Structure-based modeling and engineering of Corynebacterium glutamicum LysE transporter for efficient extrusion of L-arginine

Transporter engineering is an effective strategy for enhancing the transmembrane transfer of target substrates and alleviating feedback inhibition in microbial cells. The LysE transporter, a key indicator of both L-arginine (L-Arg) and L-lysine (L-Lys) secretion in Corynebacterium glutamicum, plays a crucial role in the efficient synthesis of these amino acids. Owing to its broad substrate specificity, a LysE mutant with high substrate specificity for L-Arg extrusion is essential for achieving high production. In this study, we constructed a structural model and identified that LysE possesses a simplified structure of the characteristic LeuT-fold pattern, including parallel discontinuous helices, three highly conserved motifs, and several critical residues within its substrate binding pocket. Molecular docking and virtual site-saturation mutagenesis identified key hotspot residues for modulating LysE transport activity, with the A156Y and A156V mutants exhibiting significantly enhanced L-Arg extrusion. Iterative saturation mutagenesis at site L49 yielded the A156VL49T mutant, which was characterized by a 32.4% increase in growth under 30 g L^-1 L-canavanine and a 17.4% reduction upon exposure to 0.3 g L^-1 H-Lys-Ala-OH. With altered substrate specificity and improved efficiency in L-Arg extrusion, the A156VL49T mutant holds promise for metabolic engineering of C. glutamicum to enhance L-Arg production.