LAUSR

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.