Fine-Regulating the Carbon Flux of <scp>l</scp>-Isoleucine Producing <i>Corynebacterium glutamicum</i> WM001 for Efficient <scp>l</scp>-Threonine Production

l-Threonine, an essential amino acid, is widely used in various industries, with an annually growing demand. However, the present <i>Corynebacterium glutamicum</i> strains are difficult to achieve industrialization of l-threonine due to low yield and purity. In this study, we engineered an l-isoleucine-producing <i>C. glutamicum</i> WM001 to efficiently produce l-threonine by finely regulating the carbon flux. First, the threonine dehydratase in WM001 was mutated to lower the level of l-isoleucine production, then the homoserine dehydrogenase and aspartate kinase were mutated to release the feedback inhibition of l-threonine, and the resulting strain TWZ006 produced 14.2 g/L l-threonine. Subsequently, aspartate ammonia-lyase and aspartate transaminase were overexpressed to accumulate the precursor l-aspartate. Next, phosphoenolpyruvate carboxylase, pyruvate carboxylase and pyruvate kinase were overexpressed, and phosphoenolpyruvate carboxykinase, oxaloacetate decarboxylase were inactivated to fine-regulate the carbon flux among oxaloacetate, pyruvate and phosphoenolpyruvate. The resulting strain TWZ017 produced 21.5 g/L l-threonine. Finally, dihydrodipicolinate synthase was mutated with strong allosteric inhibition from l-lysine to significantly decrease byproducts accumulation, l-threonine export was optimized, and the final engineered strain TWZ024/pXTuf-<i>thrE</i> produced 78.3 g/L of l-threonine with the yield of 0.33 g/g glucose and the productivity of 0.82 g/L/h in a 7 L bioreactor. To the best of our knowledge, this represents the highest l-threonine production in <i>C. glutamicum</i>, providing possibilities for industrial-scale production.