Spo0A can efficiently enhance the expression of the alkaline protease gene aprE in Bacillus licheniformis by specifically binding to its regulatory region

The expression of enzymes in Bacillus licheniformis, such as the valuable extracellular alkaline protease AprE, is highly regulated by a complex transcriptional regulation mechanism. Here, we found that the transcript abun-dance of aprE varies N343-fold in response to the supply of nutrients or to environmental challenges. To identify the underlying regulatory mechanism, the core promoter of aprE and several important upstream regulatory re-gions outside the promoter were firstly confirmed by 5 '-RACE and mutagenesis experiments. The specific pro-teins that bind to the identified sequences were subsequently captured by DNA pull-down experiments, which yielded the transcriptional factors (TFs) Spo0A, CggR, FruR, YhcZ, as well as fragments of functionally unassigned proteins. Further electrophoretic mobility shift assay (EMSA) and DNase I foot-printing experiments indicated that Spo0A can directly bind to the region from -92 to -118 nucleotides upstream of the transcription start site, and the deletion of this specific region drastically decreased the production of AprE. Taken together, these results indicated that the expression of aprE was mainly regulated by the interplay between Spo0A and its cog-nate DNA sequence, which was successfully applied to overproduce AprE in a genetically modified host harboring three aprE expression cassettes. The DNA binding proteins may serve to increase the efficiency of transcription by creating an additional binding site for RNA polymerase. The discovery of this mechanism significantly increases our understanding of the aprE transcription mechanism, which is of great importance for AprE overproduction. (c) 2020 Elsevier B.V. All rights reserved.