CN1130460C - 牻牛儿基二磷酸合酶基因 - Google Patents
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Abstract
具有通过以较Ser更大分子量的另一种氨基酸取代82位的氨基酸而源自法尼基二磷酸合酶氨基酸序列的氨基酸序列的牻牛儿基二磷酸合酶。更为具体地,为下面的重组蛋白(a)和(b)及编码它们的基因:(a)具有由SEQ ID NO:1所示表氨基酸序列的蛋白;和(b)具有通过在82位缺失、取代或添加至少一个氨基酸而源自由SEQ IDNO:1所示氨基酸序列的氨基酸序列并显示出牻牛儿基二磷酸合酶活性的蛋白。
Description
技术领域:
本发明涉及以下内容:牻牛儿基二磷酸合酶,编码该酶的基因,包含该基因的重组载体以及分别制备牻牛儿基二磷酸合酶和牻牛儿基二磷酸的方法。
背景技术:
在有机体内有重要功能的物质中、有相当数量的是由异戊二烯(2-甲基-1,3-丁二烯)单位生物合成的。这些化合物也称作类异戊二烯、类萜或萜。根据它们的碳原子数目可分为半萜(C5)、单萜(C10)、倍半萜(C15)、二萜(C20)、二倍半萜(C25)三萜(C30)四萜(C40)等。
这些物质的实际生物合成是从异戊烯基二磷酸(IPP),即活化的异戊二烯单位开始。因此,以前推测为可能的前体物质的异戊二烯真正形式是IPP,即所谓的活化的异戊二烯单位。
众所周知,二甲基烯丙基二磷酸(DMAPP),一种IPP的异构体,通过与IPP缩合可以合成活化的类异戊二烯,例如牻牛儿基二磷酸,(GPP),橙花基二磷酸、法尼基二磷酸,(FPP),牻牛儿基牻牛儿基二磷酸,牻牛儿基牻牛儿基二磷酸(GGPP),六(异戊二烯基)二磷酸(HexPP)或七(异戊二烯基)二磷酸(HepPP)。
许多复合物如天然橡胶,多萜醇,细菌萜醇(十一萜醇)或植物中所见的各种多萜醇(polyprenols)都是FPP,GPP之类的物质通过顺式-缩合反应形成的。在FPP、GPP等中,一般将所有E-构型认为是活化构型。一般认为这些复合物是通过利用焦磷酸与分子中碳骨架间的磷酸键的能量经连续缩合合成的,(因此)认为焦磷酸是缩合反应的副产物。
类异戊二烯基合酶的活化形式即顺序将IPP缩合成烯丙基底物如DMAPP,GPP,FPP,GGPP,GFPP等的酶称为异戊二烯基二磷酸合酶或异戊二烯基转移酶。异戊二烯基二磷酸合酶根据它们主要反应产物的碳原子有不同的命名。例如,催化生成有15个碳原子的法尼基二磷酸的酶称作法尼基二磷酸合酶(FPP合成酶),并且催化合成含20个碳原子的牻牛儿基牻牛儿基二磷酸的酶被称作牻牛儿基牻牛儿基二磷酸合酶(GGPP合酶)。
各种各样的戊二烯基二磷酸合酶基因已经从细菌、古细菌、真菌、植物和动物中获得。FPP合酶有关GGPP合酶、六(异戊二烯基)二磷酸合酶,七(异戊二烯基)二磷酸合酶,八(异戊二烯基)二磷酸合酶九(异戊二烯基)二磷酸合酶(茄呢基(solanesyl)二磷酸合酶)、十一(异戊二烯基)二磷酸合酶等的纯化、活性测定及基因克隆和DNA测序已有报导。
这些异戊二烯基二磷酸合酶无论是从工业角度还是从生合科学角度来看对于各种各样重要化合物的合成都是很重要的,然而这些酶一般都不稳定,并且比活性低。这样它们在工业中的应用就受到限制。然而,近几年来热稳定性的FPP合酶基因和GGPP合酶基因已经从嗜热细菌和古细菌中分离出来[A.Chen和D.Poulter(1993),生物化学杂志268(15),11002-11007,T.Koyama等,(1993),生化杂志(东京),113(3),355-363;S.-i.Ohnuma等.,(1994),生物化学杂志,269(20),14729-14797]。因此运用异戊二烯基二磷酸合酶的条件现已具备。
合成C10-25的异戊二烯基二磷酸的酶是同源二聚体。让它在体外进行反应相对而言比较容易,已有许多关于它们反应的报道。在这些酶中有一种酶具有特异合成GPP(一种C10异戊二烯基二磷酸)的活性,但它仍未被分离出来,虽有对它部分纯化的报道(L.Heide和V.Berger.1989。生物化学和生物物理文献.,273(2)331-8)。尽管已有报道说已从猪肝中成功地纯化了GPP合酶(J.K.Dorsey等.1996生物化学杂志。241(22)5353-5360),但是这个酶同时也催化FPP的合成。这样,根据现在的异戊二烯基二磷酸合成酶的定义,这种酶应被称为FPP合酶。
GPP是许多已知单萜合成过程中的第一个中间物,也是单萜生物合成途径中的最重要一个化合物。
具有代表性的单萜牻牛儿醇及其异构物橙花醇是玫瑰油主要成分中的芳香组分。另一种代表性的单萜樟脑(从香樟(Cinnamomumcamphora)中提炼出来的),常用作卫生球。
然而,GPP合酶基因至今仍未分离到。
在这种情况下就迫切需要产生一种技术,这种技术可以人工修饰嗜热菌来源的,稳定的同源二聚体型的具有高比活的异戊二烯基二磷酸合酶的氨基酸序列,这样就可以加特异催化GPP合成的同源二聚体型热稳定的异戊二烯基二磷酸合成酶。
嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)FPP合酶和嗜酸热硫化叶菌(Sulfolobus acidocaldarius)GGPP合酶二者均是嗜热的异戊烯基二磷酸合酶,并且二者均已被修饰。嗜酸热硫化叶菌(S.acidocaldarius)GGPP合酶的突变体及其基因是利用其能弥补HexPP合成缺陷型啤酒糖酵母(Saccharomyces serevisiae)(出芽酵母)甘油代谢能力作为指标筛选出来的。[S.-i.Ohnuma等.,(1996),生物化学杂志,271(31),18831-18837]嗜热脂肪芽孢杆菌(S.stearothermophilus)来源的具有GGPP合成活性的FPP合酶的突变体及其基因是通过利用蕃茄红素的合成途径作为一种指标筛选出来的。[S.-I.Ohnuma等.,(1996),生物化学杂志,271(17),10087-95]另外,18种可以按不同比例合成从GGPP到HexPP的突变酶及其基因也通过定点突变获得。这些点突变位于富含Asp的保守区-I(DDXX(XX)D)的上游第5个氨基酸处。[S.-i.Ohnuma et al(1996),JBC.chem.,271(48)30748-30750]。已经发现富含Asp的结构域保守区I(DDXX(XX)D)上游的第5个氨基酸残基是调节反应产物链长度的部位。
然而,还没有获得具有特异合成GPP活性的突变酶。
发明内容:
本发明的目的是提供牻牛儿基二磷酸合酶及编码该酶的基因。
对上述问题进行广泛和深入的研究之后本发明人成功地分离了牻牛儿基二磷酸合酶及其基因。这个牻牛儿基二磷酸合酶基因是通过替换法尼基二磷酸合酶的一段氨基酸序列得到的。这样就完成了本发明。
本发明是关于下述重组蛋白(a)或(b):
(a)一种蛋白,其氨基酸序列如SEQ NO:1所示;
(b)一种蛋白,其氨基酸序列如SEQ NO:1所示同时除了82位氨基酸外至少有一氨基酸的缺失,取代或添加并且该蛋白具有牻牛儿基二磷酸合酶活性。
而且本发明涉及编码上述重组蛋白(a)或(b)的基因。
而且本发明涉及一个牻牛儿基二磷酸合酶基因,其如SEQ ID NO:2所示的包括;
而且本发明涉及包含上述任何基因的重组载体。
而且本发明涉及上述重组载体转化的转化子。
本发明涉及一种制备牻牛儿基二磷酸合酶的方法,该方法包括将上述转化子在一种培养基中培养并且从上述培养物中回收牻牛儿基二磷酸合酶。
本发明涉及制备牻牛儿基二磷酸的方法,该方法包括将上述转化子在培养基中培养并从该培养基中回收牻牛儿基二磷酸。
本发明涉及制备牻牛儿基二磷酸的方法,包括上述转化子的培养物作用于异戊烯基二磷酸或其异构物。
下文将详细陈述本发明。
已经知道在异戊烯基二磷酸合酶的氨基酸序列中有5个保守区域,(如果合酶在其中一个亚基的氨基酸序列中是异二聚体)。[A,Chen等,(1994)蛋白质科学(Protein Sci).,3(4),600-607]。在这5个保守区中,(保守区I-V),有二个富含天冬氨酸残基的区域,据信,反应产物或反应底物结合于此。这些区域称为“天冬氨酸富含区”或“Asp-富含区”。为了区别起见在这二个区域中位于异戊二烯基二磷酸合酶氨基端的(即位于上述保守区-II)Asp-富含区称作Asp-富含区I,[序列:DDXX(XX)D,其中括号中的XX可能不存在],羧基末端的Asp-富含区(即位于上述保守区V)被命名为Asp-富含区II。
如上所述含有Asp-富含区域的异戊二烯基二磷酸合酶的具体实例包括法尼基二磷酸合酶,牻牛儿基牻牛儿基二磷酸合酶。六(异戊二烯基)二磷酸合成酶,七(异戊二烯基)二磷酸合酶,八(异戊二烯基)二磷酸合酶九(异戊二烯基)二磷酸合酶和十一(异戊二烯基)二磷酸合酶。更具体的实例包括嗜热脂肪芽孢杆菌法尼基二磷酸合酶,大肠杆菌法尼基二磷酸合酶,大鼠法尼基二磷酸合酶,人法尼基二磷酸合酶,啤酒糖酵母六(异戊二烯基)二磷酸合酶等。上述例子中细菌法尼基二磷酸合酶的保守区I-IV的氨基酸序列列于表4。在表4中“1.”代表嗜热脂肪芽孢杆菌法尼基二磷酸合酶的氨基酸序列,“2.”代表大肠杆菌法尼基二磷酸合酶的氨基酸序列。用大一点框标的区域代表Asp-富含区-I,用星号(☆)标记氨基酸残基表示Asp-富含区-I上游的第4个氨基酸残基。
本发明的特征在于用一种分子量较大的氨基酸残基取代Asp-富含区I上游第4个氨基酸残基而产生的牻牛儿基二磷酸合酶,另一个就是通过生产的牻牛儿基二磷酸合酶经过酶反应制备牻牛儿基二磷酸。更特异的是通过从用分子量大的氨基酸取代位于氨基端组成Asp富含区-I的DDXX(XX)D上游4个氨基酸处标有“☆”的图4中氨基酸残基(Ser)产生了一种牻牛儿基二磷酸合酶。(取代氨基酸为除了Gly和Ala之外的任何氨基酸,即下列任一种Val,Leu,Ile,Thr,Asp,Glu,Asn,Gln,Lys,Arg,Cys,Met,Phe,Tyr,Trp,His或Pro),上述取代氨基酸只要不是Gly或Ala就没有特别的限制。优选使用Phe。
特别地,本发明的牻牛儿基二磷酸合酶可以通过用例如Phe取代SEQ ID NO:5中所示的法尼基二磷酸合酶的第82位丝氨酸而得到。
这种取代可以通过部分修饰编码脂肪嗜热芽孢杆菌FPP合酶的基因得到,这种酶据报道有很高的热稳定性和高的比活性。(1)诱变制备靶基因:
将要导入突变的基因是脂肪嗜热芽孢杆菌编码FPP合酶的基因(下文缩写成“BstFPS”)。BstFPS基因的全序列已知(T.Koyama等.,(1993)生化杂志.,113,355-363;SEQ ID NO:4),并以许可号NO.D13293公开于遗传信息数据库如DDBJ中。
因为脂肪嗜热芽孢杆菌可以在许多菌种保藏中心,如ATCC(ATCC10149)中均可以找到,因此BstFPS的基因通过常规基因克隆手段得到。(S.Sambrook等(编)(1989)分子克隆,冷泉港实验室出版社,纽约)。
随后,这个基因片断连入适当的载体,(如pTV118N,FakaraShuzo提供)构建成进行诱变的质粒。这个质粒命名为pFPS。(2)诱变用寡核苷酸的合成及突变的引入
设计一段诱变用的寡核苷酸,这段寡核苷酸(a)BstFPS的82位Ser的密码子以除Gly,Ala,Ser外的任一氨基酸的密码子取代(如Phe)取代;(b)新引入BspHI限制酶位点(5′TCA TGA3′)。例如,可用下述的寡核苷酸。5′-CAT ACG TAC TTC TTG ATT CAT GATGAT TTG-3′(SEQ ID NO:6)设计这段寡核苷酸从而即便在引入BspHI位点之后因为密码子简并性也不改变BstFPS的氨基酸序列。因为引入了这个限制酶位点,其中导入取代突变的质粒可以在BspHI消化后用琼脂糖电泳检测鉴定出来。
寡核苷酸的合成可用常规的化学合成仪器合成。优选地,合成的寡核苷酸磷酸化,并且变性(例如70℃10min加热)。
随后,用此寡核苷酸作引物向上述质粒中引入突变。引入突变的方法并不特别限定。例如,可使用以Kunkel方法为基础的商用试剂盒(美国自然科学院院报(1985)82,488)(Takara Skuzo公司的Mutan-K试剂盒),或者也可用聚合酶链式反应(PCR)。
制备单链DNA作模板,以上述寡核苷酸做引物与模板退火,作为DNA合成的互补链引物以制备双链DNA。得到的DNA引入质粒后,以这种质粒转化大肠杆菌。
本发明的基因可以很容易地获得,例如,通过定点诱变或PCR等常规方法在合酶的天然氨基酸序列(SEQ ID NO:4)中引入突变。
这些得到的转化子克隆,及核苷酸序列可以测定。测序可以用各种常规的方法,如Maxam-Gilbert法或双脱氧法进行。通常用根据双脱氧法的自助DNA测序仪进行测序。
SEQ ID NO:2例示了本发明基因的核苷酸序列,SEQ ID NO1和3,例示了本发明中牻牛儿基二磷酸合酶的氨基酸序列,这些酶在除了82位(phe)氨基酸外可能至少有一个氨基酸突变,如缺失,替代或添加至少一个氨基酸(如一个或数个氨基酸),这些酶尽管有突变但都有牻牛儿基二磷酸合酶活性。例如,SEQ ID NO:1或3第一位Met缺失,但仍包括在本发明的牻牛儿基二磷酸合酶内。同样编码这些酶的基因也包括在本发明基因内。
这里使用的“牻牛儿基二磷酸合酶活性”是指以IPP或其异构物(如DMAPP)为底物合成GPP的催化活性。可以用上述同样的方法引入突变。
一旦本发明牻牛儿基二磷酸合酶基因的核苷酸序列已经建立起来就可以通过化学合成,或以该基因为模板进行PCR,或者通过含该基因核苷酸序列的DNA中段作为探针进行杂交的方法获得该基因。(3)载体的构建
本发明的重组载体可以通过将本发明基因连入一个合适的载体而建成。这种待插入本发明基因的载体只要是在宿主中可以复制的就可以,没有特别的限制。可用碱裂解提取法或其他改进的方法从大肠杆菌等细菌中制备可用于制备本发明载体的载体。(Birnboim,H.C.&Doly,J.(1979)核酸研究。7:1513)。作为选择,商用载体或可根据目的诱导的载体也可以使用。例如pBR322,pBR327,pKK233-2,pKK233-3或有pMB1-来源的复制起始的pTrc99A。其它的如pUC18,pUC19,pUC118,pUC119,pTV118N,pTV119N,pBluescvipt,pHSG298或pHSG396(这二种均被修饰以获得高拷贝),或者是pSC101,COlE1因子,R1质粒或F因子。再进一步,融合蛋白表达载体如pGEX或pMal载体这种利于表达产物纯化的载体也可以用。
用病毒载体(如λ噬菌体或M13噬菌体)或转化子也可以代替质粒进行基因转移。作为噬菌体DNA,M13mp18,M13mp19,λgt10、λgt11之类均可用。
将编码牻牛儿基二磷酸合酶的DNA片段掺入载体可用常规的方法,使用合适的限制酶和连接酶而进行。例如,可利用将纯化的DNA用合适的限制酶消化后插入合适载体的相应酶位点之中而连接的方法。
本发明的基因连入载体中要能显示出它的活性。因此,本发明载体可含有一个复制起点。和适用于待用宿主的表达调控序列。而且此载体要同时含转录启动子,转录终止子,核糖体结合位点等。启动子可用Ptac,Plac或Ptrc,终止子可用rrnB,核糖体结合位点可用SD序列(以5′-AGGAGG-3′为代表)。
作为按上述方法制备质粒的具体实例,pFPSm作为例子列出。(4)转化子的制备
本发明转化子可通过将本发明的重组表达载体转入宿主菌而获得,从而目的基因可得到表达。
宿主菌只要可以表达目的基因就可以,并没有特别的限制。宿主菌具体实例包括埃希氏杆菌属或芽孢杆菌属细菌如大肠杆菌和枯草芽孢杆菌,短芽孢杆菌(B.brevis),酵母属(Saccharomyos)或毕赤酵母属(Pichia)酵母,如啤酒糖酵母(S,cerevisiae),巴斯德毕赤酵母(P.pastris);曲霉曲属的丝状真菌,如半曲霉(A.oryzae),黑曲霉(A.niger);蚕的培养细胞,动物细胞如COS,CHO细胞或植物细胞。
当细菌如大肠杆菌用作宿主菌时,优选本发明的重组载体可以在此宿主内自主复制,且同时载体由转录启动子,核糖体结合位点,本发明的DNA及转录终止子所组成。载体还包括一个转录启动子的调节基因。
启始从DNA到mRNA转录的启动子序列可用一段天然序列(如lac,trp,bla,lpp,PL,PR,T3或T7)。此外已知在本发明中还可以用它们的突变体(如lacUV5)或天然启动子序列融合形成的序列(如tac,trc等)。
至于调控由mRNA到蛋白质合成能力的序列,现在已知核糖体结合位点(GAGG或相似序列)和起始密码子(ATG或GTG)之间的距离很重要。更进一步说,现在众所周知3′端的转录终止子(如rrnBT1T2)影响重组子中的蛋白合成效率。因此本发明中可通过运用这些序列使得基因表达有效地进行。
作为将外源基因导入细菌中的方法,各种将DNA导入细菌中的方法都可以用。例如用钙离子(美国自然科学院院报69:2110-2114(1972)),电穿孔等均可以。
若用酵母作宿主菌,YEp13,YEp24,YCp50等可用作表达载体。至于用于这种情形下的启动子,只要在酵母菌中可以引导目的基因表达的启动子均可用。例如,gal1启动子,gal10启动子热休克蛋白启动子,MFα1启动子等均可用。
至于将外源基因导入酵母中的方法,任何将DNA导入酵母的方法均可用,如电穿孔[酶学方法,194:182-7(1990)],原生质球法,[美国自然科学院院报84:1929-1933(1978)],醋酸锂法,[细菌学杂志(J.Bacteriod),153:163-168(1983)]等。
当用动物细胞作宿主时,pcDNAI/Amp,pcDNAI等可用作表达载体,在这种情况下,可用人细胞肥大病毒早期基因启动子等作为启动子。
作为将外源基因转入动物细胞的方法,如电穿孔,磷酸钙法,脂质体转染法等可用。
作为将外源基因转入植物细胞的方法,用土壤杆菌感染法广泛应用。直接导入的方法,如原生质体法,电穿孔法,粒子轰击法等均可以。
本发明的重组载体转入大肠杆菌DH5α,[命名为pFPSm(S82F)/DH5α],根据布达佩斯条约于1997年12月12日保藏于国立生命科学及人体技术研究所,工业科技院,(日本茨城县筑波市东1丁目1番3号),保藏号为FERM BP-6551。(5)牻牛儿基二磷酸合酶的制备
本发明的牻牛儿基二磷酸合酶可通过在培养基中培养上述转化子并从得到培养物中回收该合酶而获得。
本发明转化子在培养基中的培养从与通常宿主菌的培养方法相似的方法进行。
作为培养获自微生物宿主如大肠杆菌或酵母的转化子的培养基。只要培养基须含有可被微生物吸收的碳源、氮源及无机盐以便重组子的培养有效地进行,天然培养基或合成培养基都可以。
碳源可用碳水化合物如葡萄糖,果糖,蔗糖,淀粉,有机酸可用醋酸,丙酸,柠檬酸;醇类可用甘油,甲醇,乙醇,丙醇。
氮源可用氨,无机酸或有机酸的铵盐,如氯化铵,硫酸铵,醋酸铵,磷酸铵;或其他含氮化合物;蛋白胨;肉提取物;谷物浸汁等。
无机物可用磷酸二氢钾,磷酸氢二钾,磷酸镁,硫酸镁,氯化镁,氯化钠,硫酸亚铁(II),硫酸锰,硫酸铜,碳酸钙,氯化钙等。
当大肠杆菌用作宿主时,通常都是在需氧条件下(如摇瓶培养或通气培养)37℃培养16-24小时。在培养过程中pH值维持在6-8。pH值的调节可用无机或有机盐,碱溶液,缓冲液之类进行调节。培养过程中若有必要要加入氨苄青霉素或四环素至培养基中。
若有必要培养带有可诱导的启动子表达载体转化的微生物时,就要在培养基中加入诱导物。例如,培养若具有lac启动子载体转化的微生物时,培养基中可加入异丙基-β-D-硫代半乳吡喃糖苷(IPTG)等,若培养具有trp启动子载体转化的微生物时,可加入吲哚丙烯酸(IAA)等。
若培养宿主细胞是动物细胞的转化时,可以使用通常用的RPMI1640或DMEM培养基,或补充胎牛血清的上述之一培养基。
通常培养条件是5%CO2,37℃培养2-20天,在培养过程中加入卡那霉素或青霉素(如需要的话)。
若培养以植物细胞作宿主的转化子时,使用常用的补充卡那霉素,各种植物激素MS培养基或MS培养基。通常在20-30℃培养3至14天。
培养之后,如果合酶在微生物或细胞内,则通过裂解微生物或细胞来回收本发明的礌牛儿基二磷酸合酶。如果该合酶在微生物或细胞外产生,则通过离心法等去除微生物或细胞而收集培养上清。然后收集物(即细胞提取物或培养上清)用常规分离纯化蛋白的方法进行分离纯化。这些技术包括盐析,有机溶剂沉淀法,凝胶层析,亲和层析,硫水相互作用层析,离子交换层析。这些方法可以单独用,也可以适当组合以从培养物中分离和纯化本发明礌牛儿基二磷酸合酶。
需要指出的是,本发明的礌牛儿基二磷酸合酶在未纯化时就有活性。因此,只要它有合成酶活性细胞提取物或培养上清可作为粗酶溶液不需要纯化。(6)异戊二烯基二磷酸的制备
根据本发明通过培养从本发明DNA转化的宿主可以在培养物中积累GPP,并可通过回收此积累的GPP而制备之。
根据本发明,也可以通过本发明的酶作用于IPP或DMAPP来制备GPP,IPP和DMAPP是该酶的底物。在本方法中,本发明酶与反应底物在溶剂中反应,特别是水溶液中反应。这样目的异戊二烯基二磷酸便可在反应溶液中得到回收。反应中的酶不仅可以用纯化的酶,而且半纯化至各阶段的粗酶或含酶物如培养细胞,细胞培养物等含酶物都可以用。进一步而言,通过固定上述酶而得到的固定化酶,粗酶或各种常规方法得到的含酶物均可以用。
至于底物,可用IPP和/或DMAPP。反应溶剂可用水或水性缓冲液如Tris缓冲液或磷酸缓冲液。
附图简述
图1示突变型BstFPS和野生型BstFPS的酶活性。
图2是薄层层析谱的照片。
图3示突变型BstFPS和野生型BstFPS反应产物的特异性。
图4示法尼基二磷酸合酶的氨基酸序列比较。
完成本发明的最好方式
在下文中将通过参照实施例对本发明进行更为详细地说明。然而本发明的技术范围并不限于下列例子。
氨基酸用下文单字符或三字符表示。A;Ala;丙氨酸C;Cys;半胱氨酸D;Asp;天冬氨酸E;Glu;谷氨酸F;Phe;苯丙氨酸G;Gly;甘氨酸H;His;组氨酸I;Ile;异亮氨酸K;Lys;赖氨酸L;Leu;亮氨酸M;Met;甲硫氨酸N;Asn;天冬酰氨P;Pro;脯氨酸Q;Gln;谷氨酰胺R;Arg;精氨酸S;Ser;丝氨酸T;Thr;苏氨酸V;Val;缬氨酸W;Trp;色氨酸Y;Tyr;酪氨酸
在此文中,氨基酸残基的替换用单字符以下列顺序表示:“替换前残基”“氨基酸残基的位置”和“替换后残基”。
例如,当82位Ser由Phe替换时,该替换表示为“S82F”。实施例1:含有FPP合酶基因的质粒的制备
将嗜热脂肪芽孢杆菌-来源的FPP合酶BstFPS基因亚克隆于质粒载体pTV118N(PTV118N购于Takara Shuzo公司)的NcoI-Hind III位点之中。这个质粒DNA命名为pFPS。BstFPS基因全核苷酸序列由T·Koyama等公布(生物化学杂志(J.Biochem),113,355-363,(1993))或在遗传信息库如DDBJ中公布,许可号为D13293。实施例2:突变用寡核苷酸的合成
合成下列核苷酸以便向实施例1中获得的基因引入突变。
5′-CAT ACG TAC TTC TTG ATT CAT GAT GAT TTG -3′(SEQ ID NO:6)
设计上述寡核苷酸中从而(a)BstFPS的82位Ser的密码子替换成Phe密码子;和(b)引入了新的限制酶位点BspHI(5′-TCATGA3′)因为密码子的简并性,BspHI位点的引入并没有导致BstFPS所编码氨基酸序列的改变。因为这个限制位点的引入,可以在BspHI消化之后通过琼脂糖凝胶电泳检测到引入突变的克隆。
合成的寡核苷酸在下列反应溶液中37℃磷酸化30min。然后70℃10min使酶失活。
10pmol/μl寡核苷酸 2μl
10×激活缓冲液 1μl
1000mM Tris-Cl(pH8.0)
100mM MgCl2
70mM DTT二硫苏糖醇
10mM ATP 1μl
H2O 5μl
T4多核苷酸激酶 1μl实施例3:向BstFPS基因82位氨基酸残基的密码子引入替代突变
根据Kunkel的方法,用实施例2合成的寡核苷酸作引物,在实施例1制备的质粒中引入替代突变。本实施方法中,使用Takara Shuzo的Mutan-K试剂盒。实验操作过程如试剂盒中所带的实验手册进行。
简而言之,单链DNA用大肠杆菌CJ-236作宿主菌制备,在位于质粒pFPS DNA中的此单链DNA中胸腺嘧啶被脱氧尿嘧啶代替。
以此单链DNA作模板与用于合成互补链的引物DNA(即上述寡核苷酸)在下列溶液中退火。
单链DNA 0.6pmol
退火缓冲液 1μl
200mM Tris-Cl
(pH8.0)
100mM MgCl2
500mM NaCl
10mM DTT
引物DNA(来自实施例2) 1μl
H2O 以得到10μl的终体积
然后,在上述溶液中加入25μl延伸缓冲液,60单位大肠杆菌DNA连接酶和1单位T4DNA聚合酶,25℃2hr以合成互补链。延伸缓冲液组成如下,50mM Tris-Cl(pH8.0),60mM乙酸铵,5mM MgCl,5mM DTT,1mM NAD,0.5mM dNTP。
加入3μl0.2M EDTA(pH8.0)然后65℃处理5分钟以终止反应。实施例4:BstFPS基因82位氨基酸残基密码子发生替代突变的转化子的构建
大肠杆菌DH5α用下述CaCl2方法用实施例3中的DNA溶液进行转化。简言之,将DNA溶液加入用50mM CaCl2处理的DH5α感受态细胞悬液中。然后悬浮液置冰浴30分钟。
将得到的转化子铺于含有氨苄青霉素(转化子选择标记)的琼脂平板,并于37℃过夜培养。提取含有氨苄抗性的转化子的质粒DNA。用BspHI消化后质粒DNA进行琼脂糖凝胶电泳,然后从转化子中筛选BstFPS编码序列中有BspHI位点的替代突变子。
然后用双脱氧法对相当于BstFPS基因82位氨基酸处的密码子周围的核苷酸序列进行测定。由此获得了质粒pFPS,该质粒含有替换突变的BstFPS基因(SEQ ID NO:2),该基因82位Ser密码子(TCT)变为Phe密码子(TTC)。这个突变子命名为S82F,质粒为pFPSm。实施例5:突变体BstFPS活性的测定
用下述方法从不同的转化子中获取粗酶液,这些转化子分别包括由实施例4获得的含突变BstFPS基因和野生型BstFPS基因的转化子,和仪包括载体pTV118N的转化子。
将每个转化子细胞在2×LB培养基中隔夜培养的细胞,离心收集后重悬于细胞裂解液中[50mM Tris-Cl(pH8.0),10mM硫基乙醇,1mM EDTA]。细胞悬液超声裂解后10,000rpm4℃离心10分钟。裂解液上清55℃处理30分钟使来自细菌的异戊二烯基二磷酸合酶失活。这种上清经上述同样条件离心后得到的上清作为酶的粗提物。这种酶提取物在下列反应溶液中55℃反应15分钟。[1-14C]-IPP(1Ci/mol) 25nmol烯丙二磷酸(DMAPP或GPP或FPP) 25nmolTris-Cl(pH8.5) 50mMMgCl2 5mMNH4Cl 50mMβ-巯基乙醇 50mM酶溶液 50μl
H2O以得到1ml总体积
反应后,在反应液中加入3ml水饱和的丁醇以便将反应产物萃取到丁醇溶液层中。每1ml丁醇溶液中加入3ml液闪溶液,然后混合物用液闪计数器测定放射活性。
结果如图1所示。图1示突变体S82F BstFPS和野生型BstFPS的酶活性。样品Nos1、4和7代表由仅含载体pTV118N的宿主中制备的酶,样品Nos2、5和8代表由含突变体S82F BstFPS的宿主中制备的酶。样品Nos3、6和9代表从包括编码野生型BstFPS基因宿主中制备的酶。进一步,样品Nos1、2和3代表以DMAPP为烯丙基底物时的结果,样品Nos4、5和6代表GPP为烯丙基底物的结果。样品7,8,9代表以FPP为烯丙基底物时的结果。
从图1可以看出,野生型的酶可以以DMAPP和GPP为烯丙基底物,而不可以用FPP。相反,S82F突变体以GPP为烯丙基底物的能力是非常低的。
随后,分别如上所示制备反应液。反应后立即加入1ml土豆酸磷酸酶溶液[2mg/ml土豆酸磷酸酶,0.5M醋酸钠(pH4.7)]至反应液中,37℃进行去磷酸化,然后用3ml戊烷提取。提取物用薄层层析分析[反相TLC板:LKC18(Whatman);展层剂:丙酮/水=9/1]。展层后的去磷酸化反应产物用Bioimage Analyzer BAS2000(Fuji photofilm)分析放射性活性的位置及相对数量。
结果示于图2和3。图3显示了当作用单独的烯丙基底物时,其磷酸化的突变体PstFPS反应产物的展层方式。为了比较,从含有野生型BstFPS基因的宿主菌和只含有载体的宿主菌均制备了样品,其展层效果均得到展示。在这幅图中,“s.f.”代表溶剂前沿,“ori”代表展层起点,“GOH”代表牻牛儿醇标准品的展层位置;且“FOH”代表法尼醇标准品的展层位置。“野生型(Willd type)”表示用野生型BstFBS时的结果,“S82F”显示用突变S82F BstFPS时的结果;且“载体(Vector)”表示使用的酶是从仅含该载体的宿主菌制备的。“n.d.”表示活性未检测。图3显示了野生型BstFPS和突变型BstFPS反应产物的特异性。这个图显示了当以IPP和DMAPP为底物时GGPP,FPP和GPP的产生率。
图2和3所示结果说明,野生型BstFPS特异性催化FPP的合成而S82F突变型BstFPS已经变成特异性合成GPP。这意味着S82F突变型BstFPS已变成一种可被称为牻牛儿基二磷酸合酶的酶。
工业应用
根据本发明,现在可分别提供牻牛儿基二磷酸合酶,此合酶编码基因,含有此基因的重组载体,制备牻牛儿基二磷酸合酶和牻牛儿基二磷酸的方法。
因为本发明的酶可用于代谢工程和酶工程以合成单萜,因此本发明的酶是有用的。
序列表之外的文本:
SEQ ID NO:1:Xaa代表Val,Leu,Ile,Thr,Asp,Glu,Asn,Gln,Lys,Arg,Cys,Met,Phe,Tyr,Trp,His或Pro。
SEQ ID NO:6:根据FPP合酶的氨基酸序列设计的寡核苷酸,并含有BspHI位点。
序列列表<110>丰田自动车株式会社<120>牻牛儿基二磷酸合成酶基因<130>PH-586<140><141><150>JP97/346686<151>1997-12-16<160>6<170>PatentIn Ver.2.0<210>1<211>297<212>蛋白质<213>嗜热脂肪芽孢杆菌<220><221>肽<222>82<223>Xaa代表Val,Leu,Ile,Thr,Asp,Glu,Asn,Gln,Lys,Arg,Cys,Met,Phe,Tyr,Trp,His或Pro。<400>1
Val Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala
1 5 10 15
Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala
20 25 30
Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg
35 40 45
Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp
50 55 60
Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr
65 70 75 80
Tyr Xaa Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu
85 90 95
Arg Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Met Ala
100 105 110
Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Phe Gln Leu Ile Thr
115 120 125
Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile
130 135 140
Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln
145 150 155 160
Ala Ala Asp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu
165 170 175
Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val
180 185 190
His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu
195 200 205
Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp
210 215 220
Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val
225 230 235 240
Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser
245 250 255
Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln
260 265 270
Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile
275 280 285
Cys Glu Leu Val Ala Ala Arg AsP His
290 295<210>2<211>894<212>DNA<213>嗜热脂肪芽孢杆菌<220><221>CDS<222>(1)..(894)<400>2
gtg gcg cag ctt tca gtt gaa cag ttt ctc aac gag caa aaa cag gcg 48
Val Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala
1 5 10 15
gtg gaa aca gcg ctc tcc cgt tat ata gag cgc tta gaa ggg ccg gcg 96
Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala
20 25 30
aag ctg aaa aag gcg atg gcg tac tca ttg gag gcc ggc ggc aaa cga 144
Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg
35 40 45
atc cgt ccg ttg ctg ctt ctg tcc acc gtt cgg gcg ctc ggc aaa gac 192
Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp
50 55 60
ccg gcg gtc gga ttg ccc gtc gcc tgc gcg att gaa atg atc cat acg 240
Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr
65 70 75 80
tac ttc ttg atc cat gat gat ttg ccg agc atg gac aac gat gat ttg 288
Tyr Phe Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu
85 90 95
cgg cgc ggc aag ccg acg aac cat aaa gtg ttc ggc gag gcg atg gcc 336
Arg Arg Gly Lys Pro Thr Asn His Lys Val phe Gly Glu Ala Met Ala
100 105 110
atc ttg gcg ggg gac ggg ttg ttg acg tac gcg ttt caa ttg atc acc 384
Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Phe Gln Leu Ile Thr
115 120 125
gaa atc gac gat gag cgc atc cct cct tcc gtc cgg ctt cgg ctc atc 432
Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile
130 135 140
gaa cgg ctg gcg aaa gcg gcc ggt ccg gaa ggg atg gtc gcc ggt cag 480
Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln
145 150 155 160
gca gcc gat atg gaa gga gag ggg aaa acg ctg acg ctt tcg gag ctc 528
Ala Ala Asp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu
165 170 175
gaa tac att cat cgg cat aaa acc ggg aaa atg ctg caa tac agc gtg 576
Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val
180 185 190
cac gcc ggc gcc ttg atc ggc ggc gct gat gcc cgg caa acg cgg gag 624
His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu
195 200 205
ctt gac gaa ttc gcc gcc cat cta ggc ctt gcc ttt caa att cgc gat 672
Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp
210 215 220
gat att ctc gat att gaa ggg gca gaa gaa aaa atc ggc aag ccg gtc 720
Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val
225 230 235 240
ggc agc gac caa agc aac aac aaa gcg acg tat cca gcg ttg ctg tcg 768
Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser
245 250 255
ctt gcc ggc gcg aag gaa aag ttg gcg ttc cat atc gag gcg gcg cag 816
Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln
260 265 270
cgc cat tta cgg aac gcc gac gtt gac ggc gcc gcg ctc gcc tat att 864
Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile
275 280 285
tgc gaa ctg gtc gcc gcc cgc gac cat taa 894
Cys Glu Leu Val Ala Ala Arg Asp His
290 295<210>3<211>297<212>蛋白质<213>嗜热脂肪芽孢杆菌<400>3
Val Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala
1 5 10 15
Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala
20 25 30
Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg
35 40 45
Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp
50 55 60
Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr
65 70 75 80
Tyr Phe Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu
85 90 95
Arg Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Met Ala
100 105 110
Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Phe Gln Leu Ile Thr
115 120 125
Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile
130 135 140
Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln
145 150 155 160
Ala Ala Asp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu
165 170 175
Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val
180 185 190
His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu
195 200 205
Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp
210 215 220
Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val
225 230 235 240
Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser
245 250 255
Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln
260 265 270
Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile
275 280 285
Cys Glu Leu Val Ala Ala Arg Asp His
290 295<210>4<211>894<212>DNA<213>嗜热脂肪芽孢杆菌<220><221>CDS<222>(1)..(894)<400>4
gtg gcg cag ctt tca gtt gaa cag ttt ctc aac gag caa aaa cag gcg 48
Val Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala
1 5 10 15
gtg gaa aca gcg ctc tcc cgt tat ata gag cgc tta gaa ggg ccg gcg 96
Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala
20 25 30
aag ctg aaa aag gcg atg gcg tac tca ttg gag gcc ggc ggc aaa cga 144
Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg
35 40 45
atc cgt ccg ttg ctg ctt ctg tcc acc gtt cgg gcg ctc ggc aaa gac 192
Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp
50 55 60
ccg gcg gtc gga ttg ccc gtc gcc tgc gcg att gaa atg atc cat acg 240
Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr
65 70 75 80
tac tct ttg atc cat gat gat ttg ceg agc atg gac aac gat gat ttg 288
Tyr Ser Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu
85 90 95
cgg cgc ggc aag ccg acg aac cat aaa gtg ttc ggc gag gcg atg gcc 336
Arg Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Met Ala
100 105 110
atc ttg gcg ggg gac ggg ttg ttg acg tac gcg ttt caa ttg atc acc 384
Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Phe Gln Leu Ile Thr
115 120 125
gaa atc gac gat gag cgc atc cct cct tcc gtc cgg ctt cgg ctc atc 432
Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile
130 135 140
gaa cgg ctg gcg aaa gcg gcc ggt ccg gaa ggg atg gtc gcc ggt cag 480
Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln
145 150 155 160
gca gcc gat atg gaa gga gag ggg aaa acg ctg acg ctt tcg gag ctc 528
Ala Ala Asp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu
165 170 175
gaa tac att cat cgg cat aaa acc ggg aaa atg ctg caa tac agc gtg 576
Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val
180 185 190
cac gcc ggc gcc ttg atc ggc ggc gct gat gcc cgg caa acg cgg gag 624
His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu
195 200 205
ctt gac gaa ttc gcc gcc cat cta ggc ctt gcc ttt caa att cgc gat 672
Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp
210 215 220
gat att ctc gat att gaa ggg gca gaa gaa aaa atc ggc aag ccg gtc 720
Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val
225 230 235 240
ggc agc gac caa agc aac aac aaa gcg acg tat cca gcg ttg ctg tcg 768
Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser
245 250 255
ctt gcc ggc gcg aag gaa aag ttg gcg ttc cat atc gag gcg gcg cag 816
Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln
260 265 270
cgc cat tta cgg aac gcc gac gtt gac ggc gcc gcg ctc gcc tat att 864
Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile
275 280 285
tgc gaa ctg gtc gcc gcc cgc gac cat taa 894
Cys Glu Leu Val Ala Ala Arg Asp His
290 295<210>5<211>297<212>蛋白质<213>嗜热脂肪芽孢杆菌<400>5
Val Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala
1 5 10 15
Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala
20 25 30
Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg
35 40 45
Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp
50 55 60
Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr
65 70 75 80
Tyr Ser Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu
85 90 95
Arg Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Met Ala
100 105 110
Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Phe Gln Leu Ile Thr
115 120 125
Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile
130 135 140
Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln
145 150 155 160
Ala Ala Asp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu
165 170 175
Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val
180 185 190
His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu
195 200 205
Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp
210 215 220
Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val
225 230 235 240
Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser
245 250 255
Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln
260 265 270
Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile
275 280 285
Cys Glu Leu Val Ala Ala Arg Asp His
290 295<210>6<211>30<212>DNA<213>修饰后序列<220><223>基于FPP合成酶氨基酸序列设计的寡核苷酸,含BspHI位点。<400>6catacgtact tcttgattca tgatgatttg 30
Claims (8)
1.由SEQ ID NO:1所示的氨基酸序列所组成的具有牻牛儿基二磷酸合酶活性的重组蛋白。
2.编码由SEQ ID NO:1所示的氨基酸序列所组成的具有牻牛儿基二磷酸合酶活性的重组蛋白的基因。
3.由SEQ ID NO:2所示的核苷酸序列组成的分离的牻牛儿基二磷酸合酶基因。
4.含权利要求2或3的基因的重组载体。
5.由权利要求4的重组载体转化得到的转化体。
6.制备牻牛儿基二磷酸合酶的方法,包括在培养基中培养权利要求5中转化体以及从得到的培养物中回收牻牛儿基二磷酸合酶。
7.制备牻牛儿基二磷酸的方法,包括在培养基中培养权利要求5中转化体以及从得到的培养物中回收牻牛儿基二磷酸。
8.制备牻牛儿基二磷酸的方法,包括使权利要求5的转化体的培养物作用于异戊烯二磷酸或其异构体。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP34668697A JP3562280B2 (ja) | 1997-12-16 | 1997-12-16 | ゲラニル二リン酸合成酵素遺伝子 |
JP346686/1997 | 1997-12-16 |
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CN1252838A CN1252838A (zh) | 2000-05-10 |
CN1130460C true CN1130460C (zh) | 2003-12-10 |
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CN98804224A Expired - Fee Related CN1130460C (zh) | 1997-12-16 | 1998-12-10 | 牻牛儿基二磷酸合酶基因 |
Country Status (7)
Country | Link |
---|---|
US (1) | US6395525B2 (zh) |
EP (1) | EP0974661A4 (zh) |
JP (1) | JP3562280B2 (zh) |
KR (1) | KR20000071073A (zh) |
CN (1) | CN1130460C (zh) |
CA (1) | CA2281206C (zh) |
WO (1) | WO1999031254A1 (zh) |
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US6660507B2 (en) | 2000-09-01 | 2003-12-09 | E. I. Du Pont De Nemours And Company | Genes involved in isoprenoid compound production |
WO2007029577A1 (ja) * | 2005-09-07 | 2007-03-15 | Toudai Tlo, Ltd. | 放線菌由来のゲラニルジリン酸合成酵素及びこれを用いたゲラニルジリン酸の製造方法 |
JP5035871B2 (ja) | 2005-09-16 | 2012-09-26 | 株式会社ブリヂストン | パラゴムノキのプレニルトランスフェラーゼの遺伝子群 |
CN101896603A (zh) | 2007-06-01 | 2010-11-24 | 蓝宝石能源公司 | 使用遗传修饰的生物产生生物质降解酶 |
EP2203542A1 (en) * | 2007-09-11 | 2010-07-07 | Sapphire Energy, Inc. | Methods of producing organic products with photosynthetic organisms and products and compositions thereof |
CN101896607A (zh) * | 2007-09-11 | 2010-11-24 | 蓝宝石能源公司 | 通过光合生物产生分子 |
CA2718469C (en) | 2008-03-17 | 2017-07-04 | National Research Council Of Canada | Aromatic prenyltransferase from hop |
JP5787341B2 (ja) * | 2009-11-19 | 2015-09-30 | 国立大学法人 千葉大学 | テルペン合成酵素遺伝子のスクリーニング方法 |
US8715962B2 (en) | 2010-03-31 | 2014-05-06 | Codexis, Inc. | Production of geranyl diphosphate |
CN102876689B (zh) * | 2012-07-11 | 2014-06-18 | 浙江大学 | 茶树fps基因及其应用 |
CN111593032B (zh) * | 2020-05-26 | 2022-10-28 | 中国烟草总公司郑州烟草研究院 | 酶口袋和酶分子表面的定向五位点突变蛋白ggpps |
CN111534496B (zh) * | 2020-05-26 | 2022-10-28 | 中国烟草总公司郑州烟草研究院 | Ggpps定向单点突变蛋白ggpps-154 |
CN111500551B (zh) * | 2020-05-26 | 2022-10-28 | 中国烟草总公司郑州烟草研究院 | Ggpps定向单点突变蛋白ggpps-218 |
CN111534499A (zh) * | 2020-05-28 | 2020-08-14 | 河南大学 | Ggpps定向单点突变蛋白ggpps-233 |
CN111718916A (zh) * | 2020-06-08 | 2020-09-29 | 贵州省烟草科学研究院 | Ggpps定向单点突变蛋白及其应用 |
CN112410236B (zh) * | 2020-11-23 | 2022-08-30 | 江南大学 | 类大牻牛儿烯合成酶c突变体及构建方法与应用 |
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JP3120684B2 (ja) * | 1995-02-14 | 2000-12-25 | トヨタ自動車株式会社 | ゲラニルゲラニル二リン酸を合成する変異型ファルネシル二リン酸合成酵素及びそれをコードするdna |
JP3538998B2 (ja) * | 1995-09-01 | 2004-06-14 | トヨタ自動車株式会社 | 長鎖プレニル二燐酸合成酵素 |
JP3209103B2 (ja) * | 1996-07-03 | 2001-09-17 | トヨタ自動車株式会社 | 変異型プレニル二燐酸合成酵素 |
US5876964A (en) * | 1997-10-16 | 1999-03-02 | Washington State University Research Foundation | Geranyl diphosphate synthase from mint |
-
1997
- 1997-12-16 JP JP34668697A patent/JP3562280B2/ja not_active Expired - Fee Related
-
1998
- 1998-12-10 WO PCT/JP1998/005590 patent/WO1999031254A1/ja not_active Application Discontinuation
- 1998-12-10 CA CA002281206A patent/CA2281206C/en not_active Expired - Fee Related
- 1998-12-10 US US09/367,528 patent/US6395525B2/en not_active Expired - Fee Related
- 1998-12-10 CN CN98804224A patent/CN1130460C/zh not_active Expired - Fee Related
- 1998-12-10 EP EP98959156A patent/EP0974661A4/en not_active Withdrawn
- 1998-12-10 KR KR1019997007359A patent/KR20000071073A/ko not_active Application Discontinuation
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US6395525B2 (en) | 2002-05-28 |
CA2281206A1 (en) | 1999-06-24 |
KR20000071073A (ko) | 2000-11-25 |
JP3562280B2 (ja) | 2004-09-08 |
EP0974661A1 (en) | 2000-01-26 |
EP0974661A4 (en) | 2004-09-01 |
CN1252838A (zh) | 2000-05-10 |
WO1999031254A1 (fr) | 1999-06-24 |
CA2281206C (en) | 2003-04-29 |
JPH11169178A (ja) | 1999-06-29 |
US20010051359A1 (en) | 2001-12-13 |
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