Highly Efficient Whole-Cell Biocatalysis for the Biosynthesis of 7-Methylxanthine and Other Xanthine Derivatives

7-Methylxanthine (7-MX), a caffeine (1,3,7-trimethylxanthine) derivative, has gained significant attention as a potential drug for myopia treatment. However, the efficient production of this valuable compound poses challenges: Isolation and chemical synthesis of 7-MX are both difficult to realize due to their poor yields and high costs. Therefore, developing efficient biosynthetic pathways has emerged as a promising alternative strategy. This study aimed to establish an efficient, low-cost, and pollutant-free biosynthetic process for producing 7-MX from caffeine, in which the biosynthetic process is achieved by utilization of ndmA, ndmB, and modified ndmD genes. Moreover, the rich caffeine in coffee waste can be used as an ideal substrate for this reaction, which can reduce the production cost and treat the caffeine in coffee waste residue to promote secondary utilization. By optimizing the gene expression, constructing cofactor regeneration system composed of frmA, frmB, and FDH to regenerate NADH to remove the bottleneck, and engineering Escherichia coli for high-density fermentation, we increased the production of 7-MX to an unprecedented 8.37 g/L. This approach represents the most efficient method thus far for producing 7-MX from caffeine and provides insights into synthesizing other valuable methylxanthines.