Systematic engineering of Micromonospora echinospora cell factory for gentamicin C1a overproduction.

The clinical importance of gentamicin C1a as a broad-spectrum aminoglycoside antibiotic underscores the need for efficient biomanufacturing strategies. In this study, we developed a systematic engineering framework to enhance gentamicin C1a production. First, a genome-scale metabolic model (iFX1172) was reconstructed to pinpoint critical bottlenecks in both regulatory and biosynthetic pathways. Guided by model predictions and experimental validation, we identified genC, metK, and BldD as synergistic targets. Coordinated overexpression of these genes increased gentamicin C1a titers to 198.1 mg/L, representing a 34.3% improvement over the parental strain, and also enhanced the titers of other aminoglycoside antibiotics by up to 1.6-fold, demonstrating the universality of the strategy. Metabolic flux analysis and targeted metabolomics revealed that redox homeostasis and ATP availability are pivotal for biosynthesis. Finally, process optimization in a fed-batch bioreactor using a Bayesian framework, coupled with in situ resin adsorption, yielded 964.1 mg/L gentamicin C1a with a yield of 24.1 mg/g glucose and a productivity of 6.7 mg/L/h.