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

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.