Mixed culture biocatalytic production of the high-value biochemical 7-methylxanthine

Abstract

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 h. 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. Conclusion 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.

Electrochemical Synthesis of Added Valuable Purine Alkaloid Metabolites from Caffeine Feedstock Employing a Structure Electro-Activity Relationship (SeAR) Approach

“…[2] Caffeine metabolites have been biosynthesised, [6] synthesised via multi-step chemical procedures, [7][8] or via genetic engineering. [9][10] The use of electrochemistry to generate information on drug metabolites is a rapidly developing method of importance for establishing environmental fate. [11][12][13][14][15][16][17][18][19][20] In this study, we considered whether the application of electrochemistry [21][22][23][24][25][26] would achieve the single step synthesis of authentic caffeine metabolites and a predictive structure electroactivity relationship (SeAR) model for metabolism.…”

Highly Efficient Whole-Cell Biocatalysis for the Biosynthesis of 7-Methylxanthine and Other Xanthine Derivatives

“…coli strains either expressing ndmAD or ndmBD . Altering the ratio of these two strains led to 2.23 mM 7-MX . Therefore, the low production volume of 7-MX has become one of the key factors limiting further research on the use of this material.…”

Catalytic Conversion of Caffeine into Molecules of Valuable Interest Via N‐Demethylation Reaction

“…In bacteria, caffeine degrades by enzymatic N-demethylation to generate the related methylxanthines. [1] Thus the use of biocatalysis is another way to promote the formation of methylxanthines from caffeine, by genetic engineering using microorganisms modified through enzymatic catalysis, [12,[26][27][28][29] the active centers can be constituted by non-heme iron or cobalt. [30] Although these processes have high selectivity and are environmentally correct, there are difficulties due to caffeine inhibition in some microorganism strains, the great technological demand, the need for selection and maintenance of strains, and the limited quantity of products generated.…”