Efficient, Flexible Autoinduction Expression Systems with Broad Initiation in Bacillus subtilis

Low expression levels and inflexible induction initiation have been the main obstacles to produce proteins using bacterial quorum sensing (QS). The typical QS system in Bacillus subtilis, ComQXPA, activates the promoter P_srfA using ComX and ComA as an auto-inducer and a promoter activator, respectively. Here, we developed a series of flexible autoinduction expression systems in B. subtilis WB600 based on ComQXPA using a super-folder green fluorescent protein as the reporter. The -35 region of P_srfA was replaced with corresponding conserved sequences of σA-dependent promoters, yielding P₁ with 85% enhanced strength. We then applied a semi-rational design within the spacer between the -35 and -15 regions of P₁ to generate the QS promoter P_S1E, which generated 8.22-fold more expression than P_srfA. Based on P_S1E, we finally obtained three types of autoinduction expression systems with initiation ranging from 1.5-9.5 h by optimizing the combination of the promoters for ComX and ComA. The yield of Bacillus deramificans pullulanase generated using autoinduction expression systems in B. subtilis reached 80.2 U/mL, which was 36% more than that of the most powerful constitutive promoter P₅₆₆. Flexible autoinduction expression systems with diverse dynamic features have considerable potential for improving protein expression and metabolite production in B. subtilis.