Engineering Sanghuangporus sanghuang for enhanced (-)-aristolone production via metabolic pathway optimization and terpene synthase engineering

文献类型： 外文期刊

第一作者： Li, Yihan

作者： Li, Yihan;Xu, Jiahui;Pan, Weishan;Liang, Jian;Ma, Xiao-kui;Kang, Chuanzhi;Guo, Lanping;Zhou, Wenqing;Xia, Daofang;Ma, Xiao-kui

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关键词： Sanghuangporus sanghuang; Sesquiterpenoids; Terpenoid synthesis (TPS); Sesquiterpene synthase; (-)-Aristolone; Fermentation kinetics

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.3； 五年影响因子：5.1 ）

ISSN： 0175-7598

年卷期： 2025 年 109 卷 1 期

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收录情况： SCI

摘要： (-)-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 Sanghuangporus sanghuang 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 Delta SQS/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 Delta SQS/TPS2152D+ strain, achieving a 217% higher (-)-aristolone yield than FPPS+. Fermentation kinetics showed product accumulation from day 5, with maximal Qp on days 8 and complete squalene suppression by day 9. These results establish S. sanghuang 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.

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