芸香科植物黄皮（Clausena lansium）中含有结构多样的异戊烯基取代芳香类化合物，主要包括异戊烯基取代的香豆素、咔唑及衍生物，是黄皮次级代谢产物结构多样性和药用活性的主要来源。参与黄皮异戊烯基芳香类化合物生物合成的关键异戊烯基转移酶至今无一报道，限制了相关生物合成研究的深入开展。基于此，本研究系统开展了黄皮中芳香类化合物异戊烯基转移酶基因的挖掘和功能研究，所取得的研究结果如下：
(1)黄皮异戊烯基转移酶的克隆和功能鉴定  通过生物信息学分析从黄皮转录组数据中筛选得到5个异戊烯基转移酶候选基因，在酿酒酵母和昆虫表达系统中进行候选基因的异源表达，并通过微粒体反应进行体外酶功能鉴定。结合HPLC-MS和NMR分析发现异戊烯基转移酶ClPT3可以接受GPP为异戊烯基供体，催化伞形花内酯（7-羟基香豆素）发生C-8位的香叶基取代反应。
（2）异戊烯基转移酶ClPT3的酶学性质研究  ClPT3为首次发现的黄皮芳香类化合物异戊烯基转移酶，为了全面了解该酶的性质，本研究对该酶催化反应的最适反应时间、最适反应温度、最适pH值、金属离子选择性、KM值和底物选择性进行了全面考察。结果发现，ClPT3对异戊烯基供体和受体均有高度选择性，可特异性催化伞形花内酯C-8位的香叶基取代反应，该催化反应依赖于二价金属离子，最适反应温度为35 ℃，最适pH为11.0 (CAPS)。酶促动力学分析结果显示，KM值为76.5 μM，Vmax为124.0×10-4 µg·min-1·mg-1。
（3）异戊烯基转移酶ClPT3催化反应的活性位点初探  ClPT3为8次跨膜的强膜结合型蛋白，其结构研究难度较大。为探究ClPT3催化反应的结构基础，本研究利用Alphafold 2模拟其蛋白三维结构，结合分子对接预测催化关键氨基酸位点，进一步通过定点突变技术构建ClPT3突变体，并结合酶活性分析鉴定活性位点。经过多轮突变实验，本研究成功鉴定了ClPT3催化反应的关键活性位点。更值得一提的是，发现突变体S170A催化伞形花内酯发生C-8位香叶基化的活性较野生型增强25%，而N128A可以改变ClPT3催化异戊烯基化的位置选择性，不仅可以产生伞形花内酯的C-8位的取代产物，还可以产生C-6位的取代产物。
综上，本论文研究从黄皮中鉴定了一个新型芳香类化合物异戊烯基转移酶基因ClPT3，其重组酶可以特异性催化伞形花内酯发生C-8位香叶基化反应。本研究通过酶学性质的系统分析掌握了ClPT3催化反应的特点，通过对ClPT3催化反应的结构基础的探究，鉴定了该酶催化反应的关键活性位点，并获得活性增强以及催化位置选择性改变的酶突变体S170A和N128A。本研究所取得的结果为进一步对ClPT3开展深入的酶催化机制和生物合成应用研究打下了基础。

Clausena lansium contains a significant amount of prenylated coumarins, carbazoles and derivatives with diverse structures, which contributes to the structural diversity and medicinal activity of this plant. None of the prenyltransferases involved in the biosynthesis pathway of prenylated aromatic compounds in C. lansium has been reported so far, which has limited the related in-depth biosynthesis study. Thus, the present study was carried out to identify and investigate the function of prenyltransferases genes in C. lansium:
(1) Cloning and functional identification of coumarin prenyltransferase in C. lansium 
Five prenyltransferase candidate genes were selected from C. lansium transcriptome data through bioinformatics analysis, and then heterologously expressed in yeast and insect expression systems respectively, finally enzymatic function was identified through in vitro microsome catalysis. In combination with HPLC-MS analysis, it was found that the prenyltransferase ClPT3 could accept GPP as the prenyl donor and fuse the geranyl group to the C-8 position of umbelliferone(7-hydroxy coumarin).
(2) Enzymatic properties of prenyltransferase ClPT3 
ClPT3 is the first coumarin prenyltransferase identified from C. lansium. In order to fully understand the properties of this enzyme, the optimum reaction time, optimum reaction temperature, optimum pH value, metal ion selectivity, KM value and substrate selectivity of the enzyme were comprehensively investigated in this study. The results revealed that ClPT3 was highly selective for both the prenyl donor and acceptor and specifically catalyzed the geranyl substitution reaction at the C-8 position of umbelliferone, which was dependent on the divalent metal ion, with an optimum reaction temperature of 35 ℃ and an optimum pH of 11.0 (CAPS). The enzymatic kinetic analysis showed that the KM value was 76.5 μM and the Vmax was 124.0×10-4 µg·min-1·mg-1.
(3) Preliminary investigation of the key residues in the active site of ClPT3 
ClPT3 is a strong membrane-bound protein with 8 transmembranes, and therefore its structure is difficult to determine. In order to investigate the structural basis of ClPT3, we used Alphafold 2 to predict the three-dimensional structure of ClPT3, combined with molecular docking to predict the key amino acid sites, and further constructed ClPT3 mutants by site-directed mutagenesis, and identified the active sites by enzymatic activity analysis. After several rounds of mutation experiments, the key active sites of ClPT3 catalytic reaction were successfully identified in this study. More notably, the mutant S170A catalyzed the C-8 geranylation of umbelliferone with greatly enhanced activity compared to the wild type, while N128A could alter the position preference of ClPT3 to produce prenylated umbelliferone not only at the C-8 position, but also at the C-6 position.
In summary, this study identified a novel coumarin prenyltransferase gene from C. lansium, whose recombinant enzyme can specifically catalyse the umbelliferone prenylated at C-8 position. In this case, the enzymatic properties of ClPT3 were systematically analysed to understand the characteristics of ClPT3. The structure and activity relationship of ClPT3 was further investigated by combining structural modelling, molecular docking and targeted mutagenesis techniques, and the key active sites of the enzyme catalyzed by ClPT3 were identified. This thesis provides a paradigm for the discovery and functional study of plant-derived prenyltransferase. In addition, the results obtained in this study lay a solid foundation for further studies on the catalytic mechanism and biosynthetic applications of ClPT3.