Expression of xylanase XynB is synergistically controlled by two two-component systems in <i>Ruminiclostridium cellulolyticum</i>

Xylan, a major component of hemicellulose, is crucially targeted by xylanases for its breakdown. This study focuses on the free xylanase XynB from <i>Ruminiclostridium cellulolyticum</i> 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 <i>xynB</i> confirmed its essential role in xylan degradation by <i>R. cellulolyticum</i>. We further explored the transcription of <i>xynB</i> under various carbon sources and uncovered its regulatory mechanisms mediated by two-component systems (TCSs). We found that <i>xynB</i> 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 <i>R. cellulolyticum</i>, offering potential targets for the genetic engineering and process optimization of cellulolysis.IMPORTANCE<i>Ruminiclostridium cellulolyticum</i>, 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 <i>R. cellulolyticum</i>, particularly by detailing the synergistic effects of two two-component systems on the transcriptional regulation of <i>xynB</i>. This knowledge is essential for harnessing the full potential of <i>R. cellulolyticum</i> in the production of biofuels from lignocellulosic biomass.