Rewiring the PEP-pyruvate-oxaloacetate node through coordinated fine- tuning of gltA and pykF expression for L-threonine production in wild-type Escherichia coli

L-threonine is an essential amino acid for humans and monogastric animals, and produced industrially on a large scale by microbial fermentation. Maintaining a balanced allocation of carbon resources between cell growth and product formation is critical for achieving a high titer. Most previous studies have focused on deleting the pathways that compete with L-threonine biosynthesis, whereas few have examined fine-tuning of these pathways. Here, we rebalanced carbon distribution at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate (PPO) node by coordinately regulating citrate synthase ( gltA ) and pyruvate kinase Ⅰ ( pykF ). Growth-phase-responsive promoters maintain carbon flux through the tricarboxylic acid (TCA) cycle during exponential growth and redirected excess flux was redirected toward L-threonine biosynthesis after entry into the stationary phase. We then combined this module with reinforcement of the rate-limiting steps ( aspC , rhtC , and asd ), modulation of phosphoenolpyruvate carboxylase ( ppc ), and chromosomal integration of all expression cassettes. The resulting plasmid-free strain, WS026, required neither exogenous inducers nor antibiotics. It produced 28.3 g/L L-threonine in shake-flask culture, corresponding to a yield of 0.71 g/g glucose and an approximately 283-fold increase over the control strain. In a bioreactor, WS026 produced 101.6 g/L L-threonine. These results indicate that the fine-tuning of competing pathways, rather than deleting them, can improve flux distribution at the PPO node in E. coli . The strategy may also be useful for producing other aspartate-family amino acids.