Metabolomic and Transcriptomic Characterization of Actinokineospora bangkokensis 44EHWT: Enhancing Anti-anthracnose Production Through N-Acetylglucosamine Supplementation

N -acetylglucosamine (GlcNAc) serves as a key carbon source and regulatory signal for secondary metabolism in actinomycetes, yet its role in Actinokineospora remains poorly understood. Here, genome mining, transcriptomics, and untargeted metabolomics were integrated to investigate GlcNAc-supplemented Actinokineospora bangkokensis 44EHWT. Genome mining revealed 35 biosynthetic gene clusters (BGCs) and multiple GntR-family regulators. Untargeted metabolomics identified 2,705 features, with 493 uniquely induced by GlcNAc. Molecular networking showed significant enrichment of cyclic peptides and polyketides, including a significant increase in the relative abundance of thailandin B (fold change = 2.004, p adj = 0.00012861). In contrast, thailandin A showed only a modest and statistically non-significant change in relative abundance (fold change = 1.105, p adj = 0.390). The GlcNAc-supplemented crude extract showed a 25.92% increase in production yield compared with ISP2 alone and exhibited the largest inhibition zone against Colletotrichum gloeosporioides DoA c1060 (22.31 ± 0.50 mm), followed by C. gloeosporioides DoA c2434 (20.42 ± 0.54 mm) and Fusarium oxysporum DoA c0761 (17.06 ± 0.08 mm). Transcriptomics revealed 2,577 differentially expressed genes, though BGC regulation was heterogeneous. Notably, the Thailandin BGC was transcriptionally downregulated at Day 5 despite increased metabolite accumulation, indicating temporal offsets likely supported by precursor supply. Overall, GlcNAc acts as a context-dependent modulator that selectively triggers specific bioactive pathways rather than globally activating all BGCs. This study highlights the power of multi-omics to expand chemical diversity and harness the natural product potential of Actinokineospora .