CN108330122B - 柳杉二醇合成酶、其编码基因及在生物合成上的应用 - Google Patents
柳杉二醇合成酶、其编码基因及在生物合成上的应用 Download PDFInfo
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Abstract
本发明涉及一种柳杉二醇合成酶及其编码基因,以及柳杉二醇合成酶及编码基因在植物育种及生物合成中的运用,通过聚合酶链式反应首次克隆得到了雷公藤中新型柳杉二醇合成酶基因的cDNA全长序列,然后利用合成生物学手段,构建了包含本发明所述柳杉醇合成酶基因的酵母工程菌,实现酵母中生产柳杉二醇。
Description
技术领域
本发明涉及一种柳杉二醇合成酶及其编码基因,以及柳杉二醇合成酶及编码基因在植物育种及生物合成中的运用,属于药用成分合成生物学领域。
通过聚合酶链式反应首次克隆得到了雷公藤(Tripterygium wilfordiiHook.F.)中新型柳杉二醇合成酶基因(TwCS)的cDNA全长序列,然后利用合成生物学手段,构建酵母工程菌,在酵母中生产柳杉二醇。
背景技术
倍半萜类化合物是一类天然化合物,具有多种生理和生态功能,如抗微生物,防御昆虫和草食动物等。目前,在高等植物和低等植物中都发现了大量的倍半萜类化合物,广泛用于工农业和医药领域。倍半萜的生物合成仅来自简单的C5异戊二烯结构单元,异戊烯基焦磷酸(isopentenyl pyrophosphate,IPP)及其异构体二甲基烯丙基焦磷酸(dimethylallyl pyrophosphate,DMAPP)。三分子的C5单元在异戊烯基转移酶参与下,发生级联反应,碳链延伸,生成含有15个碳原子的焦磷酸法呢酯(arnesyl pyrophosphate,FPP),FPP既而可在倍半萜合酶催化下,环化生成结构多样的倍半萜化合物。
柳杉二醇首先在Cryptomeria japonica植物中被发现,随后在多种植物的挥发油中均有微量的柳杉二醇被检出。柳杉二醇具有一定的解痉作用,是抗痉挛药物的活性成08鉴于柳杉二醇在植物中含量较低且分离困难,一些研究者设计了其化学合成和半合成的路线,如通过金属离子催化的氧化反应,可以将(-)-elemol历经多步反应合成柳杉二醇。此外,还可以茴香酸(ilicic acid)为起始原料,采用相对简单高效的半合成工艺,经三步反应获得柳杉二醇。
然而,使用多步合成步骤相较植物体内,柳杉二醇合成途径的酶而言,是较为繁琐的。目前,通过细胞工厂的微生物发酵在单体化合物的工业生产方面具有高度竞争力。将基因元件(启动子、转录调控区域、核糖体结合位点、开放阅读框、终止子等)依据工程化目标需要,有机重构和连接起来,便形成了功能基因模块。通过对已有生物网络加以利用,同时引入新的功能基因模块,表达出天然细胞不能合成或含量极低的产物。如利用完整的青蒿酸生物合成途径在酵母工程菌中生产抗疟药青蒿素,每升发酵液可生产青蒿酸25克。
发明内容
本发明一方面提供了一种柳杉二醇合成酶,所述合成酶具有如下氨基酸序列:
(1)SEQ ID NO:2所示氨基酸序列;
(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蛋白。
本发明的另一方面提供了一种编码本发明所述柳杉二醇合成酶的基因(Tripterygium wilfordii Cryptomeridiol Synthase,TwCS):所述基因,它是下列核苷酸序列之一:
(1)SEQ ID NO:1第所示的核苷酸分子;或
(2)SEQ ID NO:1第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;或
(3)在严谨条件下与SEQ ID NO:1所示核苷酸分子杂交的核苷酸序列,所述严谨条件为:在含0.1%SDS的0.1×SSPE或含0.1%的SDS的0.1×SSC溶液中杂交。
杂交反应的“严谨度”可以容易的由本领域普通技术人员确定,而且通常根据探针长度、洗涤温度、和盐浓度凭经验计算。通常,较长的探针要求较高的温度以正确退火,而较短的探针需要较低的温度。杂交通常依赖于当互补链存在于低于其解链温度的环境中时变性DNA重新退火的能力。探针和可杂交序列之间的期望同源性程度越高,可使用的相对温度也越高。结果是,推断出较高相对温度将趋向于使反应条件更为严格,而较低温度也就较不严格。关于杂交反应严谨度的另外细节和解释,参见Ausubel等人,《Current Protocols inMolecular Biology》,Wiley Interscience Publishers,1995。
“严谨条件”或“高严谨条件”,如本文中所定义的,可如下鉴别:(1)采用低离子强度和高温进行洗涤,例如0.015M氯化钠/0.0015M柠檬酸钠/0.1%十二烷基硫酸钠,50℃;(2)在杂交过程中采用变性剂,诸如甲酰胺,例如50%(v/v)甲酰胺及0.1%牛血清清蛋白/0.1%Ficoll/0.1%聚乙烯吡咯烷酮/50mM磷酸钠缓冲液pH 6.5,含750mM氯化钠,75mM柠檬酸钠,42℃;或(3)在采用50%甲酰胺,5x SSC(0.75M NaCl,0.075M柠檬酸钠),50mM磷酸钠(pH 6.8),0.1%焦磷酸钠,5x Denhardt氏溶液,超声波处理的鲑鱼精DNA(50μg/ml),0.1%SDS,和10%硫酸右旋糖苷的溶液中于42℃杂交过夜,以及于42℃在0.2x SSC(氯化钠/柠檬酸钠)中洗涤10分钟,接着在含EDTA的0.1x SSC中于55℃进行10分钟高严格性洗涤。
本发明的再一方面,提供了一种表达载体,所述表达载体包含启动子、编码本发明所述柳杉二醇合成酶的基因、以及转录终止子。将启动子、柳杉二醇合成酶编码基因、终止子与附加型载体采用酵母同源重组的方法拼接起来,所述附加型载体为酵母表达载体,如pYX212、pYES2.0、pRS425、pRS426或p424。
在本发明的一具体实施方案中,提供了以下一些具体的表达载体:
(1)重组表达载体pYX212-TwCS:其包含启动子TPIp、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
(2)重组表达载体pYX212-ERG20+TwCS:其包含启动子TPIp、EGR20基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
(3)重组表达载体pYX212-IDI+TwCS:其包含启动子TPIp、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
(4)重组表达载体pYX212-(IDI-EGR20)+TwCS:其包含启动子TPIp、IDI基因、融合蛋白连接肽、EGR20基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
(5)重组表达载体pYX212-(EGR20-IDI)+TwCS:其包含启动子TPIp、EGR20基因、融合蛋白连接肽、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
其中所述融合蛋白为GGGS、GSG、GSGGGGS、GSGEAAAK、GSGEAAAKEAAAK或GSGMGSSSN中的任意一种,优选为GGGS,编码基因为ggtggtggttct。
本发明的又一方面,提供了一种包含本发明所述表达载体的工程菌,所述工程菌可以是选自酵母细胞或植物细胞,优选为酵母细胞。
本发明的一具体实施例中,提供了构建本发明所述工程菌的方法,所述方法为:
(1)将重组表达载体pYX212-TwCS转化到酵母BY4741菌株中,得到工程菌TE1;或(2)将重组表达载体pYX212-ERG20+TwCS转化到酵母BY4741菌株中,得到工程菌TE2;或
(3)将重组表达载体pYX212-IDI+TwCS转化到酵母BY4741菌株中,得到工程菌TE3;或
(4)将重组表达载体pYX212-(IDI-EGR20)+TwCS转化到酵母BY4741菌株中,得到工程菌TE4;或
(5)将重组表达载体pYX212-(EGR20-IDI)+TwCS转化到酵母BY4741菌株中,得到工程菌TE5;或
(6)将重组表达载体pYX212-(EGR20-IDI)+TwCS和重组表达载体p424-tHMG1转化到酵母BY4741菌株中,得到工程菌TE6;或
(7)将重组表达载体pYX212-IDI+TwCS和重组表达载体p424-tHMG1转化到酵母BY4741菌株中,得到工程菌TE7;
其中所述重组表达载体p424-tHMG1包括:酵母启动子TDH3p、截断的HMG-CoA还原酶基因tHMG1,酵母终止子TDH3t。
本发明的又一方面,提供了本发明所述柳杉二醇合成酶、或本发明所述柳杉二醇合成酶编码基因、或本发明所述重组表达载体、或本发明所述工程菌,在合成柳杉二醇及桉叶醇中的运用。发酵培所得菌株2-3d后,用正己烷提取发酵液,经GC-MS检测,可检测到倍半萜产物,经结构鉴定确定柳杉二醇为主产物。利用本发明可以通过生物合成技术来生成柳杉二醇,缓解药源缺乏问题,具有很好的应用前景。
本发明的还一方面,提供了本发明所述柳杉二醇合成酶或编码本发明所述柳杉二醇合成酶基因,在含有柳杉二醇化学成分的植物育种中的运用。运用本发明所述柳杉二醇合成酶、或其编码基因,通过将其运用到植物细胞中,可改善植物体内柳杉二醇的含量。
本发明SEQ ID No.1的DNA序列由1662个碱基组成,编码序列表中由553个氨基酸残基组成的蛋白质序列SEQ ID No.2。
附图说明
图1为TE1-TE7菌株质粒类型简图。
图2为GC-MS分析发酵产物图,其中A为菌株TE1的发酵产物,出峰位置在1,2的产物为微量的倍半萜成分(出峰位置1是桉叶醇),出峰位置在3处的产物,经鉴定为柳杉二醇,其保留时间为21.43min;B为不含TwCS表达基因的空载体表达产物图,发现没有任何倍半萜类产物;C为标准品柳杉二醇的GC-MS图,出峰位置与图1的峰3相同;D和E分别为TE1的峰3位置的产物以及标准品柳杉二醇的质谱图。
图3为柳杉二醇标准品的定量用标准曲线图。
图4为TE1-TE7菌株表达产物柳杉二醇产量测定图。
具体实施方式
以下通过优选实施例并结合附图具体说明本发明的各个方面和特征,本领域的技术人员应该理解,这些实施例只是用于说明,而不是限制本发明的范围。在不背离权利要求书范围的条件下,本领域的技术人员可以对本发明的各个方面进行各种修改和改进,这些修改和改进也属于本发明的保护范围。例如,将实施例中所实用的启动子和表达载体替换为本领域中常用的其它启动子和表达载体,是本领域的普通技术人员所能够理解并实现的。
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“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):4165-4170”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。
实施例1雷公藤悬浮细胞总RNA提取、纯化
采用改良CTAB法(CTAB Buffer:2%CTAB(W/V);100mmol·L-1Tris-HCl(pH 8.0);25mmol·L-1EDTA;2.0mol·L-1NaCl;0.5g·L-1亚精胺)提取雷公藤悬浮细胞的总RNA。利用RNA纯化试剂盒(天根生化科技有限公司),对所提RNA进行纯化。
实施例2TwCS基因全长cDNA克隆
1.引物设计
根据雷公藤转录组数据注释筛选得到基因全长序列片段,设计5’RACE和3’RACE引物,引物序列如下:
5’RACE GTACCGTAAGCATCGTATGTGTCG(SEQ ID NO:3)
3’RACE CTATGAAGAGGACGAGTCTCGG(SEQ ID NO:4)
2.PCR扩增
利用PrimeScript 1st Strand cDNA Synthesis Kit(Takara公司)试剂盒将实施例1所得RNA反转录成RACE Ready第一链cDNA。参照SMARTerTM RACE试剂盒说明书,进行cDNA末端快速扩增。
通过RACE方法得到SEQ ID No.1DNA序列的3’和5’端,然后依据序列信息设计引物,引物序列如下:
TwCS-F ATGGCAGCGACCACCCAATCCAC(SEQ ID NO:5)
TwCS-R TTAATCTTGCATTGGTATTTGTTG(SEQ ID NO:6)
以RACE Ready第一链cDNA为模板,进行PCR扩增。
PCR反应条件为98℃30s;98℃ 10s,60℃ 15s,72℃ 1min,35个循环;72℃,7min。
测序结果表明,PCR扩增产物的序列如SEQ ID No.1所示,将序列1所示的基因命名为TwCS,此DNA序列编码553个氨基酸组成的蛋白质,该蛋白命名为TwCS,该蛋白的氨基酸序列为SEQ ID No.2。
实施例3质粒构建
1、启动子和终止子的克隆
本实施例中使用的酵母启动子和终止子均可在SGD网站(https://www.yeastgenome.org)上公开获取。
采用酵母基因组提取试剂盒(天根生化科技有限公司)提取酵母BY4741的总DNA。然后以此DNA为模板,设计如下引物:
TEF1p-F ATAGCTTCAAAATGTTTCTACTC(SEQ ID NO:7)
TEF1p-R TTTGTAATTAAAACTTAGATTAG(SEQ ID NO:8)
FBA1t-F GTTAATTCAAATTAATTGATATAG(SEQ ID NO:9)
FBA1t-R AGTAAGCTACTATGAAAGACTTT(SEQ ID NO:10)
通过PCR扩增(具体步骤同实施例2)得到启动子TEF1p(TEF1SGD ID:S000006284)和终止子FBA1t(FBA1SGD ID:S000001543)片段。
以pYX212质粒为模板,通过PCR扩增(具体步骤同实施例2)得到启动子TPIp和终止子pYX212t。扩增引物如下:
TPIp-F GAATTGGGGATCTACGTATGGTC(SEQ ID NO:11)
TPIp-R AGTTTATGTATGTGTTTTTTG(SEQ ID NO:12)
pYX212t-F GAATTGGGGATCTACGTATGGTC(SEQ ID NO:13)
pYX212t-R TGCCGTAAACCACTAAATCGGAACC(SEQ ID NO:14)
2、EGR20基因(酵母FPP和GPP合酶基因)的获得
根据酵母EGR20基因序列(SGD ID:S000003703)设计引物如下:
ERG20-F:ATGGCTTCAGAAAAAGAAATTAG(SEQ ID NO:15)
ERG20-R:CTATTTGCTTCTCTTGTAAAC(SEQ ID NO:16)
通过PCR扩增(具体步骤同实施例2),获得酵母EGR20基因序列。
3、IDI基因的获得
根据酵母IDI基因序列(SGD ID:S000006038)设计引物如下:
IDI-F:ATGACTGCCGACAACAATAGTATGC(SEQ ID NO:17)
IDI-R:TTATAGCATTCTATGAATTTGCCTG(SEQ ID NO:18)
通过PCR扩增(具体步骤同实施例2),获得酵母EGR20基因序列。
4、表达模块构建
利用PCR方法构建以下模块:
TPIp-ERG20-FBA1t-TEF1p
TPIp-IDI-FBA1t-TEF1p
TPIp-IDI/ERG20-FBA1t-TEF1p
TPIp-ERG20/IDI-FBA1t-TEF1p
TEF1p-TwCS-pYX212t
TPIp-TwCS-pYX212t
构建方法:(1)混合DNA片段:将启动子,基因,终止子,启动子……按照1:3:5:7:XX:7:5:3:1的摩尔比例混合,比例为1的DNA的量为50-100ng/kb。(2)第一步PCR:将(1)的混合DNA作为模板,不加入引物,通过PCR扩增,PCR反应条件为98℃ 30s;98℃10s,60℃ 15s,72℃ 1min,15个循环;72℃,7min。(3)第二步PCR:取2μL(2)中PCR产物,作为模板,利用始端启动子的正向引物,终端终止子或启动子的反向引物进行PCR扩增(具体步骤同实施例2)。(4)利用EZNA Gel Extraction Kit(OMEGA公司),参照说明书,纯化PCR产物。(5)纯化产物,参照pEASY-Blunt Simple Cloning Kit(北京全式金生物技术有限公司)说明书,连接,转化,经过测序鉴定,得到相应模块DNA。
其中IDI和ERG20之间有GGGS(GGT GGTGGT TCT)linker连接。
5、同源重组法构建质粒
利用酵母同源重组方法,将构建的模块连接到表达载体pYX212中,具体操作如下:
(1)利用BamH I内切酶(NEB公司)对表达载体pYX212进行酶切。
酶切反应体系(50μL体系)
37℃反应2h后琼脂糖凝胶电泳,利用EZNA Gel Extraction Kit(OMEGA公司),参照说明书,纯化酶切产物。
(2)将TPIp-TwCS-pYX212t模块与(1)中所得线性表达载体pYX212混合,其中模块摩尔浓度为(100ng/kb),载体的摩尔浓度为(60-80ng/kb)。然后共同电转入酵母BY4741感受态,电转条件为2.5kV,25μF和200Ω(Bio-Rad Gene Pulsers)。
其中,酵母BY4741感受态是利用醋酸锂转化法制备。
(3)酵母菌株在各自的筛选缺陷培养基中培养2-3d,30℃。挑取单菌落,用E.Z.N.A.Yeast Plasmid Mini Kit(OMEGA公司),参照说明书,提取酵母质粒。
(4)以(3)质粒为模板,利用PCR方法筛选,筛选引物为TPIp-F和pYX212t-R(见1启动子和终止子的克隆),经过测序鉴定,得到重组质粒pYX212-TPIp-TwCS-pYX212t,简写为pYX212-TwCS。
(5)重复(1)-(4)步骤,依次将TPIp-ERG20-FBA1t-TEF1p,TPIp-IDI-FBA1t-TEF1p,TPIp-IDI/ERG20-FBA1t-TEF1p,TPIp-ERG20/IDI-FBA1t-TEF1p,TEF1p-TwCS-pYX212t模块构建到载体pYX212中,得到以下重组质粒:
pYX212-TPIp-ERG20-FBA1t-TEF1p-TwCS-pYX212t,简写为pYX212-ERG20+TwCS
pYX212-TPIp-IDI-FBA1t-TEF1p-TwCS-pYX212t,简写为pYX212-IDI+TwCS
pYX212-TPIp-IDI/ERG20-FBA1t-TEF1p-TwCS-pYX212t,简写为pYX212-(IDI-ERG20)+TwCS
pYX212-TPIp-ERG20/IDI-FBA1t-TEF1p-TwCS-pYX212t,简写为pYX212-(ERG20-IDI)+TwCS
(6)质粒p424-tHMG1是将酵母启动子TDH3p,基因tHMG1,酵母终止子TDH3t构建到质粒p424中,质粒携带HIS3marker。详细构建方法在文献“Zhou,Y.J.;Gao,W.;Rong,Q.;Jin,G.;Chu,H.;Liu,W.;Yang,W.;Zhu,Z.;Li,G.;Zhu,G.J.Am.Chem.Soc.2012,134,3234-3241.”中公开过,可依据文献记载方式获得,公众也可从首都医科大学分子生药与中药资源实验室获得。
实施例4生产柳杉二醇工程菌构建
将实施例3中的质粒pYX212-TwCS转化到BY4741菌株中,转化方法参照Frozen-EZYeast Transformation IITM(Zymo Research公司)说明书,得到工程菌TE1,如表1及附图1所示。
将实施例3中的质粒pYX212-ERG20+TwCS转化到BY4741菌株中,转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书,得到工程菌TE2,如表1及附图1所示。
将实施例3中的质粒pYX212-IDI+TwCS转化到BY4741菌株中,转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书,得到工程菌TE3,如表1及附图1所示。
将实施例3中的质粒pYX212-(IDI-ERG20)+TwCS转化到BY4741菌株中,转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书,得到工程菌TE4,如表1及附图1所示。
将实施例3中的质粒pYX212-(ERG20-IDI)+TwCS转化到BY4741菌株中,转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书,得到工程菌TE5,如表1及附图1所示。
将实施例3中的质粒pYX212-(ERG20-IDI)+TwCS和p424-tHMG1转化到BY4741菌株中,转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书,得到工程菌TE6,如表1及附图1所示。
将实施例3中的质粒pYX212-IDI+TwCS和p424-tHMG1转化到BY4741菌株中,转化方法参照Frozen-EZ Yeast Transformation IITM(Zymo Research公司)说明书,得到工程菌TE7,如表1及附图1所示。
表1本发明所涉及到的菌株的基因型和重组质粒
实施例5工程菌培养和产物鉴定
(1)工程菌培养
通过生物反应器来发酵生产倍半萜的菌株。用20g/L浓度的葡萄糖作为碳源,相应的缺陷培养基(北京泛基诺有限公司)用于预培养对应的营养缺陷型菌株。用于3L生物反应器的培养基由不含尿嘧啶和组氨酸的8g/L合成脱氮培养基组成,10g/L(NH4)2SO4,10g/LKH2PO4,1.0g/L MgSO4·7H2O。50%NH3·H2O用作pH调节剂。将菌株在摇瓶中在30℃条件下以230rpm转速预培养48h。然后在3L搅拌式生物反应器(Eppendorf BioFlo/CelliGen 115)中,用预培养细胞接种1L发酵培养基。定期喂养500g/L葡萄糖溶液以保持菌株的生长。将含有40g/L合成脱氮培养基的缺乏尿嘧啶和组氨酸和(NH4)2SO4的浓缩培养基进行发酵。
(2)产物提取和分离
发酵产物为倍半萜类成分,易溶于正己烷,因此选取正己烷为提取试剂。将发酵液离心分为菌体和菌液两部分,向菌液中加入等体积正己烷,萃取3次;菌体破碎后,用3倍体积的正己烷超声提取3次。合并有机层,加入适量的无水硫酸钠,静置片刻,除去萃取液中的水分。将萃取液用旋转蒸发仪浓缩,注意水浴温度不超过35℃(挥发性成分),最终转移到玻璃收集瓶中。
取硅胶薄层板,将浓缩产物用正己烷和乙酸乙酯以不同配比展开,显色剂为香草醛硫酸。初步分离。然后用XSelect CSH Prep C18OBD(19×150mm,5um)色谱柱分离,流动相A为0.1%(v/v)甲酸水,流动相B为乙腈.流速为20mL/min.分离浓缩富集单体化合物。
(3)结构鉴定
化合物结构通过NMR光谱解析,所有数据从BRUKER ACANCE III 600MHzspectrometer中收集,溶剂为含有TMS的氘代氯仿,最终化合物鉴定为柳杉二醇,结果如附图2所示。
实施例6工程菌生产柳杉二醇的产量比较
为了测定每种菌株的倍半萜生产量,按1:100比例接种,预培养50mL菌液作为菌种。菌株在含有20g/L葡萄糖的缺陷培养基中以230rpm转速,30℃条件下培养。振荡培养72h后,检测所有菌株的OD600。向培养液加入等体积的正己烷中并在200rpm下保持振荡2h小时,然后加入等体积正己烷超声波提取两次。合并有机层并旋转蒸发浓缩。浓缩样最终定容到1.0mL,然后取100uL制备GC-MS样品,进行GC-MS分析。使用Thermo TRACE 1310/TSQ8000气相色谱仪(不分流;注射器温度250℃),TG-5MS(30m×0.25mm×0.25μm)毛细管柱;GC条件如下:首先将烘箱温度保持在50℃恒定2分钟,然后以8℃/分钟的速度升至280℃,并在最终温度下保持10分钟。注射器和检测器的温度为50℃。使用柳杉二醇类似物β-eudesmol建立标准曲线,得到标准曲线方程为:y=3E+06x-3E+07,如附图3所示。计算得到具体的产量为:
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。
序列表
<110> 首都医科大学中医药学院
<120> 柳杉二醇合成酶、其编码基因及在生物合成上的应用
<141> 2018-02-09
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1662
<212> DNA
<213> 雷公藤(Tripterygium wilfordii)
<400> 1
atggcagcga ccacccaatc cacggaggct ccacggcggt tggccaactt cgcccctgcc 60
gtttggggtc acgatgactt tgcttctttt gcttctgatc aagattcgga gtacggatcg 120
tacacaaaga tagtggagga gttgaaagta caagtgaaag atatgttgtt gtctacaaat 180
gagattgtgg agaaagttga gttgattgac ttgttgggtc gtcttggtat ttcatatcac 240
tttgaaagtg aaattgaaga ccagcttatg caaaatctcg acatagtcaa aactaaactc 300
gtggatgaca acaatgacta cggcctatac gccgttgcac ttctgttccg cgtcttcaga 360
caacatggtt gcaaaatttc ttgtgatgtg tttgacaaat ttaagggaga tgatggaaag 420
ttgaaaatga gtctagctag tgatgtagag gggatgctaa gcttgtacga agcttctcac 480
ttgagcatgc atggagagga tgttttggat gaagcacttg gtttttcaaa aacttctctt 540
cattccgcgg tgacccaatt gaacccacac tttgcaaacc aagttgccca tgcattgcaa 600
caaccttatc aaaagggcat tccaagaatc gagtcaaggc aatacatcaa tttctatgaa 660
gaggacgagt ctcggaatga aattctgctc aaattagcga aaatcgattt taatcgagta 720
caattgttgc accaacaaga gctaagtcat gtctcaaggt ggtataaaga cttgcagatt 780
gcttcaaaat ttccttatgc aagagacaga attgctgaaa tctatatgtg gactgttggg 840
tctaactttg aaccacatta tggacgtgtc cgaatctttc ttactaaaag tgtgacaatg 900
atatcaattt tagacgacac atacgatgct tacggtacaa ttgaagaact tcgactcttg 960
actgatgcaa tagataggtg ggacattggt gccattgatc aattaccaga ttacatgaaa 1020
gttctttaca agatgattct aaatctctac gatgaattcg agaacgaatt gaaaaacgaa 1080
ggaagatctt cctgtgttgc ttatgctaga gacgcgttaa gagaaatggt gaaagcctac 1140
cacgttgaag ctgagtggtg caacaaaagt tacgtaccaa cattcgatga gtacatggag 1200
aatgcactga tcacaagctg ttatcatgca attccagctg catgttttct aggcatggga 1260
gaaattgcag ggataaaaga atttgaatgg ctcaaaagca tcccgaaaat ggttagggct 1320
tccgagatga tcggtcgtct tatggacgac ataatgtcac ataaggagga acaaaagagg 1380
gggcatgttg cctcaagcgt tgagtgcttt atgaagcaat atggtgtgtc agaagaagag 1440
gtggttaaag atttccaaaa cagggttgcg aatgcatgga aggatattaa tgaagagtgt 1500
atgagaccaa ctgctgtgtc tcttcatctt ctgatgccaa ttctgaacct aacacgcatc 1560
atcgatgttg tctacaagaa cgacgatggg tattcaaatc cagtcaattt gaaggagcat 1620
gtcaagtctt tgttcattca acaaatacca atgcaagatt aa 1662
<210> 2
<211> 553
<212> PRT
<213> 雷公藤(Tripterygium wilfordii)
<400> 2
Met Ala Ala Thr Thr Gln Ser Thr Glu Ala Pro Arg Arg Leu Ala Asn
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Phe Ala Pro Ala Val Trp Gly His Asp Asp Phe Ala Ser Phe Ala Ser
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Asp Gln Asp Ser Glu Tyr Gly Ser Tyr Thr Lys Ile Val Glu Glu Leu
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Lys Val Gln Val Lys Asp Met Leu Leu Ser Thr Asn Glu Ile Val Glu
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Lys Val Glu Leu Ile Asp Leu Leu Gly Arg Leu Gly Ile Ser Tyr His
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Phe Glu Ser Glu Ile Glu Asp Gln Leu Met Gln Asn Leu Asp Ile Val
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Lys Thr Lys Leu Val Asp Asp Asn Asn Asp Tyr Gly Leu Tyr Ala Val
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Ala Leu Leu Phe Arg Val Phe Arg Gln His Gly Cys Lys Ile Ser Cys
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Asp Val Phe Asp Lys Phe Lys Gly Asp Asp Gly Lys Leu Lys Met Ser
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Leu Ala Ser Asp Val Glu Gly Met Leu Ser Leu Tyr Glu Ala Ser His
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Leu Ser Met His Gly Glu Asp Val Leu Asp Glu Ala Leu Gly Phe Ser
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Lys Thr Ser Leu His Ser Ala Val Thr Gln Leu Asn Pro His Phe Ala
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Asn Gln Val Ala His Ala Leu Gln Gln Pro Tyr Gln Lys Gly Ile Pro
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Arg Ile Glu Ser Arg Gln Tyr Ile Asn Phe Tyr Glu Glu Asp Glu Ser
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Arg Asn Glu Ile Leu Leu Lys Leu Ala Lys Ile Asp Phe Asn Arg Val
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Gln Leu Leu His Gln Gln Glu Leu Ser His Val Ser Arg Trp Tyr Lys
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Asp Leu Gln Ile Ala Ser Lys Phe Pro Tyr Ala Arg Asp Arg Ile Ala
260 265 270
Glu Ile Tyr Met Trp Thr Val Gly Ser Asn Phe Glu Pro His Tyr Gly
275 280 285
Arg Val Arg Ile Phe Leu Thr Lys Ser Val Thr Met Ile Ser Ile Leu
290 295 300
Asp Asp Thr Tyr Asp Ala Tyr Gly Thr Ile Glu Glu Leu Arg Leu Leu
305 310 315 320
Thr Asp Ala Ile Asp Arg Trp Asp Ile Gly Ala Ile Asp Gln Leu Pro
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Asp Tyr Met Lys Val Leu Tyr Lys Met Ile Leu Asn Leu Tyr Asp Glu
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Phe Glu Asn Glu Leu Lys Asn Glu Gly Arg Ser Ser Cys Val Ala Tyr
355 360 365
Ala Arg Asp Ala Leu Arg Glu Met Val Lys Ala Tyr His Val Glu Ala
370 375 380
Glu Trp Cys Asn Lys Ser Tyr Val Pro Thr Phe Asp Glu Tyr Met Glu
385 390 395 400
Asn Ala Leu Ile Thr Ser Cys Tyr His Ala Ile Pro Ala Ala Cys Phe
405 410 415
Leu Gly Met Gly Glu Ile Ala Gly Ile Lys Glu Phe Glu Trp Leu Lys
420 425 430
Ser Ile Pro Lys Met Val Arg Ala Ser Glu Met Ile Gly Arg Leu Met
435 440 445
Asp Asp Ile Met Ser His Lys Glu Glu Gln Lys Arg Gly His Val Ala
450 455 460
Ser Ser Val Glu Cys Phe Met Lys Gln Tyr Gly Val Ser Glu Glu Glu
465 470 475 480
Val Val Lys Asp Phe Gln Asn Arg Val Ala Asn Ala Trp Lys Asp Ile
485 490 495
Asn Glu Glu Cys Met Arg Pro Thr Ala Val Ser Leu His Leu Leu Met
500 505 510
Pro Ile Leu Asn Leu Thr Arg Ile Ile Asp Val Val Tyr Lys Asn Asp
515 520 525
Asp Gly Tyr Ser Asn Pro Val Asn Leu Lys Glu His Val Lys Ser Leu
530 535 540
Phe Ile Gln Gln Ile Pro Met Gln Asp
545 550
<210> 3
<211> 24
<212> DNA
<213> Human adenovirus type 1
<400> 3
gtaccgtaag catcgtatgt gtcg 24
<210> 4
<211> 22
<212> DNA
<213> Human adenovirus type 1
<400> 4
ctatgaagag gacgagtctc gg 22
<210> 5
<211> 23
<212> DNA
<213> Human adenovirus type 1
<400> 5
atggcagcga ccacccaatc cac 23
<210> 6
<211> 24
<212> DNA
<213> Human adenovirus type 1
<400> 6
ttaatcttgc attggtattt gttg 24
<210> 7
<211> 23
<212> DNA
<213> Human adenovirus type 1
<400> 7
atagcttcaa aatgtttcta ctc 23
<210> 8
<211> 23
<212> DNA
<213> Human adenovirus type 1
<400> 8
tttgtaatta aaacttagat tag 23
<210> 9
<211> 24
<212> DNA
<213> Human adenovirus type 1
<400> 9
gttaattcaa attaattgat atag 24
<210> 10
<211> 23
<212> DNA
<213> Human adenovirus type 1
<400> 10
agtaagctac tatgaaagac ttt 23
<210> 11
<211> 23
<212> DNA
<213> Human adenovirus type 1
<400> 11
gaattgggga tctacgtatg gtc 23
<210> 12
<211> 21
<212> DNA
<213> Human adenovirus type 1
<400> 12
agtttatgta tgtgtttttt g 21
<210> 13
<211> 23
<212> DNA
<213> Human adenovirus type 1
<400> 13
gaattgggga tctacgtatg gtc 23
<210> 14
<211> 25
<212> DNA
<213> Human adenovirus type 1
<400> 14
tgccgtaaac cactaaatcg gaacc 25
<210> 15
<211> 23
<212> DNA
<213> Human adenovirus type 1
<400> 15
atggcttcag aaaaagaaat tag 23
<210> 16
<211> 21
<212> DNA
<213> Human adenovirus type 1
<400> 16
ctatttgctt ctcttgtaaa c 21
<210> 17
<211> 25
<212> DNA
<213> Human adenovirus type 1
<400> 17
atgactgccg acaacaatag tatgc 25
<210> 18
<211> 25
<212> DNA
<213> Human adenovirus type 1
<400> 18
ttatagcatt ctatgaattt gcctg 25
Claims (12)
1.一种柳杉二醇合成酶,所述合成酶如SEQ ID NO:2所示氨基酸序列。
2.编码权利要求1所述合成酶的基因,所述基因为如下中的至少一种:
(1)SEQ ID NO:1第所示的核苷酸分子;或
(2)SEQ ID NO:1第所示的核苷酸分子经取代一个或多个核苷酸且表达权利要求1所述合成酶的核苷酸序列。
3.重组表达载体,其包含启动子、权利要求2所示基因和转录终止子。
4.权利要求3所述的表达载体,其为将启动子、柳杉二醇合成酶编码基因、终止子与附加型载体采用酵母同源重组的方法拼接起来,所述附加型载体为酵母表达载体。
5.根据权利要求4所述的表达载体,其中所述酵母表达载体选自pYX212、pYES2.0、pRS425、pRS426或p424。
6.根据权利要求3所述的表达载体,其中所述载体为以下中的任意一种:
(1)重组表达载体pYX212-TwCS:其包含启动子TPIp、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
(2)重组表达载体pYX212-ERG20+TwCS:其包含启动子TPIp、EGR20基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
(3)重组表达载体pYX212-IDI+TwCS:其包含启动子TPIp、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
(4)重组表达载体pYX212-(IDI-EGR20)+TwCS:其包含启动子TPIp、IDI基因、融合蛋白连接肽、EGR20基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
(5)重组表达载体pYX212-(EGR20-IDI)+TwCS:其包含启动子TPIp、EGR20基因、融合蛋白连接肽、IDI基因、酵母终止子FBA1t、酵母启动子TEF1p、柳杉二醇合成酶表达基因TwCS、终止子pYX12t;
其中所述融合蛋白为GGGS、GSG、GSGGGGS、GSGEAAAK、GSGEAAAKEAAAK或GSGMGSSSN中的任意一种。
7.根据权利要求6所述的表达载体,其中所述融合蛋白为GGGS,编码基因为ggtggtggttct。
8.一种生产柳杉二醇工程菌,其包含权利要求2所述基因或权利要求3-7中任意一权利要求所述重组表达载体。
9.根据权利要求8所述的工程菌,其中所述工程菌选自酵母细胞。
10.根据权利要求8所述的工程菌,其构建方法为:
(1)将重组表达载体pYX212-TwCS转化到酵母BY4741菌株中,得到工程菌TE1;或
(2)将重组表达载体pYX212-ERG20+TwCS转化到酵母BY4741菌株中,得到工程菌TE2;或
(3)将重组表达载体pYX212-IDI+TwCS转化到酵母BY4741菌株中,得到工程菌TE3;或
(4)将重组表达载体pYX212-(IDI-EGR20)+TwCS转化到酵母BY4741菌株中,得到工程菌TE4;或
(5)将重组表达载体pYX212-(EGR20-IDI)+TwCS转化到酵母BY4741菌株中,得到工程菌TE5;或
(6)将重组表达载体pYX212-(EGR20-IDI)+TwCS和重组表达载体p424-tHMG1转化到酵母BY4741菌株中,得到工程菌TE6;或
(7)将重组表达载体pYX212-IDI+TwCS和重组表达载体p424-tHMG1转化到酵母BY4741菌株中,得到工程菌TE7;
其中所述重组表达载体p424-tHMG1是将酵母启动子TDH3p、截断的HMG-CoA还原酶基因tHMG1,酵母终止子TDH3t构建到质粒p424中,质粒携带HIS3marker。
11.权利要求1所述合成酶、或权利要求2所述基因、或权利要求3-7中任意一权利要求所述表达载体、或权利要求8-10中任意一权利要求所述工程菌,在合成柳杉二醇或桉叶醇中的运用。
12.权利要求1所述合成酶或权利要求2所述基因在含有柳杉二醇化学成分的植物育种中的运用。
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