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KMID : 1094720220270040640
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Biotechnology and Bioprocess Engineering
2022 Volume.27 No. 4 p.640 ~ p.651
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Biocatalytic Production and Purification of the High-value Biochemical Paraxanthine
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Mock Meredith B.
Mills Shelby Brooks
Cyrus Ashley
Campo Hailey
Dreischarf Tyler
Strock Sydney
Summers Ryan M.
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Abstract
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Paraxanthine (1,7-dimethylxanthine), a purine alkaloid derivative of caffeine (1,3,7-trimethylxanthine), is a high-value biochemical with several applications in the pharmaceutical and cosmetic industries. However, chemical synthesis of paraxanthine requires harsh conditions and frequently results in low yield mixtures of non-specifically N-methylated compounds. We have recently demonstrated that the mutant bacterial N-demethylase NdmA4 with its partner reductase NdmD is capable of producing paraxanthine as the major metabolite from caffeine. Here, we report the construction and screening of several Escherichia coli strains to produce paraxanthine from caffeine by means of whole-cell biocatalysts using varying dosages of ndmA4, ndmD, and the frmAB formaldehyde dehydrogenase genes. Preliminary resting cell assay results with the best paraxanthine-producing strain, MBM019, showed a 33% molar conversion of caffeine, from 5 mM to 3.35 mM, resulting in approximately 0.90 mM paraxanthine. However, a small amount of 7-methylxanthine was unexpectedly produced at a concentration of approximately 0.35 mM. After optimizing reaction conditions to a cellular concentration of OD600 = 50 and a caffeine concentration of 5 mM, the reaction was scaled-up to a volume of 620 mL, producing 1.02 mM paraxanthine and consuming 2.49 mM caffeine. The purified paraxanthine was then isolated via preparatory scale chromatography, resulting in 104.1 mg of product at high purity. This is the first reported strain genetically optimized for the biosynthetic production of paraxanthine.
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KEYWORD
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paraxanthine, caffeine, biocatalysis, N-demethylase
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