CN114480312B - 一种松香烷型三环二萜类化合物c-14位羟化酶 - Google Patents
一种松香烷型三环二萜类化合物c-14位羟化酶 Download PDFInfo
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Abstract
本发明涉及一种催化松香烷型三环二萜类化合物C‑14羟化的酶，所述酶为一种雷公藤细胞色素p450氧化酶CYP82D274，以及涉及编码所述酶的多核苷酸CYP82D274，所述酶催化实验证实其具有催化雷公藤甲素生物合成中间体次丹参酮二烯(miltiradiene)、脱氢松香酸(dehydroabietic acid)和雷酚萜D(triptobenzene D)C‑14位羟基化的功能，本发明进一步推动了雷公藤甲素生物合成途径解析，并为雷公藤甲素中间体合成生物学生产提供了重要基因元件，对雷公藤甲素等松香烷型三环二萜化合物的合成调控具有重要的意义。
Description
技术领域
本发明涉及一种雷公藤细胞色素P450氧化酶，以及编码所述酶的多核苷酸，所述酶可用于松香烷型三环二萜类化合物的关键C-14位生物合成，属于药用植物基因工程领域。
背景技术
雷公藤甲素是药用植物雷公藤(Tripterygium wilfordii Hook.f.)的主要活性成分之一，是一种具有3个环氧基团及一个α,β不饱和五元内酯环结构的松香烷型二萜化合物，同时也是雷公藤中活性最高的环氧二萜内酯化合物(Zhou ZL,et al.Triptolide:structural modifications,structure-activity relationships,bioactivities,clinical development and mechanisms[J].Natural Product Reports,2012,29(4):457-475)。雷公藤甲素具有显著的抗炎、免疫抑制、抗肿瘤等生物活性，细胞或动物模型实验表明其对类风湿性关节炎、系统性红斑狼疮等自身免疫系统疾病，胰腺癌、肺癌等多种癌症具有较好的疗效，同时研究表明，雷公藤甲素对老年痴呆症、帕金森病等中枢神经系统疾病展现出潜在的药用价值，基于雷公藤甲素构-效机制研发的多种衍生药物已进入临床试验阶段(Zhou ZL等,Natural Product Reports,2012,29(4)；TitovDV等,Nature ChemicalBiology,2011,7(3)；Huang J等,Molecular and Cellular Endocrinology,2015,399)。当前，雷公藤甲素的获取方式依赖传统的植物提取和化学合成，受限于植物较长的生长周期和化合物复杂的手性结构，其获取量难以满足临床科研和治疗的大量需求。近年来，利用合成生物学策略对雷公藤甲素生物合成途径进行解析，有望通过这种新颖且可持续的生产方式获取雷公藤甲素及其中间体。
当前，雷公藤甲素生物合成途径的解析，已成功表征了萜类合酶TPS7v2和TPS27v2环化形成雷公藤甲素关键母核结构——次丹参酮二烯(Su P等，Plant Journal,2018,93(1))，并揭示了其生物合成下游途径第一个细胞色素P450CYP728B70(Tu L等,NatureCommunications,2020,11(1))，完成了从二萜共同前体牻牛儿基牻牛儿基焦磷酸到次丹参酮二烯的母核环化，再经双键重排自发转化为阿松香三烯，再羧基化形成脱氢松香酸的过程。然而，从脱氢松香酸到雷公藤甲素的下游多步途径仍然未知。
本研究的对象雷公藤甲素在水中溶解度极低(约17μg·mL-1)，而对其C-14位羟基进行三步化学合成转化成相应的磷酸二钠盐后，其溶解度显著提升至61mg·mL-1，同时具有较好的药物稳定性(Patil S等,Journal of Medicinal Chemistry,2015,58(23))。由此可见，揭示雷公藤甲素的关键靶点C-14位羟基化机制对雷公藤甲素的生物合成及结构修饰具有重要意义。
发明内容
本发明的第一方面，提供了一种分离的酶，所述酶参与三环二萜类化合物的C-14位羟基化过程，尤其是松香烷型二萜类化合物的C-14位羟基化过程。
本发明中，所述分离的酶，是从药用植物雷公藤中分离得到的细胞色素P450氧化酶(以下用CYP82D274表示)，参与雷公藤松香烷型二萜类化合物C-14位羟化的合成过程、尤其是雷公藤甲素C-14位羟化的关键酶，所述酶具有SEQ ID NO：2所示的氨基酸序列，或SEQID NO：2所示氨基酸序列经取代、缺失或增加一个或多个氨基酸其功能相同的肽。
可以在CYP82D274酶中进行氨基酸序列改变以便最少破坏对生物学活性必须的高级结构。例如，当CYP82D274酶包含一个或多个螺旋时，将改变氨基酸残基以便不破坏螺旋的几何学和其中的构象改变将使一些关键功能(例如分子与其结合伙伴的结合)减弱的其它分子成分。氨基酸序列改变的效果可以通过例如以上公开的计算机模拟进行预测，或通过晶体结构分析进行测定(例如Lapthorn等，Nat.Struct.Biol.2:266-268，1995)。本领域熟知的其它技术对改变蛋白和标准分子(例如天然蛋白)的折叠进行比较，例如，可以比较变体和标准分子中的半胱氨酸分布情况。质谱和使用还原烷基化的化学修饰为测定与二硫键相关的或未形成这些键的半胱氨酸残基提供了方法(Bean等，Anal.Biochem.201:216-266,1992；Gray，Protein Sci.2:1732-1748,1993；和Patterson等，Anal.Chem.66:3727-3732，1994)。一般认为如果修饰的分子与标准分子具有不相同的半胱氨酸分布情况，则折叠受到影响。另一个用于测量这点的被接受的熟知方法是园二色谱(CD)。测量和比较修饰分子和标准分子产生的CD谱是常规的(Johnson,Proteins 7:205-214,1990)。晶体学是另一个分析折叠和结构的熟知方法，核磁共振(NMR)、消化肽作图和表位作图也是分析折叠及蛋白质和多肽之间的结构相似性的已知方法(Schaanan等，Science 257:961-964,1992)。
本发明中，CYP82D274酶的变异体形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严谨条件下能与CYP82D274酶编码基因杂交的DNA所编码的蛋白。
本发明的第二方面，提供了一种编码本发明所述酶的多核苷酸(或称雷公藤细胞色素P450氧化酶编码基因，以下可用CYP82D274表示)，所述多核苷酸优选具有SEQ ID NO：1所示核苷酸序列及其简并序列。该简并序列指位于SEQ ID NO：1序列核苷酸中，有一个或多个密码子被编码相同氨基酸的简并密码子所取代后而产生的序列。由于密码子的简并性，所以与SEQ ID NO：1核苷酸序列同源性低至约74％的简并序列也能编码出SEQ ID NO：2所述酶的氨基酸序列。还包括能在中度严谨条件下，更佳的在高度严谨条件下与SEQ ID NO：1中从核苷酸162-1566位的核苷酸序列杂交的核苷酸序列。还包括与SEQ ID NO：1的核苷酸序列的同源性至少70％，较佳地至少80％，更佳地至少90％，最佳地至少95％的核苷酸序列。还包括能编码具有与天然的雷公藤细胞色素P450氧化酶相同功能的蛋白的SEQ ID NO：1中开放阅读框序列的变异形式。这些变异形式包括(但不限于)若干个(通常为1-90个，较佳地1-60个，更佳地1-20个，最佳地1-10个)核苷酸的缺失、插入和/或取代，以及在5′和/或3′端添加数个(通常为60个以内，较佳地为30个以内，更佳地为10个以内，最佳地为5个以内)核苷酸。
本发明的第三方面，提供了一种包含本发明第二方面所述多核苷酸的表达载体，所述的表达载体，如可以是选自pESC系列的表达载体。
本发明中，可选用本领域已知的各种载体，如市售的载体，包括质粒、粘粒等。在生产本发明CYP82D274酶时，可以将CYP82D274酶编码基因的核苷酸序列可操作地连接于表达调控序列，从而形成雷公藤P450表达载体。所述“可操作地连接”当指DNA区段时表示这些区段按照一定方式排列以致它们可以协调地为其预期的目的发挥作用，例如在启动子中起始转录并前行通过编码区段到达终止子。也指这样一种状况：即线性DNA序列的某些部分能够影响同一线性DNA序列其它部分活性，例如，如果信号肽DNA作为前提表达并参与多肽的分泌，那么信号肽(分泌前导序列)DNA就是可操作地连接于多肽DNA；如果启动子控制序列转录，那么它是可操作地连接于编码序列；如果核糖体结合位点被置于能使其翻译的位置时，那么它是可操作地连接于编码序列。一般，“可操作地连接”意味着相邻，而对于分泌前导序列则意味着在阅读框中相邻。
本发明第四方面，提供了一种包含本发明第二方面所述多核苷酸、或本发明第三方面所述表达载体的重组宿主菌，所述宿主菌为酵母菌，如BY系列酵母菌、WAT系列酵母菌等。
本发明述宿主菌，包含本发明所述编码CYP82D274酶或其变体的多核苷酸分子，或在严谨条件下可与所述多核苷酸分子进行杂交的核苷酸分子，或包含本发明上述的表达载体。所述的宿主细胞选自：细菌细胞(如大肠杆菌)、真菌细胞(如酵母细胞)、昆虫细胞、哺乳动物细胞或植物细胞，优选为酵母细胞或植物细胞。
特别有意义的酵母包括酿酒酵母(Saccharomyces cerevisiae)、巴斯德毕赤酵母(Pichia pastoris)和甲醇毕赤酵母(Pichia methanolica)等。用外源DNA转化酿酒酵母细胞和从其中制备重组多肽的方法公开在例如Kawassaki，美国专利US4599311，US4931373、US4870008、US5037743、US4845075等中。通过选择标记所确定的表型，通常是药物抗性或在缺少特定养分(例如亮氨酸)时的生长能力，选择转化细胞。用于酿酒酵母的优选载体系统如可以是pESC系列表达载体。用于酵母的适宜启动子和终止子包括来自于糖酵解基因(US4599311、US4615974和US4977092)和醇脱氢酶的。用于其它酵母，包括多形汉逊氏酵母、乳克鲁维氏酵母、脆壁克鲁维氏酵母、巴斯德毕赤酵母、甲醇毕赤酵母、季也蒙氏毕赤酵母和麦芽糖假丝酵母的转化系统也是本领域已知的。
根据常规方法在含有养分和其它对于所选宿主细胞的生长必须成分的培养基中培养转化的或转染的宿主细胞。多种适宜的培养基，包括已知成分培养基和复杂培养基，是本领域已知的，一般包括碳源、氮源、必需氨基酸、维生素和矿物质。如果需要，培养基还可以含有诸如生长因子或血清这样的成分。生长培养基一般通过例如药物筛选或缺少可由表达载体携带的或共转染至宿主细胞中的选择标记补充的必需养分来选择含有外源添加DNA的细胞。通过常规方式，如振摇小三角瓶或发酵罐通气给液体培养物提供充足的空气。
本发明编码CYP82D274酶的多核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法制备cDNA库作为模板，扩增而得有关序列。当序列较长时，往往需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。此外，还可通过化学合成将突变体引入本发明蛋白序列中。除了用重组法产生之外，本发明蛋白的片段还可用固相技术，通过直接合成肽而加以生产(Stewart等，Solid-Phase Pedtide Synthesis,J.Am.Chem.Soc.1963,85:2149-2154)。在体外合成蛋白质可以用手工或自动进行。例如，可以用Applied Biosystems的431A型肽合成仪(FosterCity，CA)来自动合成肽。可以分别化学合成本发明蛋白的各片段，然后用化学方法加以连接以产生全长的分子。
本发明将克隆得到的CYP82D274基因构建表达载体，经催化实验证实其完成松香烷型三环二萜C环芳香化及C-14位羟基化过程。三环二萜类化合物C环芳香化，比如氧化次丹参酮二烯生成阿松香三烯，过去普遍认为是植物生物体内的自发反应过程，而本发明证明CYP82D274具有催化C环芳香化的作用，可大大提高生物合成效率。C14位羟基化如氧化次丹参酮二烯生成14-羟基-阿松香三烯，氧化脱氢松香酸生成14-羟基-脱氢松香酸，氧化雷酚萜D生成雷公藤宁B。
因此，本发明的第五方面，提供了CYP82D274、或其编码基因CYP82D274、或包含所述编码基因CYP82D274的表达载体、以及包含所述表达载体的宿主菌，在调节和/或合成二萜类化合物中的应用，尤其是催化松香烷型二萜类化合物C-14位羟化方面的应用。
本发明CYP82D274，或其编码基因CYP82D274、或包含所述编码基因CYP82D274的表达载体、以及包含所述表达载体的宿主菌，也可以用于催化三环二萜类化合物C环的芳香化，C环芳香化过去普遍认为是在生物体内的自发过程，比如次丹参酮二烯的C环芳香化生成阿松香三烯，这大大地限制了生物合成二萜类化合物的效率，而CYP82D274酶的这一作用，可以极大地改善这一弊端，提升二萜类化合物(如雷公藤甲素)的生物合成过程。
本发明中，所述的二萜类化合物优选为松香烷型三环二萜类化合物，如脱氢松香酸、雷酚萜D或雷公藤甲素。
本发明中，在催化C环芳香化时，所述的松香烷型二萜化合物，一个具体的实施方式是，优选的是C环具有两个双键的松香烷型三环二萜类化合物。
本发明的第六方面，提供了一种组合物，包含本发明第一方面所述的酶，以及细胞色素P450氧化还原酶(cytochrome P450 reductase,CPR)如TwCPR3。本发明中，将C-14位未羟化的三环二萜类化合物以底物喂养的方式加入到获得的重组酵母菌中/或加入到重组酵母菌表达后分离所得的蛋白中，生物合成获得C-14位羟化的松香烷型三环二萜类化合物。
本发明的第七方面，提供了一种制备C-14位羟化的松香烷型二萜类化合物的方法，所述方法包括：
(1)将CYP82D274的基因表达盒插入真核表达载体中，制备表达所述酶CYP82D274的重组酵母菌；
(2)以脱氢松香酸为底物，饲喂步骤(1)中获得的酶CYP82D274的重组酵母菌，制备14-羟基-脱氢松香酸；
类似地，如果以雷酚萜D为底物，可以制备得到C-14位羟化的产物雷公藤宁B。
类似地，以次丹参酮二烯为底物，可以制备得到C环芳香化的阿松香三烯，以及C-14位羟化的14-羟基-阿松香三烯。
在一个优选的实施方案中，所述制备松香烷型二萜类化合物的方法，其中步骤(1)所述的真核表达载体为pESC-Leu，所述酶CYP82D274的编码基因插在NotI、Spe I位点。
在步骤(2)完成后还包括对反应产物进行GC-MS分析的步骤，其中目标产物14-羟基-脱氢松香酸在第7.622分钟出现，目标产物雷公藤宁B在第8.167分钟出现。
本发明的第八方面，提供的CYP82D274和CYP82D274基因是植物中克隆获得的，其直接参与二萜类化合物C-14位羟化的生物合成，典型地化合物如脱氢松香酸和雷酚萜D(雷公藤甲素生物合成前体)，因此可以通过基因工程技术进一步验证基因对雷公藤甲素生物合成的直接作用。基因枪介导的CYP82D274基因干扰结果显示，转入含CYP82D274基因的干扰载体的雷公藤悬浮细胞对比转入空载的悬浮细胞，CYP82D274基因表达量显著下降，相对应的雷公藤甲素含量显著抑制62％；当过表达CYP82D274时，含有C-14羟化结构的松香烷型二萜化合物，如雷公藤甲素、雷酚内酯、14-羟基-脱氢松香酸含量较对照组分别出现1.36倍(P<0.001)、1.60倍(P<0.05)和1.39倍(P<0.05)提高，表明CYP82D274具有C-14位羟基化功能。CYP82D274基因可用于利用合成生物学策略来提高雷公藤甲素含量的研究和产业化中，尤其可用于中药雷公藤的品质改良和栽培育种，对于缓解雷公藤药源匮乏问题有较好的促进作用。
附图说明
图1为CYP82D274基因克隆琼脂糖凝胶电泳图(Marker为Trans2K PlusII DNAMarker，核酸分子量标准，条带由上往下分别为8000、5000、3000、2000、1000、750、500、250、100bp)，CYP82D274表示CYP82D274基因。
图2为酵母体内CYP82D274功能表征。
图3为CYP82D274体外酶活表征。
图4为14-羟基-脱氢松香酸1H NMR谱。
图5为14-羟基-脱氢松香酸13C NMR谱。
图6为GC-MS检测CYP82D274催化松香烷型二萜产物(催化雷酚萜D的C-14位羟化生成雷公藤宁B)。
图7为GC-MS检测CYP82D274催化松香烷型二萜产物。a.催化次丹参酮二烯芳香化生成阿松香三烯并C-14位羟基化；b.CYP82D274催化芳香化产物相对含量。
图8为雷公藤悬浮细胞CYP82D274基因干扰及雷公藤甲素的相对含量。
图9为雷公藤悬浮细胞CYP82D274基因过表达时相应C-14位羟化的松香烷类二萜化合物相对含量。
具体实施方式
以下结合具体实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中如未注明特殊的实验方法，通常按照常规条件或依据制造厂商建议的条件方法。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅为本发明表明基因功能所作示范之用。
下述实施例中的雷公藤悬浮细胞在文献“雷公藤4-(5’-二磷酸胞苷)-2-C-甲基-D-赤藓醇激酶基因的全长克隆与表达分析.中国中药杂志,2015,40(21)”中公开过，公众可从首都医科大学分子生药与中药资源实验室获得。
High-Fidelity DNA Polymerase、BamHI、SalI、NotI、SpeI限制性内切酶是New England Biolabs公司的产品；/>Max Master Mix、2×Rapid Taq MasterMix和/>One Step Cloning是南京诺唯赞生物科技有限公司；快速通用植物RNA提取试剂盒是北京华越洋生物科技有限公司的产品；Trans2K Plus II DNA Marker、pEASY-Blunt Simple Cloning Kit和大肠杆菌感受态细胞Trans1-T1是北京全式金生物技术有限公司的产品；Gene JET Gel Extraction Kit是Thermo Scientific公司的产品；E.Z.N.ATM plasmid mini kit I是Omega Bio-Tek公司的产品；pESC-Leu载体是Agilent公司的产品；SD-Leu-His-Ura、SD-Leu是北京泛基诺科技有限公司的产品；ZYMO RESEARCHFrozen-EZ Yeast Transformation II试剂盒Zymo Research公司的产品；脱氢松香酸标准品是上海源叶生物科技有限公司，CAS号为1740-19-8；雷公藤宁B标准品是云南西力生物技术股份有限公司，CAS号为189389-05-7。
实施例一、雷公藤CYP82D274基因克隆(图1)
首先，使用Super总RNA提取试剂盒(Promega)进行操作：
(1)取约10mg雷公藤悬浮细胞样品置于2mL的离心管中，液氮环境下粉碎均匀，迅速用混合型研磨仪(MM400，Retsch)粉碎2min；
(2)向装有粉碎样品的EP管中迅速加入500μL RNA裂解液，上下颠倒离心管3-4次，使样品充分裂解；
(3)继续加入500μL RNA稀释液，用移液枪吸打混匀，4℃，14 000rpm，离心5min；
(4)取上清液于新的无菌2mL离心管中，加入0.5倍体积无水乙醇，颠倒离心管以充分混匀；
(5)将混合液分次转入离心柱中，4℃，12 000rpm，离心1min，弃滤液；
(6)向离心柱中加入600μL RNA洗液，4℃，12 000rpm，离心45s，弃滤液；
(7)向离心柱中加入50μL DNA酶I，室温环境孵育15min；
(8)向离心柱中加入600μL RNA洗液，4℃，12 000-14 000rpm离心45s，弃滤液，重复清洗操作一次；
(9)4℃，13 000rpm，空柱离心2min；
(10)向离心柱中加入50μL预热的无核酸水(RNase-free water)，室温静置2min，4℃，13 000rpm离心1min，收集RNA溶液。
(11)经1％琼脂糖凝胶电泳检测RNA完整度后，测定RNA浓度，可直接用于cDNA反转录合成或液氮速冻置于-80℃超低温冰箱保存。
接着，利用FastQuant RT Kit(withgDNase)进行反转录，每0.5-2μg总RNA可建立20μL以下反应体系。在冰上进行操作，为提高反应体系配制准确性，可根据反应需要提前配制混合液，再分装到每个PCR反应管中。
(1)去除基因组DNA步骤：按照如下体系配制混合液，充分混匀并短暂离心。42℃孵育3min，冰上冷却；
5×gDNABuffer 2.0μL
TotalRNA 50ng-2μg
RNase-FreeddH2O 补足至10μL
(2)按照如下体系配置反转录反应混合液；
(3)第一链cDNA反转录反应体系：(2)中反转录反应混合液，加入到(1)去除DNA的反应液中，充分混匀，并于42℃，孵育15min，95℃，孵育3min；
(4)反应完成后，产物置于冰上停止反应，得到的cDNA置于-20℃保存。
最后，基因全长克隆及产物纯化
根据转录组P450基因全长cDNA序列，设计全长克隆的特异性引物(F：ATGGAGTTTCTTCTTTCACTCCCAG，R：TCAGCCCATATAGAGATGAGCTGG)，使用2×Phanta MaxMaster Mix(延伸速度1-2kb·min-1)进行PCR反应，反应体系如下：
PCR反应条件：95℃30s；95℃15s，56℃15s，72℃1.5min 35个循环；72℃7min；4℃保持。反应结束后取5μL反应产物，加1μL 6×Loading Buffer，进行1.0％琼脂糖凝胶电泳(180V，约10min)，检测目标条带位置正确后，使用1.5％琼脂糖凝胶电泳(150V，约25min)充分分离DNA并切取目标条带。
使用Thermo Scientfic GeneJET胶回收试剂盒回收纯化DNA：
(1)加入等比量的Binding Buffer(每0.1g琼脂糖凝胶加100μL Buffer)至含有胶的2mL离心管中，60℃条件下孵育凝胶混合物约10min保证完全溶解；
(2)转移凝胶溶液至GeneJET纯化柱，使用离心机12 000g离心1min，弃去收集管中废液；
(3)加入700μL Wash Buffer到纯化柱，12 000g离心1min，弃去废液；
(4)空柱12 000g，离心1min，彻底去除残余的Wash Buffer；
(5)将纯化柱转移到干净无菌的已标记的1.5mL离心管，加入60℃预热的无菌水30-50μL，室温孵育2min，12 000g离心2min彻底收集DNA；
(6)纯化的DNA可直接用于下一步实验或于-20℃储存。
实施例二、CYP82D274酵母体内功能表征
首先，构建含P450和CPR的真核表达载体
(1)pESC-LEU::TwCPR3的构建
由于P450发挥功能需要细胞色素P450氧化还原酶(CPR)提供电子，因此将雷公藤细胞色素P450氧化还原酶3(TwCPR3)构建至pESC-Leu上Gal 1启动子的多克隆位点MCS2(multiple cloning site)端。载体部分采用NEB限制性内切酶BamHI-HF和SalI-HF进行双酶切反应，反应体系为：
待反应结束后，与未酶切载体和15K DNA Marker(全式金)一同进行电泳检测，确定目标大小有条带并且与未酶切样品表现形式不同，视为酶切成功，使用1.5％琼脂糖凝胶电泳(150V，约25min)和胶回收试剂盒纯化回收线性载体条带。
DNA片段准备：以含有雷公藤TwCPR3基因全长cDNA的质粒为模板，用特异引物Uni-CPR-F/R进行基因编码区的扩增，引物见表1。采用High-Fidelity PCR超保真酶混合液进行扩增。PCR程序参数为98℃3min，1个循环；98℃10s，60℃20s，72℃60s，35个循环；72℃7min，1个循环；4℃终止反应。经电泳检测相应位置有条带，扩增产物经琼脂糖凝胶电泳分离后进行DNA片段的纯化回收。
表1真核表达载体构建引物列表
将目的片段TwCPR3和线性载体连接成环状，采用无缝拼接法(同源重组原理)，利用Ultra One Step Cloning Kit(诺唯赞)构建真核表达载体pESC-LEU::TwCPR3。核酸定量仪检测上述目的基因片段和载体片段浓度，按照目的片段与载体片段摩尔比2:1配制反应体系，加入等体积2×ClonExpress Mix溶液充分混匀后，50℃反应30min。将连接产物与50μLTransTI克隆感受态细胞混合，冰上静置30min，42℃水浴热激45s，冰上静置2-3min，加入500μL不含抗生素的LB液体培养基，置于37℃摇床中，180-200rpm培养1-2h，取100μL菌液涂布在含有100mg·L-1氨苄霉素的LB固体培养基上，倒置培养12-16h。经菌落筛选后，以37℃250rpm避光振荡培养约3-4h的菌液作为模板，以Gal 1正反引物(表1)进行阳性克隆验证，将含目的条带的菌液进行测序，获得pESC-LEU::TwCPR3真核表达载体。
(2)pESC-LEU::(CYP82D274+TwCPR3)的构建
将CYP82D274构建至上述pESC-LEU::TwCPR3真核表达载体Gal 10的MCS1端，组成pESC-LEU::(CYP82D274+TwCPR3)重组质粒。
载体准备：反应体系包含载体DNA 1.5μg，内切酶NotⅠ-HF 1μL，内切酶SpeⅠ-HF 1μL，10×Cutsmart 5μL，加无菌水至总体积50μL。37℃，酶切反应4h。以未酶切的载体和DNAMarker进行对照，经电泳检测酶切前后载体条带出现位置及表现方式不同。酶切成功的产物经大胶分离后采用胶回收试剂盒进行线性载体片段的回收。
DNA片段准备：以含有雷公藤CYP82D274基因全长cDNA的质粒为模板，用特异引物进行基因编码区的扩增，引物见表1。使用2×Phanta Max Master Mix进行扩增。PCR参数为95℃30s；95℃15s，56℃15s，72℃1.5min，35个循环；72℃7min；4℃保持。经电泳检测相应位置有条带，扩增产物经大胶分离后进行DNA片段的回收。
无缝拼接法采用Ultra One Step CloningKit构建表达载体pESC-LEU::(CYP82D274+TwCPR3)方法同上，以Gal 10正反引物(表1)进行阳性克隆验证后测序，获得pESC-LEU::(CYP82D274+TwCPR3)真核表达载体。
接着，真核表达载体转化酵母BY4741及发酵产物
将上述pESC-LEU::(CYP82D274+TwCPR3)真核表达载体和相应的空载体pESC-LEU::TwCPR3转化BY4741酿酒酵母，感受态细胞制作及转化具体方法如下：
(1)酵母感受态制备
采用Zymo Research Frozen-EZ酵母转化试剂盒II制作酵母感受态细胞，在超净台中无菌操作：
1)将BY4741酵母菌用接菌环划线YPD+2％葡萄糖(Glucose，Glc)固体平板，30℃倒置培养36-48h；
2)从平板上挑取新活化的单菌落，接种于10mL YPD+2％Glc液体培养基中，30℃，200rpm振荡培养至OD600达到0.8-1.0左右；
3)5810R型低温冷冻离心机于室温，500g离心4min，去上清；
4)加入5mL Frozen-EZ溶液1重悬菌体，室温500g离心4min，弃上清；
5)加入0.5mL Frozen-EZ溶液2重悬菌体，每管30-50μL分装至无菌干净的1.5mLEP管中，可直接用于后续质粒转化或逐级降温后于-80℃保存。
(2)转化酵母感受态
1)取0.2-1μg重组质粒pESC-LEU::(CYP82D274+TwCPR3)加入50μL感受态细胞，轻弹混合；
2)加入500μL Frozen-EZ溶液3，移液器吹打混匀；
3)30℃静置孵育1-2h，每30min颠倒混匀1次；
4)取100μL孵育菌液，涂布合成型营养缺陷型培养基(Synthetic DropoutMedium，SD)SD-Leu平板，30℃倒置培养48-72h。
(3)阳性验证及菌液发酵
随机挑取单菌落，置于10mL SD-Leu+2％Glc液体培养基中，30℃，200rpm震荡培养36-48h；
阳性验证：取1μL菌液进行阳性克隆PCR反应，检测质粒成功转化BY4741酿酒酵母。使用2×Rapid Taq Master Mix 10.0μL，Gal 10及Gal 1正反引物各0.8μL，菌液1.0μL，无菌水补足至20.0μL，反应程序为95℃，3min；95℃15s，56℃15s，72℃0.5min，35个循环；72℃，5min；4℃停止反应。反应结束后取8μL反应产物与2K DNA Marker(全式金)进行1.0％琼脂糖凝胶电泳(200V，约10min)，检测是否含有目标条带。
将验证成功的菌液，以OD约0.1接种至20mL SD-Leu+2％Glc液体培养基中，30℃，200rpm震荡培养至OD 1.0左右，离心收集菌体，以20mL SD-Leu+2％半乳糖(galactose，Gal)培养基(含50μM脱氢松香酸)重悬菌体，30℃，200rpm诱导发酵48-72h。
(4)发酵产物提取
1)收集发酵菌液，加入等体积乙酸乙酯20mL，低温超声1h，离心收集上层乙酸乙酯层，下层发酵液加入等体积乙酸乙酯继续超声提取1次；
2)混合萃取液，-20℃低温冷冻除去萃取液中的水分；
3)将萃取液使用旋转蒸发仪旋干有机溶剂，用0.8mL色谱级乙酸乙酯清洗旋蒸瓶内壁，复溶2次，合并复溶液；
4)高纯氮气吹干，用100μL甲醇复溶，加入100μL重氮甲烷(trimethylsilyldiazomethane,Tokyo Chemical Industry,Japan)，室温避光反应10min；
5)氮吹至干，100μL色谱级乙酸乙酯复溶，12 000g高速离心10min后取上清GC-MS进样。
(5)GC-MS检测条件
使用安捷伦7000C GC-TQ-MS，进样量1μL，进样口温度300℃，离子源温度250℃，电子能量70ev，对样品进行10-400m/z范围扫描。色谱柱DB-5MS(15m，0.25mm，0.1μm)检测条件：50℃起始保持1min；40℃·min-1上升至200℃；20℃·min-1上升至240℃；1.5℃·min-1上升至246℃；40℃·min-1上升至300℃，保持3min。
结果见图2。CYP82D274催化底物脱氢松香酸，生成新的羟化产物1：14-羟基-脱氢松香酸。
实施例三、CYP82D274酵母体外功能表征(图3)
将上述包含pESC-LEU::(CYP82D274+TwCPR3)和相应的空载体pESC-LEU::TwCPR3的BY47471酿酒酵母提取微粒体，以脱氢松香酸为底物，体外反应检测产物。
提取微粒体方法如下：
(1)活化菌液转接入50mL SD-Leu+2％Glc液体培养基，30℃200rpm培养16-20h，收集菌液，室温4 000g离心5min；
(2)用100mL诱导型SD-Leu+2％Gal液体培养基重悬菌体，30℃，200rpm诱导12-16h；
(3)菌液4℃，4 000g离心3min，收集菌体，用10mL TEK溶液(TE(即50mM Tris-HCl,1mM EDTA,pH 7.4)+氯化钾KCl)重悬菌体，室温静置5min；
(4)4℃，4 000g离心3min，收集菌体，用45mL预冷的TESB(TE+山梨醇Sorbitol)重悬菌体；
(5)均质机使用前打开循环水浴4℃预冷。菌体重悬液使用ATS高压细胞破碎仪以>1 000psi的压力破碎7min，收集破碎后的菌液；
(6)4℃，12 000g离心15min；
(7)取上清液(粗酶)加入0.15M氯化钠(NaCl)和0.1g·mL-1聚乙二醇4000(PEG4000)充分混匀，冰浴15min沉降微粒体；
(8)4℃，12 000g离心15min，沉淀(微粒体)用1-2mL预冷的TEG(含20％甘油的TE溶液)溶解，-80℃保存。
微粒体体外酶促反应需要辅因子1M Tris-HCl(pH 7.4)，50mM还原型烟酰胺腺嘌呤二核苷酸磷酸(Nicotinamide adenine dinucleotide phosphate，NADPH)，10mM黄素腺嘌呤二核苷酸(Flavin adenine dinucleotide，FAD)，10mM黄素腺嘌呤二核苷酸(Flavinmononucleotide，FMN)，500mM 6-磷酸葡萄糖(Glucose-6-phosphate，G-6-P)，1kU 6-磷酸葡萄糖脱氢酶(Glucose-6-phosphate dehydrogenase，G-6-P-DH)，1M二硫苏糖醇(Dithiothreitol，DTT)，底物终浓度100μM。反应体系如下：
反应条件30℃，100rpm，3-4h。
酶促产物提取加入500μL等体积色谱级乙酸乙酯萃取反应后溶液，涡旋震荡1min，萃取三次，合并萃取液，氮吹至干，100μL色谱级乙酸乙酯复溶，室温12 000g高速离心10min，取上清转移至含内插管的棕色液相小瓶中；GC-MS检测同实施例三。CYP82D274酶体外可催化底物脱氢松香酸，生成产物1：14-羟基-脱氢松香酸(图3)。
实施例四、脱氢松香酸催化产物鉴定
1.大量发酵获取催化产物
将阳性鉴定成功的包含pESC-LEU::(CYP82D274+TwCPR3)的BY4741酿酒酵母转接入500mL SD-Leu+2％Glc液体培养基，30℃200rpm震荡培养至OD 10左右，离心收集菌体，使用新鲜50mL SD-Leu+2％Glc重悬菌体，分配至20个装有500mL YPL(1％Yeast extract，2％Peptone，2％Gal)的2L三角瓶中，加入脱氢松香酸至终浓度100μM，30℃200rpm诱导发酵72h。超净台中加入15-20g HP2MGL树脂(三菱，日本)，继续30℃200rpm提取发酵产物24h。
2.催化产物提取与制备分离
提前进行树脂提取效率的预实验，分离菌体、培养基和树脂，提取三部分发酵产物并使用GC-MS检测，发现树脂将培养基中产物几乎完全吸附，发酵产物仅在菌体和树脂中检测到。因此，对上述10L发酵液使用大体积低温离心机(Sigma)4 000g离心5min，弃掉培养基，收集菌体和树脂置于烘箱中40℃烘干。加入8倍体积的无水甲醇(分析级)，低温超声提取催化产物30min，反复提取菌体和树脂固体至无水甲醇提取液无色。
合并提取液，布氏漏斗过滤提取液以除去树脂和菌体残渣。旋转蒸发仪40℃真空蒸干提取液，回收无水甲醇(可用于相同催化产物发酵液的反复提取)。使用5mL乙酸乙酯洗涤旋蒸瓶上凝结固体三次，合并复溶液，氮吹至干，以4mL色谱级甲醇复溶。进样吉尔森Gilson 281semi-preparative HPLC半制备液相，连接H322泵，GX-281自动进样器，156双波长紫外检测器和Trilution LC 2.1工作站；Xtimate C18色谱柱(30×75mm,3.0μm,Welch)，流动相A为0.1％三氟乙酸-水，流动相B为纯乙腈，梯度洗脱：起始，50％流动相B；8.0min，100％流动相B；设置流速25mL·min-1,进样量500.0μL。通过与空载对比和质谱结构确认目标催化产物峰，收集7.4-7.8min产物馏分，旋蒸除去乙腈后低温冷冻干燥获得催化产物粉末。
3.催化产物高分辨质谱和核磁鉴定
产物纯度鉴定：精密称定2.0mg催化产物粉末，加入1mL甲醇制备标准品溶液，稀释后进样LC-MS，检测产物纯度为98.23％；
产物核磁鉴定：精密称定3.0mg产物粉末，加入500μL氘代丙酮(C3D6O，Innochem)充分溶解粉末，转移至核磁管，使用800M 1H-NMR和200M 13C-NMR分析鉴定，MestReNova软件分析数据。(图4和图5)
14-羟基-脱氢松香酸NMR数据(3.0mg白色粉末)
1HNMR(C3D6O,800MHz)δ2.35(1H,d,J＝12.6Hz,H-1a),1.44-1.40(1H,m,H-1b),1.72-1.67(2H,m,H-2),2.19(1H,d,J＝12.6Hz,H-4),1.84(3H,m,H-6a),1.62(1H,dd,J＝13.2,8.0Hz,H-6b),3.31(1H,m,H-7),6.84(1H,d,J＝8.0Hz,H-11),7.00(1H,d,J＝8.0Hz,H-12),1.19(6H,dd,J＝13.2,6.9Hz,H-15,19),1.23(3H,s,H-16),2.71-2.63(1H,m,H-17a),2.89(1H,dd,J＝17.2,6.8Hz,H-17b),1.28(3H,s,H-20)；13C NMR(C3D6O,200MHz)δ38.3(C-1),18.4(C-2),36.6(C-3),44.5(C-4),46.8(C-5),21.0(C-6),26.3(C-7),121.6(C-8),148.1(C-9),36.7(C-10),115.8(C-11),123.0(C-12),130.9(C-13),151.0(C-14),22.2(C-15),24.4(C-16),24.5(C-17),178.8(C-18),22.4(C-19),16.0(C-20)。
实施例五、CYP82D274催化雷酚萜D功能表征(图6)
1.转化与阳性验证
构建pESC- LEU::(CYP82D274+TwCPR3)真核表达载体，具体方法同实施例二。将获得的pESC-LEU::(CYP82D274+TwCPR3)表达载体及不含CYP82D274基因的空载体pESC-LEU::TwCPR3分别转化BY4741酿酒酵母感受态细胞，使用营养缺陷型培养基SD-Leu筛选培养。随机挑取单菌落，置于10mL SD-Leu+2％Glc液体培养基中，30℃，200rpm震荡培养36-48h后，取1μL菌液进行阳性克隆PCR反应，检测质粒成功转化BY4741酿酒酵母。使用2×Rapid TaqMaster Mix 10.0μL，Gal 10及Gal 1正反引物各0.8μL，菌液1.0μL，无菌水补足至20.0μL，反应程序为95℃，3min；95℃15s，56℃15s，72℃0.5min，35个循环；72℃，5min；4℃停止反应。反应结束后取8μL反应产物与2K DNA Marker(全式金)进行1.0％琼脂糖凝胶电泳(200V，约10min)，检测是否含有目标条带。
2.基因诱导与酵母发酵
将验证成功的菌液，以OD约0.1接种至20mL SD-Leu+2％Glc液体培养基中，30℃，200rpm震荡培养至OD 1.0左右，离心收集菌体，以20mL SD-Leu+2％半乳糖(Gal)培养基(含50μM雷酚萜D)重悬菌体，30℃，200rpm诱导发酵48-72h。
3.发酵产物提取与检测
收集发酵菌液，加入等体积乙酸乙酯20mL，低温超声1h，共提取2次，合并提取液，并于-20℃低温冷冻除去萃取液中的水分。旋转蒸发仪旋干有机溶剂，用0.8mL色谱级乙酸乙酯清洗旋蒸瓶内壁2次，合并复溶液。高纯氮气吹干，用100μL甲醇复溶，加入100μL重氮甲烷(trimethylsilyl diazomethane,Tokyo Chemical Industry,Japan)，室温避光反应10min。氮吹至干，100μL色谱级乙酸乙酯复溶，12 000g高速离心10min后取上清作为待检测样品。使用安捷伦7000C GC-TQ-MS，进样量1μL，进样口温度300℃，离子源温度250℃，电子能量70ev，对样品进行10-400m/z范围扫描。色谱柱DB-5MS(15m，0.25mm，0.1μm)检测条件：50℃起始保持1min；40℃·min-1上升至200℃；20℃·min-1上升至240℃；1.5℃·min-1上升至246℃；40℃·min-1上升至300℃，保持3min。
结果见图6。CYP82D274催化底物雷酚萜D生成产物2与雷公藤宁B标准品具有相同的保留时间和质谱碎片。因此表明，CYP82D274具有催化雷酚萜D的C-14位羟基化生成雷公藤宁B的功能。
实施例六、高产次丹参酮二烯工程酵母体内CYP82D274功能表征
1.CYP82D274催化功能表征
(1)真核表达载体构建及转化
构建pESC- LEU::(CYP82D274+TwCPR3)真核表达载体，具体方法同实施例二。从SD-Ura-His+2％Glc固体平板挑取活化的次丹参酮二烯高产工程菌BY-PS2(为BY系酵母菌整合两个相同的SmMS-SmCPS1模块，详见Tu L等,Genome of Tripterygium wilfordii andidentification of cytochrome P450 involved in triptolide biosynthesis[J].Nat.Commun.,2020,11)，30℃，200rpm振荡培养至OD600达到0.8-1.0左右。室温环境，500g离心4min，去上清；加入5mL Frozen-EZ溶液1重悬菌体，室温500g离心4min，弃上清；加入0.5mL Frozen-EZ溶液2重悬菌体，每管30-50μL分装至无菌干净的1.5mL EP管中。向感受态细胞中加入0.2-1μg重组质粒pESC-LEU::(CYP82D274+TwCPR3)轻弹混合；加入500μLFrozen-EZ溶液3，混匀后于30℃孵育1-2h，取100μL孵育菌液，涂布SD-Leu-Ura-His+2％Glc平板，30℃倒置培养48-72h。
(2)阳性验证及菌液发酵
随机挑取SD-Leu-Ura-His+2％Glc平板单菌落，置于10mL SD-Leu-Ura-His+2％Glc液体培养基中，30℃，200rpm震荡培养36-48h；
阳性验证：取1μL菌液进行阳性克隆PCR反应，检测质粒成功转化BY-PS2酿酒酵母。使用2×Rapid Taq Master Mix，PCR反应体系如下，2×Rapid Taq Master Mix 10.0μL，Gal 10及Gal 1正反引物各0.8μL，菌液1.0μL，无菌水补足至20.0μL，反应程序为95℃3min；95℃15s，56℃15s，72℃0.5min，35个循环；72℃，5min；4℃停止反应。反应结束后取8μL反应产物与2K DNA Marker进行琼脂糖凝胶电泳，检测是否含有目标条带。
将验证成功的菌液，以OD约0.1接菌至20mL SD-Leu-Ura-His+2％Glc液体培养基中，30℃，200rpm震荡培养至OD 1.0左右，离心收集菌体，以20mL SD-Leu-Ura-His+2％Gal重悬菌体，30℃，200rpm诱导发酵48-72h。
(3)发酵产物提取
以等体积乙酸乙酯提取发酵产物两次，旋干萃取液后，以0.8mL色谱级乙酸乙酯复溶2次，氮吹干，100μL色谱级乙酸乙酯复溶，12 000g高速离心10min后取上清GC-MS进样，检测使用色谱柱DB-5MS(15m，0.25mm，0.1μm)，50℃起始保持1min；40℃·min-1上升至200℃；20℃·min-1上升至240℃；1.5℃·min-1上升至246℃；40℃·min-1上升至300℃，保持3min。
结果见图7a，CYP82D274催化底物次丹参酮二烯生成两个产物，即将次丹参酮二烯的C环芳香化生成产物3：阿松香三烯，并在其C-14位羟化生成产物4：14-羟基-阿松香三烯。
2.定量产物发酵与检测
(1)载体构建
构建组成型启动CYP82D274和TwCPR3的质粒pESC-LEU::(PPGK1-CYP82D274-TADH1-PTEF2-TwCPR3-TCYC1)，引物见表2。
载体准备：载体部分采用NEB限制性内切酶SpeⅠ-HF和SalI-HF对pESC-LEU载体进行双酶切反应，反应体系为：
待反应结束后，与未酶切载体和15K的DNA Marker一同电泳检测，确定目标大小有条带并且与未酶切样品表现形式不同，视为酶切成功，大胶分离并使用胶回收试剂盒纯化回收线性载体条带。
DNA片段准备：使用High-Fidelity PCR超保真酶分别对CYP82D274、TwCPR3和启动子PPGK1-PTEF2(PT)的质粒进行扩增，反应体系：2×Phusion HF 25.0μL，2.5μL引物F(10μM)，2.5μL引物R(10μM)，1μL质粒模板，无菌水补足至50μL。PCR程序参数为98℃3min，1个循环；98℃10s，60℃20s，72℃延伸速度1-2kb·min-1，根据条带大小设置，35个循环；72℃7min，1个循环；4℃停反应。
无缝拼接法采用Ultra One Step Cloning Kit(诺唯赞)构建表达载体pESC-LEU::(PPGK1-CYP82D274-TADH1-PTEF2-TwCPR3-TCYC1)：检测目的基因片段和载体片段浓度，按照三个目的基因与载体片段摩尔比1:1:1:1插入片段，加入等体积2×ClonExpress Mix混合均匀后，50℃反应1h。将连接产物与50μL Trans1-T1克隆感受态细胞混合，冰上静置30min，42℃水浴热激45s，冰上静置2-3min，加入500μL不含抗生素的LB液体培养基，置于37℃摇床中，200rpm培养1-2h，取100μL菌液涂布在含有100mg·L-1氨苄霉素(Amp)的LB固体培养基上，倒置培养12-16h。经菌落筛选后，分别使用PT(MCS2)和PT(MCS1)的正反引物(表2)进行阳性克隆验证后测序，获得pESC-LEU::(PPGK1-CYP82D274-TADH1-PTEF2-TwCPR3-TCYC1)真核表达载体。
表2真核表达载体及突变载体构建引物列表
(2)表达载体转化BY-PS2及产物提取检测
将构建的真核表达载体转化次丹参酮二烯高产酵母菌BY-PS2，平板随机挑取5个独立、完整、圆润的菌落，5mL SD-Leu-Ura-His+2％Glc培养基30℃200rpm培养48-72h至菌体浑浊，Nanodrop检测菌液OD，计算各菌液加入量，以相同OD 0.1起始定量转接入新鲜的20mL SD-Leu-Ura-His+2％Glc培养基，30℃200rpm黑暗震荡，10h后加入2mL(十分之一培养基体积)正十二烷进行双向发酵，共发酵72h。待发酵结束，收集发酵液，8 000g离心5min，吸取上层正十二烷层约200μL，12 000g高速离心10min后取上清100μL GC-MS进样。检测使用色谱柱DB-5MS(15m，0.25mm，0.1μm)，50℃起始保持1min；40℃·min-1上升至200℃；20℃·min-1上升至240℃；1.5℃·min-1上升至246℃；40℃·min-1上升至300℃，保持3min。
结果见图7b。CYP82D274催化芳香化生成阿松香三烯，其催化产物含量较自发反应产量提高188.44倍。表明次丹参酮二烯芳香化虽可像文献报道的自发反应，但CYP82D274可催化实现该芳香化过程，极大幅度提高阿松香三烯产量。
实施例七、基因与代谢物和松香烷型二萜化合物生物合成
1.雷公藤悬浮细胞准备
配制MS固体培养基：Murashige&Skoog with Vitamins(Caisson Labs公司)4.43g·L-1，蔗糖(AOBOX公司)30g·L-1，2,4-D 1.0mg·L-1，IBA 1.0mg·L-1，KT0.1mg·L-1，加水后调pH到5.8-6.0，加琼脂(Oxoid公司)8％，121℃，20min高压灭菌后温度降到60℃以下时倒入35mm细胞培养皿(每个培养皿3mL培养基)中凝固。将要继代的0.2g悬浮细胞转入细胞培养皿中心位置，倾压使细胞接触培养基，25℃，暗培养7d用于基因枪转化。
2.Gateway技术构建CYP82D274表达载体
(1)BP入门载体构建
构建干扰表达载体引物为正向：CACCCGAAACGTTTAAGCACATAAGAG；反向R：TCATCATCATCAAAAACGGAGAG。构建过表达片段引物为正向F：CTCCACCTGCATCGCACCATGGAGTTTCTTCTTTCACTC，反向R：GACCACCTGCATCGCCTTTCAGCCCATATAGAGATGAGC。
以含有雷公藤CYP82D274基因全长cDNA的载体质粒为模板，进行PCR扩增基因相应片段。DNA聚合酶采用高保真Phusion High-Fidelity CR Master Mix。PCR参数为98℃30s，1循环；98℃10s，60℃10s，72℃45s，35循环；72℃5min；4℃维持。扩增产物经Gene JET GelExtraction Kit胶回收。与TOPO载体(Invitrogen)22℃共反应2h后转入大肠杆菌感受态细胞Trans1-T1，于LB+Kan培养基37℃培养，阳性克隆送测序成功后，快速质粒小提试剂盒提取质粒。
(2)干扰表达载体/过表达载体构建
入门载体和干扰表达载体各300ng，加入1μL LR酶(Invitrogen)，25℃反应3-4h，反应产物转化入Trans1-T1于LB+壮观霉素(Spe)培养基37℃培养，阳性克隆送测序成功后，用Plasmid Maxi Kit(Omega公司)质粒大提试剂盒提取质粒。
入门载体和过扰表达载体各300ng，加入1μL LR酶(Invitrogen)，25℃反应3-4h，反应产物转化入Trans1-T1于LB+Spe培养基37℃培养，阳性克隆送测序成功后，用PlasmidMaxi Kit(Omega公司)质粒大提试剂盒提取质粒。
3.基因枪介导转化
实验器材要求无菌：根据器材性质分别或结合采用高温高压灭菌、紫外照射除菌和70％乙醇杀菌。
每个质粒做5个生物学重复，即打5盘细胞，每盘2枪，共10枪。将转化空载体的悬浮细胞用作对照。将20μg质粒，5mg金粉，100μL的2.5MCaCl2，40μL的0.1M亚精胺混合，震荡2-3min，静止1min后离心约2s，弃上清；加入140μL 70％乙醇，轻弹后离心弃上清；加入140μL100％乙醇，轻弹后离心弃上清；加入100μL100％乙醇，轻弹后低速震荡2-3s，置于冰中。
将消毒后的载体膜置于铁托中，每个铁托加入10μL上述配制的载体金粉预混液，按基因枪操作规范，将铁托、阻挡网、可裂膜及雷公藤悬浮细胞分别放置在基因枪设备的相应位置上，进行基因枪转化载体，每盘细胞轰击两枪。
4.代谢物含量检测
悬浮细胞样品取至2.0mL离心管，球磨机于液氮环境下充分粉碎样品，于-80℃冷冻超过4h后，放置在冷冻干燥机(德国Christ Delta 1-20LSC)中冷冻干燥36-48h。精密称重干燥后的样品粉末20mg，以1.0mL 80％甲醇4℃提取过夜，低温40kHz，90％功率超声提取30min。离心后的上清过0.22μm PTFE滤膜，取续滤液即为供试品溶液用于后续检测。
检测使用Q Exactive HF LC-MS/MS(Thermo Scientific)，ACQUITY UPLC HSS T3色谱柱(2.1mm×100mm×1.8μm,Waters)。流动相A为0.1％甲酸-水溶液(v/v),流动相B为纯乙腈溶液。起始，30％流动相B；4min，34％流动相B；8min，52％流动相B；12min，52％流动相B；23min，77％流动相B；26min，90％流动相B进行梯度洗脱。设置流速0.4mL·min-1,进样量2.0μL,柱温40℃，正负离子扫描。
结果见图8和图9所示。图8表明，转入含CYP82D274基因的干扰载体的悬浮细胞对比转入空载的悬浮细胞，CYP82D274基因表达量显著下降，相对应的雷公藤甲素含量显著抑制62％。图9结果表明，当过表达CYP82D274时，含有C-14羟化结构的松香烷型三环二萜化合物，如雷公藤甲素、雷酚内酯、14-羟基-脱氢松香酸含量较对照组分别出现1.36倍(P<0.001)、1.60倍(P<0.05)和1.39倍(P<0.05)提高，表明CYP82D274具有C-14位羟基化功能。
序列表
<110> 首都医科大学附属北京世纪坛医院
<120> 一种松香烷型三环二萜类化合物C-14位羟化酶
<130> TQZX2022-ZL027
<141> 2022-01-19
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1602
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggagtttc ttctttcact cccagcaaac accatagccc caaaaatctt tgcagtttta 60
ttactcttca tatgtttacg gatactcaca aatgtcctca aacccaagaa atccaagaca 120
tccccaccac aagccagcgg cgcctggcct ttgattggcc acctcctcca cctccgcggc 180
ccacaggcac cacacataac attaggcaag atggctgata aatacggtcc aatcttcaaa 240
ataaagctag gcgtgcatcc aactcttgtc ataagtgact ccgaggttgc caaagagtgt 300
ttgaccacac acgacatagc cttggctggc cgtccagcga ctgtggccat ggaaatcatg 360
ggatacaacc atgccatgtt cgctttcagc ccatacggcc catattggcg tcacatgcgc 420
aagctagcca ccgttgagct tctatccact caacggctcg aaacgtttaa gcacataaga 480
gaatctgagc ttaagcgttc catgaaagag atgtaccaat catgggttca taacaaaagc 540
ggtagcggcg attcgaatca tgttaccgtg gacatgacta gaattttggg agacataatc 600
gcaaacgtaa tatacaggat ggtggtagga aaagtgtatg cgagtaaggg tgaggaggat 660
gcgagatgga aacaagtagt gtgggagtat attaagttgc tgagccactt tggtgtgggt 720
gatgcgttgc catttttgag gtggttggat ttgggaggtg ttgagaagtc catgaaaaag 780
gcagccaaag aattggacat ttatgttgaa gagtggttgg aagaacataa aaagaagaga 840
tccgagcgta agagtgataa tggaattgta gaagaagact tcatggacgt tatgctctcc 900
gtttttgatg atgatgatca actggagaat tttgctcatc atagtgctca tacaattaac 960
aaggccatgt gcttggctat tatattggcg gcctctgaca ccactaagac tactttgact 1020
tgggctctat cactattact caatcaccct gacgtcatga agaaggtcca acaagagttg 1080
gctgctcaca ttggtccaga taaaccagtg aaggaatcag atgtcaaatc tttggtctac 1140
cttgaagcag ttgttaagga aacattacga ttataccctc caggtccact cgggttacca 1200
cacgagtcta tggaggactg caccgtagcc ggttaccatg ttccatcagg gactaggatt 1260
ctctacaacc tctggaagat tcaacaggat ccgcaggtgt gggagaatcc ttcagagttc 1320
aagccagaca ggtttctgac aacacataag gacgtcgatg ttaggggacg taactttgag 1380
tatttaccat ttgggagtgg tagaagaatg tgtcctggaa tgtctttcgc tcttcaagtc 1440
atggaggttt cacttgctaa tatgcttcat gggttcgatt ttgccactcc aaatggtaaa 1500
ccagtggata tgactgaagt taatggattg gtcactgacc gtgcaacgcc acttgaggcc 1560
ctcattactc cacgcctccc agctcatctc tatatgggct ga 1602
<210> 2
<211> 533
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Glu Phe Leu Leu Ser Leu Pro Ala Asn Thr Ile Ala Pro Lys Ile
1 5 10 15
Phe Ala Val Leu Leu Leu Phe Ile Cys Leu Arg Ile Leu Thr Asn Val
20 25 30
Leu Lys Pro Lys Lys Ser Lys Thr Ser Pro Pro Gln Ala Ser Gly Ala
35 40 45
Trp Pro Leu Ile Gly His Leu Leu His Leu Arg Gly Pro Gln Ala Pro
50 55 60
His Ile Thr Leu Gly Lys Met Ala Asp Lys Tyr Gly Pro Ile Phe Lys
65 70 75 80
Ile Lys Leu Gly Val His Pro Thr Leu Val Ile Ser Asp Ser Glu Val
85 90 95
Ala Lys Glu Cys Leu Thr Thr His Asp Ile Ala Leu Ala Gly Arg Pro
100 105 110
Ala Thr Val Ala Met Glu Ile Met Gly Tyr Asn His Ala Met Phe Ala
115 120 125
Phe Ser Pro Tyr Gly Pro Tyr Trp Arg His Met Arg Lys Leu Ala Thr
130 135 140
Val Glu Leu Leu Ser Thr Gln Arg Leu Glu Thr Phe Lys His Ile Arg
145 150 155 160
Glu Ser Glu Leu Lys Arg Ser Met Lys Glu Met Tyr Gln Ser Trp Val
165 170 175
His Asn Lys Ser Gly Ser Gly Asp Ser Asn His Val Thr Val Asp Met
180 185 190
Thr Arg Ile Leu Gly Asp Ile Ile Ala Asn Val Ile Tyr Arg Met Val
195 200 205
Val Gly Lys Val Tyr Ala Ser Lys Gly Glu Glu Asp Ala Arg Trp Lys
210 215 220
Gln Val Val Trp Glu Tyr Ile Lys Leu Leu Ser His Phe Gly Val Gly
225 230 235 240
Asp Ala Leu Pro Phe Leu Arg Trp Leu Asp Leu Gly Gly Val Glu Lys
245 250 255
Ser Met Lys Lys Ala Ala Lys Glu Leu Asp Ile Tyr Val Glu Glu Trp
260 265 270
Leu Glu Glu His Lys Lys Lys Arg Ser Glu Arg Lys Ser Asp Asn Gly
275 280 285
Ile Val Glu Glu Asp Phe Met Asp Val Met Leu Ser Val Phe Asp Asp
290 295 300
Asp Asp Gln Leu Glu Asn Phe Ala His His Ser Ala His Thr Ile Asn
305 310 315 320
Lys Ala Met Cys Leu Ala Ile Ile Leu Ala Ala Ser Asp Thr Thr Lys
325 330 335
Thr Thr Leu Thr Trp Ala Leu Ser Leu Leu Leu Asn His Pro Asp Val
340 345 350
Met Lys Lys Val Gln Gln Glu Leu Ala Ala His Ile Gly Pro Asp Lys
355 360 365
Pro Val Lys Glu Ser Asp Val Lys Ser Leu Val Tyr Leu Glu Ala Val
370 375 380
Val Lys Glu Thr Leu Arg Leu Tyr Pro Pro Gly Pro Leu Gly Leu Pro
385 390 395 400
His Glu Ser Met Glu Asp Cys Thr Val Ala Gly Tyr His Val Pro Ser
405 410 415
Gly Thr Arg Ile Leu Tyr Asn Leu Trp Lys Ile Gln Gln Asp Pro Gln
420 425 430
Val Trp Glu Asn Pro Ser Glu Phe Lys Pro Asp Arg Phe Leu Thr Thr
435 440 445
His Lys Asp Val Asp Val Arg Gly Arg Asn Phe Glu Tyr Leu Pro Phe
450 455 460
Gly Ser Gly Arg Arg Met Cys Pro Gly Met Ser Phe Ala Leu Gln Val
465 470 475 480
Met Glu Val Ser Leu Ala Asn Met Leu His Gly Phe Asp Phe Ala Thr
485 490 495
Pro Asn Gly Lys Pro Val Asp Met Thr Glu Val Asn Gly Leu Val Thr
500 505 510
Asp Arg Ala Thr Pro Leu Glu Ala Leu Ile Thr Pro Arg Leu Pro Ala
515 520 525
His Leu Tyr Met Gly
530
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggagtttc ttctttcact cccag 25
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tcagcccata tagagatgag ctgg 24
<210> 5
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
caaggagaaa aaaccccgat gagttcgagc tcggattt 38
<210> 6
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aaatcaactt ctgttccatg tctcaccaaa catctcgaag atatc 45
<210> 7
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gaattcaacc ctcactaaag ggcgatggag tttcttcttt cactcccag 49
<210> 8
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
catccttgta atccatcgat actcagccca tatagagatg agctgg 46
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
attttcggtt tgtattactt c 21
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gttcttaata ctaacataac t 21
<210> 11
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggtggtaatg ccatgtaata tg 22
<210> 12
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggcaaggtag acaagccgac aac 23
<210> 13
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cacccgaaac gtttaagcac ataagag 27
<210> 14
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tcatcatcat caaaaacgga gag 23
<210> 15
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ctccacctgc atcgcaccat ggagtttctt ctttcactc 39
<210> 16
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gaccacctgc atcgcctttc agcccatata gagatgagc 39
<210> 17
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cgcggtacta gtgtttagtt aatta 25
<210> 18
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ctgttttata tttgttgtaa aaagtagat 29
<210> 19
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aactaaacac tagtaccgcg atggagtttc ttctttcact cccag 45
<210> 20
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tccttgtaat ccatcgatac tcagcccata tagagatgag ctggg 45
<210> 21
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ttacaacaaa tataaaacag atgagttcga gctcggattt ggttc 45
<210> 22
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atcaacttct gttccatgtc tcaccaaaca tctcgaagat atcttc 46
<210> 23
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ggtcaatgca agaaatacat at 22
<210> 24
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gttcttaata ctaacataac t 21
<210> 25
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tctctctatc tattctactt gt 22
<210> 26
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ggcaaggtag acaagccgac aac 23
Claims (9)
1.如SEQ ID NO：2所示氨基酸序列的酶在催化松香烷型三环二萜类化合物C-14位羟基化方面的应用。
2.编码如SEQ ID NO：2所示氨基酸序列的酶的多核苷酸分子在催化松香烷型三环二萜类化合物C-14位羟基化方面的应用。
3.如权利要求2所述的应用，其中所述核苷酸分子具有如SEQ ID NO:1所示的碱基。
4.包含编码如SEQ ID NO：2所示氨基酸序列的酶的多核苷酸分子的表达载体在催化松香烷型三环二萜类化合物C-14位羟基化方面的应用。
5.包含编码如SEQ ID NO：2所示氨基酸序列的酶的多核苷酸分子的表达载体的重组宿主菌在催化松香烷型三环二萜类化合物C-14位羟基化方面的应用。
6.根据权利要求5所述的重组宿主菌，其中所述重组宿主菌为酵母菌，所述酵母菌为BY系酵母菌或WAT系酵母菌。
7.权利要求1-6中任意一权利要求所述的应用，其中所述松香烷型三环二萜类化合物为次丹参酮二烯、脱氢松香酸或雷酚萜D。
8.一种催化松香烷型三环二萜类化合物C-14位羟基化的方法，其包括：
(1)将编码如SEQ ID NO：2所示氨基酸序列的酶的基因表达盒插入真核表达载体pESC-Leu的NotI、SpeI位点，制备表达所述酶的重组酵母菌；
(2)以C-14位未羟化的松香烷型三环二萜类化合物为底物，加入到步骤(1)中所获得重组酵母菌中/或加入到步骤(1)中重组酵母菌表达后分离所得的蛋白中，制备获得C-14位羟化的松香烷型三环二萜类化合物；或
(3)以次丹参酮二烯为底物，饲喂步骤(1)中获得的表达所述酶的重组酵母菌，制备松香烷型二萜类化合物。
9.根据权利要求8所述的应用，其中步骤2中所述松香烷型三环二萜类化合物为次丹参酮二烯、脱氢松香酸或雷酚萜D。
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