Biocatalytic Production and Purification of the High-value Biochemical Paraxanthine

Mock, Meredith B.; Mills, Shelby Brooks; Cyrus, Ashley; Campo, Hailey; Dreischarf, Tyler; Strock, Sydney; Summers, Ryan M.

doi:10.1007/s12257-021-0301-0

Cited by 12 publications

(26 citation statements)

References 55 publications

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“…This resulted in an 85.6 mol% conversion of caffeine to 7-methylxanthine and a 9.4 mol% conversion to theobromine. The remaining 5 mol% of consumed caffeine could be accounted for in two unidenti ed HPLC peaks at 3.44 and 4.58 minutes not observed in a cell-only control [9].…”

Section: -Methylxanthine Puri Cation and Recoverymentioning

confidence: 90%

“…We replaced ndmA in pADP1 with ndmB to create plasmid pBDP1, which was used for the theobromine specialist cells. We chose the ndmDP1 truncated reductase gene in place of the full-length ndmD gene because cells expressing the truncated gene exhibit higher activity than cells with the full-length gene [9].…”

Section: Strain Development and Screeningmentioning

confidence: 99%

“…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.…”

Section: -Methylxanthine Puri Cation and Recoverymentioning

confidence: 99%

“…For example, 3-methylxanthine has been produced from theophylline (1,3-dimethylxanthine) using E. coli strain pDdA [5], theobromine (3,7-dimethylxanthine) has been produced from caffeine using E. coli strain pAD1dDD [6], and 7-methylxanthine has been produced from theobromine using E. coli strain pBD2dDB [7]. 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].…”

mentioning

confidence: 99%

“…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].…”

mentioning

confidence: 99%

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

Mock

Summers

2022

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Background 7-Methylxanthine, a derivative of caffeine noted for its lack of toxicity and ability to treat and even prevent myopia progression, is a high-value biochemical with limited natural availability. Attempts to produce 7-methylxanthine through purely chemical methods of synthesis are faced with complicated chemical processes and/or the requirement of a variety of hazardous chemicals, resulting in low yields and racemic mixtures of products. In recent years, we have developed engineered microbial cells to produce several methylxanthines, including 3-methylxanthine, theobromine, and paraxanthine. The purpose of this study is to establish a more efficient biosynthetic process for the production of 7-methylxanthine from caffeine. Results Here, we describe the use of a mixed-culture system composed of Escherichia coli strains engineered as caffeine and theobromine “specialist” cells. Optimal reaction conditions for the maximal conversion of caffeine to 7-methylxanthine were determined to be equal concentrations of caffeine and theobromine specialist cells at an optical density (600 nm) of 50 reacted with 2.5 mM caffeine for 5 hours. When scaled-up to 560 mL, the simple biocatalytic reaction produced 183.81 mg 7-methylxanthine from 238.38 mg caffeine under ambient conditions, an 85.6% molar conversion. Following HPLC purification and solvent evaporation, 153.3 mg of dried 7-methylxanthine powder was collected, resulting in an 83.4% product recovery. Conclusions We present the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 7-methylxanthine from caffeine using a mixed culture of E. coli strains. This process constitutes the most efficient method for the production of 7-methylxanthine from caffeine to date.

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Section: -Methylxanthine Puri Cation and Recoverymentioning

confidence: 90%

Section: Strain Development and Screeningmentioning

confidence: 99%

Section: -Methylxanthine Puri Cation and Recoverymentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Mock

Summers

2022

Preprint

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Biocatalytic production of 7‐methylxanthine by a caffeine‐degrading Escherichia coli strain

Mock

Cyrus

Summers

2022

Biotech & Bioengineering

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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 N3‐demethylation of caffeine to produce paraxanthine, also retains N1‐demethylation activity toward paraxanthine. This study 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.

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Electrochemical Synthesis of Purine Alkaloid Metabolites from Caffeine

Asra,

Jones

2024

ChemElectroChem

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The development of electrochemical approaches to the valorization of abundant natural products into high value medications and metabolites is of pharmaceutical interest. In this study, we explored the electrosynthetic behavior of the abundant legal psychoactive, caffeine, a representative member of the purine alkaloid class. Initial screening of the cyclic voltammetric behavior of eleven exemplar purine alkaloids revealed a structure electroactivity relationship (SeAR) for determining the initial oxidation site of caffeine. Optimization of the current controlled electrochemical (CCE) reaction enabled the dialing‐in/out of differential oxidative metabolic products using both undivided and divided cells. Sequential desmethylation around the purine ring was observed both by isolation and comparison to authentic metabolite reference standards via HPLC measurements. Amide, imide, and a novel N‐methyl heteroaryl oxidation mechanism were observed. Tractable quantities of the high‐value medication, theophylline, and the dietary supplement, paraxanthine, were isolated in 17 % and 8 % b.r.s.m. This approach offers a marked improvement compared to the best‐in‐class techniques (chemical 0.8 % and enzymatic 0.97 % yields) and may have potential in other natural product and drug discovery settings to prepare valuable metabolites.

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Biocatalytic Production and Purification of the High-value Biochemical Paraxanthine

Cited by 12 publications

References 55 publications

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

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

Biocatalytic production of 7‐methylxanthine by a caffeine‐degrading Escherichia coli strain

Electrochemical Synthesis of Purine Alkaloid Metabolites from Caffeine

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