Genome-scale metabolic model guided metabolic flux analysis in the endophyte Alternaria burnsii NCIM1409

Camptothecin (CPT), a monoterpene indole alkaloid widely used in anticancer therapy, faces production bottlenecks arising from its plant-based origin. In this study, we systematically investigated the metabolic network of the camptothecin-producing fungal endophyte Alternaria burnsii NCIM1409 using an integrative framework combining genome-scale metabolic modeling and ¹³C-based pathway analysis. A genome-scale metabolic model, AltGEM iDD1552, was reconstructed, encompassing 2,188 reactions, 2,148 metabolites, and 1,552 genes, and was manually curated to incorporate camptothecin biosynthetic pathways. Key enzymatic control points, including secologanin synthase and tryptophan decarboxylase, as potential targets were identified using flux balance for enhancing CPT production. Additionally, metabolic tracer analysis using 20% [U-¹³C₆] glucose and 99% [1-¹³ C] glucose labeling confirmed the active involvement of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle in central carbon metabolism. Overall, this study establishes a systems-level framework for understanding and optimizing camptothecin biosynthesis in A. burnsii NCIM1409. Collectively, this integrative systems-level analysis elucidates the metabolic capabilities of A. burnsii NCIM1409 and provides rational strategies for optimizing camptothecin biosynthesis, laying the groundwork for sustainable microbial production of this high-value anticancer compound.