CN114891840A - 荷叶o-甲基转移酶及其编码基因在苄基异喹啉生物碱和苯丙酸类化合物合成中的应用 - Google Patents
荷叶o-甲基转移酶及其编码基因在苄基异喹啉生物碱和苯丙酸类化合物合成中的应用 Download PDFInfo
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- CN114891840A CN114891840A CN202210567202.9A CN202210567202A CN114891840A CN 114891840 A CN114891840 A CN 114891840A CN 202210567202 A CN202210567202 A CN 202210567202A CN 114891840 A CN114891840 A CN 114891840A
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- acid
- methyltransferase
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- lotus leaf
- alkaloid
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Abstract
本发明属于生物医药领域,具体涉及荷叶O‑甲基转移酶及其编码基因在苄基异喹啉生物碱和苯丙酸类化合物合成中的应用。本发明首先发现荷叶O‑甲基转移酶及其编码基因可用于苄基异喹啉生物碱和苯丙酸类化合物的生物合成,从而能高效、环保地合成相关化合物,有利于为相关新药的创制提供充足的原料保障。
Description
技术领域
本发明涉及生物医药领域,具体涉及荷叶O-甲基转移酶及其编码基因在苄基异喹啉生物碱和苯丙酸类化合物合成中的应用。
背景技术
荷叶是莲科植物莲(Nelumbo nucifera Gaertn.)的干燥叶。传统药效学表明荷叶清暑化湿,升发清阳,凉血止血。国内外对荷叶化学成分和药理活性进行了广泛的研究,并取得了一定的应用成果。现代药理学通过动物实验研究表明荷叶具有抗氧化、减肥、抗疟疾、潜在抗艾滋病活性,且主要的功效成分是苄基异喹啉生物碱。
关于苄基异喹啉生物碱O-甲基转移酶,在罂粟等毛茛目植物中已有过相关报道,而苄基异喹生物碱O位发生甲基化,则能影响其药理活性。因此,通过合理利用苄基异喹啉生物碱O-甲基转移酶,可以高效合成有特定药理活性的苄基异喹啉生物碱,这将为新药创制提供充足的原料保障,并为其它药用植物研究提供思路。
不过,不同的苄基异喹啉生物碱O-甲基转移酶的有效作用底物及作用效果往往较难预期,有待利用现代药理学研究对荷叶苄基异喹啉生物碱O-甲基转移酶的应用方式进行探究,这对相关新药的生物合成途径的研究将具有重要的研究意义,且存在潜在的巨大经济价值。
发明内容
本发明基于荷叶苄基异喹啉生物碱的药效价值及化学合成困难的问题,根据代谢谱、转录组、关联分析、酶活分析和鉴定,挖掘得到荷叶苄基异喹啉生物碱O-甲基转移酶,利用生物酶的方法,一步在苄基异喹啉生物碱O-位上加甲基,合成苄基异喹啉生物碱O甲基产物,达到高效、环保合成O甲基苄基异喹啉生物碱的目的。
具体而言,本发明首先提供荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料在化合物的生物合成中的应用;所述化合物选自苄基异喹啉生物碱、苯丙酸类化合物中的至少一种;其中,所述苄基异喹啉生物碱包括单苄基异喹啉生物碱和阿朴啡类生物碱。
作为优选,所述荷叶O-甲基转移酶具有以下任意一种氨基酸序列:
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过一个或多个氨基酸残基的替换、缺失或插入获得的具有相同功能蛋白的氨基酸序列。
作为优选,荷叶O-甲基转移酶的编码基因具有以下任一种核苷酸序列:
(1)SEQ ID NO.1所示的核苷酸序列,或,
(2)SEQ ID NO.1所示的核苷酸序列经过一个或多个核苷酸的替换、缺失或插入获得的具有相同功能蛋白的编码核苷酸序列;或,
(3)在严格条件下可以与SEQ ID NO.1所示的核苷酸序列进行杂交的核苷酸序列。
作为优选,通过使生产菌中表达或过表达所述荷叶O-甲基转移酶,特异性地获取含O-甲基化修饰的化合物,或提高混合物中含O-甲基化修饰的化合物的含量。
作为优选,通过外源性地加入所述荷叶O-甲基转移酶,特异性地获取含O-甲基化修饰的化合物,或提高混合物中含O-甲基化修饰的化合物的含量。
作为优选,通过外源性地加入所述荷叶O-甲基转移酶,或人为地将荷叶O-甲基转移酶的编码基因导入宿主进行内源性表达,使不含O-甲基化修饰的化合物的O位上发生甲基化。
作为优选,通过抑制生产菌中所述荷叶O-甲基转移酶的表达,特异性地获取不含O-甲基化修饰的化合物,或降低混合物中含O-甲基化修饰的化合物的含量。
作为优选,所述含O-甲基化修饰的化合物包括去甲亚美罂粟碱(Norarmepavine)、亚美罂粟碱(Armepavine)、N-去甲基荷叶碱(N-nornuciferine)、荷叶碱(Nuciferine)、阿魏酸(Ferulic acid)、3-O-阿魏酰奎尼酸(3-O-Feruloylquinic acid)、绿原酸甲酯(Methyl chlorogenate)、4-O-阿魏酰奎尼酸(4-O-Feruloylquinic acid)中的一种或多种。
其中,去甲亚美罂粟碱(Norarmepavine)、亚美罂粟碱(Armepavine)属于单苄基异喹啉生物碱;N-去甲基荷叶碱(N-nornuciferine)、荷叶碱(Nuciferine)属于阿朴啡类生物碱;阿魏酸(Ferulic acid)、3-O-阿魏酰奎尼酸(3-O-Feruloylquinic acid)、绿原酸甲酯(Methyl chlorogenate)、4-O-阿魏酰奎尼酸(4-O-Feruloylquinic acid)属于苯丙酸类化合物。
作为优选,所述不含O-甲基化修饰的化合物包括去甲乌药碱(Norcoclaurine)、(R,S)-乌药碱((R,S)-Coclaurine)、N-甲基衡州乌药碱(N-MethylCoclaurine)、巴婆碱(Asimilobine)、N-甲基巴婆碱(N-MethlyAsimilobine)、咖啡酸(Caffeic acid)、绿原酸(Chlorogenic acid)中的一种或多种。
其中,去甲乌药碱(Norcoclaurine)、(R,S)-乌药碱((R,S)-Coclaurine)、N-甲基衡州乌药碱(N-MethylCoclaurine)属于单苄基异喹啉生物碱;巴婆碱(Asimilobine)、N-甲基巴婆碱(N-MethlyAsimilobine)属于阿朴啡类生物碱;咖啡酸(Caffeic acid)、绿原酸(Chlorogenic acid)属于苯丙酸类化合物。
作为优选,当所述不含O-甲基化修饰的化合物为单苄基异喹啉生物碱时,所述O位为6’-O位或7’-O位,更优选所述O位为6’-O位。
作为优选,当所述不含O-甲基化修饰的化合物为阿朴啡类生物碱时,所述O位为6’-O位。
作为优选,当所述不含O-甲基化修饰的化合物为苯丙酸类化合物时,所述O位为3’-O位。
在一个优选的实施方案中,所述荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料被用于使(R,S)-乌药碱((R,S)-Coclaurine)的7-O位发生甲基化,以生成去甲亚美罂粟碱(Norarmepavine)。
在一个优选的实施方案中,所述荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料被用于使去甲乌药碱(Norcoclaurine)的6-O位发生甲基化,以生成(R,S)-乌药碱((R,S)-Coclaurine),而后进一步使(R,S)-乌药碱((R,S)-Coclaurine)的7-O位发生甲基化,以生成去甲亚美罂粟碱(Norarmepavine)。
在一个优选的实施方式中,所述荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料被用于使N-甲基衡州乌药碱(N-MethylCoclaurine)的7-O位发生甲基化,以生成亚美罂粟碱(Armepavine)。
在一个优选的实施方式中,所述荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料被用于使巴婆碱(Asimilobine)的6-O位发生甲基化,以生成N-去甲基荷叶碱(N-nornuciferine)。
在一个优选的实施方式中,所述荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料被用于使N-甲基巴婆碱(N-MethlyAsimilobine)的6-O位发生甲基化,以生成荷叶碱(Nuciferine)。
在一个优选的实施方式中,所述荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料被用于使咖啡酸(Caffeic acid)的3位的酚羟基发生甲基化,以生成阿魏酸(Ferulic acid)。
在一个优选的实施方式中,所述荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料被用于使绿原酸(Chlorogenic acid)的O位发生甲基化,以生成3-O-阿魏酰奎尼酸(3-O-Feruloylquinic acid)、绿原酸甲酯(Methyl chlorogenate)、4-O-阿魏酰奎尼酸(4-O-Feruloylquinic acid)。
在一些实施方案中,本发明所述的生物材料为表达盒、载体、宿主细胞或重组菌。
基于上述技术方案,本发明的有益效果如下:
本发明首先发现荷叶O-甲基转移酶及其编码基因可用于单苄基异喹啉生物碱、阿朴啡类生物碱和苯丙酸类化合物的生物合成,从而能高效、环保地合成相关化合物,有利于为相关新药的创制提供充足的原料保障。
附图说明
图1为本发明实施例1中提取的荷叶总RNA电泳图。
图2为本发明实施例2中候选NnOMT6重组蛋白的SDS-PAGE电泳图;图注:M,蛋白maker;NnOMT6为纯化的重组蛋白。
图3为本发明实施例2中NnOMT6酶活反应的EIC图及催化反应。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例中未注明具体技术或条件者,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。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购买得到的常规产品。
实施例1基因克隆的方法和步骤
(1)植物总RNA的提取
使用天根RNA Easy Fast Plant Tissue Kit提取试剂盒提取了荷叶中的总RNA,经Nano超微量分光光度计检测,A260/A280值在1.8~2.0之间,A260/A230值大于2.0,凝胶电泳结果显示总RNA中的28S rRNA与18S rRNA的条带清晰且无明显降解情况,表明提取的荷叶总质量好,可以用于下游试验。RNA电泳结果见图1。
(2)RNA反转录为cDNA
使用天根FastKing RT Kit(With gDNase)试剂盒(Cat#KR116-02)将提取的荷叶总RNA反转录成双链cDNA,具体操作步骤如下:
按照表1的基因组DNA的去除体系配制混合液,彻底混匀。简短离心,并置于42℃,孵育3min。然后置于冰上放置。
表1 gDNA去除反应体系
表2反转录反应体系
按照表2的反转录反应体系配制混合液,将反转录反应中的Mix,加到gDNA去除步骤的反应液中,充分混匀。42℃,孵育15min。95℃,孵育3min之后放于冰上,即得到荷叶中的cDNA。
(3)目的基因的PCR扩增及胶回收
使用VazymeII One Step Cloning Kit试剂盒,利用DNA无缝克隆技术将NnOMT6编码基因克隆至pET28a表达载体上。设计目的基因扩增引物时,分别在正反向加入载体酶切位点及酶切位点两末端的15bp同源序列。设计PCR引物如表3,由上至下依次为SEQ ID NO.3~4。
表3 NnOMT1基因PCR扩增引物
以上述获得的荷叶cDNA为模板,使用KOD高保真酶,按表4配制反应体系,按表5PCR反应程序扩增目的基因,将扩增的PCR产物经1%琼脂糖凝胶电泳进行检测,所得条带大小与目标基因接近,之后采用Axysen AxyPrep DNA凝胶回收试剂盒进行胶回收。
表4 KOD高保真酶反应体系
表5 KOD高保真酶PCR反应程序
(4)线性化克隆载体的制备及胶回收
使用全式金Plasmid MiniPrep Kit试剂盒提取pET28a质粒。使用限制性内切酶BamHI对pET28a载体进行单酶切,制备线性化载体。按表6配制载体酶切反应体系,37℃孵育2h,65℃反应20min终止反应。将酶切后载体经1%琼脂糖凝胶电泳进行检测。目的条带用Axysen AxyPrep DNA凝胶回收试剂盒进行胶回收。
表6载体酶切反应体系
(5)重组连接
按照VazymeII One Step Cloning Kit试剂盒计算重组反应所需的目的基因和线性化载体的量,按表7于冰水浴中配制重组反应体系,用移液器上下轻轻吹打几次混匀各组分,置于37℃反应30min。待反应完成后,立即将反应管置于冰水浴中冷却5min,即得重组质粒。
表7重组反应体系
(6)重组产物的转化
在冰上融化DH5α大肠杆菌感受态细胞,取10μL重组产物,加入到100μL DH5α感受态细胞中,轻弹管壁混匀,置于冰上放置30min,42℃热激60s,冰水浴孵育2min。加入900μLLB培养基,置于37℃摇床中200rpm培养1h。之后将菌液置于离心机中以4500rpm离心2min,吸弃900μL上层上清液体后,将下层100μL菌液混合吹打均匀后,涂在含有Kana抗性的固体LB培养基上,用涂布器轻轻涂布均匀,待培养基表面风干后盖上盖子封口,置于37℃培养箱中培养过夜。
(7)阳性克隆子筛选
挑取5个过夜培养长出的菌落置于2mL EP管中,加入1mL含有Kana的液体LB培养基,置37℃,200rpm摇床中振荡培养5小时,取2μL菌液进行菌液PCR,检测目的片段是否与载体连接,正向引物和反向引物使用PCR扩增时的引物。反应体系如表8,反应程序如表9。菌液PCR用的酶为天根2×Taq PCR MaserMix II。菌液PCR产物经1%琼脂糖凝胶电泳进行检测,5个菌落均在目的基因大小位置有高亮条带,均为阳性克隆子。
表8菌液PCR反应体系
表9菌落PCR反应程序
(8)测序
将出现目标片段大小条带的菌液送500μL至生工生物公司测序,其余菌液加等体积50%甘油摇匀后于-80℃保存;收到测序结果后,与目标序列进行比对,结果完全一致。
NnOMT6含有1095个核苷酸,编码364个氨基酸的蛋白,命名为NnOMT6,该基因的核苷酸序列如SEQ ID NO.1所示,所编码的氨基酸序列如SEQ ID NO.2所示。
实施例2基因功能的验证
将构建的经测序验证序列正确的pET28a-NnOMT6载体,转入BL21(DE3)表达菌株中,借助原核表达系统对基因功能进行了验证。
重组蛋白的诱导、纯化、酶活分析和产物鉴定如下:
(1)重组蛋白的诱导
分别挑取pET28a-NnOMT6与pET28a单克隆菌落(阴性对照)置于3mL LB液体培养基(含Kana 50mg/L)中,37℃,200rpm过夜培养。
取过夜培养的菌液转接至300mL新鲜灭菌的LB液体培养基(含Kana 50mg/L)中置于37℃摇床(180rpm)培养至OD600为0.6。
在300mL菌液中加入900μL的IPTG(100mM,异丙基-β-D-硫代半乳苷),终浓度为0.3mM,于16℃,180rpm培养16h以上。
收集菌液至离心管中,4℃,8,000×g离心20min,弃上清,收集菌沉淀,随后进行细胞破碎。
(2)重组蛋白的纯化
利用镍柱对本实验带His标签的重组蛋白进行纯化。步骤如下:
使用上样缓冲液(50mM NaH2PO4,300mM NaCl,10mM咪唑)重悬上面收集的菌液沉淀,利用低温超高压细胞破碎仪破碎细胞,释放蛋白,4℃,10,000rpm高速离心30min后,取上清,待上样;
组装镍柱:将Ni填料(BeyoGoldTMHis-tag Purification Resin)混匀后加入层析柱中,将出液口打开,让乙醇自然流出;加入10倍柱体积的上样缓冲液平衡亲和层析柱,将粗蛋白加入柱中,平衡5min后,收集流穿液,过柱上样2遍,待样品都流过亲和柱填料后,用15倍柱体积的上样缓冲液冲洗柱中杂蛋白,之后用含不同浓度咪唑(20mM,50mM,250mM)的洗脱液洗脱目标蛋白,收集洗脱液。将纯化蛋白进行SDS-PAGE电泳检测,结果含20mM和50mM咪唑的洗脱液有纯化蛋白,目的蛋白条带与预测的重组蛋白大小一致(45.7kDa),获得的纯化蛋白(纯度大于90%)可以进一步应用于后期的活性验证(如图2)。
(3)NnOMT6的体外酶活实验
取适量的纯化蛋白,分别对Norcoclaurine、(R)-Coclaurine、(S)-Coclaurine、N-MethylCoclaurine、Norarmepavine、Armepavine、Asimilobine、N-MethlyAsimilobine、Lotusine等9种苄基异喹啉生物碱类底物和o-Coumaric Acid、p-Coumaric acid、Caffeicacid、Ferulic acid、Chlorogenic acid等5种苯丙酸类底物进行催化反应,以S-adenosyl-L-methionine为甲基供体进行酶促反应。以pET28a空载所得的粗蛋白为阴性对照。按表10配置酶促反应体系,反应配置完成后充分吹打混匀,置于37℃水浴中反应4h,之后加入100μL甲醇终止反应,随后在离心机中12000×g离心30min,取上清过0.22μm微孔滤膜后注入UPLC-QTOF-MS/MS进行检测。
色谱条件如下:色谱柱为ACQUITY UPLC CSH C18柱(2.1mm×50mm,1.7μm)。流动相A为0.1%甲酸水,B为乙腈,采用梯度洗脱,条件为:0–0.5min,2%B;0.5–1min,2%–5%B;1–5min,5%–9%B;5–12min,9%–10%B;12–16min,10%–15%B;16–20min,15%–45%B;20–22min,45%–100%B。进样体积2μL,柱温35℃,流速0.3mL/min。
质谱条件如下:离子源为Dusal ESI源;生物碱类底物采用正离子模式(PI)检测,苯酚酸类底物采用负离子模式(NI)检测,扫描范围:m/z 100-1000。鞘气温度为350℃;鞘气流速为11.0L/min;干燥气温度为350℃,干燥气流速为8L/min,雾化器压力为45psi;毛细管电压在PI模式下为4000V,NI模式下3500V,喷嘴电压在PI模式下为500V,NI模式下为1500V。MS/MS分析时碰撞能量设置为30eV,碰撞能量电压为20V。
结果,NnOMT6可以催化Norcoclaurine、(R)-Coclaurine、(S)-Coclaurine、N-MethylCoclaurine、Asimilobine、N-MethlyAsimilobine等生物碱类底物和Caffeic acid、Chlorogenic acid等苯丙酸类底物在酚羟基上发生O-甲基化反应,对Norarmepavine、Lotusine、o-Coumaric Acid、p-Coumaric acid、Ferulic acid等底物无活性。NnOMT6可以催化Norcoclaurine的6位发生O甲基化,生成Coclaurine,可以进一步催化Coclaurine在7位发生O甲基化生成Norarmepavine;可以催化N-MethylCoclaurine的7位发生O甲基化生成Armepavine。对单苄基异喹啉类生物碱在6位的活性要强于7位。NnOMT6还可以催化阿朴啡类的生物碱Asimilobine、N-MethlyAsimilobine在6位发生O甲基化,分别生成N-nornuciferine和Nuciferine。NnOMT6可以催化Caffeic acid在3位的酚羟基发生甲基化生成Ferulic acid。以上反应产物均经对照品对比确认鉴别。NnOMT6可以催化Chlorogenicacid,生成三种产物,结构鉴定为3-O-Feruloylquinic acid、Methyl chlorogenate、4-O-Feruloylquinic acid。酶促反应产物的质谱数据见表11。酶活反应的EIC图及反应过程见图3。
表10重组蛋白酶活反应体系
表11酶促反应产物的质谱数据
注:ND表示对该底物没有活性。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 中国中医科学院中药研究所
<120> 荷叶O-甲基转移酶及其编码基因在苄基异喹啉生物碱和苯丙酸类化合物合成中的应用
<130> KHP221112212.4
<160> 4
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<211> 1095
<212> DNA
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<400> 1
atgggttcga ccaagaccca gatcaaaatt gcagaccaag aagaggagga agcttgcaac 60
tacgccatgc aattggccag cgcttcggtc gtacccatgg tattgaaagc agccatcgag 120
ctagatgtac tggagatcat tgctgaagca ggcgctgggg ctcacatctc gacctctgag 180
atcgcctctc atcttcctac gcagaaccca gatgcacctg ttatgctcga ccgtatgctg 240
cgtctcctag ccagcttctc cattcttact tgctccctac gcacccacga cgatggacgg 300
gtagagagac tctacggcct tgcacccgtg tgtaagttct tggtgaagaa cgaagatgga 360
gtgtcgatgg ctccactggt gctcatgaat caagacaagg tcctcatgga aagctggtac 420
catttgaaag acgcagtgct tgatggggga attccattta acaaggccta cggcatgact 480
gcattcgaat accacggcac agaccccaga ttcaacaaag tgttcaacag gggaatgtca 540
gatcactcca ccataaccat gaagaagatt cttgagacat acaagggatt cgagggcctc 600
aactccgtgg tggacgtcgg tgggggaact ggagctacac ttaacatgat tatttccaag 660
tacccttcca tcaagggcat taacttcgat ttgcctcacg ttattgaaga tgccccatcc 720
tatcctggcg tggagcatgt tggaggagat atgtttgtta gtgtacctaa aggagatgcc 780
attttcatga aatggatatg tcatgactgg agcgatgcac attgcttgga atttttgaag 840
aactgctacc aagcgttacc ggaaaatgga aagataattg tcgtcgagtc cattcttccg 900
gtagctcccg agactaatct ttccgccaac ggtgtgttcc aacttgacaa catcatgctg 960
gcacataacc ctggaggaaa agagagagcc gagaaagact ttgaggcctt ggccaagggc 1020
gccggattcg ctggagttgg agttatgtgt cgcgctttca acagctatgt catggaattc 1080
tataaatctg cttga 1095
<210> 2
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Gly Ser Thr Lys Thr Gln Ile Lys Ile Ala Asp Gln Glu Glu Glu
1 5 10 15
Glu Ala Cys Asn Tyr Ala Met Gln Leu Ala Ser Ala Ser Val Val Pro
20 25 30
Met Val Leu Lys Ala Ala Ile Glu Leu Asp Val Leu Glu Ile Ile Ala
35 40 45
Glu Ala Gly Ala Gly Ala His Ile Ser Thr Ser Glu Ile Ala Ser His
50 55 60
Leu Pro Thr Gln Asn Pro Asp Ala Pro Val Met Leu Asp Arg Met Leu
65 70 75 80
Arg Leu Leu Ala Ser Phe Ser Ile Leu Thr Cys Ser Leu Arg Thr His
85 90 95
Asp Asp Gly Arg Val Glu Arg Leu Tyr Gly Leu Ala Pro Val Cys Lys
100 105 110
Phe Leu Val Lys Asn Glu Asp Gly Val Ser Met Ala Pro Leu Val Leu
115 120 125
Met Asn Gln Asp Lys Val Leu Met Glu Ser Trp Tyr His Leu Lys Asp
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Ala Val Leu Asp Gly Gly Ile Pro Phe Asn Lys Ala Tyr Gly Met Thr
145 150 155 160
Ala Phe Glu Tyr His Gly Thr Asp Pro Arg Phe Asn Lys Val Phe Asn
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Arg Gly Met Ser Asp His Ser Thr Ile Thr Met Lys Lys Ile Leu Glu
180 185 190
Thr Tyr Lys Gly Phe Glu Gly Leu Asn Ser Val Val Asp Val Gly Gly
195 200 205
Gly Thr Gly Ala Thr Leu Asn Met Ile Ile Ser Lys Tyr Pro Ser Ile
210 215 220
Lys Gly Ile Asn Phe Asp Leu Pro His Val Ile Glu Asp Ala Pro Ser
225 230 235 240
Tyr Pro Gly Val Glu His Val Gly Gly Asp Met Phe Val Ser Val Pro
245 250 255
Lys Gly Asp Ala Ile Phe Met Lys Trp Ile Cys His Asp Trp Ser Asp
260 265 270
Ala His Cys Leu Glu Phe Leu Lys Asn Cys Tyr Gln Ala Leu Pro Glu
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Asn Gly Lys Ile Ile Val Val Glu Ser Ile Leu Pro Val Ala Pro Glu
290 295 300
Thr Asn Leu Ser Ala Asn Gly Val Phe Gln Leu Asp Asn Ile Met Leu
305 310 315 320
Ala His Asn Pro Gly Gly Lys Glu Arg Ala Glu Lys Asp Phe Glu Ala
325 330 335
Leu Ala Lys Gly Ala Gly Phe Ala Gly Val Gly Val Met Cys Arg Ala
340 345 350
Phe Asn Ser Tyr Val Met Glu Phe Tyr Lys Ser Ala
355 360
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cagcaaatgg gtcgcggatc catgggttcg accaagacc 39
<210> 4
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
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acggagctcg aattcggatc ccaagcagat ttatagaatt ccatga 46
Claims (10)
1.荷叶O-甲基转移酶、或其编码基因、或含有其编码基因的生物材料在化合物的生物合成中的应用;
所述化合物选自苄基异喹啉生物碱、苯丙酸类化合物中的至少一种;其中,所述苄基异喹啉生物碱包括单苄基异喹啉生物碱和阿朴啡类生物碱。
2.根据权利要求1所述的应用,其特征在于,所述荷叶苄基异喹啉生物碱O-甲基转移酶具有以下任意一种氨基酸序列:
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过一个或多个氨基酸残基的替换、缺失或插入获得的具有相同功能蛋白的氨基酸序列。
3.根据权利要求1所述的应用,其特征在于,荷叶苄基异喹啉生物碱O-甲基转移酶的编码基因具有以下任一种核苷酸序列:
(1)SEQ ID NO.1所示的核苷酸序列,或,
(2)SEQ ID NO.1所示的核苷酸序列经过一个或多个核苷酸的替换、缺失或插入获得的具有相同功能蛋白的编码核苷酸序列;或,
(3)在严格条件下可以与SEQ ID NO.1所示的核苷酸序列进行杂交的核苷酸序列。
4.根据权利要求1~3中任一项所述的应用,其特征在于,通过使生产菌中表达或过表达所述荷叶O-甲基转移酶,特异性地获取含O-甲基化修饰的化合物,或提高混合物中含O-甲基化修饰的化合物的含量。
5.根据权利要求1~3中任一项所述的应用,其特征在于,通过外源性地加入所述荷叶O-甲基转移酶,特异性地获取含O-甲基化修饰的化合物,或提高混合物中含O-甲基化修饰的化合物的含量。
6.根据权利要求1~3中任一项所述的应用,其特征在于,通过外源性地加入所述荷叶O-甲基转移酶,或人为地将荷叶苄基异喹啉生物碱和苯丙酸类O-甲基转移酶的编码基因导入宿主进行内源性表达,使不含O-甲基化修饰的化合物的O位上发生甲基化。
7.根据权利要求1~3中任一项所述的应用,其特征在于,通过抑制生产菌中所述荷叶O-甲基转移酶的表达,特异性地获取不含O-甲基化修饰的化合物,或降低混合物中含O-甲基化修饰的化合物的含量。
8.根据权利要求4~7中任一项所述的应用,其特征在于,所述含O-甲基化修饰的化合物包括去甲亚美罂粟碱(Norarmepavine)、亚美罂粟碱(Armepavine)、N-去甲基荷叶碱(N-nornuciferine)、荷叶碱(Nuciferine)、阿魏酸(Ferulic acid)、3-O-阿魏酰奎尼酸(3-O-Feruloylquinic acid)、绿原酸甲酯(Methyl chlorogenate)、4-O-阿魏酰奎尼酸(4-O-Feruloylquinic acid)中的一种或多种。
9.根据权利要求6或7所述的应用,其特征在于,所述不含O-甲基化修饰的化合物包括去甲乌药碱(Norcoclaurine)、(R,S)-乌药碱((R,S)-Coclaurine)、N-甲基衡州乌药碱(N-MethylCoclaurine)、巴婆碱(Asimilobine)、N-甲基巴婆碱(N-MethlyAsimilobine)、咖啡酸(Caffeic acid)、绿原酸(Chlorogenic acid)中的一种或多种。
10.根据权利要求6或9所述的应用,其特征在于,当所述不含O-甲基化修饰的化合物为单苄基异喹啉生物碱时,所述O位为6’-O位或7’-O位;当所述不含O-甲基化修饰的化合物为阿朴啡类生物碱时,所述O位为6’-O位;当所述不含O-甲基化修饰的化合物为苯丙酸类化合物时,所述O位为3’-O位。
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