米团花二倍半萜生物合成途径关键酶Lc-GFDPS的酶学机制及生物功能研究

Plant sesterterpenoids (C25) are a special and important group of secondary metabolites with unprecedented structural complexity and fascinating bioactivities. However, our knowledge of sesterterpenoid biosynthesis, distribution and biological function in plants is rather limited. Geranylfarnesyl diphosphate synthase (GFDPS), producing the C25 prenyl diphosphate precursor to all sesterterpenoids, is the key enzyme for the biosynthesis and distribution of these compounds. Our previous study on the plant sesterterpenoid biosynthesis pathway led to the clone and functional characterization of a geranylfarnesyl diphosphate synthase (Lc-GFDPS) from the glandular trichomes of Leucosceptrum canum which harbor a large amount of defensive sesterterpenoids. Phylogenetic analysis suggested that GFDPS was probably evolved from plant geranylgeranyl diphosphate synthase (biosynthesizing C20 prenyl diphosphate precursor for diterpenoid) under the influence of positive selection by duplication and neofunctionalization. In addition, the overexpressing Lc-GFDPS Arabidopsis plants showed a phenotype with several stalks bolted from the rosettes after the initiation of flowering, in comparison to the typical single stalk that bolted from the rosette of wild-type plants. Based on these previous researches, this project aims to further investigate the enzymological mechanism of Lc-GFDPS to i) reveal the product chain-length determination mechanism of Lc-GFDPS, ii) analyze the universality of the mechanism in plants containing sesterterpenoids, and then iii) develop a reliable method to discover plant GFDPS from plant genomic or transcriptomic data based on Lc-GFDPS product chain-length determination mechanism. Moreover, the biological function of Lc-GFDPS would be analyzed in overexpressing Lc-GFDPS plants from the perspective of signal pathways, plant hormones and secondary metabolites of plant branching. The results of this project are expected to provide theoretic bases for chemodivisty and distribution of sesterterpenoid, as well as for the utilization of the biological function of Lc-GFDPS in agriculture.

植物二倍半萜(C25)结构新颖、活性显著，是次生代谢产物的重要类群，但其生物合成、分布规律和生物功能研究较少。香叶基法尼基焦磷酸酯合成酶(GFDPS)是负责二倍半萜C25直链前体合成的关键酶基因，对该类化合物的生物合成和分布起决定作用。前期从富含二倍半萜的米团花腺毛中克隆和功能鉴定了米团花香叶基法尼基焦磷酸酯合成酶(Lc-GFDPS)，可能起源于二萜C20直链前体合成酶的复制和新功能化，并发现过量表达Lc-GFDPS的转基因拟南芥呈现分枝增多的表型。拟在此基础上开展Lc-GFDPS的酶学机制及生物功能研究：解析其产物链长决定机制并分析该机制的普遍性，建立从植物组学数据出发快速精准地挖掘植物GFDPS的方法；从调控植物分枝的信号通路、激素和次生代谢三方面，揭示Lc-GFDPS在拟南芥中的生物功能，为其在农业上的应用提供参考价值，并为深入研究植物二倍半萜的化学多样性及分布规律奠定重要基础。

本项目通过分子对接、系统进化结合定点突变，研究了Lc-GFDPS产物链长决定机制和催化机制，发现30个关键氨基酸位点，构建了30个Lc-GFDPS突变酶和7个Lc-GGDPS2突变酶，并对突变酶的功能进行了分析；筛选了火把花、丹参、一串红、黄芩、欧薄荷、马铃薯的GFDPS候选基因，克隆并功能鉴定了火把花、丹参、黄芩和马铃薯的GFDPS和GGDPS。基于GFDPS功能构建了合成萜类直链前体的工程大肠杆菌和酵母菌，鉴定了4个新颖的萜类合酶，包括双功能二倍半萜/二萜合酶、多功能二倍半萜/二萜/倍半萜/单萜合酶、杜松烯合酶和雪松醇合酶，发现了8个二倍半萜化合物，包括2个首次发现的具有18元最大碳环结构的二倍半萜；实现了大环二倍半萜、杜松烯、雪松醇等萜类化合物的异源合成，并发现化合物的免疫抑制、化学防御等活性。发现了合成二倍半萜的唇形科植物宽管花和龙胆科植物新疆假龙胆，从这两种植物和火把花中分离鉴定了23个二倍半萜，并发现其免疫抑制和减脂活性。受邀在天然产物领域权威期刊Nat Prod Rep上分别发表植物二倍半萜的化学、生物活性与生物合成研究以及植物腺毛中非挥发天然产物的化学、生物功能与生物合成研究长篇综述。部分结果已在国内外高水平SCI期刊Angew Chem Int Ed、New Phytol等发表带标注论文10篇，其中IF>10的论文4篇；获授权发明专利1项，申请发明专利3项；参加国内外学术会议交流报告1次，发表会议论文2篇；项目负责人入选中科院“西部之光”青年学者A类等；培养毕业博士生2名和毕业硕士生2名。研究结果为植物GFDPS的开发应用以及新颖萜类合酶（尤其是二倍半萜合酶）的功能鉴定奠定了重要基础，同时也为植物二倍半萜的化学多样性、分布规律、生物功能和生物合成研究提供了科学参考。

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