Expression of xylanase XynB is synergistically controlled by two two-component systems in Ruminiclostridium...

Xylan, a major component of hemicellulose, is crucially targeted by xylanases for its breakdown. This study focuses on the free xylanase XynB from Ruminiclostridium cellulolyticum to elucidate its expression and regulatory mechanisms. We successfully achieved heterologous expression and purification of recombinant XynB, verifying its enzymatic activity specifically against xylan. The mutation of xynB confirmed its essential role in xylan degradation by R. cellulolyticum. We further explored the transcription of xynB under various carbon sources and uncovered its regulatory mechanisms mediated by two-component systems (TCSs). We found that xynB transcription is activated by the xylan-sensing TCS (XuaDRS) and repressed by the cellobiose-sensing TCS (CuaDRS). This research enriches our understanding of the regulatory mechanisms governing the activity and expression of free xylanases like XynB from R. cellulolyticum, offering potential targets for the genetic engineering and process optimization of cellulolysis.IMPORTANCERuminiclostridium cellulolyticum, an anaerobic, mesophilic, and cellulolytic gram-positive bacterium, is a model organism for the microbial degradation of plant cell wall polysaccharides and a promising host for biofuel production from lignocelluloses. The degradation process of lignocellulosic materials is complex due to their intricate structure and interlocking complexity. XynB, a GH11 family xylanase, plays a significant role in the breakdown of xylan, a major constituent of hemicelluloses. Our study reveals the molecular mechanisms that link the specific adaptation of xylan utilization with the general stress response in the regulatory network of R. cellulolyticum, particularly by detailing the synergistic effects of two two-component systems on the transcriptional regulation of xynB. This knowledge is essential for harnessing the full potential of R. cellulolyticum in the production of biofuels from lignocellulosic biomass.