S267P mutation of OxyR regulator in Zymomonas mobilis: mechanism of oxidative stress tolerance and applications in cellulosic hydrolysate fermentation and oxidative stress monitoring

The peroxide-sensing transcriptional regulator OxyR plays a pivotal role in bacterial oxidative stress responses. The OxyR S267P mutation was previously identified in Zymomonas mobilis ZMNP, a mutant strain with enhanced hydrolysate tolerance. To investigate the biological function of revert strain and the corresponding mutant, phenotypic, transcriptomic, structural, and functional analyses were performed. The fermentation performance of strain ZMNPC in toxic xylose mother liquor had similar slow substrate utilization profile as that of the wild-type strain ZM4 with an approximate 20 h lag behind strain ZMNP. Transcriptomic analysis revealed constitutive downregulation of genes involved in reactive oxygen species (ROS) detoxification, cysteine biosynthesis, redox homeostasis, and macromolecular repair in strain ZMNPC, which is consistent with higher intracellular ROS levels and reduced survival under H 2 O 2 . Protein structural modeling demonstrated that S267P relieves steric hindrance near disulfide bond-forming cysteines, promoting OxyR activation, molecular dynamics simulations indicated that the OxyR mutation stabilizes the oxidized conformation, binding reactions showing enhanced DNA-binding affinity of OxyR S267P , and gene expression results confirmed OxyR S267P upregulates ROS clearance genes under different inhibitor stress conditions. We further developed oxidative-stress biosensors OxyR S267P –P1732 and OxyR S267P –P1753 based on this regulatory module. Our study confirms OxyR S267P as a key determinant of robust oxidative stress tolerance in Z. mobilis , providing mechanistic insights to guide the development of novel regulatory tools for biosensing and metabolic engineering.