Raethong N.Jeennor S.Anantayanon J.Wannawilai S.Vongsangnak W.Laoteng K.Mahidol University2025-08-032025-08-032025-07-01International Journal of Molecular Sciences Vol.26 No.14 (2025)16616596https://repository.li.mahidol.ac.th/handle/123456789/111496Cordycepin, a bioactive adenosine analog, holds promise in pharmaceutical and health product development. However, large-scale production remains constrained by the limitations of natural producers, Cordyceps spp. Herein, we report the reconstruction of the first genome-scale metabolic model (GSMM) for a cordycepin-producing strain of recombinant Aspergillus oryzae. The model, iNR1684, incorporated 1684 genes and 1947 reactions with 93% gene-protein-reaction coverage, which was validated by the experimental biomass composition and growth rate. In silico analyses identified key gene amplification targets in the pentose phosphate and one-carbon metabolism pathways, indicating that folate metabolism is crucial for enhancing cordycepin production. Nutrient optimization simulations revealed that chitosan, D-glucosamine, and L-aspartate preferentially supported cordycepin biosynthesis. Additionally, a carbon-to-nitrogen ratio of 11.6:1 was identified and experimentally validated to maximize production, higher than that reported for Cordyceps militaris. These findings correspond to a faster growth rate, enhanced carbon assimilation, and broader substrate utilization by A. oryzae. This study demonstrates the significant role of GSMM in uncovering rational engineering strategies and provides a quantitative framework for precision fermentation, offering scalable and sustainable solutions for industrial cordycepin production.Chemical EngineeringChemistryBiochemistry, Genetics and Molecular BiologyComputer ScienceArticleSCOPUS10.3390/ijms261469062-s2.0-10501184972314220067