(246c) Biosynthetic Production of 1-Methylxanthine Production By Biocatalytically Versatile N-Demethylases

The discovery of caffeine-degrading enzymes found in the soil bacterium Pseudomonas putida CBB5 has led to the ability to biosynthetically produce a variety of high-value biopharmaceutical derivatives of caffeine (1,3,7-trimethylxanthine) previously difficult to access. These compounds can be found in the pharmaceutical and cosmetic industries, but their chemical synthesis requires harsh conditions and frequently results in low yield mixtures of non-specifically methylated compounds. P. putida CBB5 degrades caffeine via sequential N-demethylation to theobromine (3,7-dimethylxanthine), 7-methylxanthine, and ultimately xanthine using five enzymes, NdmABCDE. We have recently generated a mutant enzyme, NdmA4, swapping the positional-specificity of the NdmA enzyme from the N₁- to the N₃-methyl group, which allowed us to produce paraxanthine and 7-methylxanthine from caffeine. Here, we have extended the application of our methylxanthine biosynthetic platform to produce 1-methylxanthine from theophylline (1,3-dimethylxanthine).

Initial enzyme screening demonstrated that the mutant enzymes NdmA3 and NdmA4 are both capable of producing 1-methylxanthine from theophylline. We have constructed strains of Escherichia coli expressing the ndmA3 or ndmA4 genes, and identified strain MBM020 as the optimal 1-methylxanthine producing strain. MBM020 contains multiple copies of ndmA3, along with a formaldehyde degradation pathway to regenerate NADH. We have successfully used MBM020 to produce 6 mM 1-methylxanthine from 10 mM theophylline using whole-cell biocatalysts. This yield represents a 6-fold increase compared with production of paraxanthine from caffeine in a similar system. Following optimization of small-scale resting cell assays, we scaled-up the reaction to bench scale using 4-L induced cells. The supernatant was collected and the biocatalytically-generated 1-methylxanthine was be purified by HPLC separation, concentrated and dried to a powder. Purity and identity of the product were confirmed by ¹H-NMR and HPLC, indicating a highly-pure product. To our knowledge, this is the first report of biocatalytically-produced and isolated 1-methylxanthine and includes yields higher than any of our previous methylxanthine producing processes.