**Background:** Macrotermitinae termites have cultivated Termitomyces fungi as a food source for millions of years, yet the biochemical mechanisms underlying this mutualistic symbiosis remain poorly understood. Volatile organic compounds (VOCs) are hypothesized to serve as chemical mediators between termites and their fungal crop, but the VOC repertoire of Termitomyces in different life stages had not been systematically characterized. Drimane-type sesquiterpenes are widespread in nature with diverse biological activities including antimicrobial, cytotoxic, and antifeedant properties, but their role in the termite-fungus symbiosis was unknown.
**Methods:** Four sample types were collected from Macrotermes natalensis colonies: (1) fungus comb interspersed with Termitomyces mycelium, (2) fungus comb with emerging 4-day old mushrooms, (3) mushrooms separated from comb, and (4) axenic agar plate cultures of Termitomyces sp. T153. VOCs were captured using closed-loop-stripping analysis (CLSA) over 24 hours and analyzed by GC-MS with NIST 2017 library annotation. Five drimane sesquiterpenes were isolated from 40 PDA plates (2 L) after 2-4 weeks of growth using flash chromatography and reverse-phase HPLC, with structures elucidated by 1D/2D NMR, HR-MS, and X-ray crystallography. A focused library of drimane derivatives was synthesized from (+)-sclareolide and trans,trans-farnesol. Three candidate terpene synthase genes (DS1-3) were identified by BLAST search against the AstC HAD-like terpene cyclase from Aspergillus oryzae. Heterologous expression was performed in E. coli BL21(DE3) using pET28a and pOPIN M vectors. Antimicrobial activity was tested by disc diffusion assays against bacterial and fungal strains at 1 mg/mL compound concentration.
**Key Results:** Mushroom VOC profiles were dominated by sesquiterpenes (β-barbatene, β-cubebene, brasiladienes) with drimenol, intermedeol, african-1-ene, and α-amorphene in lower abundances, while fungus comb samples emitted mainly aliphatic acids, ketones, and alcohols with β-barbatene as the only detectable terpene. Five drimane derivatives were isolated: compound 2 (3-hydroxy-drimenol, [α]D −4.78), compound 3 (3-oxo-drimenol, [α]D −54.2°), compound 4 (3,12-dihydroxy-drimenol, [α]D −23.51°), compound 5 (drimane lactone, [α]D −6.63°), and compound 6 (dihydroxylated drimanol, [α]D −8.0). X-ray crystallography of compound 2 confirmed the structural assignment (orthorhombic, space group P ī, a=9.9807 Å, b=12.4732 Å, c=21.7124 Å). Total synthesis yielded drimenol (1), drimanol (12), conjugated diene (10), drimenal (11), 3-S-hydroxy-drimanol (18), and 3-oxo-drimanol (19). Three candidate terpene synthase genes (DS1-3) were identified; DS1 (59.5 kDa) and DS2 (66.0 kDa) required MBP fusion for solubility, while DS3 (57.2 kDa) was obtained as soluble His6-tagged protein. DS3-His6 catalyzed formation of two monocyclic sesquiterpenes, nectrianolin C (22, 0.3 mg) and nectrianolin D (23, 0.9 mg), from FPP, but did not produce drimenol or any bicyclic drimane skeleton. Antimicrobial testing showed drimenol (1) and drimenal (11) had moderate antifungal activity against Penicillium notatum and Candida albicans, while compounds 1 and 2 showed antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium vaccae. No growth modulation of Termitomyces sp. T153 or antifeedant activity against Spodoptera littoralis was observed.
**Clinical Implications:** While this study is primarily ecological and biochemical rather than clinical, the antimicrobial activities of drimenol and its derivatives against human-relevant pathogens (S. aureus, P. aeruginosa, C. albicans) suggest potential for development of antimicrobial agents from drimane sesquiterpenes. The structure-dependent activity patterns provide a foundation for structure-activity relationship studies. The finding that drimenol is primarily emitted from mushrooms rather than vegetative mycelium suggests context-dependent antimicrobial defense of fruiting bodies, which could inform natural product discovery strategies targeting specific fungal life stages. The inability to identify the complete drimenol biosynthetic pathway despite genomic mining indicates that additional biosynthetic enzymes remain to be discovered, which is relevant for future heterologous production of these bioactive compounds.