本氏烟草中角鲨烯的异源合成及其在木栓烷型三萜化合物合成的应用

Heterologous bioynthesis of squalene in Nicotiana benthamiana and its application in the bioynthesis of friedelane-type triterpenoids

中文关键词:本氏烟草  产量优化  角鲨烯  氧化鲨烯环化酶  雷公藤

keywords:Nicotiana benthamiana  yield optimization  squalene  oxidosqualene cyclase  Tripterygium wilfordii

中文摘要:目的：利用本氏烟草异源合成三萜骨架前体角鲨烯，提升其产量并进一步利用其作为前体合成雷公藤活性三萜化合物。方法：采用农杆菌介导的瞬时转染法浸染本氏烟草叶片，筛选萜类前体基因组合、同工酶等，考察取样时间、农杆菌侵染浓度等方法逐步提升目标化合物产量，亚细胞定位分析确定基因在细胞中表达区室。结果：生成角鲨烯的最佳前体基因组合为TwDXS、TwFPS2和AstHMGR共表达，且在浸染后6天收取样品的角鲨烯产量最高，可达14.53 mg·g-1。基于此进一步整合活性最佳的TwOSC1后木栓烷型三萜化合物木栓酮产量达2.04 mg·g-1，通过继续整合TwCYP712K1和TwCYP716C52进一步实现美登木酸和雷公藤酸C的异源合成。结论：本研究异源高效合成了三萜重要线性前体角鲨烯，并利用其进一步合成雷公藤中木栓烷型三萜活性成分，为其他三萜类活性成分异源获取提供了方法借鉴。

Abstract:Objective: Heterologous biosynthesis of the triterpene skeleton precursor squalene in Nicotiana benthamiana to increase its yield and further utilize it to biosynthesize of bioactive triterpenoids from Tripterygium wilfordii. Methods: Agrobacterium-mediated transient transformation was used to infiltrate N. benthamiana leaves to screen for combinations of terpene precursor genes and isozymes. Sampling time and Agrobacterium infiltration concentration were systematically optimized to progressively improve the yields of target compounds. Subcellular localization analysis was performed to determine the expression compartment of the genes within plant cells. Results: The optimal combination of precursor genes for squalene production was the co-expression of TwDXS, TwFPS2, and AstHMGR, which yielded 14.53 mg·g-1 squalene at six days post-infiltration. Co-expression of this combination with the optimally active TwOSC1, the yield of the friedelane-type product friedelin reached 2.04 mg·g-1. Subsequent integration of TwCYP712K1 and TwCYP716C52 further enabled the heterologous biosynthesis of polpunonic acid and wilforic acid C. Conclusion: This study achieved efficient heterologous biosynthesis of linear triterpene precursor squalene, and was further utilized to biosynthesize bioactive friedelane-type triterpenoids from T. wilfordii. This strategy provides a methodological framework for the heterologous production of other bioactive triterpenoids.