7‐Methylxanthine, a derivative of caffeine (1,3,7‐trimethylxanthine), is a high‐value compound that has multiple medical applications, particularly with respect to eye health. Here, we demonstrate the biocatalytic production of 7‐methylxanthine from caffeine using Escherichia coli strain MBM019, which was constructed for production of paraxanthine (1,7‐dimethylxanthine). The mutant N‐demethylase NdmA4, which was previously shown to catalyze N3‐demethylation of caffeine to produce paraxanthine, also retains N1‐demethylation activity toward paraxanthine. This study demonstrates that whole cell biocatalysts containing NdmA4 are more active toward paraxanthine than caffeine. We used four serial resting cell assays, with spent cells exchanged for fresh cells between each round, to produce 2,120 μM 7‐methylxanthine and 552 μM paraxanthine from 4,331 μM caffeine. The purified 7‐methylxanthine and paraxanthine were then isolated via preparatory‐scale HPLC, resulting in 177.3 mg 7‐methylxanthine and 48.1 mg paraxanthine at high purity. This is the first reported strain genetically optimized for the biosynthetic production of 7‐methylxanthine from caffeine.

Biocatalytic production of 7‐methylxanthine by a caffeine‐degrading Escherichia coli strain

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.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.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. 

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

Caffeine is a metabolite derived from purine nucleotides, typically accounting for 2–5% of the dry weight of tea and 1–2% of the dry weight of coffee. In the tea and coffee plants, the main synthesis pathway of caffeine is a four-step sequence consisting of three methylation reactions and one nucleosidase reaction using xanthine as a precursor. In bacteria, caffeine degradation occurs mainly through the pathways of N-demethylation and C-8 oxidation. However, a study fully and systematically summarizing the metabolism and application of caffeine in microorganisms has not been established elsewhere. In the present study, we provide a review of the biosynthesis, microbial degradation, gene expression, and application of caffeine microbial degradation. The present review aims to further elaborate the mechanism of caffeine metabolism by microorganisms and explore the development prospects in this field.

Sydney Strock

Papers

Biocatalytic Production and Purification of the High-value Biochemical Paraxanthine