Unveiling the Spatiotemporal Landscape of Ganoderma lingzhi: Insights into Ganoderic Acid Distribution and Biosynthesis

Ganoderma lingzhi ( G. lingzhi ), renowned as the “mushroom of immortality” in traditional Chinese medicine, has emerged as a powerful medicinal model organism. Ganoderic acids (GAs) from G. lingzhi , are potent triterpenoids with substantial therapeutic properties. However, the GA biosynthetic pathway remains poorly understood, which limits its large-scale commercialization. Therefore, this study utilized high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to spatially map GA distribution in both the primordium and fruiting body, revealing predominant accumulation in the G. lingzhi shell. Additionally, the telomere to telomere (T2T) genomes of two haploid G. lingzhi strains were assembled, achieving the highest integrity and quality reported to date. A T2T-genome-based high-resolution single-cell transcriptome atlas of the G. lingzhi fruiting body was constructed, six distinct cell types were identified, and the developmental trajectory of shell was reconstructed. A multi-omics analysis was used to annotate a novel cytochrome P450 enzyme (GlCYP512A3) that catalyzes the oxidation of the GA 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA) into ganolucidic acid E and F. Overall, this study provides a comprehensive spatiotemporal multi-omics framework for G. lingzhi , providing crucial insights for deciphering complex GA biosynthesis and regulatory networks.