CN114317631A - 单胺氧化酶在制备托品酮中的应用 - Google Patents
单胺氧化酶在制备托品酮中的应用 Download PDFInfo
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- CN114317631A CN114317631A CN202111549936.6A CN202111549936A CN114317631A CN 114317631 A CN114317631 A CN 114317631A CN 202111549936 A CN202111549936 A CN 202111549936A CN 114317631 A CN114317631 A CN 114317631A
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- tropinone
- monoamine oxidase
- gly
- ala
- leu
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Abstract
本发明公开了单胺氧化酶在制备托品酮中的应用，属于酶催化和合成生物学技术领域。本发明提供的单胺氧化酶的氨基酸序列如SEQ ID NO.1所示，本发明提供了该酶在制备托品酮中的应用，通过催化4‑(1‑甲基‑2‑吡咯烷基)‑3‑氧代丁酸转化为托品酮。本发明制备托品酮的方法绿色温和，环境友好，其相较于P450的催化效率有显著的提升，具有应用前景。
Description
技术领域
本发明属于酶催化和合成生物学技术领域，更具体地，涉及一种单胺氧化酶在制备托品酮中的应用。
背景技术
单胺氧化酶(monoamine oxidase，MAO)是一种依赖黄素的胺类氧化酶，最初被发现于动物，在生物体内广泛存在。该酶能够催化单胺类物质代谢，氧化脱氨，并生成代谢产物过氧化氢，在机体的胺类代谢过程中起着重要作用，因而备受关注(Iacovino,L.etal.2018,The structure of monoamine oxidases:past,present,and future.JournalOf Neural Transmission,125,1567-1579.)。1995年从微生物黑曲霉发现的单胺氧化酶MAO-N是迄今为止发现的第一例非脊椎动物来源的单胺氧化酶，能氧化简单的末端直链脂肪胺，对R型底物体现出明显偏好性(Schilling,B.et al.Cloning,sequencing andheterologous expression of the monoamine oxidase gene from aspergillusniger.Molecular&General Genetics Mgg 1995,247,430-438.)。之后，Turner等课题组通过酶定向进化提供高了该酶的活性，以及对底物宽泛性和立体选择性，并将其用于各种光学纯手性胺类化合物的制备(Dunsmore,C.et al.A chemo-enzymatic route toenantiomerically pure cyclic tertiary amines.Journal Of The American ChemicalSociety,2006,128,2224-2225；Rowles,Ⅰ.et al.Directed evolution of the enzymemonoamine oxidase(MAO-N):highly efficient chemo-enzymatic deracemisation ofthe alkaloid(±)-Crispine A.Chemcatchem,2012,4,1259-1261.)。
托品酮(Tropinone)是一类植物来源含有叔胺结构的莨菪烷类生物碱，可以用于合成阿托品等天然托品烷类药物，其化学名称为8-甲基-8氮杂双环[3.2.1]辛烷-3-酮。目前托品烷类药物主要是从植物中提取得到。虽然化学家们很早就已经实现了托品酮的化学全合成(Robinson,R.LXⅠⅠⅠ.Asynthesis of tropinone.Chem.Soc.,Trans.,1917,111,762-768.)，但是因难以控制后续合成托品烷类药物的手性结构，环境污染等问题而没有得到很好的应用。因此，研究者们开始关注托品烷类天然产物的生物合成途径解析以及生物合成方法，期望能获得更加绿色高效的合成方法。
近年来发展的合成生物学技术，通过将目标化合物生物合成途径上相关的基因导入微生物底盘中，实现了一些植物来源重要药用化合物的异源高效合成(Kotopka,B.J.etal.Synthetic biology strategies toward heterologous phytochemicalproduction.Natural product reports 2018,35,902-920；Li,S.et al.Strategies formicrobial synthesis of highvalue phytochemicals.Nature Chemistry 2018,10,395-404.)。随着托品酮生物合成途径被阐明，合成生物学家们也通过模拟托品酮的天然生物合成途径，将相关的合成基因导入异源微生物底盘中，实现了托品酮的微生物合成(图2)，然而效率都十分的低下(Huang,J.et al.Tropane alkaloids biosynthesis involves anunusual typeⅠⅠⅠpolyketide synthase and non-enzymatic condensation.NatureCommunications,2019,10,4036；Ping,Y.et al.Building Microbial Hosts forHeterologous Production of N-Methylpyrrolinium.[J].ACS Synthetic Biology,2019,8,257-263；Ping,Y.et al.De Novo Production of the Plant-Derived Tropineand Pseudotropine in Yeast.ACS Synthetic Biology,2019,8,1257-1262；CN111826355 A)。已有研究表明，无论在植物还是微生物代谢工程中，托品酮生物合成均是托品烷类天然产物生物合成的关键限速步骤，极大地限制了下游药用托品烷生物碱的积累。而托品酮生物合成的最关键的限速步骤就是细胞色素P450介导的催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸形成托品酮这一步。植物的P450为膜蛋白，具有表达量低下、不够稳定，以及难以利用酶工程改造等缺陷，因此发展一个更高效的将4-(1-甲基-2-吡咯烷基)-3-氧代丁酸转化成托品酮的生物催化反应，将是提高托品酮的生物合成产量，进而实现托品类药物微生物生产的突破性技术。基于上述背景，本发明创新性利用单胺氧化酶替代传统的细胞色素P450酶，从而实现了4-(1-甲基-2-吡咯烷基)-3-氧代丁酸到托品酮的转化，为托品类药物微生物生产工艺的发展提供新的路线。
发明内容
本发明的目的在于针对托品酮生物合成的限速步骤——细胞色素P450介导4-(1-甲基-2-吡咯烷基)-3-氧代丁酸到托品酮的转化所存在的问题，提供一种单胺氧化酶在制备托品酮中的应用。本发明利用筛选的单胺氧化酶作为生物催化剂可用于替代P450酶催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸的环合反应，于托品类药物微生物生产具有较好的应用价值。
本发明的目的通过下述技术方案实现：
一种单胺氧化酶在制备托品酮中的应用，所述的单胺氧化酶具有催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸转化成托品酮的活性(图1)，其能够生物转化或体外催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸转化成托品酮。将单胺氧化酶的编码基因导入微生物体内进行异源表达，通过生物转化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸形成托品酮，或从微生物体内纯化出单胺氧化酶，加入辅因子FAD和/或过氧化酶，通过体外酶学催化方式催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸形成托品酮。
所述的单胺氧化酶包括但不局限于下述蛋白质：
(1)氨基酸序列如SEQ ID NO.1所示的蛋白质；
(2)将SEQ ID NO.1所示的氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加形成的由(1)衍生的蛋白质；
(3)与SEQ ID NO.1所示的氨基酸序列有80％以上同源性的蛋白质，优选地95％以上同源性，更优选地98％以上同源性。
一种上述单胺氧化酶的编码基因在制备托品酮中的应用。其中，上述氨基酸序列如SEQ ID NO.1所示的单胺氧化酶的编码基因的核苷酸序列优选如SEQ ID NO.2所示。
一种含有上述单胺氧化酶的编码基因的重组质粒在制备托品酮中的应用。所述的重组质粒转化到微生物体内能够异源表达上述单胺氧化酶。
一种含有上述重组质粒的重组菌株在制备托品酮中的应用。所述的重组菌株为能够异源表达上述单胺氧化酶的生物细胞，所述的生物细胞包括但不局限于大肠杆菌、酵母。
本发明具有如下优点和有益效果：本发明筛选出具有特异催化活性的单胺氧化酶作为生物催化剂，用于4-(1-甲基-2-吡咯烷基)-3-氧代丁酸到托品酮的转化，使得该步合成效率相较于P450催化的环合反应有了显著提升，解决了托品酮生物合成的限速步骤，具有较高的应用价值。
附图说明
图1：单胺氧化酶催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸(式I)生成托品酮(式II)反应；
图2：托品酮传统微生物合成途径以及本发明途径；
图3：单胺氧化酶体外表达的蛋白胶图；
图4：单胺氧化酶催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸生成托品酮的LC-MS检测结果图。
具体实施方式
下面通过具体的实施方案叙述本发明方法。下述实施例中的实验方法，如无特殊说明，均为常规方法。实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的，分子生物学实验包括蛋白表达，培养基和试剂配制等等，如无特殊说明，主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。
本发明中涉及菌株：大肠杆菌(Escherichia coli BL21)购自北京全式金生物技术有限公司、单胺氧化酶基因序列(SEQ ID NO.2所示)合成及单胺氧化酶表达质粒pET28a-MAO-N构建由金唯智生物科技有限公司完成。
LB培养基：酵母浸粉0.5g、胰蛋白胨1.0g、氯化钠1.0g、去离子水100mL；
LA培养基：酵母浸粉0.5g、胰蛋白胨1.0g、氯化钠1.0g、琼脂2.0g、去离子水100mL；
M9培养基：1M硫酸镁溶液2mL、1M氯化钙溶液100mL、七水磷酸钠12.8g、磷酸二氢钾3g、氯化钠0.5g、氯化铵1g、20％D-Glucose 20mL、去离子水1000mL；
Binding Buffer：HEPES 50mM、300mM NaCl、10％甘油，pH＝7.5；
脱盐Buffer：HEPES 50mM、NaCl 100mM、10％甘油，pH＝7.5。
实施例1：单胺氧化酶异源表达菌株的构建
将大肠杆菌感受态细胞E.coli BL21从-80℃冰箱中取出并置于冰上，用移液枪吸取1微升构建的单胺氧化酶表达质粒pET28a-MAO加入感受态中，轻轻混匀后，冰浴30分钟，然后放于42℃水浴锅热激90s，立刻放于冰上冰浴2min，向转化体系中加入1mL LB。37℃摇床培养40-60min后取出低速离心3min，弃去大部分上清，用移液枪轻轻吹打重悬菌体，将其涂布在含50μg/mL卡那霉素抗生素的LA平板上，37℃培养箱中培养8-12h，获得的带有卡那霉素抗性的转化子即为单胺氧化酶异源表达菌株。
实施例2：单胺氧化酶的异源表达
挑去实施例1中获得的单克隆转化子接种到4mL LB(含50μg/mL卡那霉素抗生素)中，37℃培养至OD600＝0.6，转接至400mL LB(含50μg/mL卡那霉素抗生素)中，37℃摇床震荡培养至OD600＝0.6，加入终浓度为0.1mM的异丙基-β-D-硫代半乳糖苷(IPTG)，18℃诱导表达18-20h。
实施例3：生物转化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸生成托品酮
取实施例2中异源表达完成的菌株，离心收集菌体，用4mL M9培养基重悬，加入终浓度为1mM的底物4-(1-甲基-2-吡咯烷基)-3-氧代丁酸，4℃转化120h，加入8mL乙酸乙酯，萃取，离心取上清乙酸乙酯层，挥干后用1mL乙腈重悬，离心取上清用LC-MS检测托品酮的生成(质谱结果如图4)，结果显示该反应的转化率高达90％。
LC-MS检测条件：
仪器：SHIMADZU LCMS-2020UPLC-MS；
分析柱：R227-32015-03Shim-pack Velox Biphenyl(2.7μm，2.1x100 mm，岛津公司)，流速为0.4mL min-1，检测的流动相为(A＝去离子水，B＝含0.1％甲酸的CH3CN)。
实施例4：单胺氧化酶的体外蛋白纯化
取实施例2中异源表达完成的菌株，离心收集菌体，使用20mL Binding Buffer重悬菌液。放在冰上超声破碎至澄清，将破碎后的菌体溶液高速离心60分钟，收集上清。并将处理好的2mL镍柱倒入上清中，冰浴下摇2h，使目标蛋白与镍柱充分结合。用预冷的BindingBuffer洗一次，收集液体。分别用10mL不同浓度的咪唑Buffer从低到高的浓度洗脱，用2mL的EP管接收。
分别取20μL各个浓度咪唑洗脱的蛋白液，加入5μL的5×loading buffer混匀，煮沸10min使蛋白变性。离心取10μL上清点样。配制12.5％的SDS蛋白凝胶，用凝胶电泳仪电压90V浓缩样品，160V分离不同大小的蛋白。跑胶完成后，将蛋白电泳胶放入蛋白胶染色仪中，检查染色仪中染色液与脱色液是否足够，开启染色仪染色，结束后查看结果，合并55kDa大小的纯蛋白，4℃浓缩后，加入脱盐Buffer，反复离心浓缩三次，至500μL，放入EP管中，测定浓度并跑SDS蛋白凝胶验证纯度(结果如图3)，保存于-80℃。
实施例5：体外催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸生成托品酮
反应条件如下：200μL反应体系含1mM 4-(1-甲基-2-吡咯烷基)-3-氧代丁酸，1mMFAD，0.1mg/mL过氧化物酶，0.5mg/L的实施例4纯化获得的单胺氧化酶，100mM pH＝8.0的磷酸钾缓冲液。反应温度为4℃，反应120h后，200μL乙腈淬灭反应，离心取上清用LC-MS检测托品酮的生成(条件同实施例3)，结果显示该反应的转化率高达90％。
本发明通过上述方法表达了包括氨基酸序列如SEQ ID NO.1所示的MAO-N在内的11种不同来源的单胺氧化酶，采用体外生物转化方式对这些单胺氧化酶的活性进行测试，发现只有MAO-N可以催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸形成托品酮。
序列表
<110> 武汉大学
<120> 单胺氧化酶在制备托品酮中的应用
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Claims (5)
1.一种单胺氧化酶在制备托品酮中的应用，其特征在于：所述的单胺氧化酶能催化4-(1-甲基-2-吡咯烷基)-3-氧代丁酸转化成托品酮；
所述的单胺氧化酶包括下述蛋白质：
(1)氨基酸序列如SEQ ID NO.1所示的蛋白质；
(2)将SEQ ID NO.1所示的氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加形成的由(1)衍生的蛋白质；
(3)与SEQ ID NO.1所示的氨基酸序列有80％以上同源性的蛋白质。
2.一种权利要求1中所述的单胺氧化酶的编码基因在制备托品酮中的应用。
3.根据权利要求2所述的应用，其特征在于：氨基酸序列如SEQ ID NO.1所示的单胺氧化酶的编码基因的核苷酸序列如SEQ ID NO.2所示。
4.一种重组质粒在制备托品酮中的应用，其特征在于：所述的重组质粒含有权利要求2或3中所述的单胺氧化酶的编码基因。
5.一种重组菌株在制备托品酮中的应用其特征在于：所述的重组菌株含有权利要求4中所述的重组质粒。
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