Mixed culture biocatalytic production of the high-value biochemical 7-methylxanthine

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Background 7-Methylxanthine, a derivative of caffeine noted for its lack of toxicity and ability to treat and even prevent myopia progression, is a high-value biochemical with limited natural availability. Attempts to produce 7-methylxanthine through purely chemical methods of synthesis are faced with complicated chemical processes and/or the requirement of a variety of hazardous chemicals, resulting in low yields and racemic mixtures of products. In recent years, we have developed engineered microbial cells to produce several methylxanthines, including 3-methylxanthine, theobromine, and paraxanthine. The purpose of this study is to establish a more efficient biosynthetic process for the production of 7-methylxanthine from caffeine.Results Here, we describe the use of a mixed-culture system composed of Escherichia coli strains engineered as caffeine and theobromine "specialist " cells. Optimal reaction conditions for the maximal conversion of caffeine to 7-methylxanthine were determined to be equal concentrations of caffeine and theobromine specialist cells at an optical density (600 nm) of 50 reacted with 2.5 mM caffeine for 5 h. When scaled-up to 560 mL, the simple biocatalytic reaction produced 183.81 mg 7-methylxanthine from 238.38 mg caffeine under ambient conditions, an 85.6% molar conversion. Following HPLC purification and solvent evaporation, 153.3 mg of dried 7-methylxanthine powder was collected, resulting in an 83.4% product recovery.Conclusion We present the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 7-methylxanthine from caffeine using a mixed culture of E. coli strains. This process constitutes the most efficient method for the production of 7-methylxanthine from caffeine to date.

7-methylxanthine, Caffeine, Biocatalysis, N-demethylase, Mixed-culture, FORM DEPRIVATION MYOPIA, CAFFEINE DEMETHYLASE, FERMENTATION, DEGRADATION, XANTHINES, Biochemical Research Methods, Biotechnology & Applied Microbiology
Mock, M. B., & Summers, R. M. (2023). Mixed culture biocatalytic production of the high-value biochemical 7-methylxanthine. In Journal of Biological Engineering (Vol. 17, Issue 1). Springer Science and Business Media LLC. https://doi.org/10.1186/s13036-022-00316-6