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 tran-scriptional 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 po-tential negative regulatory factor binding site in the genome. The AprE enzyme activity in shake flasks of the genomic mutant BL Delta 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 tran-scriptional regulatory network of aprE and provide important theoretical guidance for further enhancing the yield of AprE.

Automated summary

Bacillus licheniformis 2709 is an important industrial producer of alkaline protease (AprE), and this study identified the UP elements of the aprE gene and demonstrated the lack of significant effect of CggR, FruR, and YhcZ transcription factors on aprE expression. Enhanced aprE expression and enzyme activity were achieved by deleting a negative regulatory factor binding site in the genome. These findings provide valuable insights into the high-level production of proteins in other Bacillus species.