Bacterial N-demthylases as biocatalysts for the production of methylxanthines
Inventors
Summers, Ryan M. • Mills, Shelby • Mock, Meredith
Assignees
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Publication Number
US-12448633-B2
Publication Date
2025-10-21
Expiration Date
Abstract
The present disclosure relates to mutant enzymes and bacterial strains constructed to produce paraxanthine and 7-methylxanthine from caffeine and methods for the manufacture and use thereof.
Core Innovation
The document describes mutant bacterial N-demethylase/reductase systems for biocatalytic conversion of caffeine and related methylxanthines into paraxanthine (1,7-dimethylxanthine) and/or 7-methylxanthine. It specifies bacterial whole-cell biocatalysts, including engineered Escherichia coli strains expressing NdmA and NdmB N-demethylase mutants together with the essential reductase NdmD, and it also describes optional frmAB for NADH/formaldehyde recycling in these systems.
The document reports that specific NdmA mutants and NdmA variants can shift regioselectivity toward N3-demethylation to increase formation of paraxanthine. It highlights examples such as NdmA N282Q/F286L and a loop-swapped NdmA4 variant, while indicating that NdmB mutants remain poor for N1-demethylation. The overall system is described in the context of contacting a bacterial strain with caffeine, theobromine, or theophylline to produce the target methylxanthines.
The document further describes production improvements in paraxanthine generation through example optimization and scale-up of the engineered system. It reports whole-cell assay results and quantitative outcomes using HPLC quantification, with identity confirmation by HPLC retention time and 1H-NMR, and it additionally reports preparative purification and a reported scale process yielding paraxanthine powder with HPLC recovery.
Claims Coverage
The independent claim provides a core method framework for preparing paraxanthine (1,7-dimethylxanthine) and/or 7-methylxanthine using a bacterial strain with N-demethylase (or mutant) activity, and it requires simultaneous overexpression of frmAB. Across the dependent claims, the independent claim is refined with additional limitations including the bacterial host, the specific contacting substrate, and an isolating/recovery step for the target xanthines.
Paraxanthine and/or 7-methylxanthine preparation using N-demethylase with frmAB overexpression
A method of preparing paraxanthine (1,7-dimethylxanthine) and/or 7-methylxanthine by contacting a bacterial strain expressing N-demethylase or a mutant thereof with caffeine, theobromine, or theophylline, wherein the bacterial strain also overexpresses frmAB.
Using an E. coli bacterial strain
The method further comprises that the bacterial strain is E. coli.
Contacting with caffeine
The method further comprises contacting the strain with caffeine.
Contacting with theobromine
The method further comprises contacting the strain with theobromine.
Isolating paraxanthine and/or 7-methylxanthine
The method further comprises isolating paraxanthine (1,7-dimethylxanthine) and/or 7-methylxanthine.
Overall, the claim set centers on a bacterial biocatalytic contacting step using N-demethylase (or mutants) acting on caffeine, theobromine, or theophylline, with required frmAB overexpression, and it further narrows the bacterial host, substrate selection, and inclusion of an isolating step for paraxanthine and/or 7-methylxanthine.
Stated Advantages
Improves paraxanthine generation/regioselectivity toward N3-demethylation to increase formation of paraxanthine.
Provides whole-cell assay and scale process outcomes with reported quantitative paraxanthine generation and HPLC recovery.
Documented Applications
Biocatalytic preparation of paraxanthine (1,7-dimethylxanthine) and/or 7-methylxanthine by contacting an engineered bacterial strain with caffeine, theobromine, or theophylline, with frmAB overexpressed.
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