In this study, surfactant-induced mediated cell membrane permeabilization was explored as a strategy to increase the production of menaquinone-7 (MK-7) by an engineered strain, BS168-ΔSinR. Various surfactants were screened for their effectiveness in improving the MK-7 production of BS168-ΔSinR. Cell morphology was observed by scanning electron microscopy (SEM), and cell membrane permeability was detected by flow cytometry and fluorescence microscopy. RNA-seq was used to analyze the expression levels of genes related to MK-7 production. The results showed that the addition of 0.7% Brij-58 significantly increased the yield of MK-7, resulting in a 71.95% and 332.29% increase in the yield of total and extracellular MK-7 when compared with the control strain, respectively. The results from SEM, flow cytometry, and fluorescence microscopy demonstrate that Brij-58-mediated membrane permeabilization was significantly associated with enhanced MK-7 production. RNA-seq analysis showed that the expression levels of genes involved in MK-7 biosynthesis and the antioxidant defense system were generally up-regulated, while the expression levels of genes involved in spore formation were down-regulated. The results of this study provide a theoretical basis for the industrial production of MK-7, and reveal the mechanism by which surfactant-mediated membrane permeabilization increases the yield of MK-7.

To obtain alkaline protease with enhanced thermal stability and catalytic activity, this study focused on the alkaline protease AprEbl derived from Bacillus licheniformis B66. Using molecular dynamic simulations, residues N183, G186, S265, S267, and Y320 in AprEbl were identified as highly flexible regions. Computer-aided design was employed to propose mutation sites, and five single-point mutant enzymes were generated using site-directed mutagenesis. Their enzymatic properties were subsequently investigated. Two advantageous mutants were selected for a second round of combinatorial mutagenesis. The results demonstrated that the double mutant S265H/S267F exhibited significantly improved thermal stability compared with the wild-type enzyme, although their specific activities were on par with each other. The half-life of this mutant increased by 7.35-fold at 55 ℃ and 5.01-fold at 75 ℃. Other single-point mutants also displayed better high-temperature tolerance than the original enzyme. This study provides a theoretical foundation for improving the enzymatic properties of alkaline proteases via protein engineering to meet industrial demands.