Biocatalytic Production of 7-Methylxanthine by a Caffeine-Degrading Escherichia coli Strain

Abstract

7-Methylxanthine, a derivative of caffeine (1,3,7-trimethylxanthine), is a high-value compound that has multiple medical applications, particularly with respect to eye health. Here, we demonstrate the biocatalytic production of 7-methylxanthine from caffeine using Escherichia coli strain MBM019, which was constructed for production of paraxanthine (1,7-dimethylxanthine). The mutant N-demethylase NdmA4, which was previously shown to catalyze N-demethylation of caffeine to produce paraxanthine, also retains N-demethylation activity toward paraxanthine. This work demonstrates that whole cell biocatalysts containing NdmA4 are more active toward paraxanthine than caffeine. We used four serial resting cell assays, with spent cells exchanged for fresh cells between each round, to produce 2,120 μM 7-methylxanthine and 552 μM paraxanthine from 4,331 μM caffeine. The purified 7-methylxanthine and paraxanthine were then isolated via preparatory-scale HPLC, resulting in 177.3 mg 7-methylxanthine and 48.1 mg paraxanthine at high purity. This is the first reported strain genetically optimized for the biosynthetic production of 7-methylxanthine from caffeine.

Mixed Culture Biocatalytic Production of the High-Value Biochemical 7-Methylxanthine

“…Recently, we reported on our ability to synthesize 7-methylxanthine from caffeine via paraxanthine using a mutant N-demethylase, NdmA4 [9]. However, we were only able to demonstrate complete conversion of 5 mM caffeine to 7-methylxanthine after four rounds of reaction, each time supplemented with fresh cells [10]. For the present work, we theorized that production of 7-methylxanthine from caffeine would be improved by using the wild-type ndm genes operating in tandem to convert caffeine rst to theobromine instead of paraxanthine, speci cally expressing ndmA and ndmB in individual cell cultures and combining the cultures together in one reaction.…”

“…During our previous optimization of paraxanthine production, we noted that higher concentrations of cells gave a greater conversion e ciency, but also started exhibiting a secondary degradation step resulting in an additional product (7-methylxanthine) at the expense of our compound of interest [9]. We were further able to produce 7-methylxanthine using E. coli strain MBM019, but the process was very time-and laborintensive, requiring four rounds of cell growth and resting cell reactions [10]. For this reason, we tested a range of cell densities from the combination of pADP1 and pBDP1 cells to determine the optimal total cell concentration for the production of 7-methylxanthine from caffeine that would most effectively minimize any side products or additional degradation.…”

“…Preparation of the reaction by 0.2 µm ltration and the addition of methanol to a nal concentration of 5% for HPLC puri cation resulted in 542.85 mL supernatant, which was separated by preparatory-scale HPLC as described previously [9,10]. Given that the nal concentration of 7-methylxanthine produced was 2.14 mM, the theoretical maximum amount of 7-methylxanthine that could be recovered from this process was 183.8 mg.…”

“…We recently generated a mutant of NdmA, known as NdmA4, that is capable of carrying out N 3 -demethylation of caffeine to generate paraxanthine (1,7-dimethylxanthine) as the primary metabolite [8,9] while also retaining N 1 -demethylation activity toward paraxanthine (Fig. 1) [10]. Genetic strain optimization resulted in the creation of E. coli strain MBM019 utilizing simultaneous expression of ndmA4 and ndmDP1, an N-terminally truncated version of the NdmD reductase (Fig.…”

“…S1). Using strain MBM019, we have established optimized processes for the biocatalytic production and puri cation of paraxanthine and 7-methylxanthine from caffeine [9,10]. However, these processes are limited by the low reaction rate of the NdmA4 mutant enzyme and result in low yields over longer time periods when compared to processes using wild-type enzymes [5][6][7].…”