Synonymous mutation of the N-terminal coding sequence (NCS) has been used to regulate gene expression. We here developed a statistical model to predict the effect of the NCSs on protein expression in Bacillus subtilis WB600. First, a synonymous mutation was performed within the first 10 residues of a superfolder green fluorescent protein to generate a library of 172 NCS synonymous mutants with different expression levels. A prediction model was then developed, which adopted G/C frequency at the third position of each codon and minimum free energy of mRNA as the independent variables, using multiple regression analysis between the 11 sequence parameters of the NCS and their fluorescence intensities. By designing the NCS of the 10 signal peptides de novo according to the model, the extracellular yield of B. subtilis pullulanase fused to each signal peptide was up-regulated by up to 515% or down-regulated by at most 79%. This work provided a candidate tool for fine-tuning gene expression or enzyme production in B. 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 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.

Although quorum sensing (QS) promoters that can autonomously activate gene expression have been identified and engineered in Bacillus subtilis, researchers focus on quantifying individual promoters while ignoring the interaction between other genetic regulatory elements. Here, we constructed the autoinduction expression modules consisting of promoters responsive to QS ComQXPA, ribosome binding sites (RBSs), and terminators. Using superfolder green fluorescent protein (sfGFP) as a reporter gene, three individual element libraries were generated from 945 promoters, 12,000 RBSs, and 425 terminators by random mutation, de novo design, and database mining strategies, respectively. Then, the efficiency of three libraries in regulating gene expression was further enhanced by engineering the core region of each optimal element. After hybridizing the element libraries, the generated expression modules exhibited a 627-fold range in regulating gene expression without significantly affecting the autoinduction initiation. Here, the hybrid modules with broad expression strength may benefit the application of QS-based autoinduction systems in B. subtilis.

The extracellular protease-deficient strain Bacillus subtilis WB600 is commonly used as a chassis cell for the production of industrial proteins. However, B. subtilis WB600 exhibits an increased susceptibility to cell lysis and a reduction in biomass. Inhibition of cell lysis by knocking out lytic genes will impair physiological function. Here, we dynamically inhibited cell lysis in B. subtilis WB600 to balance the impairment of physiological function with the accumulation of biomass. First, the inducible protein degradation systems (IPDSs) were constructed and used to investigate the effects of inhibiting cell lysis on biomass, cell morphology, and protein production at different times (using pullulanase as a test). The highest pullulanase activity was obtained at 20 h of inhibiting cell lysis, 184.8 U/mL, which was 44% higher than the activity of B. subtilis WB600. Then, to avoid addition of inducers, we introduced orthogonal quorum sensing and constructed autoinduction protein degradation systems (AIPDSs). The optimized AIPDS showed similar pullulanase activity to the optimal IPDS (20 h), 181.3 U/mL. Next, we constructed dual-signal input autoinduction protein degradation systems (DSI-AIPDSs) via AND gate to further address two deficiencies of AIPDS, onetime activation and damage to new cells. These DSI-AIPDSs were controlled by quorum sensing and stationary phase promoters that respond to population density and single-cell physiological state, respectively. Finally, the OD 600 and pullulanase activity of the strain with optimal DSI-AIPDS were 51% and 115% higher than those of B. subtilis WB600 in pullulanase production, respectively. We provided a B. subtilis chassis strain with considerable potential for biomass accumulation and enhanced protein production.