Production of 3-methylxanthines by Metabolically Engineered E. coli
Methylxanthines are naturally occurring purine alkaloids that are used in pharmaceutical preparations as diuretics, for asthma treatment, and as cardiac, pulmonary, and neurological stimulants. By using E. coli, metabolically engineered with N-demethylase genes as a biocatalyst, we were able to produce 100 mg of 3-methylxanthine by N-demethylation of theophylline at 30 oC and ambient atmospheric pressure. The conversion of theophylline is was 100%, 80% of which was 3-methylxanthine and 20% 1-methylxanthine.
It was previously reported that Pseudomonas putida CBB5 degrades caffeine via sequential N-demethylation to theobromine or paraxanthine, then to 7-methylxanthine, and further to xanthine. CBB5 contains five novel N-demethylase genes, ndmA, ndmB, ndmC, ndmD, and ndmE, which are responsible for caffeine degradation. Three genes, ndmA, B and D have been cloned, and expressed in E. coli. N-demethylase A (NdmA) and N-demethylase B (NdmB) are Rieske monooxygenases that catalyze position-specific N1- and N3- demethylations, respectively in the presence of NdmD. Both enzymes receive reducing equivalents from NADH via a redox-center-dense Rieske reductase, NdmD
Three E. coli strains was metabolically engineered using different combinations of ndmA, B and D genes. Strain pDdA which has one copy of each of NdmA and NdmD genes was grown overnight in 4 L super broth to produce 20 g wet cell paste. That cell paste was washed with potassium phosphate buffer pH 7.0 and used as a biocatalyst in 1.3 L reaction volume that contained 1 mM theophylline. The temperature and shaker speed used were 30 oC and 250 rpm respectively. Theophylline completely disappeared after two hours with 80% conversion to 3-methylxanthine as the major product. 3-methylxanthine was separated from post-reaction mixture by preparative chromatography by using BDS Hypersil C18 column (250 x 21.2 mm) as the stationary phase and 5% methanol as the mobile phase. The optimized solvent flow rate and the amount of post reaction mixture injected were found to be 2.5 mL/min and 25 mL respectively. 3-methylxanthine solution was concentrated by evaporation under vacuum at 70 oC and the resulting concentrated solution was freeze-dried. The purified product, 100 mg 3-methylxanthine was confirmed by LC-MS and NMR with respect to authenticity and purity.