Bacillus licheniformisglobal nitrogen homeostatic regulator TnrA is a direct repressor ofpgsBCAAtranscription in Poly-γ-glutamic acid synthesis

Abstract

AbstractPoly-γ-glutamic acid (γ-PGA) is a multifunctional and naturally occurring biopolymer made from D- and L-glutamate as monomers, which is mainly produced byBacillus. Few reports have been focused on the regulation network of γ-PGA synthesis in recent years. In this study, we have demonstrated thatBacillus licheniformisglobal nitrogen homeostatic regulator TnrA is a direct repressor of γ-PGA synthase PgsBCAA in γ-PGA synthesis. First, our results confirmed that TnrA repressed γ-PGA synthesis, deficiency oftnrAled to a 22.03% increase of γ-PGA production, and the γ-PGA yield was decreased by 19.02% in the TnrA overexpression strain. Transcriptional level assay illustrated that the γ-PGA synthase gene clusterpgsBCAAtranscriptional level were increased in thetnrAdeficient strain WXΔtnrA, indicating that γ-PGA synthase PgsBCAA was negatively regulated by TnrA. Furthermore, electrophoretic mobility shift assay (EMSA) and enzyme expression assays confirmed that TnrA directly repressedpgsBCAAexpression by binding topgsBCAApromoter, and the TnrA-binding site “CGTCGTCTTCTGTTACA” in thepgsBCAApromoter was identified by sequence and software analysis. Finally, computer analysis confirmed that the transcription regulations of γ-PGA synthase PgsBCAA by TnrA were highly conserved in other well-studiedBacillusspecies (B.licheniformis,Bacillus subtilisandBacillus amyloliquefaciens). Collectively, our results implied that TnrA was a direct repressor forpgsBCAAexpression in γ-PGA synthesis, and this research provided a novel regulatory mechanism underlying γ-PGA synthesis, and a new approach that deficiency oftnrAincreases γ-PGA production.Importanceγ-PGA is an important biopolymer with many applications, which is mainly produced byBacillusspecies. Glutamic acid is the precursor for γ-PGA synthesis, which is catalyzed by the γ-PGA synthase PgsBCAA. Previously, the expression of PgsBCAA was reported to be regulated by ComA-ComP and DegS-DegU, DegQ and SwrA systems, however, few researches were focused on the regulation network of γ-PGA synthesis in recent years. In our research, the γ-PGA synthase PgsBCAA was confirmed to be negatively regulated by the nitrogen metabolism regulator TnrA, and the TnrA binding site in thepgsBCAApromoter was identified inB. licheniformisWX-02. Furthermore, computer analysis implied that TnrA-mediated regulation effect onpgsBCAAexpression was highly conserved inBacillus. Collectively, our research provided a novel regulatory mechanism underlying γ-PGA synthesis, and a new approach that deficiency oftnrAincreases γ-PGA production.