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Browsing Theses and Dissertations by Subject "1-Methylxanthine"

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    Characterization and Optimization of the Biosynthetic Production of High-Value Methylxanthines
    (University of Alabama Libraries, 2023) Mock, Meredith Bush; Summers, Ryan M; Thompson, Matthew
    Paraxanthine, 7-methylxanthine, and 1-methylxanthine are purine alkaloid derivatives of caffeine with several applications in the pharmaceutical and cosmetic industries, but their chemical synthesis requires harsh conditions and frequently results in low-yield, racemic mixtures. The N-demethylase enzymes, NdmABCDE, isolated from the caffeine-degrading soil bacterium Pseudomonas putida CBB5, offer a biosynthetic alternative for producing these costly methylxanthines. We have developed and optimized biocatalytic processes for the production and purification of paraxanthine, 7-methylxanthine, and 1-methylxanthine from caffeine and theophylline using genetically modified E. coli cells expressing N-demethylase genes.NdmABCDE, initially characterized in vivo, were initially thought to be highly specific in their N-demethylation activity. We have identified differences between their in vitro and in vivo activity. These results reveal promiscuity of NdmABCDE towards methylxanthine substrates that can be used to engineer new enzymes for production of high-value methylxanthines.Whole-cells of E. coli strain MBM019 expressing a mutant N-demethylase, ndmA4,produced 1.02 mM paraxanthine from 2.49 mM caffeine, yielding 104.1 mg of purified paraxanthine powder. Recycling the reaction supernatant with fresh MBM019 cells over four cycles resulted in production of approximately 2.6 mM 7-methylxanthine and 0.2 mM PX from 5 mM caffeine, generating 177.3 mg 7-methylxanthine and 48.1 mg paraxanthine powder.We further optimized 7-methylanthine production by use of the wild-type N-demethylase enzymes NdmAB, through which 7-methylxanthine is generated via theobromine rather than iiiparaxanthine. This process required a novel mixed-culture of whole-cell, yielding 2.14 mM 7-methylxanthine while consuming 2.5 mM caffeine, resulting in 183.81 mg of dried product.Finally, to generate 1-methylxanthine from theophylline we replaced the copies of ndmA4in strain MBM019, with ndmA3, resulting in the 1-methylxanthine-optimized E. coli strain MBM020. This strain is capable of converting 100% of 10 mM theophylline to 1-methylxanthine in less than 3.5 hours in the optimized biocatalytic process and constitutes our most efficient Ndemethylation reaction to date.All compounds of interest generated in this work were purified by HPLC, and subsequently identified by 1H-NMR. Together, these biocatalytic processes represent novel pathways for the safe and efficient synthesis of high-value methylxanthines from inexpensive, natural sources.