(113f) Production of Paraxanthine and 7-Methylxanthine from Caffeine Using Genetically Modified E. coli

Paraxanthine (1,7-dimethylxanthine) and 7-methylxanthine are purine alkaloid derivatives of caffeine (1,3,7-trimethylxanthine). These high-value biochemicals have several applications 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. Despite the bactericidal properties of caffeine, the soil bacterium Pseudomonas putida CBB5 has been identified and characterized as capable of surviving on caffeine as a sole carbon and nitrogen source. P. putida CBB5 degrades caffeine via sequential N-demethylation to theobromine (3,7-dimethylxanthine) and 7-methylxanthine using five enzymes, NdmABCDE. However, we recently generated a mutant version of NdmA that is capable of producing paraxanthine (major product) and 7-methylxanthine (minor product) from caffeine.

In this project, we have expressed the mutant ndmA gene in E. coli BL21(DE3) to produce paraxanthine from caffeine using whole-cell biocatalysts. Preliminary resting cell assay results showed a 33% molar conversion of caffeine, from 5 mM to 3.35 mM, resulting in approximately 0.90 mM paraxanthine. However, a small amount of 7-methylxanthine was unexpectedly produced at a concentration of approximately 0.35 mM. After optimizing reaction conditions, the reaction was scaled-up to a volume of 620 mL for purification of paraxanthine via prep HPLC, producing 0.98 mM paraxanthine and consuming 2.6 mM caffeine. The purified paraxanthine was then concentrated, dried and collected, resulting in 104.1 mg of product.

We further investigated the production of 7-methylxanthine by recycling the supernatant from one small-scale resting cell assay as the substrate for a second reaction. This was repeated for a total of four rounds of reaction resulting in approximately 2.6 mM 7-methylxanthine. Although 7-methylxanthine concentration increased, the paraxanthine concentration remained relatively constant at around 0.75 - 1.0 mM and began to slowly decline once the concentration of caffeine reached approximately 1.0 mM, resulting in nearly complete conversion of caffeine. This recycling process was subsequently scaled-up for HPLC purification, resulting in the production and recovery of 177.3 mg 7-methylxanthine and 42.1 mg paraxanthine. All products were analyzed using H¹ NMR to confirm the identity and purity of each compound. This is the first reported strain genetically optimized for the biosynthetic production of paraxanthine and 7-methylxanthine.