Enhanced biosynthesis of hypoxanthine and its derivatives from glucose

Hypoxanthine and its derivatives, including allantoin and xanthine, are valuable purine compounds with broad applications in pharmaceuticals and cosmetics. However, their bio-production is hampered by insufficient precursor supply and byproduct accumulation. Here, we developed a microbial platform in Escherichia coli for the production of these compounds. We first developed a hypoxanthine-overproducing strain by systematic metabolic engineering, yielding 5.08 g/L in shake flasks. To expand the platform, we introduced xanthine dehydrogenase XdhABC and uricase for allantoin biosynthesis, achieving 5.42 g/L, the highest reported to date. Additionally, we applied protein engineering to improve the substrate specificity of XdhABC for xanthine biosynthesis. The engineered R332K/T464E mutant significantly improved xanthine production and reduced byproduct urate formation. Introducing this mutant into the hypoxanthine-overproducing strain enabled the first de novo biosynthesis of xanthine, reaching 0.42 g/L. This work demonstrates an efficient microbial platform for producing hypoxanthine and its derivatives, with potential for industrial-scale purine biomanufacturing.