(448e) Production of High-Value Methylxanthines Using Engineered Whole-Cell Biocatalysts
AIChE Annual Meeting
2022
2022 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Fermentation and Process Engineering in Food and Bioprocess Industries
Wednesday, November 16, 2022 - 9:12am to 9:30am
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 âspecialistâ 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.