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

Abstract

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.

Reducing Cellular Autolysis of Bacillus subtilis to Improve Keratinase Production

“…In this study, we selected the peptidoglycan hydrolase genes lytC , lytD , lytE , lytF , lytG , lytH , cwlC , and cwlS and their associated transcriptional regulator sigD , which act during the growth phase, and the prophage-related peptidoglycan hydrolase genes xlyA , xpf , cwlA , and pcfA as well as the self-feeding-related genes skfA and spbC to monitor for their transcript levels using qRT-PCR. ,− It can be seen from the changes in transcription levels that the expression of these genes is upregulated to varying degrees in the middle and late stages of fermentation (Figure ), which is consistent with the autolysis phenotype of B. subtilis cells.…”