Stress response regulation to extracellular polymeric substances biosynthesis in Bacillus licheniformis

The diverse metabolic mechanisms underlying bacterial extracellular polymeric substances give rise to a wide array of components with distinct functionalities, including exopolysaccharides (EPS) and poly-γ-glutamic acid (γ-PGA). The coordinated synthesis of various types of extracellular polymeric substances necessitates comprehensive investigation from a global regulatory perspective. In this study, we examined the impact of multiple environmental stressors on Bacillus species, revealing that the EPS and γ-PGA produced respond to stress through metabolic and cellular process reorganization. The overexpression of global transcriptional regulators influenced the production of EPS and γ-PGA differently. Specifically, quorum sensing-related global regulators such as rsbRA, rapA, and the carbon utilization regulator ccpA-2 were found to enhance EPS synthesis. Conversely, positive global transcriptional regulators associated with γ-PGA synthesis included carbon and nitrogen utilization-related regulators ccpA-2, cggR, and nrgB. Notably, the global regulators nrgB and cggR increased γ-PGA production by 33.64% and 44.14%, respectively, while this enhancement was accompanied by a concomitant reduction in EPS production. In B. licheniformis, omics analyses have elucidated critical pathways and metabolites implicated in stress response mechanisms that induce alterations in amino acid metabolism, carbon source utilization, alongside the activation of global regulatory elements. These studies indicated that nrgB predominantly governs downstream genes associated with carbon metabolism, energy metabolism, signal transduction, and membrane transport processes. This work combines stress induction strategies and global transcription machinery engineering for investigating the coordinated synthesis of various types of extracellular polymeric substances, which has not been explored before. The insights gained from our research contribute to a deeper understanding of the regulatory networks governing the competition between γ-PGA and EPS, thereby providing a theoretical basis for the engineered modification of Bacillus licheniformis aimed at optimizing the production of extracellular polymeric substances.