Poly-γ-Glutamic Acid Production by Engineering a DegU Quorum-Sensing Circuit in Bacillus subtilis

Abstract

As a natural biological macromolecule, γ-polyglutamic acid (γ-PGA) plays a significant role in medicine, food, and cosmetic industries owing to its unique properties of biocompatibility, biodegradability, water solubility, and viscosity. Although many strategies have been adopted to increase the yield of γ-PGA in Bacillus subtilis, the effectiveness of these common approaches is not high because the strong viscosity affects cell growth. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. A modular PhrQ-RapQ-DegU QS system is developed based on promoter PD4, which is upregulated by phosphorylated DegU (DegU-P). In this study, first, we analyzed the DegU-based gene expression regulation system in B. subtilis 168. We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters and degU region. Furthermore, we constructed a PhrQ-RapQ-DegU QS system to dynamically control the synthesis of γ-PGA in BS168. Cell growth and efficient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of γ-PGA to 6.53-fold of that by static regulation in a 3 L bioreactor, which verified the effectiveness of this strategy. In summary, the PhrQ-RapQ-DegU QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which can be stretched to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes

“…subtilis that governs the expression of numerous genes and operators crucial for the development of biofilms and spores . The DegU regulon is extensive and encompasses genes associated with motility, including flagellum synthesis and biofilm development . ComA plays a crucial role in the quorum sensing mechanism controlled by the comXQPA genes.…”

“…32 The DegU regulon is extensive and encompasses genes associated with motility, including flagellum synthesis and biofilm development. 33 ComA plays a crucial role in the quorum sensing mechanism controlled by the comXQPA genes. ComA ∼ P, which is phosphorylated, regulates the production of certain secondary metabolites and the advancement of cellular functionalities.…”

Spatiotemporal Gene Expression by a Genetic Circuit for Chemical Production in Escherichia coli

“…Unlike natural cells, the engineered strain faces more rigorous environmental fluctuation (nutrient, high concentration of the target compound, toxicity of metabolites, and oxidative stress), which needs the reconstitution of the metabolic network spatiotemporally. A series of QS-based dynamic regulation strategies for metabolic flux rewriting has been applied in chemical production, ,, but different strengths of dynamic promoters are still missing during pathway reconstruction, especially those metabolic rate-limiting nodes that need to be extensively enhanced. For example, the esaR promoter and luxR promoter could dynamically control protein expression at the transcriptional level, but the strength of these promoters cannot satisfy the engineered pathway.…”

Application of Quorum Sensing in Metabolic Engineering

“…Similarly, Hu et al built a modular PhrQ-RapQ-DegU QS system employing DegU as the phospho-transduction pathway to synthesize γ-polyglutamic acid (γ-PGA) in B. subtilis . In conjunction with the promoter library, the yield of γ-PGA increased 6.53-fold more than that by static regulation …”

“…In conjunction with the promoter library, the yield of γ-PGA increased 6.53-fold more than that by static regulation. 47 Compared with the well-studied LuxI/LuxR system, the EsaI/EsaR and Phr60-Rap60-Spo0A systems are dual-function QS regulatory systems and suited to perform up-regulation and down-regulation in complex metabolic pathways. As a natural system exists in microorganisms, QS system is not evolved to control the synthesis of target products.…”