LAUSR

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, one-time 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 OD600 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.