Comprehensive O -Phosphoproteomic Landscape of Corynebacterium glutamicum

Abstract Corynebacterium glutamicum serves as a pivotal industrial platform organism, accounting for an annual global amino acid output of several million tons. Elucidating its adaptive mechanisms under diverse environmental perturbations during high-density fermentation is fundamental to advanced metabolic engineering and strain optimization. Despite the critical role of protein phosphorylation in cellular adaptation, the phosphoproteome of C. glutamicum remains largely unexplored. With fewer than 50 phosphoproteins currently identified, this knowledge gap hinders the elucidation of regulatory networks underlying industrial stress tolerance. In this study, we implemented a highly robust workflow for O-phosphopeptides enrichment utilizing titanium-based immobilized metal affinity chromatography (Ti-IMAC). By integrating an offline reversed-phase prefractionation strategy and a combination of Electron-Transfer/Higher-Energy Collision Dissociation (EThcD) and Higher-Energy Collision Dissociation (HCD) mass spectrometry fragmentation techniques, we established the most comprehensive phosphoproteomic data set for this species to our knowledge. We identified 874 phosphoproteins and 2,137 phosphorylation sites, representing a more than 20-fold increase over existing literature. Furthermore, we delineated the dynamic phosphoproteome under nitrogen starvation, uncovering novel site-specific phosphorylation events on key proteins such as OdhI (Ser2/Thr7), FtsZ (Thr387), and EF-G (Thr48). This study provides a high-quality, global phosphoprotein resource, that elucidates the molecular insights into the orchestration of environmental adaptation and metabolic regulation of C. glutamicum.