47% higher than in the original strain, while the aprE transcription level increased 3.16 times. The protocol established in this study provides a valuable reference for the high-level production of proteins in other Bacillus species. At the same time, it will help reveal the molecular mechanism of the transcriptional regulatory network of aprE and provide important theoretical guidance for further enhancing the yield of AprE.