Engineering strong and stress-responsive promoters in Bacillus subtilis by interlocking sigma factor binding motifs

Prokaryotic gene expression is largely regulated on transcriptional levels with the involvement of promoters, RNA polymerase and sigma factors. Developing new promoters to customize gene transcriptional regulation becomes increasingly demanded in synthetic biology and biotechnology. In this study, we designed synthetic promoters in the Gram-positive model bacterium Bacillus subtilis by interlocking the binding motifs of σ^A for house-keeping gene expression and that of two alternative sigma factors σ^H and σ^B which are involved in responding post-exponential growth and general stress, respectively. The developed promoters are recognized by multiple sigma factors and hence generate strong transcriptional strength when host cells grow under normal or stressed conditions. With green fluorescent protein as the reporter, a set of strong promoters were identified, in which the transcription activities of P_HA-1, P_HAB-4, P_HAB-7 were 18.6, 4.1, 3.3 fold of that of the commonly used promoter P₄₃, respectively. Moreover, some of the promoters such as P_HA-1, P_HAB-4, P_HAB-7, P_BA-2 displayed increased transcriptional activities in response to high salinity or low pH. The promoters developed in this study should enrich the biotechnological toolboxes of B. subtilis.