(448e) Production of High-Value Methylxanthines Using Engineered Whole-Cell Biocatalysts

Methylxanthines are natural products with many applications in the food, pharmaceutical, and cosmetic industries. While some methylxanthines, such as caffeine (1,3,7-trimethylxanthine) and theophylline (1,3-dimethylxanthine), are commonly found in nature, other compounds are not generated in sufficient quantities for economical production via extraction from natural sources. Instead, these rare compounds are produced synthetically using harsh conditions that result in racemic mixtures of products and low yields. Paraxanthine (1,7-dimethylxanthine) has potential as a treatment for Parkinson’s disease, while 7-methylxanthine can serve to reduce progression of myopia, but retail price of these compounds is over 31,000- and 13,000-fold higher, respectively, than caffeine. The purpose of this project is to develop microbial strains and processes for the biocatalytic production of paraxanthine and 7-methylxanthine.

We have constructed 19 strains of Escherichia coli capable of producing paraxanthine from caffeine using a mutant N-demethylase (NdmA4) from the soil bacterium Pseudomonas putida CBB5. Strains were grown, harvested by centrifugation, and used as whole-cell biocatalysts in a resting cell reaction. The strain with the highest activity, MBM019, was used in a bench-scale reaction to produce 0.98 mM paraxanthine from 2.6 mM caffeine, with subsequent purification of paraxanthine, resulting in 104 mg of powdered product. We also demonstrate a newly-discovered promiscuous activity of NdmA4 toward paraxanthine, resulting in generation of 7-methylxanthine when paraxanthine concentration reaches approximately 1 mM. Taking advantage of the promiscuity of NdmA4, we carried out a series of four reactions by swapping spent cells with fresh cells to produce 2.6 mM 7-methylxanthine from 4.7 mM caffeine and isolated 177 mg pure 7-methylxanthine powder.

To improve 7-methylxanthine production, we have developed a co-culture reaction system using two distinct “specialist” strains. Strain MBM020 produced theobromine (3,7-dimethylxanthine) from caffeine using the wild-type N₁-demethylase NdmA, while strain MBM021 converted theobromine to 7-methylxanthine using NdmB, the wild-type N₃-demethylase. When the strains are mixed at a 1:1 ratio, we observed rapid and efficient conversion of caffeine to 7-methylxathine in a single reaction. The mixed-culture reaction system produced 2.23 mM 7-methylxanthine from 2.29 mM caffeine over 5 h, resulting in 153 mg pure 7-methylxanthine isolated from a 500 mL reaction. In summary, we have developed strains and reaction processes to produce the high-value biochemicals paraxanthine and 7-methylxanthine using caffeine as a feedstock, which will enable efficient production of these compounds and facilitate their use as pharmaceutical components.