Optimizing phosphate feeding through metabolomics and capacitance-based real‑time control enhances L‑threonine production in Escherichia coli

L-threonine is an essential amino acid in high global demand, and its efficient biosynthesis is a major goal of industrial biotechnology. In industrial fermentation, phosphate is typically supplied through fixed or manual feeding strategies that fail to accommodate real-time changes in cell growth and metabolic demand, representing a key process bottleneck. To address this, we first investigated the effect of different initial phosphate concentrations on L‑threonine production in a 5 L bioreactor. The optimal phosphate concentration improved L-threonine titer by 21.9% compared to the control group. Metabolomics and 13 C metabolic flux analysis revealed that this concentration reprograms central carbon metabolism, increasing the pentose phosphate pathway flux by 28.9% to enhance precursor supply and NADPH availability, two critical factors for L-threonine biosynthesis. Building on these mechanistic insights, we further established a more precise phosphate feeding strategy based on real-time on-line capacitance signals, enabling precise matching between phosphate feeding and cell growth status. When scaled up to a 50 L bioreactor, this capacitance-guided strategy achieved an L-threonine titer of 168.41 g/L, representing a 14.3% improvement over conventional manual feeding control. Overall, this work establishes a scalable and physiology-driven nutrient control framework for microbial cell factories and elucidates the metabolic basis of phosphate-mediated regulation, providing a reference for optimizing other industrial fermentation processes.