Exploring the biochemical landscape of bacterial medium with pyruvate as the exclusive carbon source for NMR studies.

The use of Escherichia coli for recombinant protein production is a cornerstone in structural biology, particularly for nuclear magnetic resonance (NMR) spectroscopy studies. Understanding the metabolic behavior of E. coli under different carbon sources is critical for optimizing isotope labeling strategies, which are essential for protein structure determination by NMR. Recent advancements, such as mixed pyruvate labeling, have enabled improved backbone resonance assignment in large proteins, making selective isotopic labeling strategies more important than ever for NMR studies. In this study, we aimed to investigate the metabolic adaptations of E. coli when grown on pyruvate as the sole carbon source, a common condition used to achieve selective labeling for NMR spectroscopy. Using NMR-based metabolomics, we tracked key metabolic shifts throughout the culture process to better understand how pyruvate metabolism affects protein production and isotopic labeling. Our results reveal that pyruvate is rapidly depleted before IPTG induction, while acetate and lactate accumulate due to overflow metabolism. These byproducts persist after induction, indicating that pyruvate is diverted into waste pathways, which limits its efficient use in isotope incorporation. This metabolic inefficiency presents a challenge for isotopic labeling protocols that rely on pyruvate as a carbon source for NMR studies. Our results highlight the need to fine-tune pyruvate supplementation to improve metabolic efficiency and isotopic labeling, making this study directly relevant to optimizing protocols for NMR studies involving protein structure determination. These insights provide valuable guidance for enhancing the quality and yield of isotopically labeled proteins in NMR spectroscopy.