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Rieske non-heme iron oxygenases (ROs) possess the ability to catalyze a wide range of reactions. Their ability to degrade aromatic compounds is a unique characteristic and makes ROs interesting for a variety of potential applications. However, purified ROs can be challenging to work with due to low stability and long, complex electron transport chains. Whole cell biocatalysis represents a quick and reliable method for characterizing the activity of ROs and harnessing their metabolic potential. In this protocol, we outline a step-by-step protocol for the overexpression of ROs for whole cell biocatalysis and characterization. We have utilized a caffeine-degrading, N-demethylation system, expressing the RO genes ndmA and ndmD, as an example of this method., (Copyright © 2024. Published by Elsevier Inc.)

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. [ABSTRACT FROM AUTHOR]

1-Methylxanthine is a high-value derivative of caffeine of limited natural availability with many potential pharmaceutical applications. Unfortunately, production of 1-methylxanthine through purely chemical methods of synthesis are unfavorable due to lengthy chemical processes and the requirement of hazardous chemicals, ultimately resulting in low yields. Here, we describe a novel biosynthetic process for the production of 1-methylxanthine from theophylline using engineered Escherichia coli whole-cell biocatalysts and reaction optimization. When scaled-up to 1590 mL, the simple biocatalytic reaction produced approximately 1188 mg 1-methylxanthine from 1444 mg theophylline, constituting gram-scale production of 1-methylxanthine in as little as 3 hours. Following HPLC purification and solvent evaporation, 1163 mg of dried 1-methylxanthine powder was collected, resulting in a 97.9 wt% product recovery at a purity of 97.8%. This is the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 1-methylxanthine from theophylline. This process is also the most robust methylxanthine N -demethylation process featuring engineered E. coli to date, capable of gram-scale production. • Novel, biosynthetic pathway for production of 1-methylxanthine at gram scale. • Engineered E. coli for high-value biochemical production. • High performance liquid chromatography (HPLC) separation for high purity. • Co-factor (NADH) recycle through by-product (formaldehyde) degradation. [ABSTRACT FROM AUTHOR]