Fine-Regulating the Carbon Flux of l -Isoleucine Producing Corynebacterium glutamicum WM001 for Efficient l -Threonine Production

l -Threonine, an essential amino acid, is widely used in various industries, with an annually growing demand. However, the present Corynebacterium glutamicum strains are difficult to achieve industrialization of l -threonine due to low yield and purity. In this study, we engineered an l -isoleucine-producing C. glutamicum 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- thrE 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 C. glutamicum, providing possibilities for industrial-scale production.