Engineered Pseudomonas putida monoculture system for green synthesis of 7-methylxanthine

7-Methylxanthine (7-MX) is a clinically proven safe drug to treat myopia. The chemical synthesis of 7-MX is hindered due to low specificity, demanding sustainable biological production using renewable, cost-effective feedstocks. To this end, we systematically engineered robust P. putida EM42 to produce 7-MX using caffeine and glycerol in minimal salt media. Removing the transcriptional repressor (glpR), genome integration of heterologous N-demethylase and its reductase, ndmABD, and overexpression of native fdhA to balance the redox cofactors enabled the selective conversion of caffeine to 7-MX with 100% yield. We discovered a native transporter, PP_RS18750, that efficiently takes up caffeine, facilitating the conversion in glycerol-containing media. We achieved 9.2 ± 0.42 g L⁻¹ of 7-MX in a 3-L bioreactor by process-level optimization, the highest titer reported to date. Our techno-economic analysis indicates that this novel engineered monoculture approach can produce pharmaceutical-grade 7-MX commercially for $328 per kg, with remarkably low E-factor and Process Mass Intensity (PMI) values, demonstrating the sustainable green valorization of caffeine into high-value methylated xanthine.