中药五味子是五味子（Schisandra chinensis (Turcz.) Baill.）的干燥成熟果实，因其具有酸、苦、甘、辛、咸五味而得名。现代药理学研究证明，联苯环辛烯类木脂素，具有联苯键和环辛二烯环，是五味子的主要活性成分，具有保肝、抗氧化、抗炎、抗肿瘤、抗病毒等良好活性，临床需求量大。但是五味子植物生长周期长，有效成分含量低，提取分离纯化步骤繁琐，野生资源越来越少且人工栽培起步较晚，所以亟需开发新的资源供给模式。近些年来，由于中药资源的可持续利用一直是个热门话题，植物天然产物合成生物学应运而生，越来越多的中药活性成分已经通过合成生物学策略实现了新的供给模式。综上所述，基于联苯环辛烯类木脂素结构独特和活性显著的特点，引起了团队对此类化合物生物合成途径解析的极大兴趣。

2007年，日本京都大学的学者Shiro Suzuki和Toshiaki Umezawa对各种结构类型木脂素类化合物的生物合成途径进行了总结和推测，其中对联苯环辛烯类木脂素生物合成途径的推测是：前体物质松柏醇被催化生成异丁香酚，接下来异丁香酚发生二聚化反应生成渥路可脂素，渥路可脂素接着发生开环反应生成二氢愈创木脂酸，最后二氢愈创木脂酸经分子内芳基偶联反应形成联苯键产生联苯环辛烯类木脂素戈米辛A。有学者从矮牵牛（Petunia × hybrida）中分离出两个酶PhCFAT和PhIGS1，前者能够催化松柏醇生成乙酸松柏酯，后者能够催化乙酸松柏酯生成异丁香酚。因此，松柏醇酰基转移酶（coniferyl alcohol acyltransferase, CFAT）和异丁香酚合酶（isoeugenol synthase, IGS）作为继松柏醇之后的前两个限速酶，它们对于联苯环辛烯类木脂素的积累以及生物合成途径的解析至关重要。

为了鉴定五味子中编码CFAT和IGS的基因，本研究基于不同发育时期五味子果实的转录组数据库进行了基因家族的鉴定和生物信息学分析，不仅对五味子基因家族的功能进化有了系统的认识，还筛选得到了关键候选基因，并通过体外酶促反应和底物饲喂的方式从五味子中鉴定到两个具有催化活性的酶ScCFAT和ScIGS1。

现将主要研究内容及结果叙述如下：

1. 首先为了明确五味子中是否存在编码CFAT和IGS的基因，本研究使用气质联用仪对五味子果实乙酸乙酯提取物和异丁香酚标准品溶液进行了检测，通过比对发现五味子果实中富含大量顺、反式异丁香酚混合物，以反式异丁香酚为主。因此，可以明确五味子中存在编码CFAT和IGS的基因，另一方面也进一步证实了联苯环辛烯类木脂素生物合成途径的可靠性。

2. 为了对五味子BAHD酰基转移酶家族有系统的了解并发掘编码CFAT的候选基因，本论文基于五味子果实转录组数据库对BAHD酰基转移酶家族成员进行了系统的鉴定和生物信息学分析。一共鉴定出37条ScBAHD氨基酸序列，与其它物种中共计75条BAHD酰基转移酶序列共同构建了系统发育树。结果显示，所有BAHD酰基转移酶划分为五大分支，除了第IV分支之外，37条ScBAHD在其它分支中均有分布。其中，ScBAHD1、ScBAHD3和ScBAHD7分布在第III分支中，与PhCFAT的亲缘关系最近。表达模式分析表明，只有ScBAHD1表达水平的变化符合联苯环辛烯类木脂素在五味子不同发育时期果实中的积累模式，因此被选为候选基因用于后续研究。

3. 为了验证ScBAHD1是否具有松柏醇酰基转移酶的催化活性，本研究通过同源重组技术将其开放阅读框（open reading frame, ORF）连接在原核表达载体上，以PhCFAT作为阳性对照，通过体外酶促反应发现ScBAHD1能够催化松柏醇和乙酰辅酶A产生与阳性对照组相同的产物乙酸松柏酯，所以将其命名为ScCFAT。此外，对ScCFAT进行底物多样性研究发现它只对醇羟基具有乙酰化活性，对酚羟基尚未检测到催化活性。ScCFAT的亚细胞定位分析结果表明其主要定位于烟草叶片的细胞质中。同源建模所得ScCFAT的三维晶体结构符合BAHD酰基转移酶家族成员三维结构的基本特征。最后，通过丙氨酸筛查的方式明确了His158是ScCFAT的关键残基，突变之后会导致其完全失去催化活性。

4. 为了对五味子PIP还原酶家族有系统的了解并发掘编码IGS的候选基因，本研究基于五味子果实转录组数据库对PIP还原酶家族成员进行了系统的鉴定和生物信息学分析。一共鉴定出9条ScPIP氨基酸序列，与其它物种中的32条PIP还原酶序列共同构建了系统发育树。结果显示，所有PIP还原酶一共划分为四大分支，9条ScPIP散布在四个分支中。其中，ScPIP1处于第IV分支中，与PhIGS1的亲缘关系最近。ScPIP2和ScPIP4位于第III分支中，该分支中包含一些丁香酚合酶（eugenol synthase, EGS）成员。实时荧光定量PCR（real-time quantitative PCR, RT-qPCR）检测结果表明，ScPIP1和ScPIP2表达水平的变化趋势与ScCFAT的表达模式基本一致，ScPIP4却一直呈现出较低的表达水平。最终，本研究选择对ScPIP1、ScPIP2和ScPIP4这三条基因进行克隆及功能研究。

5. 为了验证ScPIP1、ScPIP2和ScPIP4是否具有异丁香酚合酶或者丁香酚合酶的催化活性，本研究将它们的ORF序列连接在原核表达载体上，将松柏醇饲喂孵育重组质粒的大肠杆菌，利用大肠杆菌内源性酰基转移酶乙酰化松柏醇生成乙酸松柏酯作为底物。检测发现，ScPIP1组和阳性对照PhIGS1组的大肠杆菌在培养基中释放出了异丁香酚，但是ScPIP2、ScPIP4以及空载体组既没有产生异丁香酚，也没有产生丁香酚，所以将ScPIP1命名为ScIGS1。亚细胞定位分析结果显示，ScIGS1主要定位于烟草叶片的细胞质及细胞核内。同源建模所得ScIGS1的三维晶体结构符合PIP还原酶家族成员三维结构的基本特征。通过定点突变实验发现，110位和113位氨基酸都会影响ScIGS1催化产物的组成及比例，His113突变为Tyr不会改变产物组成，Lys157突变为Ala会导致ScIGS1完全失去催化活性。最后，对ScIGS1及其5个突变体的酶转换数（k_cat）进行预测发现5个突变体的k_cat值均小于ScIGS1的k_cat值，表明突变之后酶的催化活性降低。

综上所述，本研究首次基于前人对联苯环辛烯类木脂素生物合成途径的推测开展了一系列基因家族鉴定和生物信息学分析，从五味子中克隆得到联苯环辛烯类木脂素生物合成途径上编码CFAT和IGS的cDNA全长基因，并且进行了功能验证，是联苯环辛烯类木脂素生物合成途径解析的开拓性进展。此外，这两个基因将有利于后续通过共表达分析结合基因家族分析发掘出更多可能参与下游催化反应的候选基因，旨在早日完全解析联苯环辛烯类木脂素的生物合成途径，从而实现利用微生物细胞工厂异源生产联苯环辛烯类木脂素以及通过分子选育开发优质新品种的目标。

Schisandrae Chinensis Fructus, the dried ripe fruits of Schisandra chinensis (Turcz.) Baill., is commonly known as “Wuweizi” for its sour, bitter, sweet, pungent, and salty flavors. Modern pharmacological research has shown that the major bioactive components of Wuweizi are dibenzocyclooctadiene lignans, with biphenyl bonds and cyclooctadiene rings. These lignans exhibit a wide variety of biological activities, such as hepatoprotective, anti-inflammation, anti-oxidant, anti-tumor, anti-virus, and so on. As a result, they are in high clinical demand. However, S. chinensis has a long growth cycle, low content of dibenzocyclooctadiene lignans, cumbersome extraction, separation, and purification steps, limited wild resources, and a late start in artificial cultivation. Therefore, there is an urgent need to develop new resource supply models. In recent years, as the sustainable utilization of traditional Chinese medicine resources has been a hot topic, synthetic biology of plant natural products has emerged as the times require. More and more active ingredients of traditional Chinese medicine have realized new acquisition modes through synthetic biology strategy. In summary, based on the unique structure and significant activities, we have aroused great interest in the elucidation of the biosynthetic pathway of dibenzocyclooctadiene lignans.

In 2007, Shiro Suzuki and Toshiaki Umezawa, scholars from Kyoto University in Japan, summarized and speculated on the biosynthetic pathways of various structural types of lignans. Among them, they speculated that the biosynthetic pathway of dibenzocyclooctadiene lignans was as followed. The precursor, coniferyl alcohol, is catalyzed to form isoeugenol, and then isoeugenol is dimerized to form verrucosin, which undergoes ring-opening reactions to generate dihydroguaiaretic acid. Finally, dihydroguaiaretic acid forms the biphenyl bond through an intramolecular aryl-aryl coupling reaction to generate gomisin A. There are two enzymes isolated from Petunia × hybrida named PhCFAT and PhIGS1. The former can catalyze the formation of coniferyl acetate from coniferyl alcohol and the latter can catalyze the formation of isoeugenol from coniferyl acetate. Therefore, coniferyl alcohol acyltransferase (CFAT) and isoeugenol synthase (IGS), as the first two enzymes following coniferyl alcohol, are crucial for the accumulation of dibenzocyclooctadiene lignans and the elucidation of their biosynthetic pathway.

To identify the genes encoding CFAT and IGS in S. chinensis, gene family identification and bioinformatics analyses based on the transcriptome datasets of S. chinensis fruits at different development stages were carried out in this study. The findings of this study offer valuable insights into the functional evolution of the gene family, while also highlighting the identification of key candidate genes. Two catalytic enzymes ScCFAT and ScIGS1 were identified from S. chinensis through in vitro enzymatic reaction and in vivo functional characterization.

The main research contents and results are described as follows:

1. Firstly, to determine whether there are genes encoding CFAT and IGS in S. chinensis, we detected the ethyl acetate extract of S. chinensis fruits and isoeugenol standard solution through gas chromatography-mass spectrometry. The results indicate that S. chinensis fruits produce a large amount of isoeugenol including E-isoeugenol and Z-isoeugenol, mainly the former. Therefore, the presence of isoeugenol can confirm the presence of genes encoding CFAT and IGS in S. chinensis. On the other hand, it further confirms the reliability of the biosynthetic pathway of dibenzocyclooctadiene lignans.

2. In order to gain a comprehensive understanding of the BAHD acyltransferase family in S. chinensis and to investigate genes encoding CFAT, we undertook a systematic identification of the BAHD acyltransferase family members based on the transcriptome database of S. chinensis and conducted bioinformatics analyses. A total of 37 ScBAHD protein sequences were identified in this study and we constructed a phylogenetic tree of them along with 75 biochemically characterized BAHD acyltransferase members from other species. The result showed that all BAHD acyltransferase members were divided into five major clades, with 37 ScBAHD distributed in all other branches except for clade IV. Among them, ScBAHD1, ScBAHD3, and ScBAHD7 were clustered into Clade III and closely related to PhCFAT. The results of expression pattern analysis showed that only the changes in the expression level of ScBAHD1 were consistent with the accumulation pattern of dibenzocyclooctadiene lignans in S. chinensis fruits at different development stages, so it was selected as a candidate gene for subsequent research.

3. To verify whether ScBAHD1 has the catalytic activity of CFAT, the open reading frame (ORF) of ScBAHD1 was connected to the prokaryotic expression vector. With PhCFAT as the positive control, it was found that ScBAHD1 could catalyze coniferyl alcohol and acetyl-CoA to produce the same product coniferyl acetate as the positive control group through in vitro enzymatic reaction, so it was designated as ScCFAT. In addition, substrate diversity research on ScCFAT revealed that it functionally prefers alcohol hydroxyl acetylation rather than phenol hydroxyl. The results of subcellular localization analysis indicated that ScCFAT is mainly localized in the cytoplasm of tobacco leaves. The three-dimensional crystal structure of ScCFAT obtained through homologous modeling conforms to the basic characteristics of the three-dimensional structure of members in the BAHD acyltransferase family. Finally, it was confirmed through alanine screening that His158 is a key residue of ScCFAT, and mutation led to its complete loss of catalytic activity.

4. In order to achieve a comprehensive understanding of the PIP reductase family in S. chinensis and explore candidate genes encoding IGS, we systematically identified the PIP reductase family members based on the transcriptome database of S. chinensis and conducted bioinformatics analyses. A total of 9 ScPIP protein sequences were identified in this study and we constructed a phylogenetic tree of them along with 32 biochemically characterized PIP reductase members from other species. The results showed that all PIP reductases were divided into 4 major clades, with 9 ScPIPs scattered throughout the 4 clades. Among them, ScPIP1 was clustered into clade IV and closely related to PhIGS1. ScPIP2 and ScPIP4 were clustered into clade III, which includes some EGS (eugenol synthase) members. The results of real-time quantitative PCR (RT-qPCR) revealed that the expression patterns of ScPIP1 and ScPIP2 were consistent with that of ScCFAT, while ScPIP4 exhibited low expression all the time. We eventually chose ScPIP1, ScPIP2, and ScPIP4 for further clone and functional characterization.

5. To verify whether ScPIP1, ScPIP2, and ScPIP4 have the catalytic activity of IGS or EGS, their ORF sequences were connected to the prokaryotic expression vector. We fed the Escherichia coli incubating the recombinant plasmids with coniferyl alcohol, which could be acetylated by the endogenous acyltransferase of E. coli to form coniferyl acetate as substrate. It was found that E. coli in the groups of ScPIP1 and PhIGS1 released isoeugenol into the liquid medium, while the groups of ScPIP2, ScPIP4, and empty vector did not produce isoeugenol, so ScPIP1 was designated as ScIGS1. The results of subcellular localization analysis indicated that ScIGS1 is located in the cytoplasm and nucleus of tobacco leaves. The three-dimensional crystal structure of ScIGS1 obtained through homologous modeling conforms to the basic characteristics of the three-dimensional structure of the PIP reductase family members. Through the site-directed mutagenesis experiments of ScIGS1, it was found that the amino acids at positions 110 and 113 could affect the composition and proportion of products. The mutation of His113 to Tyr didn’t change the product composition, and the mutation of Lys157 to Ala resulted in the complete deactivation of ScIGS1. Finally, the results of the prediction of the enzyme turnover numbers (k_cat) of ScIGS1 and its five mutants revealed that the k_cat values of all five mutants were lower than that of ScIGS1, indicating a decrease in enzyme activity after the mutation.

In conclusion, a series of gene family identification and bioinformatics analyses were carried out in this study for the first time according to the previous speculation on the biosynthetic pathway of dibenzocyclooctadiene lignans. We cloned the full-length cDNA genes of CFAT and IGS from S. chinensis and carried out functional verification for the first time, which is pioneering progress in the elucidation of the biosynthetic pathway of dibenzocyclooctadiene lignans. In addition, these two genes will facilitate the subsequent discovery of candidate genes that may participate in downstream catalytic reactions through co-expression analyses and gene family identification, aiming to completely elucidate the biosynthetic pathway of dibenzocyclooctadiene lignans as soon as possible, to achieve the goals of heterogenous production of dibenzocyclooctadiene lignans using microbial cell factories and cultivation of superior varieties through molecular breeding.