Preparation and Bioactivity Evaluation of Novel Dihydrotanshinone I Derivatives via Biotransformation by Ganoderma lingzhi

15,16-Dihydrotanshinone I (DHT) is a prominent lipophilic diterpenoid from Salvia miltiorrhiza with significant pharmacological potential, though its therapeutic application is limited by poor aqueous solubility. In this study, a microbial biotransformation strategy using Ganoderma lingzhi, known for its wide variety of enzyme, was employed to diversify the chemical structure of DHT and improve its bioactivity profile. Through systematic screening and optimization of fermentation conditions, seven transformation products were isolated and characterized. Among these, five are reported as novel compounds: 17-hydroxy-salvianone (A), 18,19-dihydroxy-danshinspiroketallactone (B-2), epi-18,19-hydroxy-danshinspiroketallactone (B-3), 20-hydroxy-salvianone (C), and 19-hydroxy-danshinspiroketallactone (D). Biological evaluations demonstrated that these derivatives possess multi-target therapeutic potential, including moderate cytotoxic effects against 4T1 and A549 cancer cell lines, alongside anti-inflammatory and neuroprotective activities. However, no significant antibacterial activity was observed for any of the derivatives against six common pathogens. Specifically, compound A significantly inhibited nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells, while B-3 protected SH-SY5Y cells against H2O2-induced oxidative stress. Transcriptomic profiling of the biotransformation process identified 2221 differentially expressed genes (DEGs), showing significant enrichment in cytochrome P450-mediated metabolism and oxidative stress response pathways, which were further validated by qPCR. These results establish G. lingzhi as an efficient biocatalyst for the structural modification of tanshinones and provide a library of novel DHT derivatives for drug discovery.