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 2705 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 2577 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 .
Ophiocordyceps sinensis is a highly prized medicinal and edible fungus, yet its commercial cultivation is hindered by unclear regulatory mechanisms of fruiting body development. Primordium (PR) formation is the initial and critical stage of fruiting body development. Here, we employed 4D label-free quantitative phosphoproteomics to elucidate phosphorylation dynamics during the sclerotium (ST)-to-PR transition. In PR vs. ST, 642 differentially phosphorylated peptides were identified (477 with significantly elevated and 165 with significantly reduced phosphorylation levels), corresponding to 400 differentially phosphorylated proteins (DPPs), most of which showed increased phosphorylated level during PR initiation. Functional enrichment analysis revealed key pathways involved in PR development, including mitogen-activated protein kinase (MAPK) signaling, exocytosis, glycolysis, glycerophospholipid metabolism, and oxidative stress. Moreover, 30 core DPPs were identified, including the exocyst component Sec8, the SNARE protein Sec9, lysophospholipase Nte1, MAPK Fus3, and MAPKK Ste7. The Ste7-Fus3 module coordinates cell wall remodeling and oxidative stress responses to facilitate PR initiation. Knockout of the ste7 ortholog in Cordyceps militaris completely abolished PR formation. Transcriptomic and qPCR analyses confirmed that Ste7 activates cell-wall remodeling and oxidative stress-related genes while repressing vegetative growth-related genes. The conservation of Ste7 and phosphoproteomic data support a conserved mechanism underlying fungal PR development, which needs further validation in O. sinensis . Furthermore, yeast two-hybrid assays and AlphaFold 3.0 server suggest a physical interaction between O. sinensis Ste7 and Fus3, with Ser362 and Thr388 on Ste7 representing candidate phosphorylation sites that may mediate this interaction. These findings provide novel insights into phosphorylation-modulated PR development in O. sinensis .
Ophiocordyceps sinensis is a highly prized medicinal and edible fungus, yet its commercial cultivation is hindered by unclear regulatory mechanisms of fruiting body development. Primordium (PR) formation is the initial and critical stage of fruiting body development. Here, we employed 4D label-free quantitative phosphoproteomics to elucidate phosphorylation dynamics during the sclerotium (ST)-to-PR transition. In PR vs. ST, 642 differentially phosphorylated peptides were identified (477 with significantly elevated and 165 with significantly reduced phosphorylation levels), corresponding to 400 differentially phosphorylated proteins (DPPs), most of which showed increased phosphorylated level during PR initiation. Functional enrichment analysis revealed key pathways involved in PR development, including mitogen-activated protein kinase (MAPK) signaling, exocytosis, glycolysis, glycerophospholipid metabolism, and oxidative stress. Moreover, 30 core DPPs were identified, including the exocyst component Sec8, the SNARE protein Sec9, lysophospholipase Nte1, MAPK Fus3, and MAPKK Ste7. The Ste7-Fus3 module coordinates cell wall remodeling and oxidative stress responses to facilitate PR initiation. Knockout of the ste7 ortholog in Cordyceps militaris completely abolished PR formation. Transcriptomic and qPCR analyses confirmed that Ste7 activates cell-wall remodeling and oxidative stress-related genes while repressing vegetative growth-related genes. The conservation of Ste7 and phosphoproteomic data support a conserved mechanism underlying fungal PR development, which needs further validation in O. sinensis. Furthermore, yeast two-hybrid assays and AlphaFold 3.0 server suggest a physical interaction between O. sinensis Ste7 and Fus3, with Ser362 and Thr388 on Ste7 representing candidate phosphorylation sites that may mediate this interaction. These findings provide novel insights into phosphorylation-modulated PR development in O. sinensis.
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 2705 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, padj = 0.00012861). In contrast, thailandin A showed only a modest and statistically non-significant change in relative abundance (fold change = 1.105, padj = 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 2577 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.
