CN107058418B - 雷公藤二萜合酶TwCPS1在制备松香烷型二萜化合物中的应用 - Google Patents
雷公藤二萜合酶TwCPS1在制备松香烷型二萜化合物中的应用 Download PDFInfo
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- CN107058418B CN107058418B CN201611144657.0A CN201611144657A CN107058418B CN 107058418 B CN107058418 B CN 107058418B CN 201611144657 A CN201611144657 A CN 201611144657A CN 107058418 B CN107058418 B CN 107058418B
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- Prior art keywords
- diterpene
- twcps1
- tripterygium wilfordii
- smksl1
- synthase
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Abstract
一种雷公藤二萜合酶基因Twcps1在催化合成二萜化合物中的应用，通过原核表达的雷公藤二萜合酶TwCPS1与丹参SmKSL1联合使用，以GGPP为底物合成松香烷型二萜前体，证实了TwCPS1为雷公藤二萜类次生代谢途径中一个关键酶。雷公藤TwCPS1与丹参SmKSL1的组合相对于丹参SmCPS1与丹参SmKSL1的组合，合成效率更高，具有广阔的产业应用前景。
Description
技术领域
本发明涉及雷公藤主要活性成分雷公藤二萜类化合物生物合成，具体涉及雷公藤二萜合酶TwCPS1在制备松香烷型二萜化合物中的应用，属于药用植物基因工程领域。
背景技术
药用植物雷公藤(Tripterygium wilfordii.Hook.f.)是一味中草药，被广泛用于类风湿性关节炎和炎症的治疗(Raphaela G M，Mildred W，Roy F，et al.Comparison ofTripterygium wilfordii Hook F Versus Sulfasalazine in the Treatment ofRheumatoid Arthritis：A Randomized Trial[J].Annals of Internal Medicine，2009，151(4)：229-240.Tao X L，Lipsky P E.The Chinese anti-inflammatory andimmunosuppressive herbal remedy Tripterygium wilfordii Hook F.[J].RheumaticDisease Clinics of North America，2000，26(1)：29-50.)。萜类成分为雷公藤的主要活性成分，包括雷公藤甲素(triptolide)、雷酚内酯(triptophenolide)和雷公藤红素(celastrol)等。从中药中的活性成分开发新药是一种很有潜力的方式，然而由于植物的生长缓慢，再加上这些有效成分在植物体中的含量不多，因而大大限制了它的发展。通过探寻和阐释萜类成分在雷公藤中的生物合成途径及其调控机制，有助于为药材品质的形成提供理论基础，同时为利用生物技术提高目标成分含量或直接生产有效成分或中间体带来广阔的应用空间。
雷公藤甲素具有抗肿瘤、抗炎、抗排异、抗生育等药理作用，与丹参酮类化合物同属松香烷型二萜。目前已证实丹参中的SmCPS与SmKS基因能催化GGPP形成松香烷型二萜化合物前体次丹参酮二烯，然而将SmCPS与SmKS联合使用制备次丹参酮二烯合成效率低，严重制约了产业上的大规模应用、影响了其他松香烷型二萜化合物的合成研究。由于雷公藤甲素、丹参酮等松香烷型二萜化合物的良好应用前景，产业上迫切需要一种高产率的、不依赖于天然提取途径的松香烷型二萜化合物前体合成方法。
发明内容
本发明将克隆得到的雷公藤二萜合酶基因Twcps1和丹参中的Smksl1基因分别构建表达载体，成功生成了次丹参酮二烯。进一步证实了Twcps1基因在合成雷公藤甲素等松香烷型二萜合成中的作用，且提供了一种合成松香烷型二萜前体的方法，为雷公藤甲素的生物合成奠定基础。
次丹参酮二烯是一种重要的松香烷型二萜化合物前体，由于其发现于丹参，因此将丹参中的SmCPS1与SmKSL1用于制备丹参酮。除了丹参以外，其他的植物中尽管也存在二萜合酶KS、CPS，也可能产生二萜类化合物，但通常不能催化合成次丹参酮二烯或合成量远远低于丹参自身SmCPS1与SmKSL1的组合。本发明在研究雷公藤甲素合成代谢途径的过程中，意外的发现雷公藤二萜合酶中的TwCPS1，与SmKSL1组合使用时能够成功合成次丹参酮二烯，且产量高于SmCPS1与SmKSL1的组合。即：本发明用雷公藤二萜合酶TwCPS1能够用于生产松香烷型二萜化合物前体，并且以其代替丹参二萜合酶SmCPS1，取得了产量提高的技术效果。值得注意的是，其他植物来源的二萜合酶(如拟南芥AtCPS)、雷公藤来源的其他种类的二萜合酶(如TwCPS2、TwCPS3)等与丹参SmKSL1组合则不能催化合成次丹参酮二烯。本发明所述TwCPS1+SmKSL1的组合合成松香烷型二萜化合物前体次丹参酮二烯的技术方案具有广阔的应用前景。
本发明提供了一种合成雷公藤松香烷型二萜类化合物前体合成的方法：具体将Twcps1基因的cDNA克隆到原核表达载体pMAL-c2X，构建带有Twcps1基因的重组表达载体；转入E.coli表达宿主菌，通过诱导后，表达产物经初步纯化后，加入到以GGPP为底物的体外酶促反应体系中，正己烷萃取反应产物，采用GC-MS可同时检测到产物。通过将Twcps1基因的ORF克隆到真核表达载体pESC-Trp，构建带有Twcps1基因的重组表达载体；并通过定点突变等方法，通过酵母BY-T20发酵生成产物，利用气相色谱-质谱联用技术，分析Twcps1基因的功能。研究结果表明，本发明与雷公藤二萜类化合物合成有关的基因具有二萜合酶基因的DXDD特征结构域，酶促反应分析发现该基因催化雷公藤二萜类化合物的早期合成步骤，对于培育高品质的药用植物品种特别是培育具有高雷公藤甲素含量的雷公藤品种具有重要的理论及实际意义。
本发明提供雷公藤二萜合酶或其基因在合成二萜类化合物中的用途，所述雷公藤二萜合酶的氨基酸序列如SEQ ID NO：2所示，所述雷公藤二萜合酶基因的序列如SEQ IDNO：1所示。
本发明所述雷公藤二萜合酶或其基因在合成二萜类化合物中的用途，其中所述二萜类化合物包括雷公藤甲素或其前体、丹参酮或其前体等。
本发明所述雷公藤二萜合酶或其基因在合成二萜类化合物中的用途，其中所述二萜类化合物为松香烷型二萜类化合物前体，更具体的，所述二萜类化合物是次丹参酮二烯。
本发明所述雷公藤二萜合酶或其基因在合成二萜类化合物中的用途，其特征在于将雷公藤合酶或其基因与丹参二萜合酶SmKSL1或其基因联合使用。
本发明所述雷公藤二萜合酶或其基因在合成二萜类化合物中的用途，其特征在于雷公藤合酶或其基因与丹参二萜合酶SmKSL1或其基因同时使用或先后使用，
本发明所述雷公藤二萜合酶或其基因在合成二萜类化合物中的用途，其特征在于先使用雷公藤合酶或其基因将GGPP催化形成CPP，再使用丹参二萜合酶SmKSL1或其基因催化CPP合成松香烷型二萜类化合物前体。
本发明还提供一种合成松香烷型二萜类化合物前体的方法，其包括：
(1)将雷公藤二萜合酶基因Twcps1表达盒插入第一原核表达载体中，制备表达外源雷公藤二萜合酶TwCPS1的重组大肠杆菌，超声破碎菌体，取含有重组TwCPS1蛋白的上清液；
(2)将丹参二萜合酶基因Smksl1表达盒插入第二原核表达载体中，制备表达外源丹参二萜合酶SmKSL1的重组大肠杆菌，超声破碎菌体，取含有重组SmKSL1蛋白的上清液；
(3)以GGPP为底物，以步骤(1)中获得的含重组TwCPS1蛋白的上清液为催化剂，催化GGPP产生CPP；
(4)以步骤(3)获得的CPP为底物，以步骤(2)获得的含重组SmKSL1蛋白的上清液为催化剂，催化CPP产生松香烷型二萜类化合物前体。
本发明所述合成松香烷型二萜类化合物前体的方法，步骤(1)所述的原核表达载体为pMAL-c2X，所述Twcps1基因插入BamH I和Sal I之间。
本发明所述合成松香烷型二萜类化合物前体的方法，在步骤(3)的催化反应完成后还包括脱磷的步骤。
本发明所述合成松香烷型二萜类化合物前体的方法，在步骤(4)完成后还包括对反应产物进行GC-MS分析的步骤，其中在第13.06分出现的峰为次丹参酮二烯。
附图说明
图1 雷公藤二萜合酶原核表达载体构建
图2 宿主菌E.coli TransB(DE3)诱导表达产物SDS-PAGE蛋白电泳分析(C：阴性对照即空表达载体pMAL-c2X表达产物；M：蛋白分子量标准，条带由上往下分别为200、116、97.2、66.4、29.0、20.1、14.3、6.5KDa；箭头表示目的重组蛋白；1：重组质粒pMALTwCPS1的表达；2：重组质粒pMALTwCPS3的表达产物；3：重组质粒pMALTwKS的表达产物；4：重组质粒pMALTwCPS2的表达产物；5：重组质粒pMALTwGES2表达产物；6：重组质粒pMALTwGES1表达产物)
图3 定点突变引物设计
图4 雷公藤二萜合酶真核表达载体构建
图5 CPS催化底物GGPP形成产物提取离子色谱图，第13.83分的峰为CPP，质谱荷质比为275m/z。由上而下依次为TwCPS1、TwCPS2、TwCPS3、SmCPS、AtCPS、空载体。
图6 (CPS+KS)催化GGPP形成产物提取离子色谱图，第13.06分的峰为次丹参酮二烯，第13.03分的峰为对映贝壳杉烯，第13.83分的峰为CPP，第13.96分的峰为16α-贝壳杉烷，第13.46分的峰为GGOH。
图7TwCPS2C311D、TwKST318D催化GGPP形成产物提取离子色谱图，第13.46分的峰为GGOH。
图8BY-T20/pESC-Trp-TwCPS3+TwKS、BY-T20/pESC-Trp-TwCPS3+TwKSA608M、BY-T20/pESC-Trp发酵产物提取离子色谱图，第13.03分的峰为对映贝壳杉烯。
图9 BY-T20/pESC-Trp、BY-T20/pESC-Trp-TwCPS1、BY-T20/pESC-Trp-TwCPS3+TwKS、BY-T20/pESC-Trp-TwCPS3+TwKSA608M发酵产物提取离子色谱图第12.99分的峰为泪杉醇((+)-manool)，第13.03分的峰为对映贝壳杉烯，第13.83分的峰为CPP，第13.96分的峰为16α-贝壳杉烷。
具体实施方式
以下结合具体实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
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。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
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。
SMARTerTM RACE cDNA Amplification Kit，PrimeSTAR GXL DNA Polymerase购自Takara公司；pEASY-Blunt Simple Cloning Kit购自北京全式金生物技术有限公司，GGPP购自Sigma公司，产品目录号为G6025。
实施例一、雷公藤Twcps1原核表达载体构建(图1)
以含有雷公藤Twcps1基因全长cDNA的载体pMD-19-TPS质粒为模板，用含酶切位点引物，进行PCR扩增基因编码区(引物序列见表3-1)。DNA聚合酶采用高保真DNA聚合酶(PrimeSTAR HS DNA Polymerase)。PCR参数为98℃3min，1循环；98℃10s，60℃10s，72℃2min30s，30循环；72℃7min；4℃维持。扩增产物经Gene JET Gel Extraction Kit胶回收(方法如下)。
Gene JET Gel Extraction Kit胶回收步骤：
(1)取PCR产物与6×loading buffer预混合，在1.5％琼脂糖凝胶上以低电压(约5Vcm-1)电泳30-60min；
(2)用解剖刀或剃刀片切割含有DNA片段的凝胶，尽可能的靠近DNA片段切割，以减小凝胶的含量，将胶片放在事先称重的1.5mL离心管并称重。记录胶片的重量。注意避免长时间暴露在紫外灯下，损害DNA而影响后续实验；
(3)加1∶1量的Binding Buffer到胶片中(量以重量计，如每100毫克琼脂糖凝胶加100微升的Binding Buffer)；
(4)在50-60℃的条件下温育凝胶混合物10min，期间颠倒混匀2-3次，促进胶融化，保证胶全部溶解，在上柱前将凝胶混合物快速涡旋混匀一次；
(5)转移最多800μL凝胶溶解液到基因回收纯化柱，13000g离心1min，弃去流出液，然后将柱放回相同的收集管；
(6)加入700μL Wash Buffer(已用乙醇稀释)到Gene JET纯化柱。13000g离心1min，弃去流出液，然后将柱放回相同的收集管；
(7)离心空GeneJET纯化柱13000g离心1min，彻底去除残留的Wash Buffer；
(8)将Gene JET纯化柱转移到一个干净的1.5mL离心管，加30-50μ L ddH2O(可60℃预热)于纯化柱膜，13000g离心1min；
(9)丢掉Gene JET纯化柱并储存纯化的DNA在-20℃。
经纯化后的PCR产物用限制性内切酶进行双酶切，采用NEB公司T4DNA连接酶定向连入经相同双酶切的表达载体pMAL-c2X中(方法如下)，连接产物经转化、蓝白菌落筛选及阳性克隆的初步筛选，并送样测序鉴定，得到经测序核苷酸序列无突变重组质粒pMALTPS，并将经的重组质粒pMALTPS转化至大肠杆菌E.coli TransB(DE3)表达感受态细胞。与此同时，将拟南芥中功能明确的AtCPS(催化GGPP形成ent-CPP)、AtKS(催化ent-CPP形成对映贝壳杉烯)及丹参中功能明确的SmCPS1(催化GGPP形成nor-CPP)、SmKSL1(催化nor-CPP形成次丹参酮二烯)构建到pMAL-c2X原核表达载体上(SmCPS1、SmKSL1利用实验室保存的重组质粒pET32SmCPS1及pET32SmKSL1)，将雷公藤TwCPS1、TwKS分别与其他植物来源的CPS、KS进行对比。
采用TaKaRa QuickCut酶进行双酶切反应：
表1：双酶切反应体系
双酶切反应(50μL体系)
内切酶1酶切完成后，加入1μL内切酶2继续酶切。注意：最适酶切温度较低的内切酶应先进行酶切，若最适酶切温度相同，可同时加入同时酶切。
采用NEB T4DNA快速连接试剂盒进行DNA片段连接反应：
表2：连接反应体系
连接反应(20μL体系)
＊DNA与载体摩尔比约为3∶1-10∶1
25℃连接5min(可适当延长连接时间)。
表3构建原核表达载体引物序列
实施例二、重组蛋白诱导表达
诱导表达
100mmol·L-1IPTG：称取238.3mg的IPTG用10mL的ddH2O溶解，过滤分装-20℃保存；LB培养基：Trytone 1.0％，Yeast Extract 0.5％，NaCl 1.0％，Agar1.5％，pH 7.0。
操作步骤：
(1)将酶切鉴定正确且经测序验证的含目的基因的重组质粒，取1μL转化至50μlTransB(DE3)感受态细胞中，涂板LB+Amp(氨苄青霉素钠)固体平板，37℃倒置培养12-16h；
(2)挑取单克隆菌落经酶切鉴定后转到含100μg·mL-1 Amp的2mL LB液体培养基中，37℃振荡培养至OD600到0.6-1.0；
(3)取1mL菌液5000g 4℃离心5min收集菌体，用新鲜LB+Amp液体培养基混悬菌体，转接到100mL LB+Amp液体培养基中；
(4)37℃培养至宿主菌密度(OD600)达到0.6-1.0时，加入适量IPTG诱导剂(终浓度约0.4mM)，在低温下(16℃)诱导培养8h；
(5)4℃3000g离心20min收获菌体，预冷的5mL HEPES缓冲液(50mM HEPES，100mMKCl，7.5mM MgCl2，5mM DTT，1mM PMSF，5％甘油，pH 7.2)重悬；
(6)置冰浴中超声破菌(30％功率，超声5s，间隔5s，持续3min)；大肠杆菌破碎液在4℃，15000g离心30min，取上清液；
(7)上清液经蛋白超滤管浓缩(4℃5000g 40min)至1.5mL。
取上清进行十二烷基硫酸钠-聚丙烯酰胺(SDS-PAGE)凝胶电泳检测。
以空质粒pMAL-c2X转化TransB(DE3)表达菌进行表达为对照。
实施例三、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(图2)
试剂配制
30％丙烯酰胺贮存液(神经毒性，操作时配戴口罩及手套)：在通风橱中，称取丙烯酰胺29.2g，甲叉双丙烯酰胺0.8g，加ddH2O溶解后，定容到100mL。针头滤器过滤后置棕色瓶中，4℃保存；
pH 8.8 Tris-HCl分离胶缓冲液：配制1.5M Tris-HCl，并调节pH至8.8，4℃保存；
pH 6.8 Tris-HCl浓缩胶缓冲液：配制1M Tris-HCl，并调节pH至6.8，4℃保存；
10％SDS：称取SDS 1.0g，蒸馏水10mL溶解，4℃保存；
10％过硫酸铵(APS)：取APS 1.0g，蒸馏水10mL溶解，4℃保存；
TEMED(四乙基乙二胺)原液；
5×样品缓冲液(10mL)：0.6mL 1mol·L-1的Tris-HCl(pH 6.8)，5mL50％甘油，2mL10％的SDS，0.5mL巯基乙醇，1mL 1％溴酚蓝，0.9mL蒸馏水。可在4℃保存数周，或在-20℃保存数月；
10×电泳缓冲液：称取Tris 30.38g，甘氨酸144g，SDS 10.8g，加蒸馏水约900mL，调pH 8.3后，用蒸馏水定容至1000mL，置4℃保存，临用前稀释10倍。
实施例四、样品制备
将蛋白质样品与5×样品缓冲液在一个Eppendorf管中混合，放入100℃加热5-10min，常温12000g离心3min，取上清点样。
实施例五、电泳
操作步骤
(1)将玻璃板、样品梳用洗涤剂洗净，用ddH2O冲洗数次，晾干；
(2)安装玻璃板；
(3)按如下体积配制10％分离胶15mL(配制两块胶)，混匀；
(4)向玻璃板间灌制分离胶，立即加入1mL ddH2O压平胶面，大约20min后胶即可聚合；
(5)按如下体积配制5％浓缩胶5mL(配制两块胶)，混匀；
(6)将上层ddH2O倾去，滤纸吸干，灌制浓缩胶，插入样品梳；
(7)装好电泳系统，加入电泳缓冲液，上样5-10μL；
(8)恒流40mA，溴酚蓝跑出分离胶30min后，停止电泳；
(9)卸下胶板，剥离胶，染色，照胶。
实施例六、考马斯亮蓝染色
试剂配制
考马斯亮蓝R250染色液：0.25％考马斯亮蓝R250(W/V)，45％甲醇(V/V)，10％冰乙酸；
脱色液(1000mL)：100mL冰乙酸，250mL乙醇，蒸馏水补足至1000mL。
染色步骤
室温染色45min-60min(或微波炉快染，高火20s，两次)；用蒸馏水清洗3-5遍；加入脱色液，置于100rpm摇床上脱色，每20min更换一次脱色液至胶透明，脱色完成后，用UMAXPowerlook 2100XL扫描仪进行照胶。
实施例七、酶促反应
以GGPP为底物的酶促反应
取实施例二、步骤(7)获得的浓缩重组CPS蛋白上清进行酶促反应。并以pMAL-c2X空载体、pMAL-c2X-AtCPS、pET32a(+)-SmCPS1转化大肠杆菌TransB(DE3)表达感受态细胞，所破碎浓缩得到的重组蛋白上清进行酶促反应作为对照。
酶促反应体系如下：
浓缩重组蛋白上清 182μL
底物GGPP(200μM) 18μL
反应产物充分混合(枪头吹打)，在室温(25℃)、黑暗的环境下反应2h；反应结束后用正己烷抽提3次，每次加入0.5mL，弃有机相，留水相(留一层正己烷覆盖水相，防止产物氧化)；然后用N2将水相中的正己烷彻底吹干，以免影响下一步的脱磷反应。
脱磷反应体系如下：
反应产物充分混合(枪头吹打)，在37℃下反应4h；脱磷后的产物再用正己烷抽提3次，每次加入0.5mL，将抽提所得有机相汇合在一起；用N2将提取液吹干，并加入60μL正己烷溶解，用于GC-MS分析。
以CPP为底物的酶促反应
对TwKS进行功能分析的酶促反应是以经CPS酶催化GGPP所产生的产物(CPP)作为反应底物，分析TwKS酶催化所得产物的化学结构，从而鉴定TwKS功能。具体实验过程如下：
(1)配制如下酶促反应体系，在室温(25℃)黑暗条件下反应2小时，使GGPP充分转化成CPP；
浓缩重组KS蛋白上清 182μL
底物GGPP(200μM) 18μL
(2)向上述反应混合液中加入等体积的TwKS酶，并补充MgCl2至10mM，在室温(25℃)黑暗条件下，反应过夜(12-16h)；
(3)反应结束后，体系用正己烷抽提3次(每次0.5mL)，所得正己烷相用N2吹干，然后加入60μL正己烷溶解进行GC-MS分析。
实施例八、反应产物GC-MS检测
GC-MS分析条件为：取1μL的进样，无分流的模式下，50℃保持2min，20℃·min-1升至300℃，保持20min；进样口温度250℃，离子源温度250℃，电子能量70ev，对样品进行20-650m/z范围扫描。GC-MS仪器为Thermo Scientific公司Thermo TRACE 1310/TSQ 8000gaschromatograph，色谱柱为DB-5ms(30m×0.25mm)。(图5、6)
实施例九、定点突变
采用全式金试剂盒Fast Mutagenesis System对TwCPS2、TwKS进行定点突变操作。
定点突变引物设计
除突变位点外，两条引物长度大约25-30bp，5’端重叠区包含15-20bp，3’端延伸区包含至少10bp；突变位点位于两条引物上，分别位于正向突变引物重叠区下游、紧邻重叠区，反向突变引物5’端(见图3)。
根据以上原则设计定点突变引物，在‘DXDD’功能结构域处将TwCPS2DTDC(311)突变成DTDD(311)，TwCPS2C311D即，将TwKS DADT(318)突变成DADD(318)，即TwKST318D；在TwKS第608位氨基酸处，将氨基酸A突变成M，即TwKSA608M，定点突变引物序列见表4。
表4 定点突变引物序列
定点突变反应
定点突变反应体系：
(1)PCR反应条件：
(2)取10μLPCR产物，1.0％琼脂糖凝胶电泳检测。即使观察到多条扩增条带，如果目的条带大小正确，可继续用DMT消化及转化反应；
(3)加1μLDMT酶于PCR产物中，混匀，37℃孵育1h；
(4)加入2-5μLDMT酶消化产物于50μLDMT感受态细胞中，轻轻混匀，冰浴30min；
(5)42℃准确热激45s，立即置于冰上2min；
(6)加入250μL平衡至室温的LB培养基，37℃225rpm培养1h；
(7)取200μL菌液涂板，37℃倒置培养过夜。
(8)蓝白菌落筛选及阳性克隆的初步筛选，方法同2.2.3.1.4，并送样测序验证能突变是否成功。
实施例十、酵母发酵
雷公藤二萜合酶真核表达载体构建
采用双酶切方法分别将二萜合酶基因TwCPS1、TwGES2及组合TwCPS3+TwKS、TwCPS3+TwKSA608M构建到真核表达载体pESC-Trp上，方法同3.2.1。先将TwCPS1、TwGES2、TwKS、TwKSA608M PCR产物(纯化)及载体质粒pESC-Trp用限制性内切酶进行双酶切，采用NEB公司T4DNA连接酶定向连接，连接产物经转化、蓝白菌落筛选及阳性克隆的初步筛选，并送样测序鉴定，得到经测序核苷酸序列无突变重组质粒pESCTwCPS1、pESCTwGES2、pESCTwKS及pESCTwKSA608M，再将TwCPS3 PCR产物(纯化)及重组质粒pESCTwKS、pESCTwKSA608M用限制性内切酶进行双酶切，采用NEB公司T4DNA连接酶定向连接，连接产物经转化、蓝白菌落筛选及阳性克隆的初步筛选，并送样测序鉴定，得到经测序核苷酸序列无突变重组质粒pESC-(TwCPS3+TwKS)、pESC-(TwCPS3+TwKSA608M)。见图4，引物序列见表5。
表5 构建真核表达载体引物序列
酵母感受态制备
SD-His固体平板：SD-His+2％葡萄糖+4％Agar；不加Agar则成为相应液体培养基；SD-His-Trp固体平板：SD-His-Trp+2％葡萄糖+4％Agar；不加Agar则成为相应液体培养基。
BY-T20酵母菌是在缺陷型酵母菌株BY4741(基因型：MATa his3Δ1 leu2Δ0met15Δ0 ura3Δ0)基础上进行改造，致使其高效合成GGPP，并能回补His，故将BY-T20酵母菌涂布SD-His(缺His)固体平板，30℃倒置培养72h。
采用ZYMO RESEARCH Frozen-EZ Yeast Transformation II试剂盒做酵母感受态细胞：
(1)从SD-His平板上挑取新活化的单菌落，接种于10mL SD-His液体培养基中，30℃下振荡培养至OD600＝0.8-1.0左右；
(2)室温，500g离心4min，去上清；
(3)加入10mL Frozen-EZ Solution 1悬浮菌体，室温，500g离心4min，去上清；
(4)加入1mL Frozen-EZ Solution 2悬浮菌体，分装至灭菌的1.5mL EP管中，每管50μL；
(5)禁止用液氮速冻感受态细胞，应缓慢降温至-70℃(4℃，1h；-20℃，1h；-40℃，1h；-70℃保存)。转化
(1)取0.2-1μg重组质粒(少于5μL)与50μL感受态细胞混合；
(2)加入500μL Frozen-EZ Solution 3，剧烈混匀；
(3)30℃孵育45min，期间在此混匀2-3次；
(4)取50-150μL孵育的菌液，涂布相应缺陷型SD平板(SD-His-Trp)，晾干后，置于30℃倒置培养48-96h。
发酵
酵母质粒提取及鉴定
挑取SD-His-Trp固体平板上长出来的单菌落，置于10mLSD-His-Trp液体培养基中，30℃250rpm 48h；提取质粒，用特异性引物PCR验证重组质粒是否转入BY-T20酵母菌。
酵母质粒提取(天根公司酵母质粒小提试剂盒)：
(1)柱平衡：向吸附柱CP2中(吸附柱放入收集管中)加入500μL的平衡液BL，12000rpm离心1min，倒掉收集管中的废液，将吸附柱重新放回收集管中(当天处理当天使用)；
(2)取1-5mL酵母培养物，12000rpm离心1min，尽量吸除上清(菌液较多时可以通过多次离心将菌体收集到一个离心管中)；
(3)向菌体中加入250μL溶液YP1(已加入RNaseA)重悬沉淀，彻底悬浮菌体，加入直径为0.45-0.55mm的酸洗玻璃珠，涡旋振荡10min；
(4)向管中加入250μL溶液YP2，温和地上下翻转6-8次，使菌体充分混匀，室温放置5-10min(避免剧烈震荡，以免污染基因组DNA)；(5)向管中加入350μL溶液YP3，立即温和地上下翻转6-8次，充分混匀，此时会出现白色絮状沉淀，12000rpm离心20min；取上清部分，再次离心12000rpm离心20min，以得到无微小白色沉淀的上清液；
(6)小心将上清液加入吸附柱CP2中(吸附柱放入收集管中)，12000rpm离心1min，倒掉废液，将吸附柱CP2放入收集管中；
(7)向吸附柱CP2中加入500μL缓冲液PD，12000rpm离心1min，倒掉废液；
(8)向吸附柱CP2中加入600μL漂洗液(已加入无水乙醇)，12000rpm离心1min，倒掉废液，将吸附柱CP2放入收集管中；
(9)重复步骤8；
(10)将吸附柱CP2放入收集管中置于12000rpm离心2min，去除残余的漂洗液；
(11)将吸附柱CP2置于一个干净的1.5mLEP管中，向吸附膜的中间部位滴加50-100μLddH2O，室温放置2min，12000rpm离心2min将质粒溶液收集到EP管中，保存-20℃。
所提取得质粒用于PCR鉴定重组质粒是否转入BY-T20酵母菌中。
PCR反应体系：
PCR反应条件：
(备注：＊表示PCR产物的片段＞3kb，且每加1kb则延伸温度延长1min)
1.0％琼脂糖凝胶电泳检测，若出现目的片段条带，则可以证明重组质粒成功转入菌体。
半乳糖诱导发酵
诱导型SD-His-Trp液体培养基：SD-His-Trp+2％半乳糖(过滤除菌)(1)将PCR鉴定的阳性菌液(即重组质粒成功转入的BY-T20酵母菌)，转接入50mL SD-His-Trp液体培养基中，30℃250rpm培养12-16h；(2)室温5000g 5min离心收集菌体，转接到100mL诱导型SD-His-Trp液体培养基中，30℃250rpm诱导72h；
发酵产物提取
目标成分为萜类化合物，脂溶性，易溶于正己烷，因此选取正己烷为溶剂萃取目标萜类化合物。萃取步骤如下：
(1)收集发酵完成菌液，加入等体积的正己烷；
(2)超声破菌1h，期间多次振荡混摇；
(3)分液漏斗萃取两次；混合两次的萃取液，加入适量的无水硫酸钠(120℃烘干30min)，边加边摇，出去萃取液的水分；
(4)旋转蒸发仪上浓缩至近干，
(5)吸取浓缩液，过0.22μm PTFE针头滤器，过滤液储存于液相小瓶中，封口膜密闭，保存于4℃冰箱。
提取物GC-MS检测
GC-MS分析条件为：取1μL的进样，无分流的模式下，50℃保持2min，20℃·min-1升至300℃，保持20min；进样口温度250℃，离子源温度250℃，电子能量70ev，对样品进行20-650m/z范围扫描。GC-MS仪器为Thermo Scientific公司Thermo TRACE 1310/TSQ 8000gaschromatograph，色谱柱为DB-5ms(30m×0.25mm)。(图7、8、9)
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
<120> 雷公藤二萜合酶TwCPS1在制备松香烷型二萜化合物中的应用
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 2792
<212> DNA
<213> Tripterygium wilfordii. Hook. f.
<400> 1
atatgcatag tttgttaatg aaaaaggtga tcatgtactc ctctcaaaca acccacgtct 60
tctcttcccc tctccactgc accatcccaa aatcttcttc cttcttcctc gatgccccgg 120
tggtgcgtct ccattgtcta agtggccacg gtgctaagaa gaaacgactt cactttgaca 180
ttcaacaggg aagaaatgct ataagcaaga ctcacactcc agaagactta tatgccaaac 240
aggaatacag tgtgccagag attgtgaagg atgacgacaa ggaagaggaa gtggtcaaga 300
ttaaagaaca tgttgacatc attaagtcca tgttgagctc aatggaggat ggagagatta 360
gcatttcagc ttatgacact gcttgggttg cactcataca agacattcac aacaatggtg 420
ctcctcaatt cccatcgagc ctgctctgga ttgctgaaaa tcagctaccg gacgggtcat 480
ggggtgacag tcgcgtattt ttagcatttg atcgaataat caatacatta gcttgtgttg 540
ttgctctcaa gtcatggaat gttcaccctg ataaatgcga aagaggaatc tcctttttga 600
aagaaaatat aagcatgcta gaaaaagacg attccgagca catgcttgtt ggctttgaat 660
ttggtttccc tgtgttgctt gacatggctc gaagattagg catcgatgtt cctgatgatt 720
cgccattctt gcaagctatc tacgtccaga gagacctgaa actcaaaagg ataccaaaag 780
acatattgca caatgtgccc acaacactgc tccatagctt ggaagcaata ccagacctgg 840
attggacaaa gcttctaaaa ctacaatgtc aagatggttc actcttgttc tccccatcct 900
ccactgccat ggcattcatc aacactaagg acgaaaactg cttgagatat ctaaattacg 960
tggtccagag attcaatggt ggagccccaa cggtttaccc ctacgacttg tttgagcaca 1020
attgggcggt tgatcgccta caacgcttgg gaatctcaag attttttcag ccagagatta 1080
gagaatgtat gagctatgtt tacaggtatt ggactaaaga tgggatcttc tgcacaagaa 1140
attcacgggt tcatgatgtt gacgatacag ccatgggatt caggttgctt agattgcacg 1200
gctatgaagt tcaccctgat gcatttaggc agtttaagaa gggatgcgag tttatatgct 1260
atgaaggcca atcgcatccg acagtcactg taatgtataa cctgtacagg gcttctcagt 1320
tgatgtttcc tgaagaaaag attcttgatg aggccaagca gttcacagaa aaattcctgg 1380
gagaaaaacg atctgctaat aagctcctag ataagtggat cataactaag gatctgccag 1440
gggaggtggg gttcgcattg gatgttccgt ggtatgtatc cttgcctaga gtagaggcaa 1500
gattcttcat tcaacactac ggcggcgaag atgatgtgtg gcttgacaag gccctttaca 1560
ggatgcccta cgttaacaac aatgtgtatc tagagctagc aaagcttgat tacaactatt 1620
gccaggcatt gcatggaacc gaatggggcc gtatacaaaa gtggtatgaa gaatgtaaac 1680
caagagactt tgggataagc agagaatgcc ttctccgtgc atattttatg gctgctgcca 1740
gtatatttga gccagaaagg tcgatggagc gcctcgcctg ggctaagact gcgatcttgc 1800
tggaaattat agtgtcttat ttcaatgagg ttggaaattc cacagagcag aggatagcct 1860
tcacaactga attttcgata cgtgcaagtc ctatgggagg ctacataaat ggaagaaagt 1920
tagataagat tggtacgacc caagaactta ttcagatgct actcgcaacc atcgatcagt 1980
tttcgcagga tgcattcgcg gcatatgatc acgacattac tcgccattta cacaactctt 2040
ggaaaatgtg gctgctaaag tggcaagaag aaggagatag atggctggga gaagcagagt 2100
tagtgataca aactataaat ctcatggcgg atcataagat cgctgagaag ctctttatgg 2160
gacatactaa ctatgaacaa ctcttcagcc tcaccaacaa agtttgctat agtcttggtc 2220
atcatgagct ccaaaacaac agagaactgg agcatgacat gcaaagacta gtgcagttgg 2280
tgctgacgaa ttcctcagat ggtatcgact ctgatatcaa gaaaacgttt ctcgcagtcg 2340
caaagagatt ctactatact gcctttgtcg accctgagac cgtgaatgtc cacattgcca 2400
aagtactctt tgagagagta gattaacact gttgatgatg atcttgattt tcaaatattg 2460
agtagtacta ctacttgtga acaaaatctt atgggtgcta tgaacgaaga acaacaatat 2520
ggacgaagac aagtagattt aattttattt gctaatggtc agtacatagg gatgtacctc 2580
tttcaaccat gtagcaactt gctgccttcc ctgccttact ctcacgcttc gtaggggaaa 2640
aggtctcaag gatatatatg agaatgaagt ctgttgtgta tgtaactgac tgaaagtcga 2700
ttttcgatat aaaaagtagg catggatgat ggatcgttca tacatggaaa gttcctttct 2760
ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 2792
<210> 2
<211> 807
<212> PRT
<213> Tripterygium wilfordii. Hook. f.
<400> 2
Met His Ser Leu Leu Met Lys Lys Val Ile Met Tyr Ser Ser Gln Thr
1 5 10 15
Thr His Val Phe Ser Ser Pro Leu His Cys Thr Ile Pro Lys Ser Ser
20 25 30
Ser Phe Phe Leu Asp Ala Pro Val Val Arg Leu His Cys Leu Ser Gly
35 40 45
His Gly Ala Lys Lys Lys Arg Leu His Phe Asp Ile Gln Gln Gly Arg
50 55 60
Asn Ala Ile Ser Lys Thr His Thr Pro Glu Asp Leu Tyr Ala Lys Gln
65 70 75 80
Glu Tyr Ser Val Pro Glu Ile Val Lys Asp Asp Asp Lys Glu Glu Glu
85 90 95
Val Val Lys Ile Lys Glu His Val Asp Ile Ile Lys Ser Met Leu Ser
100 105 110
Ser Met Glu Asp Gly Glu Ile Ser Ile Ser Ala Tyr Asp Thr Ala Trp
115 120 125
Val Ala Leu Ile Gln Asp Ile His Asn Asn Gly Ala Pro Gln Phe Pro
130 135 140
Ser Ser Leu Leu Trp Ile Ala Glu Asn Gln Leu Pro Asp Gly Ser Trp
145 150 155 160
Gly Asp Ser Arg Val Phe Leu Ala Phe Asp Arg Ile Ile Asn Thr Leu
165 170 175
Ala Cys Val Val Ala Leu Lys Ser Trp Asn Val His Pro Asp Lys Cys
180 185 190
Glu Arg Gly Ile Ser Phe Leu Lys Glu Asn Ile Ser Met Leu Glu Lys
195 200 205
Asp Asp Ser Glu His Met Leu Val Gly Phe Glu Phe Gly Phe Pro Val
210 215 220
Leu Leu Asp Met Ala Arg Arg Leu Gly Ile Asp Val Pro Asp Asp Ser
225 230 235 240
Pro Phe Leu Gln Ala Ile Tyr Val Gln Arg Asp Leu Lys Leu Lys Arg
245 250 255
Ile Pro Lys Asp Ile Leu His Asn Val Pro Thr Thr Leu Leu His Ser
260 265 270
Leu Glu Ala Ile Pro Asp Leu Asp Trp Thr Lys Leu Leu Lys Leu Gln
275 280 285
Cys Gln Asp Gly Ser Leu Leu Phe Ser Pro Ser Ser Thr Ala Met Ala
290 295 300
Phe Ile Asn Thr Lys Asp Glu Asn Cys Leu Arg Tyr Leu Asn Tyr Val
305 310 315 320
Val Gln Arg Phe Asn Gly Gly Ala Pro Thr Val Tyr Pro Tyr Asp Leu
325 330 335
Phe Glu His Asn Trp Ala Val Asp Arg Leu Gln Arg Leu Gly Ile Ser
340 345 350
Arg Phe Phe Gln Pro Glu Ile Arg Glu Cys Met Ser Tyr Val Tyr Arg
355 360 365
Tyr Trp Thr Lys Asp Gly Ile Phe Cys Thr Arg Asn Ser Arg Val His
370 375 380
Asp Val Asp Asp Thr Ala Met Gly Phe Arg Leu Leu Arg Leu His Gly
385 390 395 400
Tyr Glu Val His Pro Asp Ala Phe Arg Gln Phe Lys Lys Gly Cys Glu
405 410 415
Phe Ile Cys Tyr Glu Gly Gln Ser His Pro Thr Val Thr Val Met Tyr
420 425 430
Asn Leu Tyr Arg Ala Ser Gln Leu Met Phe Pro Glu Glu Lys Ile Leu
435 440 445
Asp Glu Ala Lys Gln Phe Thr Glu Lys Phe Leu Gly Glu Lys Arg Ser
450 455 460
Ala Asn Lys Leu Leu Asp Lys Trp Ile Ile Thr Lys Asp Leu Pro Gly
465 470 475 480
Glu Val Gly Phe Ala Leu Asp Val Pro Trp Tyr Val Ser Leu Pro Arg
485 490 495
Val Glu Ala Arg Phe Phe Ile Gln His Tyr Gly Gly Glu Asp Asp Val
500 505 510
Trp Leu Asp Lys Ala Leu Tyr Arg Met Pro Tyr Val Asn Asn Asn Val
515 520 525
Tyr Leu Glu Leu Ala Lys Leu Asp Tyr Asn Tyr Cys Gln Ala Leu His
530 535 540
Gly Thr Glu Trp Gly Arg Ile Gln Lys Trp Tyr Glu Glu Cys Lys Pro
545 550 555 560
Arg Asp Phe Gly Ile Ser Arg Glu Cys Leu Leu Arg Ala Tyr Phe Met
565 570 575
Ala Ala Ala Ser Ile Phe Glu Pro Glu Arg Ser Met Glu Arg Leu Ala
580 585 590
Trp Ala Lys Thr Ala Ile Leu Leu Glu Ile Ile Val Ser Tyr Phe Asn
595 600 605
Glu Val Gly Asn Ser Thr Glu Gln Arg Ile Ala Phe Thr Thr Glu Phe
610 615 620
Ser Ile Arg Ala Ser Pro Met Gly Gly Tyr Ile Asn Gly Arg Lys Leu
625 630 635 640
Asp Lys Ile Gly Thr Thr Gln Glu Leu Ile Gln Met Leu Leu Ala Thr
645 650 655
Ile Asp Gln Phe Ser Gln Asp Ala Phe Ala Ala Tyr Asp His Asp Ile
660 665 670
Thr Arg His Leu His Asn Ser Trp Lys Met Trp Leu Leu Lys Trp Gln
675 680 685
Glu Glu Gly Asp Arg Trp Leu Gly Glu Ala Glu Leu Val Ile Gln Thr
690 695 700
Ile Asn Leu Met Ala Asp His Lys Ile Ala Glu Lys Leu Phe Met Gly
705 710 715 720
His Thr Asn Tyr Glu Gln Leu Phe Ser Leu Thr Asn Lys Val Cys Tyr
725 730 735
Ser Leu Gly His His Glu Leu Gln Asn Asn Arg Glu Leu Glu His Asp
740 745 750
Met Gln Arg Leu Val Gln Leu Val Leu Thr Asn Ser Ser Asp Gly Ile
755 760 765
Asp Ser Asp Ile Lys Lys Thr Phe Leu Ala Val Ala Lys Arg Phe Tyr
770 775 780
Tyr Thr Ala Phe Val Asp Pro Glu Thr Val Asn Val His Ile Ala Lys
785 790 795 800
Val Leu Phe Glu Arg Val Asp
805
<210> 3
<211> 36
<212> DNA
<213> 人工序列
<400> 3
cgcggatcca tgcatagttt gttaatgaaa aaggtg 36
<210> 4
<211> 38
<212> DNA
<213> 人工序列
<400> 4
gcactgcagt taatctactc tctcaaagag tactttgg 38
<210> 5
<211> 39
<212> DNA
<213> 人工序列
<400> 5
cgcggatcca tgtctcttca gtatcatgtt ctaaactcc 39
<210> 6
<211> 37
<212> DNA
<213> 人工序列
<400> 6
gcactgcagc tagacttttt gaaacaagac tttggag 37
<210> 7
<211> 32
<212> DNA
<213> 人工序列
<400> 7
cgcggatcca tgtctatcaa ccttcgctcc tc 32
<210> 8
<211> 35
<212> DNA
<213> 人工序列
<400> 8
cgcgtcgact caagttaaag attcttcctg taagc 35
<210> 9
<211> 32
<212> DNA
<213> 人工序列
<400> 9
cgcagcttcg cgataccgat gataccgcca tg 32
<210> 10
<211> 36
<212> DNA
<213> 人工序列
<400> 10
tcatcggtat cgcgaagctg cgaatttctt gtccag 36
<210> 11
<211> 35
<212> DNA
<213> 人工序列
<400> 11
gagatatttt ccgatgctga cgattgtgcc atggc 35
<210> 12
<211> 37
<212> DNA
<213> 人工序列
<400> 12
atcgtcagca tcggaaaata tctcttcatc tccctgc 37
<210> 13
<211> 32
<212> DNA
<213> 人工序列
<400> 13
gttggatttg ctaaagtcta tgttaaggga ag 32
<210> 14
<211> 32
<212> DNA
<213> 人工序列
<400> 14
atagacttta gcaaatccaa ccacatctca at 32
<210> 15
<211> 35
<212> DNA
<213> 人工序列
<400> 15
cgcggatccg atgcatagtt tgttaatgaa aaagg 35
<210> 16
<211> 36
<212> DNA
<213> 人工序列
<400> 16
tcggggccca aatctactct ctcaaagagt actttg 36
<210> 17
<211> 37
<212> DNA
<213> 人工序列
<400> 17
cgcggatccg atggtgatca tgtcctctca tcaaatc 37
<210> 18
<211> 35
<212> DNA
<213> 人工序列
<400> 18
tcggggccca acactctttc aaagagtact ttggc 35
<210> 19
<211> 37
<212> DNA
<213> 人工序列
<400> 19
atttgcggcc gcaatgatgt ctctatcgca tcccaat 37
<210> 20
<211> 37
<212> DNA
<213> 人工序列
<400> 20
cccatcgata ctagagagat gggttcacgt atcaaag 37
<210> 21
<211> 37
<212> DNA
<213> 人工序列
<400> 21
atttgcggcc gcaatgatgt ctctatcgca tcccaat 37
<210> 22
<211> 37
<212> DNA
<213> 人工序列
<400> 22
cccatcgata ctagagagat gggttcacgt atcaaag 37
<210> 23
<211> 34
<212> DNA
<213> 人工序列
<400> 23
cgcggatccg atggattttt cagattcctc aatt 34
<210> 24
<211> 35
<212> DNA
<213> 人工序列
<400> 24
tcggggccca aaatgaaaca tggtgagaat tttgg 35
Claims (7)
1.雷公藤二萜合酶TwCPS1与丹参二萜合酶SmKSL1在提高松香烷型二萜类化合物前体次丹参酮二烯合成效率中的用途，所述雷公藤二萜合酶的氨基酸序列如SEQ ID NO：2所示，所述雷公藤二萜合酶基因的序列如SEQ ID NO：1所示。
2.如权利要求1所述雷公藤二萜合酶TwCPS1与丹参二萜合酶SmKSL1的用途，其特征在于雷公藤二萜合酶与丹参二萜合酶SmKSL1同时使用或先后使用。
3.如权利要求1所述雷公藤二萜合酶TwCPS1与丹参二萜合酶SmKSL1的用途，其特征在于先使用雷公藤合酶将GGPP催化形成CPP，再使用丹参二萜合酶SmKSL1催化CPP合成松香烷型二萜类化合物前体次丹参酮二烯。
4.一种提高松香烷型二萜类化合物前体次丹参酮二烯合成效率的方法，其包括：
(1)将雷公藤二萜合酶基因Twcps1表达盒插入第一原核表达载体中，制备表达外源雷公藤二萜合酶TwCPS1的重组大肠杆菌，超声破碎菌体，取含有重组TwCPS1蛋白的上清液；
(2)将丹参二萜合酶基因Smksl1表达盒插入第二原核表达载体中，制备表达外源丹参二萜合酶SmKSL1的重组大肠杆菌，超声破碎菌体，取含有重组SmKSL1蛋白的上清液；
(3)以GGPP为底物，以步骤(1)中获得的含重组TwCPS1蛋白的上清液为催化剂，催化GGPP产生CPP；
(4)以步骤(3)获得的CPP为底物，以步骤(2)获得的含重组SmKSL1蛋白的上清液为催化剂，催化CPP产生松香烷型二萜类化合物前体。
5.如权利要求4所述提高松香烷型二萜类化合物前体次丹参酮二烯合成效率的方法，步骤(1)所述的原核表达载体为pMAL-c2X，所述Twcps1基因插入BamH I和Sal I之间。
6.如权利要求4所述提高松香烷型二萜类化合物前体次丹参酮二烯合成效率的方法，在步骤(3)的催化反应完成后还包括脱磷的步骤。
7.如权利要求4所述提高松香烷型二萜类化合物前体次丹参酮二烯合成效率的方法，在步骤(4)完成后还包括对反应产物进行GC-MS分析的步骤，其中在第13.06分出现的峰为次丹参酮二烯。
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