Bioassay for Determining the Concentrations of Caffeine and Individual Methylxanthines in Complex Samples

Abstract

Caffeine and other methylxanthines are stimulant molecules found in formulated beverages, including sodas and energy drinks, and in brewed beverages, such as coffee and teas. Previously, we developed a bioassay for caffeine that involves monitoring the growth of a ΔguaB mutant of Escherichia coli defective in de novo guanine biosynthesis. When supplemented with a plasmid expressing the genes for an N-demethylation pathway from Pseudomonas putida CBB5, these bacteria demethylate caffeine (1,3,7-trimethylxanthine) and other methylxanthines into xanthine, which is then converted into guanine to support cell growth. A major limitation of this bioassay was that it could only measure the total concentration of all methylxanthines in a mixture. Therefore, it could not be used to measure the caffeine content of beverages like teas, which contain substantial quantities of multiple methylxanthines. To overcome this limitation, we created seven new plasmids containing all subsets of the three demethylase genes (ndmA, ndmB, and ndmC). We show that strains of ΔguaB E. coli containing each plasmid are able to demethylate specific subsets of methylxanthines and that they can be used to determine the concentrations of individual methylxanthines in complex mixtures containing multiple methylxanthines, including coffee doped with an additional methylxanthine. While validating this assay, we also discovered an unexpected demethylation event at the 1-methyl position when NdmB and NdmC were expressed in the absence of NdmA. The improved cell-based bioassay is inexpensive, is easy to use, and gives results comparable to standard high-performance liquid chromatography methods for measuring methylxanthine concentrations. IMPORTANCE Caffeine (1,3,7-trimethylxanthine) is the dominant neurostimulant found in coffee, teas, sodas, and energy drinks. Measuring the amount of caffeine and other methylxanthines in these beverages is important for quality assurance and safety in food science. Methylxanthines are also used in medicine and as performance-enhancing drugs, two contexts in which accurately determining their concentrations in bodily fluids is important. Liquid chromatography is the standard method for measuring methylxanthine concentrations in a sample, but it requires specialized equipment and expertise. We improved a previous bioassay that links E. coli growth to methylxanthine demethylation so that it can now be used to determine the amounts of individual methylxanthines in complex mixtures or beverages, such as coffee.

Engineering Alternate Ligand Recognition in the PurR Topology: A System of Novel Caffeine Biosensing Transcriptional Antirepressors

“…Recently, several caffeine biosensors have been developed; , however, none are based on allosteric transcription factors. The benefit of engineering a system of allosteric transcription factors that respond to caffeine would not only serve as the foundation for a biosensor, but could also seamlessly integrate into our growing collection of engineered TFs and our emerging transcriptional programming edifice. , Accordingly, our design goals for the resulting system of novel antirepressors was evident i.e.…”

“…52 In previous work, most caffeine-responsive gene switches required the enzymatic conversion of caffeine to theophylline for translational control in yeast. 53 However, several caffeine biosensors have been developed in recent years, 54,55 although none are based on transcription factors, likely because existing caffeine-inducible TFs are rare and poorly characterized. 56 We posited that PurR could serve as a scaffold for engineering a caffeine responsive transcription factor given the similarity in chemical structure of the putative ligand (caffeine) to the native ligand (hypoxanthine).…”