Engineering homoserine kinase for competitive inhibition release to enhance l-threonine production in Corynebacterium...

l-threonine is widely used in the food, pharmaceutical, cosmetic, and feed industries. However, the activity of homoserine kinase (HK) in Corynebacterium glutamicum is competitively inhibited by l-threonine, restricting the efficient production of l-threonine. To address this inhibition, we identified a critical residue Arg²¹² and subsequently generated an optimal mutant R212Q via site-directed saturation mutagenesis. Kinetic characterization revealed that the HK^R212Q variant exhibited both enhanced binding affinity for l-homoserine and a 2-fold improvement in catalytic efficiency. Molecular docking simulations demonstrated that the mutated substrate-binding pocket sterically hindered l-threonine binding while accommodating l-homoserine. Further mechanistic insights were obtained through molecular dynamics simulations, which uncovered ligand selectivity determinants mediated by dynamic flexibility changes in three functional regions. Finally, we achieved 86.4 g/L of l-threonine with a yield of 0.28 g/g glucose by overexpressing the efflux transporter ThrE in the engineered strain R212Q/pXTuf-thrE, representing the highest l-threonine production to date. The Arg²¹² residue identified in this study represents the first characterized site capable of relieving competitive inhibition in HK family. The mechanistic elucidation provides valuable insights for engineering other kinase-family proteins to overcome similar metabolic constraints.