CN116334014A - 一种催化美登木酸c2位羟化的cyp716c52蛋白及其编码基因与应用 - Google Patents
一种催化美登木酸c2位羟化的cyp716c52蛋白及其编码基因与应用 Download PDFInfo
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- CN116334014A CN116334014A CN202211571171.0A CN202211571171A CN116334014A CN 116334014 A CN116334014 A CN 116334014A CN 202211571171 A CN202211571171 A CN 202211571171A CN 116334014 A CN116334014 A CN 116334014A
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Abstract
本发明涉及一种细胞色素P450氧化酶CYP716C52蛋白,以及所述CYP716C52蛋白编码基因,所述蛋白可催化美登木酸C2位羟基化生成雷公藤酸C,继而参与雷公藤红素生物合成。
Description
技术领域
本发明通过聚合酶链式反应首次克隆得到雷公藤细胞色素P450合酶CYP716C52基因及其编码产物,涉及雷公藤红素生物合成的CYP716C52蛋白及其编码基因与应用,属于药用植物基因工程领域。
背景技术
卫矛科雷公藤属植物雷公藤Tripterygium wilfordii Hook.f.是一种传统的药用植物,可用于治疗类风湿性关节炎、系统性红斑狼疮和癌症等疾病。雷公藤中含有多种三萜类化合物,其中雷公藤红素是一种具有代表性的木栓烷型三萜,于1936年首次从雷公藤中提取分离得到(Tu S H.The predicament and countermeasure of Tripterygiumwilfordii in treating rheumatoid arthritis.Chinese Journal ofIntegratedTraditional andWestern Medicine.2009,29:104–105.)。雷公藤红素是雷公藤多甙片和雷公藤片等治疗类风湿性疾病药物制剂的有效成分之一。此外,雷公藤红素在抗肿瘤、康抗肥胖和抗中枢神经系统疾病等方面也具有非常显著的药理活性,对于研发治疗癌症、炎症和中枢神经系统疾病的新药具有重要意义(Lu,Y.,Liu,Y.,Zhou,J.,et al.Biosynthesis,total synthesis,structural modifications,bioactivity,and mechanism of actionof the quinone-methide triterpenoid celastrol[J].Medicinal research reviews,2021,41(2):1022-1060.)。目前从原植物中提取分离获取雷公藤红素的传统获取方式具有很多局限性且会对野生药用资源造成极大的破坏,因此通过解析其生物合成途径并利用合成生物学策略改造微生物来生产雷公藤红素的方法将成为一种极具潜力的生产方式。
雷公藤红素的上游生物合成途径与其他三萜一致,都是由位于胞质的甲羟戊酸(MVA)途径和质体的2-C-甲基-D-赤藓糖醇-4-磷酸(MEP)途径,产生异戊烯基焦磷酸(IPP)及异构体二甲烯丙基焦磷酸(DMAPP),再通过一系列酶的催化作用生成2,3-氧化鲨烯。2,3-氧化鲨烯在三萜环化酶(oxidosqualene cyclase,OSC)的作用下产生多种三萜骨架,其中木栓酮是雷公藤红素生物合成的骨架结构(Zhou J.,Hu T.,Gao L.,et al.Friedelane-type triterpene cyclase in celastrol biosynthesis from Tripterygium wilfordiiand its application for triterpenes biosynthesis in yeast.New Phytol.2019,223(2):722-735.)。随后细胞色素P450(CYP450)和糖基转移酶等对三萜骨架进行后修饰从而产生结构各异的三萜化合物。据推测,雷公藤红素的下游生物合成途径主要是由CYP450参与的(Hansen NL,Miettinen K,Zhao Y,et al.Integrating pathway elucidation withyeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol.Microbial cellfactories 2020;19(1):15.)。CYP712K4是首个报道的参与雷公藤红素下游生物合成途径的CYP450,研究表明其具备催化木栓酮C-29位连续三步氧化生成美登木酸的功能(Bicalho KU,Santoni MM,Arendt P,et al.CYP712K4Catalyzes the C-29Oxidation of Friedelin in the Maytenus ilicifolia QuinoneMethide Triterpenoid Biosynthesis Pathway.Plant Cell Physiol 2019;60(11):2510-22.),随后,研究者又鉴定出具有相同催化功能的CYP712K1,CYP712K2和CYP712K3(Hansen NL,Miettinen K,Zhao Y,et al.Integrating pathway elucidation withyeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol.Microb Cell Fact.2020,28;19(1):15.),因此筛选可以进一步催化美登木酸生成后续雷公藤红素中间体的CYP450非常重要。
本发明从雷公藤中克隆得到一条CYP716C52基因,功能表征结果显示其具有催化雷公藤红素的中间体——美登木酸C2位羟基化生成雷公藤酸C的功能,基因干扰实验进一步证明CYP716C52基因参与了雷公藤红素的生物合成,并通过分子对接和定点突变实验获得了活性更优的突变体(Q141S、L144I和I221L)。该基因是首次从雷公藤中得到的,在本发明被公布之前,尚未有任何公开或报道过本专利申请中所提及的雷公藤CYP450基因及其氨基酸序列。
发明内容
本发明提供了一种分离的蛋白,所述蛋白参与雷公藤红素的生物合成,可催化美登木酸的C美登木酸C2位羟基化生成雷公藤酸C。
本发明中,所述分离的蛋白是一种细胞色素P450氧化酶,以下用CYP716C52,所述蛋白具有:
(1)SEQ ID NO:2所示的氨基酸序列;或
(2)SEQ ID NO:2所示的氨基酸序列经取代、缺失或增加一个或多个氨基酸且功能相同的肽。
CYP716C52变体或具有实质上相似的序列一致性的多肽的特征在于具有一个或多个氨基酸替换、缺失或插入。这些改变优选在性质上较小,即保守氨基酸替代(见表1)和不显著影响多肽的折叠或活性的其它替代;小的缺失,典型地一个至约30个氨基酸的缺失;氨基或羧基端延伸,例如氨基端甲硫氨酸残基,不超过约20-25个残基的小的接头肽或亲和标签。本发明因此提供包含与SEQ ID NO:2至少有70%,优选至少有90%、更优选95%、96%、97%、98%、99%或更多一致的序列的多肽。
可以确定包含对于维持结构完整性关键的区域或域的氨基酸残基。可以确定在这些区域中或多或少耐受改变而保持分子的整个三级结构的特定残基。分析序列结构的方法包括,但不限于具有高氨基酸或核苷酸一致性的多个序列的比对、二级结构倾向(propensity)、二级元图(binary patterns)、互补堆积(complementary packing)和隐蔽的极性相互作用(Barton,Current Opin.Struct.Biol.5:372-376,1995;和Cordes等,Current Opin.Struct.Biol.6:3-10,1996)。一般地,当设计分子修饰或鉴定特定片段时,将在确定结构的同时评价修饰的分子的活性。
一个具体的实施方案中,所述分离的蛋白变体为将SEQ ID NO:2所示氨基酸序列中的一个或多个氨基酸进行如下突变:
(1)将第141位的Gln(Q)突变为Ser(S)、Glu(E)、Asn(N)或Pro(P);和/或
(2)将第144位的Leu(L)突变为Val(V)、Ser(S)、Ile(I)或Thr(T);和/或
(3)将第221位Ile(I)突变为Phe(F)、Val(V)、Leu(L)或Gly(G)。
一个更为具体的实施方案中,本发明提供了变体,所述变体为对SEQ ID NO:2所示氨基酸序列的CYP716C52进行定点突变,具体为CYP716C52Q141S、或CYP716C52L144I、或CYP716C52I221L,三个变体,具有更好的活性。
本发明再一目的提供了一种编码所述细胞色素P450氧化酶CYP716C52的多核苷酸序列。
所述的多核苷酸序列,是一种与美登木酸C2位羟基化相关的基因(以下用CYP716C52表示),它是下列核苷酸序列之一:
1)序列表中SEQ ID No.1核苷酸序列;或
2)与序列表中SEQ ID No.1限定的核苷酸序列具有一个或几个碱基突变,且编码相同功能蛋白质的DNA序列;或
本发明的还一目的在于提供一种表达载体,所述表达载体包含编码本发明CYP716C52蛋白或其突变体的多核苷酸,所述表达载体还包含启动子和终止子,其中所述启动子与所述多核苷酸可操作地连接,并且所述多核苷酸与所述转录终止子可操作地连接。
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。在生产本发明雷公藤三萜合酶多肽时,可以将雷公藤三萜合酶基因的核苷酸序列可操作地连接于表达调控序列,从而形成雷公藤三萜合酶表达载体。所述“可操作地连连接”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。
本发明的再一目的,在于提供一种宿主细胞,所述宿主细胞,包含本发明所述编码CYP716C52蛋白或其变体的多核苷酸分子,或包含本发明上述描述的表达载体。所述的宿主细胞选自:细菌、原核细胞(如大肠杆菌、)真菌细胞、酵母细胞、昆虫细胞、哺乳动物细胞或植物细胞,优选地,为酵母细胞或植物细胞。
特别有意义的酵母包括酿酒酵母、巴斯德毕赤酵母和Pichiamethanolica。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pYES2表达载体。用于酵母的适宜启动子和终止子包括来自糖酵解基因(US4599311、US4615974和US4977092)和醇脱氢酶的那些。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、PichiaMethanolica、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的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喷气给液体培养物提供充足的空气。
本发明的CYP716C52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,尤其是开放阅读框序列来设计引物,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,常常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.85:2149-2154,1963)。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(Foster City,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。
本发明中,术语“CYP716C52蛋白编码基因”或“CYP716C52基因”可互换使用;术语“CYP716C52”、“细胞色素P450氧化酶CYP716C52”、“CYP716C52蛋白”可互换使用。术语“核苷酸分子”或“核苷酸序列”可互换使用。
术语“分离的”或“纯化的”,多肽或蛋白质是指在非其天然环境的条件下,例如离开血液或组织存在的多肽或蛋白质。在优选形式中,分离的多肽或蛋白基本上不含有其他多肽或蛋白,尤其是动物来源或植物来源的其它多肽或蛋白。优选提供高纯度化形式,即纯度大于95%、更优选纯度大于99%的多肽;“分离的”或“纯化的”DNA是指,该DNA或片段已从天然状态下位于其两侧的序列中分离出来,或指该DNA或片段已经与天然状态下伴随核酸的组分分开,而且已经在与细胞中伴随其的蛋白质分开。
本发明提供的CYP716C52蛋白及CYP716C52蛋白编码基因是首次从雷公藤中克隆制备的,其催化美登木酸C2位羟基化,生成雷公藤酸C。基因枪介导的CYP716C52干扰结果显示,本发明克隆基因CYP716C52经基因枪介导转化至雷公藤悬浮细胞后,其基因表达量明显降低,伴随着细胞中雷公藤红素含量降低,进一步证明CYP716C52参与了雷公藤红素的生物合成。CYP716C52基因可用于利用转基因技术来提高雷公藤红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,对于缓解雷公藤药源匮乏问题有较好的促进作用,可用于雷公藤育种。
本发明的一个实施方案中,提供了本发明所述CYP716C52蛋白或CYP716C52基因在催化美登木酸C2位羟基化生物合成中的应用,进而参与雷公藤红素的生物合成。
本发明的一个实施方案中,提供了一种通过运用本发明所述CYP716C52蛋白或CYP716C52基因催化美登木酸C2位羟化生成雷公藤酸C的方法。包括:(1)雷公藤悬浮细胞的培养;(2)雷公藤悬浮细胞RNA提取;(3)雷公藤悬浮细胞转录组测序;(4)CYP716C52基因全长cDNA获取;(5)质粒和菌株构建;(6)工程菌培养;(7)工程菌表达产物提取和分离。
本发明SEQ ID No.1的DNA序列由1455个碱基组成,编码序列表中由484个氨基酸残基组成的蛋白质序列SEQ ID No.2。
附图说明
图1.雷公藤红素生物合成途径推导示意图。
图2.CYP716C52催化产物UPLC/Q-TOF-MS检测结果图,其中:(a)CYP716C52功能表征色谱图(提取476.36m/z);(b)产物(峰1)质谱裂解规律。
图3.雷公藤酸C的1HNMR谱(800MHz,溶剂CD3OD)。
图4.雷公藤酸C的13C NMR谱(200MHz,溶剂CD3OD)。
图5.雷公藤酸C的COSY谱(溶剂CD3OD)。
图6.雷公藤酸C的HSQC谱(溶剂CD3OD)。
图7.雷公藤酸C的HMBC谱(溶剂CD3OD)。
图8.雷公藤酸C的NOESY谱(溶剂CD3OD)。
图9.雷公藤酸C的1H NMR谱(800MHz,溶剂C5D5N)。
图10.雷公藤酸C的13C NMR谱(200MHz,溶剂C5D5N)。
图11.CYP716C52基因RNAi结果(n=3),其中:(a)CYP716C52基因RNAi载体示意图;(b)RNAi载体转入雷公藤悬浮细胞验证结果;(c)RT-PCR检测对照组(CK)及RNAi实验组雷公藤悬浮细胞中CYP716C52基因的相对表达量;(d)CK组及RNAi组雷公藤悬浮细胞中雷公藤红素的含量(*代表P<0.05)。
图12.野生型CYP716C52(WT)及其突变体生产雷公藤酸C的相对产量比较(n=3,**代表P<0.01)。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的定量试验,均设置三次重复实验,结果取平均值。
下述实施例中的雷公藤(Tripterygium wilfordii Hook.f.)悬浮细胞在文献“Zhao Y.,ZhangY.,Su P.,et al.Tripterygium wilfordii Genetic TransformationSystem for Woody Plant and Its Application to Product Natural Celastrol[J].Front Plant Sci,2017,8:2221.”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。
下述实施例中的pEASY-Blunt Simple Cloning Kit、-Basic SeamlessCloning and Assembly Kit、Top Green qPCR SuperMix和Fast MutagenesisSystem试剂盒均购自北京全式金生物技术有限公司;Phusion High-Fidelity PCR MasterMix和限制性内切酶NotI、SacI、SpeI、PacI、KpnI和EcoRI均购自New England Biolabs公司;Super总RNA提取试剂盒购自Promega公司;RACE 5’/3’Kit购自Takara公司;Frozen-EZ Yeast转化II KitTM购自美国Zymo Research生物科技公司快速质粒小提试剂盒、FastQuant RT Kit均购自天根生化科技有限公司。Plasmid maxi Kit质粒大提试剂盒购自美国OMEGABio-tek公司;Gene JET Gel Extraction Kit试剂盒购自美国Thermo Scientific公司;LR Clonase Enzyme Mix购自美国Invitrogen公司;
实施例1、雷公藤CYP716C52全长cDNA的克隆
1.雷公藤悬浮细胞总RNA提取
2.cDNA第一链的获得
3.引物设计
根据雷公藤转录组数据筛选得到基因序列,设计CYP716C52-F和CYP716C52-R引物如下:
CYP716C52-F:ATGGCATTTTATCAAACACTTTCCG(SEQ ID NO:3)
CYP716C52-R:TTAATTGGCTGAGTGAAGGCGG(SEQ ID NO:4)
4.PCR扩增及克隆载体连接
以步骤2获得的第一链cDNA为模板,以CYP716C52-F和CYP716C52-R为引物,利用高保真酶Phusion High-Fidelity Master Mix进行PCR扩增,PCR反应程序为:98℃30s;98℃10s,56℃15s,72℃1min,35个循环;72℃7min;4℃hold。取PCR扩增产物进行1.5%琼脂糖凝胶电泳,切取大小正确的条带,并用Gene JET Gel Extraction Kit试剂盒进行切胶回收,然后按照pEASY-Blunt Simple Cloning Kit说明书操作方法将回收产物连接至克隆载体,并转化至Trans1-T1感受态细胞,涂板,对阳性克隆进行测序验证。
测序结果表明:PCR扩增产物的序列如序列1所示,将序列1所示的基因命名为CYP716C52,ORF为1455bp,编码由484个氨基酸残基组成的蛋白质,该蛋白命名为CYP716C52,该蛋白的氨基酸序列为序列2。该克隆载体命名为pEASY-Blunt Zero::CYP716C52,并于-20℃冰箱保存。
实施例2、雷公藤CYP716C52基因功能研究
1.真核表达载体构建
(1)pESC-Leu::(TwOSC1+TwCPR3)载体构建
使用无缝拼接的方法把来自雷公藤的木栓酮合酶基因TwOSC1构建至pESC-Leu::TwCPR3真核表达载体的多克隆位点1(Multiple Cloning Site 1,MCS1)处(Zhou J.,HuT.,Liu Y.,et al.Cytochrome P450 catalyses the 29-carboxyl group formation ofcelastrol[J].Phytochemistry,190.)。
设计带有同源臂的无缝拼接引物:TwOSC1-Leu-F和TwOSC1-Leu-R,以pEASY-BluntZero::TwOSC1为质粒模板(Zhou J.,Hu T.,Gao L.,et al.Friedelane-type triterpenecyclase in celastrol biosynthesis from Tripterygium wilfordii and itsapplication for triterpenes biosynthesis in yeast.New Phytol.2019,223(2):722-735.),利用高保真酶Phusion High-Fidelity Master Mix进行PCR扩增,PCR程序为:98℃30s;98℃10s,56℃15s,72℃2min,35个循环;72℃7min;4℃hold。取PCR扩增产物进行1.5%琼脂糖凝胶电泳,切取大小正确的条带,并用Gene JET Gel Extraction Kit试剂盒进行切胶回收,得到带有同源臂的目的片段。
TwOSC1-Leu-F:CCTCACTAAAGGGCGGCCGCATGTGGAAGCTCAAAGTTGC(SEQ ID NO:5)
TwOSC1-Leu-R:GAATTGTTAATTAAGAGCTCTCAATAGCCTTTGGATGGTA(SEQ ID NO:6)
用限制性内切酶NotI和SacI对载体pESC-Leu::TwCPR3进行双酶切,酶切产物进行1.5%琼脂糖凝胶电泳,切取大小正确的条带,并用Gene JET Gel Extraction Kit试剂盒进行切胶回收,得到线性化质粒。
经测序鉴定,得到经测序核苷酸序列无突变的重组质粒pESC-Leu::(TwOSC1+TwCPR3)。
(2)pESC-His::CYP712K1载体构建
使用如上无缝拼接的方法将美登木酸合酶基因CYP712K1构建至pESC-His真核表达载体的MCS1处,设计带有同源臂的无缝拼接引物:CYP712K1-His-F和CYP712K1-His-R,以pYES2::CYP712K1为质粒模板(Zhou J.,Hu T.,Liu Y.,et al.Cytochrome P450catalyses the 29-carboxyl group formation ofcelastrol[J].Phytochemistry,190.),利用高保真酶Phusion High-Fidelity Master Mix进行PCR扩增,PCR扩增产物进行1.5%琼脂糖凝胶电泳,切取大小正确的条带,并用Gene JET Gel Extraction Kit试剂盒进行切胶回收,得到带有同源臂的目的片段。
CYP712K1-His-F:AGGGCGGCCGCACTAGTATGGCCACCATCACTGACATSEQ ID NO:7)
CYP712K1-His-R:GAAGAATTGTTAATTAATTAACCGGCAAATGGATTGASEQ ID NO:8)
用限制性内切酶SpeI和PacI对载体pESC-His进行双酶切,酶切产物进行1.5%琼脂糖凝胶电泳,切取大小正确的条带并用Gene JET Gel Extraction Kit试剂盒进行切胶回收,得到线性化质粒。
经测序鉴定,得到经测序核苷酸序列无突变的重组质粒pESC-His::CYP712K1。
(3)pYES2::CYP716C52载体构建
使用如上无缝拼接的方法将CYP716C52构建至pYES2真核表达载体上,设计带有同源臂的无缝拼接引物:CYP716C52-pYES2-F和CYP716C52-pYES2-R,以pEASY-Blunt Zero::CYP716C52为模板,利用高保真酶Phusion High-Fidelity Master Mix进行PCR扩增,PCR程序为:98℃30s;98℃10s,56℃15s,72℃1min,35个循环;72℃7min;4℃hold。取PCR扩增产物进行1.5%琼脂糖凝胶电泳,切取大小正确的条带,并用Gene JET Gel Extraction Kit试剂盒进行切胶回收,得到带有同源臂的目的片段。
CYP716C52-pYES2-F:TATTAAGCTTGGTACATGGCATTTTATCAAACACT(SEQ ID NO:9)
CYP716C52-pYES2-R:GATGGATATCTGCAGTTAATTGGCTGAGTGAAGGC(SEQ ID NO:10)
用限制性内切酶KpnI和EcoRI对载体pYES2进行双酶切,酶切产物进行1.5%琼脂糖凝胶电泳,切取大小正确的条带,用Gene JET Gel Extraction Kit试剂盒进行切胶回收,得到线性化质粒。
经测序鉴定,得到经测序核苷酸序列无突变的重组质粒pYES2::CYP716C52。
2.真核表达载体转化至羊毛甾醇缺陷酵母菌株
(1)按照Frozen-EZ Yeast转化II KitTM试剂盒操作步骤制作羊毛甾醇缺陷型酵母(ATCC,cell line number:4021900,-erg7,-ura)感受态细胞。
(2)将质粒pESC-Leu::(TwOSC1+TwCPR3)和pESC-His::CYP712K1共同转化至步骤(1)的酵母感受态细胞中,涂板于Sc-Leu-His固体培养基(含2%葡萄糖)上,30℃倒置培养2-3天。挑取单菌落,再按照Frozen-EZ Yeast转化II KitTM试剂盒操作步骤制作含有上述两种质粒的酵母感受态细胞。
(3)向步骤(2)所制作的酵母感受态细胞中再转化进第三个质粒pYES2::CYP716C52,涂板于Sc-Leu-His-Ura固体培养基(含2%葡萄糖)上,30℃倒置培养2-3天。此外,用同样的方法将空载体pYES2也转化至转化到步骤(2)所制作的酵母感受态细胞中作为对照组,用相同的条件进行培养。
3.菌株发酵及产物检测
(1)分别从每个板子上挑取单菌落至3mL Sc-Leu-His-Ura液体培养基(含2%葡萄糖)中活化约24h至菌液浑浊,取1mL活化的菌液扩大培养至30mL后,振荡培养至OD600达到0.8-1.0,4000×g离心2min,在生物安全柜内弃去培养基,用等体积YPL液体培养基诱导3天(30℃,200rpm)。
(2)离心收集菌体(4000×g,2min),用20mL蒸馏水重悬,再用等体积乙酸乙酯超声提取两次,合并有机层,用旋转蒸发仪除去溶剂后,用1mL乙酸乙酯复溶两次,再用氮气吹干,最后用300μL质谱甲醇溶解,高速离心取上清液进样超高效液相色谱/离子淌度/四级杆-飞行时间质谱(UPLC/Q-TOF-MS):使用ACQUITYUPLC HSS T3色谱柱(2.1mm 100mm,1.8μm,USA),在正离子模式下的Xevo G2-S QTOF MS(Waters Corp.,Milford,MA,USA)系统进行质谱检测,毛细管电压1.0kV,锥孔电压40V,离子源温度120℃,溶媒挥散温度450℃,锥体气流50L/h,脱溶剂气流速800L/h,扫描范围设置为50-1500m/z,扫描时间为0.2s;液相条件:A为0.1%甲酸-水,B为乙腈,液相梯度见表1。进样完成后,用MassLynx软件处理数据。
表1 UPLC/Q-TOF-MS检测液相条件
结果如图2所示,与对照组相比,CYP716C52可以催化底物美登木酸生成[M+H]+为473.36的产物(峰1),分子量相比于美登木算增加了16m/z,因此推测CYP716C52可以催化底物进行羟基化。
4.CYP716C52产物制备及核磁鉴定
通过对产物进行富集、制备以及核磁鉴定来确定其化学结构。
(1)产物大量富集
将上述转化了pESC-Leu::(TwOSC1+TwCPR3)、pESC-His::CYP712K1和pYES2::CYP716C52这三种质粒的羊毛甾醇缺陷酵母菌株命名为LY1,并将该菌株在10-L生物反应器中进行高密度发酵,具体步骤为:挑取单克隆接种到含有40mL Sc-Leu-His-Ura液体培养基(含2%葡萄糖)中活化约24h后,再扩大培养至400mL,振荡培养约16h,测定OD600值。将二次活化的菌液以初始OD600为0.5接入含有3L Sc-Leu-His-Ura液体培养基(含2%葡萄糖)的生物反应器中,温度设置为30℃,溶氧(DO)设置为30%,pH设置为5.5。此外设置pH与碱泵相关联,溶氧(DO)与搅拌转速以及通气量相关联。按0.1mLmin-1的流速匀速补料6×Sc-Leu-His-Ura培养基,培养48h;从48h开始,按0.1mL min-1的流速匀速补料40%半乳糖进行诱导培养,发酵周期为96h。
发酵结束后,收集菌体和培养基,用乙酸乙酯进行多次超声提取,重复发酵提取6次,合并,富集产物。
(2)产物制备及分离纯化
把(1)中富集到的产物溶解到约30mL质谱甲醇中,用质谱引导的制备液相色谱(安捷伦1260二极管阵列检测器,单四极杆质谱检测器)对样品进行粗分离,色谱柱为SunFirePrep C18 OBD色谱柱(19×150mm,5.0μm,Waters),液相条件为A相(纯水),B(乙腈),按表2中的制备液相梯度,每次进样200uL,紫外检测范围为190-400nm,同时设置DAD信号201nm。单元泵流动相为0.1%甲酸-水,流速1mLmin-1,液相:质谱分流比为100:1,质谱条件:正离子模式下扫描TIC,质量范围为100-500m/z,同时设置SIM模式检测母离子[MS+]=473以及子离子[MS+]=455,收集27.5-29.5min的馏分,合并,旋转蒸发除去溶剂,用30mL质谱甲醇复溶,得到产物粗品。
表2质谱引导的制备液相色谱检测液相条件
把上述初步分离得到的样品,进样质谱引导的制备型高压液相色谱仪(Waters,型号Mass3100),流行相为A(0.05%氨水),B(乙腈),C18色谱柱:(30×100mm,5.0μm,Gemini),按表3中的液相梯度进样1mL,质谱条件为负离子模式,使用SIM模式检测[MS]-=471,根据出峰时间,收集10.2-11.5min的馏分,合并所得馏分,旋转蒸发除去溶剂后,再用冷冻干燥除去水分获得产物纯品约5mg,产物经ELSD和质谱检测器检测,纯度为98%。
表3质谱引导的制备型高压液相色谱仪检测液相条件
(3)产物结构核磁鉴定
称取约2.0mg纯化产物,加入200μL氘代甲醇充分溶解后,15000×g高速离心5min,取上清转移至核磁管,用800兆核磁共振谱仪系统(Bruker,AVANCE III)检测分析1H-NMR和13C-NMR,以及二维谱:COSY谱、HSQC谱、HMBC谱和NOESY谱,核磁数据用MestReNova软件进行分析,产物的核磁1H-NMR、13C-NMR及二维谱如图3~8所示,产物被鉴定为雷公藤酸C(wilforic acid C),结构见图3,是美登木酸C2位的羟基化产物,核磁归属信息见表4。
表4产物核磁信号归属(CD3OD)
查阅文献发现,雷公藤酸C是雷公藤根皮中分离出的一种三萜(Kunhua L,Hongquan D,Kazuyoshi K,Takaishi Y.Terpenoids from Tripterygiumwilfordii.Phytochemistry.1997,45:791-796.),为了与文献中报道的核磁数据(溶剂为氘代吡啶)进行比对,将溶剂更换为氘代吡啶。再次进样800兆核磁共振谱仪系统检测1H-NMR谱和13C-NMR谱,用MestReNova软件分析核磁数据。结果显示,产物的1H NMR谱(图9)和13CNMR谱数据(图10)与文献报道数据一致,进一步确定了该结果。
实施例3、雷公藤CYP716C52基因干扰研究
1.雷公藤悬浮细胞培养
配制MS固体培养基:(MS 4.43g L-1,2,4-D 1.0mg L-1,KT 0.1mg L-1,蔗糖:30g L-1,调pH至5.8-6.0后加入琼脂8g L-1),高压灭菌后,冷却至60℃左右,取3mL液体培养基至小培养皿中,凝固后取本实验室继代培养了20天左右且生长状态良好的雷公藤悬浮细胞,称取0.2g转移至MS固体培养基中,在恒温恒湿培养箱中25℃暗培养7天。
2.干扰载体构建
干扰片段的设计及扩增:根据CYP716C52基因序列,本地Blast比对雷公藤基因组,找出CYP716C52特异性片段(300-500bp),设计引物:RNAi-CYP716C52-F:CACCGCAAAAGCTTGTCAAAGTCTGG;RNAi-CYP716C52-R:CACTAATCGCAACAGCCGCC,以pEASY-Blunt Zero::CYP716C52质粒为模板,用高保真酶扩增出CYP716C52特异片段(429bp)。PCR程序为:98℃30s;98℃10s,56℃15s,72℃1min,35个循环;72℃7min;4℃hold。PCR扩增结束后,跑1.5%琼脂糖凝胶电泳,切取大小正确的条带,用Gene JET Gel Extraction Kit回收干扰片段。
BP反应连接入门载体:将上述纯化得到的干扰片段连接至pENTRTM SD/D-载体,配制BP反应体系如下:干扰片段2μL,pENTRTM SD/D-载体0.5μL,Salt solution0.5μL。放于PCR仪中,22℃孵育2h。将PCR产物全部转化至Trans1-T1感受态细胞,涂板LB+Kana固体平板,37℃倒置培养,阳性克隆送测序成功后,用快速质粒小提试剂盒提取入门载体质粒。
LR反应连接表达载体:用LR反应连接干扰表达载体pK7GWIWG2D,体系为:入门载体质粒300ng,pK7GWIWG2D 100-300ng,LR Clonase Enzyme Mix 1μL,TE Buffer1μL。于PCR仪中25℃孵育4h,将PCR产物全部转化至50μL Trans1-T1感受态细胞中,涂板LB+Spe固体平板,37℃倒置培养,阳性克隆送测序成功后,按照Plasmid maxi Kit质粒大提试剂盒说明书提取质粒,质粒浓度需达到1000ng/uL以上。
3.基因枪介导转化雷公藤悬浮细胞
用基因枪转化系统把空载质粒pK7GWIWG2D(简称PK7)和RNAi-CYP716C52质粒分别转化至雷公藤悬浮细胞。配制每种质粒的混合体系:金粉5mg,2.5M CaCl2100μL,0.1M亚精胺40μL,质粒20μg,对质粒进行包埋处理。然后在通高纯氦气(大于1000psi)的条件下,用基因枪把质粒包埋的金粉经高压轰击至悬浮细胞内。
4.验证质粒成功转入雷公藤悬浮细胞
上述基因枪介导转化的悬浮细胞,继续放置于恒温恒湿培养箱中25℃暗培养7天后取样,用滤纸吸干表面水分后放于2mL EP管中,用液氮速冻后于-80℃保存。取一部分细胞,使用Super总RNA提取试剂盒(Promega)提取总RNA,再用FastQuant RTKit试剂盒进行RNA的反转录合成第一链cDNA。以cDNA为模板,使用特异性引物EGFP-F:TTACAGCTCGTCCTTCTTGTAC和EGFP-R:TTACAGCTCGTCCTTCTTGTAC扩增出条带,用1%琼脂糖凝胶电泳检测条带大小是否正确(约为800bp),以此来确定质粒是否成功转入细胞内。
5.RT-PCR检测基因表达量
加无菌水稀释上述所得的第一链cDNA至浓度为100ngμL-1备用。用96孔板点样,按照向每个孔加入20μL体系:2×Top Green qPCR SuperMix 10uL,引物F/R各0.4uL,cDNA 1uL,Rox Dye II 0.4uL,PCR-grade water 7.8uL,做三个技术重复,以EF1α为内参基因,用ABI QuantStudio5实时荧光定量PCR仪进行检测,RT-PCR程度为:94℃30s,94℃5s,56℃15s,72℃10s,45个循环;Dissociation Stage。用2-△△Ct法分析基因的相对表达量。
6.雷公藤悬浮细胞化学含量测定
用球磨机将剩余细胞研磨成粉末,冷冻干燥3天后,精密称取20mg样品,加入800μL80%(v/v)甲醇浸泡过夜,超声提取样品2h,12000×g离心3min后,取上清液过0.22μm的微孔滤膜,收集续滤液用于Agilent 6490三重四级杆液质联用系统进行检测,色谱柱使用WatersAcquity UPLC HSS T3分析柱(2.1×100mm,1.8μm)。液相条件如表5所示:A为0.1%甲酸-水,B为乙腈,流速0.3mL min-1,柱温40℃,进样量5μL,使用MRM模式检测雷公藤红素含量,碰撞能量20eV,定量离子对为451→215,定性离子对为451→201。
表5三重四级杆液质联用检测雷公藤红素的流动相梯度
结果如图11所示,通过Gateway技术构建的CYP716C52基因干扰载体,用基因枪遗传转化体系分别将空载PK7以及CYP716C52基因干扰载体转化至雷公藤悬浮细胞后,PCR扩增出长为799bp的Egfp片段,证明载体成功转化至细胞中。RT-PCR检测CYP716C52基因的相对表达量,发现干扰组(RNAi组)基因表达量与对照组(CK组)相比明显下调了,是CK组的0.38倍,且具有统计学差异(P<0.05)。此外,测定雷公藤红素含量发现,RNAi组雷公藤红素含量与CK组相比明显降低,是CK组的0.45倍,同样具有统计学差异(P<0.05),以上结果证明CYP716C52参与了雷公藤红素的生物合成。
实施例4、雷公藤CYP716C52蛋白定点突变研究
1.突变体质粒构建
(1)按照Fast Mutagenesis System试剂盒说明书设计突变引物,以pYES2::CYP716C52质粒为模板进行定点突变,PCR反应体系为:2×FastPfu PCRSuperMix 10.0μL,引物F/R各0.5μL,pYES2::CYP716C52质粒1.0μL,ddH2O 8.0μL;PCR程序为:94℃5min;94℃20s,55℃20s,72℃3.5min,25个循环;72℃10min;4℃hold。反应结束后加入1uLDMT酶消化产物,37℃反应1h。取5μL消化后的产物于50μL DMT感受态中,涂布于LB+AMP固体平板,阳性克隆送测序成功后,提取质粒。
(2)部分突变体质粒用上述试剂盒的方法没有构建成功,因此采用无缝拼接的方法进行构建,设计带有突变位点的无缝拼接引物,以突变体Q141S为例进行说明:分别配制PCR体系1和体系2,用高保真酶进行扩增,PCR程序为:98℃30s;98℃10s,56℃15s,72℃1min,35个循环;72℃7min;4℃hold。扩增结束后分别对体系1和体系2的目的片段进行切胶回收。
PCR体系1:
PCR体系2:
用限制性内切酶KpnI和EcoRI对载体pYES2进行双酶切,酶切产物进行1.5%琼脂糖凝胶电泳,切取大小正确的条带并进行切胶回收,得到线性化质粒。按照-BasicSeamless Cloning andAssembly Kit说明书操作方法将PCR回收片段1和2连接至线性化载体。
(注:片段1、片段2和线性化载体摩尔比为1:1:1)
连接体系放于PCR仪中,50℃孵育30min后转化至Trans1-T1感受态细胞,涂板,对阳性克隆进行测序验证,测序验证无误后,提取质粒。
2.突变体菌株发酵及产物定量检测
分别将突变体质粒转化至PA1酵母感受态,具体操作步骤详见Frozen-EZ Yeast转化II KitTM试剂盒说明书,此外用相同的方法向感受态细胞中转入原始质粒pYES2::CYP716C52,作为野生型对照。
分别从每个板子上随机挑取单菌落至3mL含2%葡萄糖的Sc-Leu-His-Ura液体培养基中,设置3个生物重复,30℃,200rpm活化约24h,测定OD600值,以初始OD600=0.1接入30mL Sc-Leu-His-Ura液体混糖培养基(0.2%葡萄糖和1.8%半乳糖)中,30℃,200rpm培养4天。
直接向发酵体系中加入30mL乙酸乙酯,超声提取30min,重复两次,合并有机层,用旋转蒸发仪旋蒸除去溶剂,用1mL乙酸乙酯复溶两次,再用氮气吹干,最后用300μL质谱甲醇溶解,高速离心取上清液进样液相色谱-高分辨质谱联用仪(Q Exactive HF)。
Q Exactive HF进样条件如下:A相(0.1%甲酸-水),B相(乙腈),使用ACQUITYUPLCVR HSS T3色谱柱(2.1mm 100mm,1.8μm,USA)进行分离,柱温40℃,液相梯度见表6。使用正离子模式下的靶向-SIM定量方法进行检测,质谱检测条件为:扫描时间0.2s,鞘气流速45,毛细管温度320℃,电喷雾电流0.4μA,检测结果用Xcalibur软件进行分析。
表6Q Exactive HF检测液相条件
结果如图12所示,分别将CYP716C52蛋白的第141位谷氨酰胺(Q)分别突变成丝氨酸(S)、谷氨酸(E)、天冬酰胺(N)和脯氨酸(P)之后,除突变体CYP716C52Q141S产量提高至野生型的2.98倍外,其余3个突变体活性均有不同程度的降低;将第144位亮氨酸(L)分别突变为缬氨酸(V)、丝氨酸(S)、异亮氨酸(I)和苏氨酸(T)后,CYP716C52L144V突变体活性丧失,CYP716C52L144I突变体产量提高至野生型的1.53倍,其余2个突变体活性均降低;将第221位异亮氨酸(I)分别突变为苯丙氨酸(F)、缬氨酸(V)、亮氨酸(L)和甘氨酸(G)后,CYP716C52I221F突变体活性与野生型比没有明显变化,而CYP716C52I221L突变体产量也提高了,是野生型的2.22倍,其余2个突变体活性降低。综上,通过蛋白突变研究,得到了三个活性更优的突变体:CYP716C52Q141S、CYP716C52L144I和CYP716C52I221L。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,作出的变化、改型、添加或替换,也应属于本发明的保护范围,本发明的保护范围以权利要求书为准。
Claims (9)
1.分离的蛋白,所述蛋白为(1)SEQ ID NO:2所示的氨基酸序列;或
(2)SEQ ID NO:2所示的氨基酸序列经取代、缺失或增加一个或多个氨基酸且功能相同的多肽。
2.根据权利要求1所述的蛋白,其中所述蛋白为将SEQ ID NO:2所示氨基酸序列中的一个或多个氨基酸进行突变:
(1)将第141位的Gln(Q)突变为Ser(S)、Glu(E)、Asn(N)或Pro(P);或
(2)将第144位的Leu(L)突变为Val(V)、Ser(S)、Ile(I)或Thr(T);或
(3)将第221位Ile(I)突变为Phe(F)、Val(V)、Leu(L)或Gly(G)。
3.编码权利要求1或2所述蛋白的多核苷酸。
4.根据权利要求3所述多核苷酸,所述多核苷酸为如下中的至少一种:
(1)SEQ ID NO:1第1-1455位所示的核苷酸分子;或
(2)SEQ ID NO:1第1-1455位所示的核苷酸分子经取代、缺失或增加一个或多个核苷酸且表达相同功能蛋白的核苷酸序列。
5.表达载体,其包含启动子、权利要求3或4所示多核苷酸和转录终止子,其中所述启动子与所述多核苷酸可操作地连接,并且所述多核苷酸与所述转录终止子可操作地连接。
6.重组宿主细胞,其包含权利要求3或4所述多核苷酸分子、或权利要求5所述表达载体,所述细胞选自:细菌、酵母细胞、真菌细胞、昆虫细胞、哺乳动物细胞和植物细胞。
7.权利要求1或2所述蛋白、或权利要求3或4所述多核苷酸,或权利要求5所述表达载体、或权利要求6所述细胞在催化美登木酸C2位羟基化中的应用。
8.权利要求1或2所述蛋白、或权利要求3或4所述多核苷酸,或权利要求5所述表达载体、或权利要求6所述细胞在合成雷公藤酸C和/或雷公藤红素中的应用。
9.权利要求1或2所述蛋白、或权利要求3或4所述多核苷酸、或权利要求5所述表达载体、或权利要求6所述细胞在雷公藤植物育种中的应用。
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