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Abstract：
BACKGROUND: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids.
RESULTS: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach.
CONCLUSIONS: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.
RESULTS: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach.
CONCLUSIONS: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.
摘要：
背景：角鲨烯环氧酶是膜甾醇和三萜类生物合成途径中的限速酶之一。该酶催化氧化角鲨烯的形成，它是甾醇和三萜类化合物的常见前体。
结果：在这项研究中，在Poriacocos中评估了角鲨烯环氧酶基因（PcSE）。在PcSE和角鲨烯之间进行分子对接并鉴定活性氨基酸。基于活性位点残基设计sgRNA。对P.cous中三萜合成的影响与超高效液相色谱-四链体飞行时间-双质谱（UHPLC-QTOF-MS/MS）分析的结果一致。结果表明，PcSE的缺失抑制了三萜的合成。使用PEG介导的原生质体转化方法进行PcSE功能的体内验证。
结论：本研究的发现为进一步研究假单胞菌次生代谢产物的异源生物合成奠定了基础。
结果：在这项研究中，在Poriacocos中评估了角鲨烯环氧酶基因（PcSE）。在PcSE和角鲨烯之间进行分子对接并鉴定活性氨基酸。基于活性位点残基设计sgRNA。对P.cous中三萜合成的影响与超高效液相色谱-四链体飞行时间-双质谱（UHPLC-QTOF-MS/MS）分析的结果一致。结果表明，PcSE的缺失抑制了三萜的合成。使用PEG介导的原生质体转化方法进行PcSE功能的体内验证。
结论：本研究的发现为进一步研究假单胞菌次生代谢产物的异源生物合成奠定了基础。
