CN116656642A - 延胡索来源的氧甲基转移酶Cy4’OMT及其应用 - Google Patents
延胡索来源的氧甲基转移酶Cy4’OMT及其应用 Download PDFInfo
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- CN116656642A CN116656642A CN202210151411.5A CN202210151411A CN116656642A CN 116656642 A CN116656642 A CN 116656642A CN 202210151411 A CN202210151411 A CN 202210151411A CN 116656642 A CN116656642 A CN 116656642A
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Abstract
本发明公开了延胡索来源的氧甲基转移酶Cy4’OMT及其应用。本发明所要保护的一个技术方案是蛋白质及其相关的生物材料，所述蛋白质的氨基酸序列可为序列表中序列1或序列1的第166‑514位。实验证明，Cy4’OMT重组酶可以催化(S)‑3’‑hydroxy‑N‑methylcoclaurine的C4’位羟基甲基化，同时、兼具催化1‑BIA型化合物C6、C4’位及原小檗碱型化合物的C3位羟基甲基化的功能，后者在BIA途径中是本发明首次发现的。该酶可应用于四氢巴马汀、(S)‑牛心果碱和/或(S)‑乌药碱的生产，为苄基异喹啉生物碱的生物合成提供元件。
Description
技术领域
本发明涉及生物技术领域，具体涉及延胡索来源的氧甲基转移酶Cy4’OMT及其应用。
背景技术
苄基异喹啉生物碱(benzylisoquinoline alkaloids，BIAs)是一类具有重要研究及药用价值的次生代谢产物，包括吗啡(Morphine)、可待因(Codeine)，血根碱(Sanguinarine)、黄连素(Berberine chloride hydrate)等。异喹啉类生物碱活性与立体结构严格相关，对于结构复杂的BIAs类化合物，提取纯化和化学合成都有很大局限，因此次生代谢物的合成生物学研究即利用分子生物学技术和代谢工程手段，对生物合成途径进行分析，并构建高产的基因工程菌的方法受到广泛关注。近些年BIAs的微生物代谢工程研究取得了许多突破性的进展，在微生物中已构建出蒂巴因、氢可待、诺斯卡品、延胡索乙素等生物碱的完整通路，极大促进苄基异喹啉的异源生产。
中药延胡索是罂粟科紫堇属延胡索(Corydalis yanhusuo)的干燥块茎，具有活血、行气、止痛的功效，用于治疗胸胁、脘腹疼痛，胸痹心痛，经闭痛经，产后瘀阻，跌扑肿痛。延胡索的主要成分为苄基异喹啉生物碱，其中延胡索乙素，延胡索甲素和延胡索丑素被认为是延胡索的主要止痛成分。近年来，越来越多的药理证据显示出原小檗碱型化合物具有开发为非成瘾性止痛药物的潜力以及治疗物质使用障碍(substance use disorders)的潜力，如缓解使用吗啡类药物引发的成瘾综合征等。因此，传统中药延胡索是一个极具前景的止痛药物的资源库，值得进一步的开发研究。
延胡索含有丰富的小檗碱型和原小檗碱型化合物，生物合成途径主体较为清晰：去甲乌药碱合酶NCS通过催化多巴胺和4-HPAA两分子间形成C-C键并脱去一个分子的水产生中间体去甲乌药碱((S)-norcoclaurine)，再经过3个甲基转移酶(6OMT，CNMT，4’OMT)以及一个P450酶(NMCH)生成重要中间体牛心果碱((S)-reticuline)，它是吗啡类、原小檗碱类和苯并菲啶类化合物重要的共同中间体。在原小檗碱途径上，BBE催化牛心果碱((S)-reticuline)形成金黄紫堇碱((S)-scoulerine)，接着甲基转移酶SOMT、CoOMT进一步催化金黄紫堇碱((S)-scoulerine)形成延胡索乙素((S)-tetrahydropalmatine)。原小檗碱型化合物如(S)-tetrahydropalmatine在STOX酶作用下氧化成小檗碱型化合物。
氧甲基转移酶在BIA途径中发挥重要作用，它们有些具有高度的底物专一性，有些具有杂泛性，能同时催化多个位置的甲基化。BIAs生物合成途径中已报道的氧甲基转移酶包括6OMT、4’OMT、SOMT、N7OMT等。OMT的重要性体现在两方面，一是对于关键中间体的形成，如6OMT，4’OMT基因的特异性催化对中间体牛心果碱(reticuline)的形成至关重要；另一方面是OMT的杂泛性促进BIA化合物的多样性。
发明内容
本发明所要解决的技术问题是如何获得兼具催化1-BIA类化合物结构的C4’位、C6位和催化原小檗碱型化合物结构的C3位羟基甲基化的氧甲基转移酶。
为了解决上述技术问题，本发明首先提供了蛋白质。所述蛋白质可为如下A1)、A2)、A3)或A4)的蛋白质：
A1)氨基酸序列是序列表中序列1的蛋白质。
A2)氨基酸序列是序列表中序列1的166-514位的蛋白质。
A3)在A1)或A2)所示的蛋白质的羧基端或/和氨基端融合蛋白标签得到的融合蛋白。
A4)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由A1)或A2)衍生的或与A1)或A2)所示的蛋白质具有80％以上的同一性的蛋白质。
上述蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述蛋白质中，所述蛋白标签(protein-tag)是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述蛋白标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述蛋白质中，同一性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性，如NCBI主页网站的BLAST网页。例如，可在高级BLAST2.1中，通过使用blastp作为程序，将Expect值设置为10，将所有Filter设置为OFF，使用BLOSUM62作为Matrix，将Gap existence cost，Per residue gap cost和Lambda ratio分别设置为11，1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算，然后即可获得同一性的值(％)。
上述蛋白质中，所述80％以上的同一性可为至少81％、82％、85％、86％、88％、90％、91％、92％、95％、96％、98％、99％或100％的同一性。
为了解决上述技术问题，本发明还提供了蛋白质在作为氧甲基转移酶或在制备苄基异喹啉(类)生物碱中的应用，所述蛋白质可为上文所述的蛋白质。
上文所述产品中，所述苄基异喹啉(类)生物碱可为四氢巴马汀(tetrahydropalmatine)、(S)-牛心果碱((S)-reticuline)、(S)-乌药碱((S)-coclaurine)和/或(S)-4’-Methyl-norcoclaurine。
上文所述蛋白质相关的生物材料也属于本发明的保护范围。所述生物材料可为下述D1)至D6)中的任一种：
D1)编码上文所述蛋白质的核酸分子。
D2)含有D1)所述核酸分子的表达盒。
D3)含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体。
D4)含有D1)所述核酸分子的重组微生物、或含有D2)所述表达盒的重组微生物、或含有D3)所述重组载体的重组微生物。
D5)促进或提高上文所述蛋白质表达的核酸分子。
D6)含有D5)所述核酸分子的表达盒、重组载体或重组微生物。
上文所述的生物材料中，D1)所述核酸分子可为如下d1)、d2)或d3)所示的所述蛋白质的编码基因：
d1)编码序列是序列表中序列2的第496-1545核苷酸的cDNA分子或DNA分子。
d2)核苷酸是序列表中序列2的cDNA分子或DNA分子。
d3)与d2)限定的cDNA或DNA分子杂交且编码具有相同功能的蛋白质的cDNA分子或DNA分子。
上述杂交可为在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或，0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述生物材料中，D2)所述的含有核酸分子的表达盒，是指能够在宿主细胞中表达上文所述蛋白质的DNA。所述表达盒还可包括表达上述任意一种蛋白的核酸分子所必需的所有调控序列的单链或双链核酸分子。所述调控序列在其相容条件下能指导编码序列在合适的宿主细胞中表达上述任一种蛋白质。所述调控序列包括，但不限于，前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号序列和转录终止子。最低限度，调控序列要包括启动子以及转录和翻译的终止信号。为了导入载体的特定限制性酶位点以便将调控序列与编码蛋白质的核酸序列的编码区进行连接，可以提供带接头的调控序列。调控序列可以是合适的启动子序列，即可被表达核酸序列的宿主细胞识别的核酸序列。启动子序列含有介导蛋白质表达的转录调控序列。启动子可以是在所选宿主细胞中有转录活性的任何核酸序列，包括突变的、截短的和杂合的启动子，可以得自编码与宿主细胞同源或异源的胞外或胞内蛋白质的基因。调控序列还可以是合适的转录终止序列，即能被宿主细胞识别从而终止转录的一段序列。终止序列可操作连接在编码蛋白质的核酸序列的3’末端。在所选宿主细胞中可发挥功能的任何终止子都可以用于本发明。调控序列还可以是合适的前导序列，即对宿主细胞的翻译十分重要的mRNA非翻译区。前导序列可操作连接于编码蛋白质的核酸序列的5’末端。在所选宿主细胞中可发挥功能的任何前导序列均可用于本发明。调控序列还可以是信号肽编码区，该区编码一段连在蛋白质氨基端的氨基酸序列，能引导编码蛋白质进入细胞分泌途径。能引导表达后的蛋白质进入所用宿主细胞的分泌途径的信号肽编码区都可以用于本发明。添加能根据宿主细胞的生长情况来调节蛋白质表达的调控序列可能也是需要的。调控系统的例子是那些能对化学或物理刺激物(包括在有调控化合物的情况下)作出反应，从而开放或关闭基因表达的系统。调控序列的其他例子是那些能使基因扩增的调控序列。在这些例子中，应将编码蛋白质的核酸序列与调控序列可操作连接在一起。
为了解决上述技术问题，本发明还提供了上文所述的蛋白质和/或上文所述的生物材料的下述任一种应用：
F1、上文所述的蛋白质和/或上文所述的生物材料在催化(S)-3’-羟基-氮-甲基衡州乌药碱((S)-3’-hydroxy-N-methylcoclaurine)的C4’位羟基甲基化中的应用。
F2、上文所述的蛋白质和/或上文所述的生物材料在催化(S)-去甲乌药碱((S)-norcoclaurine)的C6位羟基甲基化及C4’位羟基甲基化中的应用。
F3、上文所述的蛋白质和/或上文所述的生物材料在催化四氢药根碱(tetrahydrojatrorrhizine)的C3位羟基甲基化中的应用。
F4、上文所述的蛋白质和/或上文所述的生物材料在制备苄基异喹啉(类)生物碱中的应用。
F5、上文所述的蛋白质和/或上文所述的生物材料在制备苄基异喹啉(类)生物碱产品中的应用。
为了解决上述技术问题，本发明还提供了一种制备延胡索来源的(S)-3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT的方法。所述方法可包括如下步骤：将上文所述蛋白质的编码基因在原核微生物中进行表达得到所述延胡索来源的(S)-3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT。
上文所述方法中，将上文所述蛋白质的编码基因在原核微生物中进行表达可包括将上文所述蛋白质的编码基因导入受体微生物，得到表达所述延胡索来源的4’位氧甲基转移酶Cy4’OMT的重组微生物，培养所述重组微生物，表达得到所述延胡索来源的4’位氧甲基转移酶Cy4’OMT。
上文所述方法中，所述表达可为诱导表达。
上文所述方法中，所述原核微生物可为大肠杆菌。
为了解决上述技术问题，本发明还提供了含有上文所述的蛋白质和/或上文所述的生物材料的下述任一种产品：
P1、生产苄基异喹啉(类)生物碱的产品。
P2、制备催化3’-羟基-氮-甲基衡州乌药碱((S)-3’-hydroxy-N-methylcoclaurine)的C4’位羟基甲基化的产品。
P3、制备催化去甲乌药碱((S)-norcoclaurine)的C6位及C4’羟基甲基化的产品。
P4、制备催化四氢药根碱(tetrahydrojatrorrhizine)的C3位羟基甲基化的产品。
P5、生产延胡索来源的(S)-3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT的产品。
上文所述产品中，所述苄基异喹啉(类)生物碱可为四氢巴马汀(tetrahydropalmatine)、(S)-牛心果碱((S)-reticuline)、(S)-乌药碱((S)-coclaurine)和/或(S)-4’-Methyl-norcoclaurine。
本发明通过基因的筛选、异源表达以及酶活性的体外检测等技术对延胡索的1个OMT基因进行了功能鉴定，将其命名为胡索来源的3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT。实验证明，该酶可以催化(S)-3’-hydroxy-N-methylcoclaurine的C4’位羟基甲基化，同时具有催化(S)-norcoclaurine C6和C4’位和tetrahydrojatrorrhizine的C3位羟基甲基化的功能。Cy4’OMT兼具催化1-BIA型化合物C6，C4’位，及原小檗碱型化合物的C3位羟基甲基化的功能在BIA途径中是本次申请中首次发现。
附图说明
图1为重组质粒pET32a-Cy4’OMT的PCR鉴定。M：DNA分子量标准(DL2000)；P：PCR产物。
图2为重组酶Cy4’OMT以(S)-3’-hydroxy-N-methylcoclaurine为底物的酶促反应产物UPLC-TOF分析。A：重组酶Cy4’OMT催化(S)-3’-hydroxy-N-methylcoclaurine图示；B：重组酶Cy4’OMT与空载体粗酶催化(S)-3’-hydroxy-N-methylcoclaurine反应产物色谱图；1对应的峰代表底物(S)-3’-hydroxy-N-methylcoclaurine，2对应的峰代表酶促产物(S)-reticuline；C：(S)-reticuline的标样二级质谱图；D：重组酶Cy4’OMT催化产物峰(S)-reticuline的二级质谱图。横坐标为质荷比，纵坐标为相对丰度。
图3为重组酶Cy4’OMT以(S)-norcoclaurine为底物的酶促反应产物UPLC-TOF分析。A：重组酶Cy4’OMT催化(S)-norcoclaurine图示；B：重组酶Cy4’OMT与空载体粗酶催化(S)-norcoclaurine反应产物色谱图；1对应的峰代表底物(S)-norcoclaurine，2对应的峰代表产物(S)-coclaurine，3对应的峰代表副产物，无标准品比对；C：(S)-coclaurine的标样二级质谱图；D：重组酶Cy4’OMT催化产物峰(S)-coclaurine的二级质谱图。E：重组酶Cy4’OMT催化产物峰3对应的二级质谱图，根据裂解规律为(S)-4’-Methyl-norcoclaurine。横坐标为质荷比，纵坐标为相对丰度。
图4为重组酶Cy4’OMT以tetrahydrojatrorrhizine为底物的酶促反应产物UPLC-TOF分析。A：重组酶Cy4’OMT催化tetrahydrojatrorrhizine图示；B：重组酶Cy4’OMT与空载体粗酶催化tetrahydrojatrorrhizine反应产物色谱图；1对应的峰代表底物tetrahydrojatrorrhizine，2对应的峰代表酶促产物tetrahydropalmatine；C：tetrahydropalmatine的标样二级质谱图；D：重组酶Cy4’OMT催化产物峰tetrahydropalmatine的二级质谱图。横坐标为质荷比，纵坐标为相对丰度。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。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。
本发明实施例中所用标准品的来源：
(S)-3’-hydroxy-N-methylcoclaurine((S)-3’-羟基-氮-甲基衡州乌药碱)：CAS号1936-17-0，加拿大TRC。
(S)-norcoclaurine((S)-去甲乌药碱)：CAS号22672-77-1，加拿大TRC。
tetrahydrojatrorrhizine(四氢药根碱)：CAS号27313-86-6，上海源叶生物科技有限公司产品。
tetrahydropalmatine(四氢巴马汀)：CAS号6024-85-7，上海源叶生物科技有限公司产品。
(S)-reticuline((S)-牛心果碱)：CAS号486-39-5，加拿大TRC。
(S)-coclaurine((S)-乌药碱)：CAS号22672-77-1，上海源叶生物科技有限公司产品。
甲基供体SAM(S-(5’-腺苷)-L-甲硫氨酸对甲苯磺酸盐)为Sigma公司产品，货号A2408，CAS号52248-03-0。
实施例一、重组酶Cy4’OMTs的获得及其催化功能分析
1、Cy4’OMT基因全长的克隆及载体构建
采用高通量测序技术，提取延胡索叶和块茎RNA，送北京诺禾致源科技股份有限公司进行转录组测序，筛选延胡索来源的3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT(氨基酸序列为序列表中序列1的第166-514位氨基酸)，根据基因的核苷酸序列(序列表中序列2的第496-1545位核苷酸)，设计扩增出完整编码阅读框的引物，并在上游和下游引物上引入载体重叠序列(Cy4’OMT基因特异性引物序列如表1所示)。以cDNA为模板进行PCR反应，PCR反应参数为：首先95℃变性5min；其次，98℃变性10sec，55℃退火15sec，72℃衍生2min，30个循环；最后70℃延伸10min。(PCR扩增的高保真酶：Takara公司，PrimeStarHS DNA polymerase,R010B)经PCR扩增后，利用无缝拼接技术(无缝拼接酶：TransGen公司，Basic Seamless Cloning and Assembly Kit CU201)将克隆得到的Cy4’OMT基因构建到原核表达载体pET32a(+)(索莱宝公司，货号P3100)上，测序分析，成功获得Cy4’OMT基因的重组表达载体pET32a-Cy4’OMT，pET32a-Cy4’OMT含有序列表中序列2的第496-1545位核苷酸所示的Cy4’OMT基因的CDS序列，能表达序列表中序列1所示的重组酶Cy4’OMT。
将重组基因表达载体pET32a-Cy4’OMT扩大培养后提取重组质粒pET32a-Cy4’OMT，随后将所提重组质粒pET32a-Cy4’OMT转化进入表达感受态细胞，转化所用细胞为大肠杆菌BL21(DE3)感受态细胞(TransGen公司，BL21(DE3)Chemically Competent Cell,CD601)，得到重组大肠杆菌BL21(DE3)/pET32a-Cy4’OMT。同时将pET32a(+)空载质粒转化BL21(DE3)感受态细胞，得到重组大肠杆菌BL21(DE3)/pET32a。
表1 Cy4’OMT基因特异性引物序列
2.Cy4’OMT蛋白的诱导表达与蛋白提取
将步骤1中获得的重组大肠杆菌BL21(DE3)/pET32a-Cy4’OMT和空载对照重组大肠杆菌BL21(DE3)/pET32a分别涂布于含100μg/mL氨苄青霉素的LB固体培养基中，37℃过夜培养。挑取单克隆于5mL含100μg/mL氨苄青霉素的LB液体培养基中过夜培养后，取1mL菌液转接至100mL含100μg/mL氨苄青霉素的LB液体培养基，37℃，200rpm培养，直到菌液OD600nm达到0.6-0.8时，得到未诱导pET32a-Cy4’OMT全菌液和未诱导空载全菌液BL21(DE3)/pET32a。待菌液降温后加入isopropyl-β-D-thiogalactopyranoside(IPTG)至终浓度为0.5mmol/L，17℃诱导表达16h后，收集发酵液，将重组大肠杆菌BL21(DE3)/pET32a-Cy4’OMT诱导表达得到的发酵液命名为诱导pET32a-Cy4’OMT全菌液，将重组大肠杆菌BL21(DE3)/pET32a诱导表达得到的发酵液命名为诱导空载全菌液。将诱导pET32a-Cy4’OMT全菌液和诱导空载全菌液4℃下以5000rpm转速离心10min，弃取上清后以灭菌水再次清洗并离心收集菌体，确保培养基清除干净。随后，用10mL缓冲液Tris-HCl(100mmol/L Tris-HCl,300mmol/L NaCl,pH7.4)重悬，加Phenylmethyanesulfonyl Fluoride(PMSF)至终浓度1mmol/L超声破碎细胞(Branson digital sonifer，USA；10％amplitude，10min，3s ON，3s OFF)。12000rpm离心15min，取上清即为粗酶，分别得到诱导pET32a-Cy4’OMT上清和诱导空载上清，该诱导pET32a-Cy4’OMT上清(氨基酸序列为序列表中序列1)，即重组酶Cy4’OMT。
3、重组酶Cy4’OMT的催化分析
酶促反应的体系为：100μM底物，包括(S)-3’-羟基-氮-甲基衡州乌药碱((S)-3’-hydroxy-N-methylcoclaurine)、(S)-去甲乌药碱((S)-norcoclaurine)和四氢药根碱(tetrahydrojatrorrhizine)；500μM甲基供体SAM，50μg步骤2得到的重组酶Cy4’OMT，Tris-HCl缓冲液(100mmol/L，pH 9)构成反应体系200μL，在温度＝37℃，时间＝180min下，使底物与酶进行反应，反应结束后加入400uL甲醇终止反应。最后用UPLC-QTOF-MS(WatersTechnologies，Milford，MA，USA)检测其产物，色谱柱为T3柱(2.1mm×100mm，2.7μm)，流动相：A相为0.1％甲酸-水，B相为0.1％甲酸-乙腈；梯度为：0-6min，5％-30％B，6-12min，30％-60％B，12-13.5min，60％-90％B，13.5-15min，90％-5％B，15-17min，5％B。进样体积为1μL，柱温为35℃，流动相流速为0.5mL/min。电喷雾离子源(ESI)，在正离子模式下进行扫描并采集MS数据；scan range为50-1500Da；scan time为0.1s；Ramp collision energy为30-50V。UPLC-QTOF-MS检测的催化反应色谱峰如图2-图4所示。
4、产物鉴定
依据质谱裂解规律，根据特征碎片及保留时间确定各反应的催化产物。
Cy4’OMT位点特异性催化(S)-3’-羟基-氮-甲基衡州乌药碱((S)-3’-hydroxy-N-methylcoclaurine)的C4’位羟基甲基化生成(S)-牛心果碱((S)-reticuline)(图2中B的峰1为底物(S)-3’-hydroxy-N-methylcoclaurine，峰2为产物(S)-reticuline)。
Cy4’OMT催化(S)-去甲乌药碱((S)-norcoclaurine)产生两个产物，主要产物是(S)-去甲乌药碱的C6位羟基甲基化产物(S)-乌药碱((S)-coclaurine)(图3中B峰2所指的产物峰)，另一个产物是图3中B峰3所指的产物峰，依据质谱裂解规律(如图3中E所示)该峰为(S)-4’-Methyl-norcoclaurine。
Cy4’OMT催化四氢药根碱(tetrahydrojatrorrhizine)的C3位羟基甲基化生成四氢巴马汀(tetrahydropalmatine)(图4中B的峰1为底物tetrahydrojatrorrhizine，峰2为产物tetrahydropalmatine)。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
序列表
<110> 中国中医科学院中药研究所
<120> 延胡索来源的氧甲基转移酶Cy4'OMT及其应用
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Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
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Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
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Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
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Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
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Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
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Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln
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His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met
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Ala Asp Ile Gly Ser Met Gly Val Asn Asp Ile Ala Glu Ala Gln Asp
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Val Asp Ile Lys Ala Gln Ala His Leu Trp Asn Ile Ile Tyr Gly Phe
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Ala Asp Ser Leu Val Leu Arg Cys Ala Val Glu Leu Gly Ile Ala Asp
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Ile Ile Asn Ser Asn Asn Gly Thr Val Thr Ile Ser Asp Ile Ala Ser
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Lys Leu Pro Val Asp Asn Val Asn Glu Glu Asn Leu Tyr Arg Val Leu
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Arg Tyr Leu Val Tyr Met Gly Leu Leu Lys Glu Ser Gln Asp Lys Cys
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Tyr Ser Leu Glu Pro Val Ala Thr Leu Leu Leu Lys Asp Ala Gln Arg
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Ser Met Val Pro Ile Ile Leu Gly Met Thr Gln Lys Asp Phe Met Val
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Pro Trp Phe Phe Met Lys Glu Gly Leu Gly Ser Gly Ser Thr Thr Ala
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Phe Glu Lys Gly Met Gly Met Thr Leu Trp Glu Tyr Leu Glu Gly His
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Pro Asp Gln Ser Gln Leu Phe Asn Glu Gly Met Ala Gly Glu Thr Arg
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Phe Pro
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cgatacttag tctacatggg tctcttgaaa gagtcccaag ataagtgtta ctcattagaa 780
ccagttgcta ctttgctctt gaaagatgct cagagaagta tggttcctat cattctagga 840
atgactcaaa aggatttcat ggttccttgg tttttcatga aagaaggatt gggtagtggg 900
agtactactg catttgagaa aggaatggga atgactcttt gggagtattt ggaaggacac 960
ccagatcaga gtcagttgtt taatgaagga atggctggtg aaactagact tttgacttct 1020
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Claims (10)
1.蛋白质，其特征在于：所述蛋白质是如下A1)、A2)、A3)或A4)的蛋白质：
A1)氨基酸序列是序列表中序列1的蛋白质；
A2)氨基酸序列是序列表中序列1的166-514位的蛋白质；
A3)在A1)或A2)所示的蛋白质的羧基端或/和氨基端融合蛋白标签得到的融合蛋白；
A4)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由A1)或A2)衍生的或与A1)或A2)所示的蛋白质具有80％以上的同一性的蛋白质。
2.蛋白质在作为氧甲基转移酶或在制备苄基异喹啉生物碱中的应用，所述蛋白质为权利要求1中所述的蛋白质。
3.与权利要求1中所述蛋白质相关的生物材料，其特征在于：所述生物材料为下述D1)至D6)中的任一种：
D1)编码权利要求1中所述蛋白质的核酸分子；
D2)含有D1)所述核酸分子的表达盒；
D3)含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体；
D4)含有D1)所述核酸分子的重组微生物、或含有D2)所述表达盒的重组微生物、或含有D3)所述重组载体的重组微生物；
D5)促进或提高权利要求1中所述蛋白质表达的核酸分子；
D6)含有D5)所述核酸分子的表达盒、重组载体或重组微生物。
4.根据权利要求3所述的生物材料，其特征在于：D1)所述核酸分子为如下d1)、d2)或d3)所示的所述蛋白质的编码基因：
d1)编码序列是序列表中序列2的第496-1545核苷酸的cDNA分子或DNA分子；
d2)核苷酸是序列表中序列2的cDNA分子或DNA分子；
d3)与d2)限定的cDNA或DNA分子杂交且编码具有相同功能的蛋白质的cDNA分子或DNA分子。
5.权利要求1中所述的蛋白质和/或权利要求3或4中所述的生物材料的下述任一种应用：
F1、权利要求1中所述的蛋白质和/或权利要求3或4中所述的生物材料在催化(S)-3’-羟基-氮-甲基衡州乌药碱的C4’位羟基甲基化中的应用；
F2、权利要求1中所述的蛋白质和/或权利要求3或4中所述的生物材料在催化(S)-去甲乌药碱C6位羟基甲基化及C4’位羟基甲基化中的应用；
F3、权利要求1中所述的蛋白质和/或权利要求3或4中所述的生物材料在催化四氢药根碱C3位羟基甲基化中的应用；
F4、权利要求1中所述的蛋白质和/或权利要求3或4中所述的生物材料在制备苄基异喹啉生物碱中的应用；
F5、权利要求1中所述的蛋白质和/或权利要求3或4中所述的生物材料在制备苄基异喹啉生物碱产品中的应用。
6.一种制备延胡索来源的(S)-3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT的方法，包括如下步骤：将权利要求1中所述蛋白质的编码基因在原核微生物中进行表达得到所述延胡索来源的(S)-3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT。
7.根据权利要求6所述的方法，其特征在于：将权利要求1中所述蛋白质的编码基因在原核微生物中进行表达包括将权利要求1中所述蛋白质的编码基因导入受体微生物，得到表达所述延胡索来源的(S)-3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT的重组微生物，培养所述重组微生物，表达得到所述延胡索来源的4’位氧甲基转移酶Cy4’OMT。
8.根据权利要求6或7所述的方法，其特征在于：所述表达为诱导表达。
9.含有权利要求1中所述的蛋白质和/或权利要求3或4中所述的生物材料的下述任一种产品：
P1、生产苄基异喹啉生物碱的产品；
P2、制备催化3’-羟基-氮-甲基衡州乌药碱的C4’位羟基甲基化的产品；
P3、制备催化去甲乌药碱的C6位及C4’位羟基甲基化的产品；
P4、制备催化四氢药根碱的C3位羟基甲基化的产品；
P5、生产延胡索来源的(S)-3’-羟基-氮-甲基衡州乌药碱4’位氧甲基转移酶Cy4’OMT的产品。
10.根据权利要求9所述的产品，其特征在于：所述苄基异喹啉生物碱为四氢巴马汀、(S)-牛心果碱和/或(S)-乌药碱。
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