Plasmid-Free, High-Titer De Novo Adenine Production in Escherichia coli via Modular Pathway Engineering and Adaptive Evolution

, and adaptive laboratory evolution for product tolerance. The resulting plasmid-free and antibiotic-free strain demonstrated superior genetic stability and industrial robustness, achieving an adenine titer of 4.76 g/L (0.18 g/L/h) during a 26-h fed-batch fermentation without selection pressure. This system maintained high cell density and minimal byproduct formation, with a conversion efficiency of 0.025 g/g glucose. To our knowledge, this is the first report of a high-level, plasmid-free adenine production system, establishing a robust platform for the scalable biobased manufacturing of nucleobase-derived ingredients.