Identification of multiple regulatory genes involved in TGase production in DSM 40587.

Microbial transglutaminase (TGase) is a protein that is secreted in a mature form and finds wide applications in meat products, tissue scaffold crosslinking, and textile engineering. is the only licensed producer of TGase. However, increasing the production of TGase using metabolic engineering and heterologous expression approaches has encountered challenges in meeting industrial demands. Therefore, it is necessary to identify the regulatory networks involved in TGase biosynthesis to establish a stable and highly efficient TGase cell factory. In this study, we employed a DNA-affinity capture assay and mass spectrometry analysis to discover several transcription factors. Among the candidates, eight were selected and found to impact TGase biosynthesis. Notably, SMDS_4150, an AdpA-family regulator, exhibited a significant influence and was hence named AdpA . Through electrophoretic mobility shift assays, we determined that AdpA regulates TGase biosynthesis by directly repressing the transcription of and indirectly inhibiting the transcription of . The latter gene encodes a LytR-family positive regulator of TGase biosynthesis. Additionally, AdpA exhibited negative regulation of its own transcription. To further enhance TGase production, we combined the overexpression of with the repression of , resulting in a remarkable improvement in TGase titer from 28.67 to 52.0 U/mL, representing an 81.37% increase. This study establishes AdpA as a versatile regulator involved in coordinating enzyme biosynthesis in species. Furthermore, we elucidated a cascaded regulatory network governing TGase production.