Biocatalytic Production of 7-Methylxanthine by a Caffeine-Degrading
Escherichia coli Strain
Abstract
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
N 3-demethylation of caffeine to produce
paraxanthine, also retains N 1-demethylation
activity toward paraxanthine. This work 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.