Zou_2025_Bioresour.Technol_436_133024

Corynebacterium glutamicum serves as an attractive cell factory for protein secretion, surface display and glutamate production. Nevertheless, its complex cell wall structure imposes a bottleneck on biomolecule export. Although cell wall engineering has shown potential to overcome this limitation, how engineering cell wall biosynthesis affects the export of diverse biomolecules remains poorly understood. This study systematically investigated the effects of engineering cell wall biosynthesis on protein and glutamate export in C. glutamicum, using the MtrAB two-component system (TCS) as a model target. To mitigate the growth defect caused by mtrAB deletion, an inositol-silencing system was employed to dynamically regulate mtrAB expression. Perturbation of mtrAB significantly improved secretion of multiple recombinant proteins under varied conditions. Further optimization of the inositol-silencing system in an inositol-catabolism-deficient strain prevented inducer consumption while maintaining high-performance. This strategy increased the activity of leaf-branch-compost cutinase variant (LCC-A2) by 2.58-fold in fed-batch fermentation, and also enhanced protein surface display and glutamate excretion in shake flasks. Proteomic profiling, reverse engineering, biochemical analyses and cell wall structural characterization indicate that cell wall peptidases play a pivotal role in enhancing cell wall permeability via regulation of cell wall remodelling, thereby promoting protein and glutamate export across the cell envelope. Overall, this work provides deep insights and useful targets for development of more efficient chassis cells through engineering cell wall biosynthesis.