Efficient, Flexible Autoinduction Expression Systems with Broad Initiation in .

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 , ComQXPA, activates the promoter P using ComX and ComA as an auto-inducer and a promoter activator, respectively. Here, we developed a series of flexible autoinduction expression systems in WB600 based on ComQXPA using a super-folder green fluorescent protein as the reporter. The -35 region of P was replaced with corresponding conserved sequences of σA-dependent promoters, yielding P with 85% enhanced strength. We then applied a semi-rational design within the spacer between the -35 and -15 regions of P to generate the QS promoter P, which generated 8.22-fold more expression than P. Based on P, 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 pullulanase generated using autoinduction expression systems in reached 80.2 U/mL, which was 36% more than that of the most powerful constitutive promoter P. Flexible autoinduction expression systems with diverse dynamic features have considerable potential for improving protein expression and metabolite production in .