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.

1-甲基黄嘌呤是一种高价值的咖啡因衍生物，其天然利用率有限，具有许多潜在的药物应用。不幸的是，由于化学过程冗长且需要危险化学品，通过纯化学合成方法生产1-甲基黄嘌呤是不利的，最终导致收率低。在这里，我们描述了一种利用工程大肠杆菌全细胞生物催化剂和反应优化从茶碱生产 1-甲基黄嘌呤的新型生物合成工艺。当放大至 1590 mL 时，简单的生物催化反应可从 1444 mg 茶碱中产生约 1188 mg 1-甲基黄嘌呤，在短短 3 小时内即可实现克级规模的 1-甲基黄嘌呤生产。经过 HPLC纯化和溶剂蒸发后，收集到 1163 mg 干燥的 1-甲基黄嘌呤粉末，产物回收率为 97.9 wt%，纯度为 97.8%。这是专门为从茶碱中生产和纯化高价值生化物质 1-甲基黄嘌呤而设计的生物催化工艺的第一份报告。该工艺也是迄今为止最强大的甲基黄嘌呤N-去甲基化工艺，采用工程大肠杆菌，能够进行克级生产。