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

Abstract

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 PsrfA 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 PsrfA 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 PS1E, which generated 8.22-fold more expression than PsrfA. Based on PS1E, 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.

Autoinduction Expression Modules for Regulating Gene Expression in Bacillus subtilis

“…These complex interactions increased the difficulty of predicting how elements affect gene expression in B. subtilis , making it practical to screen for powerful expression modules by combining elements. Compared with constitutive expression systems, autoinduction expression systems have more advantages in balancing cell growth and protein synthesis . Previously, by replacing the promoters of ComX and ComA in B. subtilis WB600, we generated the autoinduction expression systems varied in the autoinduction initiation time without significantly affecting the expression intensity .…”

“…Compared with constitutive expression systems, autoinduction expression systems have more advantages in balancing cell growth and protein synthesis. 38 Previously, by replacing the promoters of ComX and ComA in B. subtilis WB600, we generated the autoinduction expression systems varied in the autoinduction initiation time without significantly affecting the expression intensity. 24 Notably, the expression cassettes of ComX and ComA have been integrated into the genome of B. subtilis WB600.…”

Autoinduction AND Gate Inhibits Cell Lysis to Enhance Protein Production in Bacillus subtilis Controlled by Population Density and Cell Physiological State

“…The plasmids and strains used in this study are listed in Table S3 and S4. The plasmid P xylA -NprB encoding the xylose operon and plasmid pP43NMK-srfA/sfGFP encoding the P srfA promoter, superfolder green fluorescent protein (sfGFP), and mCherry were stored in our lab . The B. deramificans pullulanase (Pul) expression plasmid pH33/Pul was constructed as described in our previous work .…”

“…The resulting plasmid was transformed into B. subtilis WB600 containing native endogenous ComQXPA QS to construct engineering bacteria No. 1; the fragment containing the luxIR gene cluster was cloned into the plasmid constructed above, and transformed into B. subtilis WB600 and previously constructed modified B. subtilis WB600(SX) with enhanced ComQXPA (expressing comX and comA by strong promoter P sigW and P xynA , respectively) 24 to construct engineering bacteria No. 2 and 3, respectively (Figure 3B and 3C).…”

“…The plasmid P xylA -NprB encoding the xylose operon and plasmid pP43NMK-srfA/sfGFP encoding the P srfA promoter, superfolder green fluorescent protein (sfGFP), and mCherry were stored in our lab. 24 The B. deramificans pullulanase (Pul) expression plasmid pH33/Pul was constructed as described in our previous work. 33 The pUC-lux genes encoding luxI, luxR, and PluxI were constructed by Sangon Biotech (Shanghai, China).…”