<p>(-)-Aristolone, a sesquiterpene with promising therapeutic properties such as antidiabetic and vasorelaxant effects, currently suffers from limited availability due to inefficient chemical synthesis and lack of viable extraction methods. This study presents a novel strategy for high-yield microbial (-)-aristolone production using <i>Sanghuangporus sanghuang</i> DM989 as a fungal chassis. Genome mining identified nine sesquiterpene synthases, among which TPS2152 was functionally linked to (-)-aristolone biosynthesis. TPS2152 harbors a rare DQxxD motif, diverging from the canonical DDxxD motif in plants, suggesting unique catalytic properties in fungi. Overexpression of farnesyl pyrophosphate synthase (FPPS) increased FPP precursor supply, resulting in a 78.79% rise in squalene content (1.18 mg/g) and enabling de novo (-)-aristolone synthesis (0.42 mg/g) in the FPPS⁺ strain. To enhance FPP flux toward (-)-aristolone, the ΔSQS/TPS2152⁺ strain was constructed by co-overexpressing TPS2152 and silencing squalene synthase (SQS), yielding a 210% increase in (-)-aristolone (1.30 mg/g) and 56.78% reduction in squalene compared to FPPS⁺. Further, site-directed mutagenesis converted DQxxD to DDxxD, producing TPS2152D, which retained substrate binding affinity (docking score: − 9.1 kcal/mol) and exhibited a 2.57-fold increase in catalytic efficiency. Integration of TPS2152D with SQS silencing produced the ΔSQS/TPS2152D⁺ strain, achieving a 217% higher (-)-aristolone yield than FPPS⁺. Fermentation kinetics showed product accumulation from day 5, with maximal <i>Q</i>_p on days 8 and complete squalene suppression by day 9. These results establish <i>S. sanghuang</i> as a robust microbial platform for sesquiterpene production and demonstrate the feasibility of combining fungal pathway engineering and motif-based enzyme optimization for scalable biosynthesis of high-value terpenoids.</p> Graphical Abstract <p></p>