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

Adenine, a fundamental purine nucleobase, is a vital precursor for the biosynthesis of numerous bioactive nucleosides and nucleotides used in pharmaceutical development and biomedical research. However, sustainable microbial production of adenine is hindered by strict metabolic regulation and product toxicity. Here, we developed a modular engineering strategy in Escherichia coli combining removal of catabolic and transcriptional repression, reinforcement of flux from PRPP to adenine, blocking of competing salvage/branch pathways, growth-phase-dependent control of guaB, feedback-resistant prs/purF, 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.