苄基异喹啉生物碱（BIA）是一类含有丰富药用活性物质的生物碱，在植物中广泛存在。其中，集中分布在木兰类、毛茛目和睡莲目植物中。目前对于BIA合成途径的研究主要集中在毛茛目和山龙眼目中，木兰类的植物研究较少。近几年，多个木兰类植物的基因组已经得到测序组装，木兰类植物BIA的合成途径也逐渐得到关注。北马兜铃（Aristolochia contorta Bunge.）是木兰类胡椒目植物，富含BIA类生物碱，因其含有毒性成分马兜铃酸而被2020版药典删去。马兜铃酸属于BIA衍生物，合成途径位于BIA生物合成途径下游。解析北马兜铃中BIA的合成机制，不仅对北马兜铃及马兜铃酸类药材的现代化开发具有重要科学价值，同时可为BIA合成生物学研究体系的优化提供理论支撑。

本篇论文，我们关注到北马兜铃BIA途径中两个具有构型选择性的酶基因，入口酶基因去甲乌药碱合酶（NCS）和CYP80B8基因。围绕这两个关键酶基因的具体研究如下：

（1）全基因组鉴定AcNCS酶基因，并对其理化性质、进化等进行分析。从全基因组水平鉴定了北马兜铃NCS基因，共鉴定出15条全长AcNCS基因。对AcNCS的理化性质、保守结构域和保守motif进行分析。结合多序列比对，进化树分析和去甲乌药碱含量的组织分布情况等，筛选出AcNCS2，AcNCS4，AcNCS5，AcNCS14，AcNCS15作为北马兜铃NCS候选基因，进行下一步的功能验证。

（2）体外酶法研究AcNCS的功能。克隆了AcNCS2，AcNCS4，AcNCS5，AcNCS14，AcNCS15基因，构建体外原核表达载体。诱导、提取融合蛋白，纯化后进行体外酶促反应。体外酶法验证了AcNCS2，AcNCS4，AcNCS5，AcNCS14，AcNCS15均可以催化去甲乌药碱的生成。且与毛茛目NCS不同，与莲NnNCS相似，AcNCS2，AcNCS4，AcNCS5，AcNCS14，AcNCS15可以同时生成S构型和R构型的去甲乌药碱，且S构型产量大于R构型。测定酶动力学参数，发现具有两个串联结构域的AcNCS14，AcNCS15的酶活性大于只具有一个结构域的AcNCS2，AcNCS4，AcNCS5。

（3）体外酶法研究AcCYP80B8功能。利用酵母表达体系获得AcCYP80B8微粒体蛋白，进行体外酶促反应，发现其可以催化(R)-N-甲基乌药碱反应，生成3’-羟基-N-甲基乌药碱，即AcCYP80B8是北马兜铃N-甲基乌药碱3’羟基化酶（AcNMCH）。它与毛茛目植物中特异反应(S)-N-甲基乌药碱的NMCH基因在构型选择上不同。AcCYP80B8反应(R)-N-甲基乌药碱的最适温度和pH分别为30 ℃和7.5。对AcCYP80B8的底物构型特异性进行分析，发现AcCYP80B8不能催化(S,R)-N-甲基乌药碱生成3’-羟基-N-甲基乌药碱。利用多序列比对和分子对接对关键催化位点进行预测，结合单碱基突变技术获得突变体。测定突变体的酶促反应变化，发现AcCYP80B8^I215L突变对功能影响不大，AcCYP80B8^M219L突变影响酶活力十分明显。

综上，本研究对北马兜铃中AcNCS酶基因进行了全基因组水平鉴定、筛选、进化分析与功能验证，并对AcCYP80B8进行了功能研究和关键催化位点鉴定，得到一个影响反应的关键位点M219。在此之外，探究了北马兜铃不依赖组织培养体系的遗传转化系统。以上研究结果为苄基异喹啉类生物碱（BIA）合成生物学提供了重要的候选酶资源，为解析 BIA 合成途径中的手性控制机制提供了参考依据，同时为解析北马兜铃中马兜铃酸的合成奠定了基础，并为其遗传转化提供了具有可行性的方法。

Benzyl isoquinoline alkaloids (BIA) are a class of alkaloids containing rich medicinal active substances and are widely found in plants. It is concentrated in Magnoliids, Ranales and Proteales. At present, the research on the BIA synthesis pathway mainly focuses on Ranales and Proteales, and there are few studies on Magnoliids. In recent years, the genomes of many Magnoliids plants have been sequenced and assembled, and the synthetic pathway of BIA in magnolia plants has gradually received attention. Aristolochia contorta Bunge. belongs to magnoliids clade, Piperales order and is rich in BIA alkaloids. It was deleted from the 2020 edition of the pharmacopoeia because it contains the toxic component aristolochic acid. Aristolochic acid is a BIA derivative and its synthesis pathway is located downstream of BIA synthesis pathway. The analysis of the synthetic mechanism of BIA in A. contorta not only has important scientific value for the modern development of A. contorta and medicinal materials containing aristolochic acid, but also provides theoretical support for the optimization of BIA synthetic biology research system.

In this paper, we focus on two configuration-selective enzyme genes in the BIA pathway of A. contorta, the first enzyme genes Norcoclaurine Synthase Gene (NCS) in BIA synthesis pathway and CYP80B8. The research around these two key enzyme genes is as follows:

(1) The whole AcNCS enzyme gene was identified in the whole genome level, and its physicochemical properties and evolution were analyzed. NCS genes of A. contorta were identified at the whole genome level, and 15 AcNCS genes were identified. The physicochemical properties, conserved domains and conserved motifs of AcNCS were analyzed. Combined with multiple sequence alignment, evolutionary tree analysis and tissue distribution of norcoclaurine content, AcNCS2，AcNCS4，AcNCS5，AcNCS14，AcNCS15 were selected as NCS candidate genes of A. contorta for further functional verification.

(2) The function of AcNCS was studied by enzymatic method in vitro. AcNCS2，AcNCS4，AcNCS5，AcNCS14，AcNCS15 genes were cloned and prokaryotic expression vectors were constructed in vitro. The fusion protein was induced, extracted and purified for enzymatic reaction in vitro. AcNCS2，AcNCS4，AcNCS5，AcNCS14，AcNCS15 can catalyze the formation of norcoclaurine. Unlike NCS of Ranales, but similar to NnNCS of lotus, AcNCS2，AcNCS4，AcNCS5，AcNCS14，AcNCS15 can produce both S-configuration and R-configuration norcoclaurine, and the yield of S-configuration is greater than that of R configuration. The activity of AcNCS14, AcNCS15 with two tandem domains was higher than that of AcNCS2，AcNCS4，AcNCS5 with only one domain.

(3) Study the function of AcCYP80B8. AcCYP80B8 microsome protein was obtained by yeast expression system, and it was found that AcCYP80B8 could catalyze the (R)-N-methycoclaurine reaction to produce 3'-hydroxy-N-methycoclaurine ，which means its 3' hydroxylase of N-methycoclaurine in A. contorta (AcNMCH). It is different from the NMCH that is specific for (S)-N-Methylcoclaurine in Ranales. The optimum temperature and pH for the reaction of (R)-N-Methylcoclaurine with AcCYP80B8 are 30 ℃ and 7.5, respectively. The substrate configurational specificity of AcCYP80B8 was analyzed, and it was found that AcCYP80B8 could not react (S,R)-N-Methylcoclaurine. Multiple sequence alignment and molecular docking were used to analyze the key catalytic sites, mutate them, and measure the changes of enzymatic reaction. AcCYP80B8^I215L mutation had little effect on function, while AcCYP80B8^M219L mutation had significant effect on enzyme activity.

In summary, this study conducted genome-wide identification, screening, evolutionary analysis and functional verification of the AcNCS enzyme gene in A. contorta, and carried out functional research and key catalytic site identification on AcCYP80B8, obtaining a key site M219 that affects the reaction. In addition, the genetic transformation system of A. contorta that does not rely on the tissue culture system was explored. The above research results provide important candidate enzyme resources for the synthetic biology of benzylisoquinoline alkaloids (BIA), offer a reference basis for understanding the chiral control mechanism in the BIA synthetic pathway, lay a foundation for the synthesis of aristolochic acid in A. contorta, and provide a feasible method for its genetic transformation.