CN1308457C - 发酵生产s－腺苷甲硫氨酸的方法 - Google Patents
发酵生产s－腺苷甲硫氨酸的方法 Download PDFInfo
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- CN1308457C CN1308457C CNB2004100074938A CN200410007493A CN1308457C CN 1308457 C CN1308457 C CN 1308457C CN B2004100074938 A CNB2004100074938 A CN B2004100074938A CN 200410007493 A CN200410007493 A CN 200410007493A CN 1308457 C CN1308457 C CN 1308457C
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- adenosylmethionine
- val
- gly
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- 229960001570 ademetionine Drugs 0.000 title claims abstract description 117
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Abstract
一种发酵生产S-腺苷甲硫氨酸的方法，包括在一种培养基中培养可得自一种起始菌株、且与该起始菌株相比具有增强的S-腺苷甲硫氨酸合成酶活性的细菌菌株，该细菌菌株将S-腺苷甲硫氨酸分泌到该培养基中，以及自该培养基中收集该S-腺苷甲硫氨酸。
Description
技术领域
本发明涉及使用超量生产S-腺苷甲硫氨酸合成酶的细菌菌株而发酵生产S-腺苷甲硫氨酸的方法。
背景技术
S-腺苷甲硫氨酸(S-Adenosylmethionine，SAM)是代谢中最重要的甲基供体，在药学方面可用于治疗抑郁症、肝脏疾病及关节炎。己公开的生产S-腺苷甲硫氨酸的方法，包括在含有前体物质L-甲硫氨酸条件下生长酵母(施伦克F.及德帕尔玛R.E.，生化期刊1037-1050页(1957)；盐崎S.等人，农业生化53，3269-3274页(1989))以及在提取细胞裂解物后利用层析法纯化生产S-腺苷甲硫氨酸(美国专利US 4562149)。此方法的缺点，特别是复杂的纯化所生产的S-腺苷甲硫氨酸的过程，因为细胞必须先被破碎，且S-腺苷甲硫氨酸必须从其他细胞成分，如氨基酸、糖类、脂肪、核酸、蛋白质、辅因子、以及其他大分子量和小分子量化合物中收集出来。因此，如果所生产的S-腺苷甲硫氨酸可选择性地被分泌至培养物上清液中并因此使得纯化方法更为简化，这样发展出的用来发酵生产S-腺苷甲硫氨酸的方法能够具有空前的效率。而且培育物上清液中也常仅含少许物质，所以S-腺苷甲硫氨酸分泌至培养物上清液中将成为初步的纯化步骤，易于进一步的纯化。
GB 1436509描述了通过酵母，如热带假丝酵母(Candida tropicalis)生产S-腺苷甲硫氨酸的胞外生产方法。这方法的问题是，事实上，所用的生产菌株不是普通的真菌，其不具GRAS(通常被认为是安全的)状态，甚至有时被归类为致病菌。此外，该生物体也难以运用常用的遗传方法操作且它的代谢也尚未明了。因此，它缺乏可通过代谢工程进行改良的两种必备先决条件。相反地，细菌在遗传方面易于使用，其多数以上的代谢途径已经被详细研究，且许多具有GRAS状态的非致病菌。所以，通过细菌生产S-腺苷甲硫氨酸将是非常理想。但是，至今尚不了解通过细菌进行胞外生产S-腺苷甲硫氨酸的方法。
在大肠杆菌中S-腺苷甲硫氨酸的合成，曾被特别广泛研究过(格林，R.C.，甲硫氨酸的生物合成：奈德哈德F.C.，大肠杆菌及鼠伤寒沙门氏菌(Salmonella typhimurium)，细胞及分子生物学。二版，ASM出版，华盛顿DC(1996)，542-560页，在此并入作为参考)。S-腺苷甲硫氨酸的合成仅需由L-甲硫氨酸及腺嘌呤核苷三磷酸盐(ATP)进行一步合成，而L-甲硫氨酸则是由复杂且高度调控下方可合成。在合成过程中，ATP上的三个磷酸基将被释放出来，而提供无机磷酸及焦磷酸。整个反应是由S-腺苷甲硫氨酸合成酶(EC 2.5.1.6，甲硫氨酸腺苷转移酶，S-腺苷甲硫氨酸合成酶)所催化，其在大肠杆菌中是由metK基因所编码，此酶在生化及基因方面皆有完整的定性研究，它也具有很强的反馈调节，例如，在过多的S-腺苷甲硫氨酸存在下，此酶的活性就会被强有力地抑制(马卡姆等人，生化期刊，9082-9092页(1980))。所谓的反馈调节作用，可避免不必要的耗能的S-腺苷甲硫氨酸合成，以及细胞内的S-腺苷甲硫氨酸水平过高，可能伤及细胞，但也因此阻碍了S-腺苷甲硫氨酸的发酵超量生产。其他生物体(如啤酒酵母、詹氏甲烷球菌(Methanococcusjanaschii)，大鼠)的S-腺苷甲硫氨酸合成酶，也曾被研究过，而且S-腺苷甲硫氨酸同样对其具有抑制能力，但这些例子中表现的抑制作用却不及大肠杆菌中的抑制作用那样明显(派克等人，生物有机医疗化学，2179-2185页(1996)；陆及马卡姆，生化期刊，16624-16631页(2002))欧登及克拉基，生物化学，2978-2986页(1983))。
相比于其他生物体，如酵母，细菌本身并不具有S-腺苷甲硫氨酸的运输系统，也使得细菌无法从培养基吸收S-腺苷甲硫氨酸。因此S-腺苷甲硫氨酸合成酶成为细菌必需的酶。当大肠杆菌中S-腺苷甲硫氨酸合成酶超量生产，其胞内酶量也因此增加(马卡姆等人，生化期刊，9082-9092页(1980))。但是，对于是否酶超量生产后，进而也增加胞内S-腺苷甲硫氨酸的含量，则尚未明了。但此情况是不被期待的，因为胞内S-腺苷甲硫氨酸的累积，会被先前提到的S-腺苷甲硫氨酸合成酶反馈调节所抑制，因此S-腺苷甲硫氨酸合成酶活性受到调节时，也会限制S-腺苷甲硫氨酸在胞内的生产量。
相对地，在大肠杆菌中超量生产大鼠肝中的S-腺苷甲硫氨酸合成酶，其胞内的S-腺苷甲硫氨酸则明显地增加(阿尔瓦雷斯等人，生物化学期刊，557-561页(1994)；欧洲专利EP 0647712A1)。但是这是有可能的，因为它与大肠杆菌的同源酶并不相似，而且此S-腺苷甲硫氨酸酶并不会受到严格的反馈调节，因此细菌调控也得以规避。但是，关于S-腺苷甲硫氨酸可在胞外累积，则尚未观察到。
发明内容
本发明的目的是提供一种可通过细菌进行发酵生产S-腺苷甲硫氨酸的方法，此方法也明显简化了S-腺苷甲硫氨酸的纯化方式。
该目的可通过以下方法而实现，该方法包含在一种培养基中培养一种细菌菌株，该菌株可得自一种具有S-腺苷甲硫氨酸合成酶的起始菌株，且与该起始菌株相比，该菌株具有增强的S-腺苷甲硫氨酸合成酶活性，该菌株分泌S-腺苷甲硫氨酸至所述的培养基中，并将S-腺苷甲硫氨酸自所述的培养基中收集出来。
附图说明
图1为本发明的pKP481质粒的构建图，metK基因被置于tac启动子的调控下。
具体实施方式
本发明的目的是提供一种可通过细菌进行发酵生产S-腺苷甲硫氨酸的方法，此方法也明显简化了S-腺苷甲硫氨酸的纯化方式。
该目的可通过以下方法而实现，该方法包含在一种培养基中培养一种细菌菌株，该菌株可得自一种具有S-腺苷甲硫氨酸合成酶的起始菌株，且与该起始菌株相比，该菌株具有增强的S-腺苷甲硫氨酸合成酶活性，该菌株分泌S-腺苷甲硫氨酸至所述的培养基中，并将S-腺苷甲硫氨酸自所述的培养基中收集出来。
如上所述，由于在细菌中，特别是大肠杆菌中，S-腺苷甲硫氨酸合成酶受到严格的反馈调节，在此却很惊讶地发现，随着酶活性增加，S-腺苷甲硫氨酸的产量也增加。
特别地，超量生产的S-腺苷甲硫氨酸却完全意外地被分泌至培养物上清液中。如同先前所提及的，无任何细菌先例说明，细菌可将发酵生产的S-腺苷甲硫氨酸释放至培养物上清液中。特别地，细菌中没有任何已知的S-腺苷甲硫氨酸运输系统，也不能从培养基中吸收S-腺苷甲硫氨酸。而对于如S-腺苷甲硫氨酸这样的大分子量而且带电的分子来说，向细胞外进行被动扩散几乎是不可能的。因此S-腺苷甲硫氨酸在胞外浓缩，令本领域人员感到十分惊奇。
本发明提供的方法，其优点在于可增加S-腺苷甲硫氨酸的产量，及使分离净化培养物上清液变得更容易。这个方法也使得S-腺苷甲硫氨酸合成酶可增加S-腺苷甲硫氨酸的生成量，通常S-腺苷甲硫氨酸的生产易受到严格的产物抑制作用以防止S-腺苷甲硫氨酸在细胞中的积累，但当S-腺苷甲硫氨酸得以被分泌至培养物上清液中，也就不会抑制S-腺苷甲硫氨酸合成酶。
另外，惊奇地发现，与既有技术对比，D，L-甲硫氨酸，在本发明的方法中，也可取代L-甲硫氨酸作为前体物质，前者较便宜，也使得生产成本显著降低。
因此本发明还涉及一种方法，其包括在一种培养基中培养可得自一种具有S-腺苷甲硫氨酸合成酶的起始菌株的细菌菌株，相比于该起始菌株，该细菌菌株具有升高的S-腺苷甲硫氨酸合成酶活性，该细菌菌株将S-腺苷甲硫氨酸分泌至该培养基中，及自该含有D，L-甲硫氨酸的培养基中将S-腺苷甲硫氨酸收集起来。
优选地使用一种蛋白质或其功能性变体作为本发明的方法所使用的S-腺苷甲硫氨酸合成酶，该蛋白质具有SEQ ID NO：1的序列，所述的功能性变体具有的序列与SEQ ID NO：1的相似性大于40％，优选地大于60％，特别优选地大于80％。
在本发明中所提及的所有同源性的数值，皆是使用BESTFIT演算法分析(GCG威斯康辛软件包，遗传学电脑组(GCG)麦迪逊，威斯康辛)，本发明也与上述S-腺苷甲硫氨酸合成酶相关。
优选地使用本发明方法中所用的S-腺苷甲硫氨酸合成酶的基因，在以下的论述中，则称为metK基因，此基因的序列为SEQ ID NO：2或该基因的功能性变体。
所谓的功能性变体指的是依照本发明通过核苷酸缺失作用、插入作用或取代作用自SEQ ID NO：2中描述的序列衍生的DNA序列，仍保留由该基因编码的S-腺苷甲硫氨酸合成酶的酶活性。
活性的增加意味着依照本发明所用的细菌菌株，其在细菌细胞内的S-腺苷甲硫氨酸合成酶的活性，比相应的起始菌株增加了两倍，优选地是至少增加五倍。
在本发明方法所用的细菌菌株，通常是野生型菌株使用标准的分子生物技术改良产生的，它的S-腺苷甲硫氨酸合成酶活性比起始菌株高。
S-腺苷甲硫氨酸合成酶的所有基因皆是由许多起始菌株中所鉴定的。在本发明方法中所用的细菌菌株，优选地是从可通过基因重组方法进行改良、可用发酵方法培养，且能将S-腺苷甲硫氨酸分泌至培养基中的原核生物体起始菌株来制备。优选为肠杆菌科的细菌菌株，特别是埃希氏菌属的菌株，特别优选地为大肠杆菌菌株，优选地使用不具外源性基因的大肠杆菌菌株。
原则上，可通过不同的方式来获得比起始菌株更高的S-腺苷甲硫氨酸合成酶的活性。
一方面，S-腺苷甲硫氨酸合成酶的基因可被修饰由此使得其编码的酶具有比起始酶更高的酶活性。例如为由非特异性或特异性的突变方式，来改变S-腺苷甲硫氨酸合成酶的基因。非特异性的突变法，如使用化学剂(如1-甲基-3-硝基-1-亚硝基胍、甲基磺酸乙酯，等等)和/或通过物理方法和/或在特殊条件下的聚合酶链反应下进行，及/或在突变菌株(如XLI-Red，Stratagene，阿姆斯特丹，荷兰)中进行DNA的扩增。在DNA片段引发特定位置的突变，为已知的方法。其他可能产生与起始酶相比具有较高S-腺苷甲硫氨酸合成酶活性的方法，是综合以上所提及的方式。
获得比起始菌株更高的S-腺苷甲硫氨酸合成酶活性的另外一种可能的方式，是使编码此酶的基因进行过表达。过表达意味着，依照本发明，S-腺苷甲硫氨酸合成酶基因，能表达至少比起始菌株多两倍的量，优选地至少是5倍。
细菌菌株可通过增加metK基因的拷贝数目以达到在该菌株中的过表达，及/或通过适当的启动子来增加metK基因在的表达。
在起始菌株的细胞中，可通过一些本领域人员熟知的方法，来实现增加起始菌株的细胞内metK基因的拷贝数目。例如，metK基因可被克隆至质粒载体上并将其引入菌株，该质粒载体在每个细胞内具有多个拷贝(例如大肠杆菌中的pUC19、pBR322及pACYC184)。也可多次将metK基因整合入细胞的染色体中。整合的方法可利用已知的系统，即利用温性噬菌体或整合质粒，或是利用同源性重组方式进行整合。
优选地将metK基因克隆入质粒载体中置于启动子的控制下而增加基因的拷贝数目。优选地将metK基因克隆入pBR322的衍生物如pJF118ut(其由pJF118EH衍生，菲尔斯特等人，基因，119-131页(1986))中以增加大肠杆菌中的拷贝数目。
用于表达由质粒编码的metK基因的适当的调控区是该metK基因的天然启动子及操纵子区，但可通过其他的启动子来增强metK基因的表达。相应的使得S-腺苷甲硫氨酸合成酶基因能够以组成型或控制型、诱导型表达的启动子系统是本领域人员已知的，例如，gapA基因的组成型GAPDH启动子或大肠杆菌中的诱导型lac、tac、trc、Lamda、ara或tet启动子。这些构建体可按已知的方式用于质粒或染色体上。
一个特别优选的克隆metK基因的实施方式利用已含有可增强表达的启动子的质粒，如大肠杆菌的诱导型tac-启动子系统。
此外，也可通过翻译起始信号如核糖体结合位点或以特定构建体的最佳序列形式存在的该基因的起始密码子来实现基因表达的增强，或通过依据常用密码子使用规则将少见的密码子以更为常见的密码子进行取代，或通过优化使mRNA稳定的序列来实现基因表达的增强。
在本发明的方法所使用的细菌菌株，优选地含有metK基因的质粒及上述经修饰过的调节信号。
可通过以下步骤将metK基因克隆至质粒载体，例如，以覆盖完整的metK基因的特异性的引物进行聚合酶链反应以特异性扩增metK基因，然后与载体的DNA片段进行连接。
本发明可用额外的措施使细菌发酵生产S-腺苷甲硫氨酸更为有效且经济，即加强本发明的方法中所使用的菌株的内源性甲硫氨酸合成而不添加L-甲硫氨酸或D，L-甲硫氨酸。为此，例如可用下述菌株，所述的菌株中不表达metJ基因，而该metJ基因编码甲硫氨酸和S-腺苷甲硫氨酸代谢基因的阻抑物(日本专利JP 2000139471A)，或所述的菌株中的甲硫氨酸合成是增强的，原因在于其具有活性增强的高丝氨酸转琥珀酰酶(homoserine transsuccinylase)。(日本专利JP 2000139471A，德国专利DE-A-10247437、DE-A-10249642)。
使用通常的转化方法，如电穿孔法、氯化钙法(CaCl2、)，将带有metK基因的质粒送至起始细菌株中，然后通过例如抗生素抗性进行筛选携带质粒的克隆。
依本发明生产S-腺苷甲硫氨酸所用的细菌菌株，优选地是培养在文献公开的含最基本盐类的培养基中。
原则上，主要的碳源为任何可利用的糖类、糖醇、有机酸或其盐类，以及淀粉水解物、糖蜜(molasses)或其他物质。优选地是用葡萄糖或甘油。许多不同碳源的组合性补料也是可行的。适当的氮源，如尿素、氨及其盐类，硝酸盐及其他的氮源。可能的氮源也包括复合氨基酸的混合物，如酵母提取物、蛋白胨、麦芽提取物、大豆蛋白胨、酪蛋白氨基酸、玉米浸液或NZ胺类(酪蛋白的酶促水解物)。
此外，也可加入特别的成分，如维生素、盐类、酵母提取物、氨基酸及微量元素至培养基中，促进细胞生长。
此外，L-甲硫氨酸也可被加入至培养基，作为合成S-腺苷甲硫氨酸的特异性前体物质，其浓度为0.05至25克/升，优选地，L-甲硫氨酸浓度为1至5克/升。
在本发明的一个特别优选的方法中，是将D，L-甲硫氨酸而不是L-甲硫氨酸加入培养基中，其浓度为0.05至25克/升，优选地，D，L-甲硫氨酸浓度为1至5克/升。
菌株优选地是在有氧条件下培养16至150小时，而特定的菌株则在最适生长温度范围内培养。
最适培养温度范围在15℃至55℃，在30℃至37℃尤佳。
菌株可在振荡式三角瓶或发酵罐中培养，对培养体积的大小没有限制。进行培养的方法可用分批法、补料分批法或连续式培养法。
可用本领域人员已知的方法，如离心法去除细胞后，从培养基中收集到S-腺苷甲硫氨酸，然后进行层析法纯化、络合、过滤如交叉流过滤法，或用沉淀法收集。
在本发明的方法，可通过例如层析仪器(如HPLC)对S-腺苷甲硫氨酸的生成进行检测及定量。
以下的实验例作为对本发明做进一步说明，实施例中所用的细菌菌株大肠杆菌W3110/pKP481，依据布达佩斯条约保藏于德国微生物及细胞培养物保藏中心(DSMZ，D-38142 Braunschweig，德国)，编号为DSM15426。
所有运用的分子生物学方法，如聚合酶链反应、DMA的分离及纯化、限制酶、Klenow片段及连接酶的DNA修饰作用、转化作用等以本领域人员已知的方式实施或依文献公开或特定厂商所提供的操作说明进行。
实验例1：建构pKP481质粒
A.扩增metK基因
大肠杆菌的metK基因，可通过本领域人员常用的操作法，将大肠杆菌起始菌株W3110野生菌株(ATCC 27325)的染色体做为模板，用TaqDNA聚合酶进行聚合酶链反应得以扩增metK基因，所用的引物为寡核苷酸metK2，其序列为
5’-GGTTAATTAATGTCTGTTGTGGTTGGTGT-3’(SEQ ID NO：3)，及metK4(序列为
5’-GGAATTCTCTTTAGGAGGTATTAAATATG-3’)(SEQ ID NO：4)。由聚合酶链反应可得到约1.2kb的DNA片段，通过QIAprep SpinMiniprep试剂盒中(Qiagen，希尔登，德国)的DNA吸附管柱进行纯化，操作步骤依据使用说明。
B.克隆metK基因至pJF118ut载体
通过引物metK4可将一个EcoRI限制核酸内切酶的酶切位点引入至聚合酶链反应的片段中。所以纯化的聚合酶链反应的片段可被EcoRI限制核酸内切酶在厂商指定的条件下作用，然后磷酸化，通过琼脂糖凝胶进行分离后，再通过QIAquick凝胶提取试剂盒(Qiagen)将聚合酶链反应的片段从琼脂糖凝胶中分离出来，操作步骤依据使用说明。
载体pJF118ut是由克隆及表达载体pJF1183EH所衍生出来的(菲因斯特等人，基因，119-131页(1986))，其含有不同的基因元件，使其得以控制任意基因的表达。这载体有一个由pBR质粒家族衍生的复制起点。克隆基因的表达，则是由tac启动子所控制，被lacIq阻抑物所抑制，并且由乳糖或IPTG诱导表达。
在厂商指定的条件下，利用限制酶EcoRI及PstI将pJF128ut载体酶切后，克隆得到metK基因。PstI作用后所产生的3’突出端，可再以本领域人员已知的方式通过Klenow酶作用成齐平未端。质粒的5’端则用碱性磷酸酶进行去磷酸化反应，然后用QIAquick胶体提取试剂盒(Qiagen)进行纯化。聚合酶链反应的片段与经过酶切及已去磷酸化的载体，用T4-DNA连接酶接合在一起。通过电穿孔法也可将接合后的DNA转化至大肠杆菌菌株DH5α中。转化作用后的混合物则倒至含氨苄青霉素的LB固态琼脂培养基上(10克/升胰蛋白胨；5克/升酵母提取物；5克/升氯化纳；15克/升琼脂；20毫克/升四环素)，在37℃下隔夜培养。
通过QIAprep Spin Miniprep试剂盒(Qiagen，希尔登，德国)分离收集质粒，再用限制酶的酶切分析鉴定并测序分析，确定所需的转化子。
图1说明如何获得pKP481质粒的方法，metK基因被置于tac启动子的调控下。
实验例2：S-腺苷甲硫氨酸生产菌的制备
在实验例1中所描述的pKP481质粒，通过氯化钙的方法转化至大肠杆菌W3110(ATCC 27325)中，再通过含有20毫克/升四环素的LB固体琼脂培养基进行筛选后重新分离自其中的一个转化子，再用限制核酸内切酶作用及检查，得到适合用于S-腺苷甲硫氨酸生产的菌株，并命名为W3110/pKP481。
实验例3：S-腺苷甲硫氨酸的发酵生产
A.S-腺苷甲硫氨酸的生产
W3110/pKP481菌株用于发酵生产S-腺苷甲硫氨酸，而不具有质粒的W3110起始菌株(ATCC 27325)则培养在相同条件下，做比较用。
以下为所用的培养基：一升中含有0.0147克的二水氯化钙，0.3克七水硫酸镁，0.15毫克二水钼酸钠，2.5毫克硼酸，0.7毫克六水氯化钴，0.25毫克五水硫酸铜，1.6毫克四水氯化锰，0.3毫克七水硫酸锌，3克磷酸二氢钾，12克磷酸氢二钾，5克硫酸铵，0.6克氯化钠，0.002克七水硫酸亚铁，1克二水柠檬酸钠，15克葡萄糖，1克胰蛋白胨，及0.5克酵母提取物，培养W3110/pKP481则需再加入20微克/毫升的四环素至培养基，在表中有标示处为额外再加入0.5克/升L-甲硫氨酸或1克/升D，L-甲硫氨酸至培养基中。
首先，将10毫升的培养基放入100毫升的三角烧瓶中，植入菌株，37℃，160转的震荡培养16小时，得到用于培养生产菌的预培养物。由此制备的细胞最终转接培养至50毫升的培养基中(用300毫升的三角瓶)进行培养，直到OD600吸光值(在600nm波长吸光值)达0.1，再将其在160转的震荡培养48小时。S-腺苷甲硫氨酸合成酶的基因是通过0.1mM的异丙基-β-硫代半乳糖苷(IPTG)，在OD600为0.6下进行诱导表达，在24及48小时后分别取样，通过离心将细胞从培养基中除去。
B.定量所生产的S-腺苷甲硫氨酸
可通过高效液相层析法(HPLC)定量在培养物上清液中所含有的S-腺苷甲硫氨酸的量(Develosil RP-Aqueous C 30柱，5微米，250×4.6毫米)(购自普亨奥孟克斯公司，阿赫亚芬贝格，德国)，加入10微升的培养物上清液，在等度洗脱下进行分离，洗脱液为在一升水中含3毫升85％的磷酸，常温，0.5毫升/分钟流速，在260nm波长下用真空二极管探测器定量S-腺苷甲硫氨酸。表一列出在特定的一种培养物上清液中S-腺苷甲硫氨酸的含量。
表一：
| 菌株 | S-腺苷甲硫氨酸(mg/l) | |||||
| 无甲硫氨酸的培养 | 含0.5克/升L-甲硫氨酸的培养 | 含1克/升D，L-甲硫氨酸的培养 | ||||
| 24小时 | 48小时 | 24小时 | 48小时 | 24小时 | 48小时 | |
| W3110 | 0 | 0 | 0 | 0 | 0 | 0 |
| W3110/pKP481 | 3 | 12 | 61 | 71 | 34 | 31 |
实验例4：pMSRLSSK质粒的建构
A.RLSS基因的扩增
大鼠肝中的S-腺苷甲硫氨酸合成酶(RLSS)基因(马投等人，药理治疗，265-280页(1997))可通过使用Taq DNA聚合酶，进行本领域人员常用的聚合酶链反应，进行扩增。大鼠(Rattus nozvegicus)的cDNA做为模板。使用的引物为寡核苷酸RLSS1，其序列为
5’-CTAGCAGGAGGAATTCACCATGGGACCTGTGGATGGC-3’(SEQ IDNO：5)
及RLSS2，其序列为
5’-GGGTACCCCGCTAAAACACAAGCTTCTTGGGGACCTCCCA-3’(SEQ ID NO：6)。
在聚合酶链反应中得到一大约1.2kb的DNA片段，再通过QIAprep SpinMiniprep试剂盒的DNA吸附管柱(Qiagen，希尔登，德国)(依照说明操作)进行纯化及磷酸化反应。
B.RLSS基因克隆入载体
在此用来构建本发明的质粒的基础质粒为pACYC184的衍生质粒pACYC184-LH，其保藏于德国微生物及细胞培养物保藏中心，编号为DSM 10172。GAPDH启动子的序列被插入至本质粒中：用寡核苷酸GAPDHfw及GAPDH revII作为引物，通过聚合酶链反应依本领域人员已知的规则使扩增GAPDH启动子，GAPDHfw的序列为
5’-GTCGACGCGTGAGGCGAGTCAGTCGCGTAATGC-3’(SEQ ID NO：7)，GAPDHrevII的序列为
5’-GACCTTAATTAAGATCTCATATATTCCACCAGCTATTTGTTAG-3’(SEQ ID NO：8)，并以大肠杆菌菌株W3110(ATCC 27325)的染色体作为底物。产物通过电泳法分离及用QIAquick胶体提取用试剂盒(Qiagen)进行纯化(依照说明操作)及用限制酶MluI及PacI作用。通过T4-连接酶的帮助插入已被相同限制酶酶切后的pACYC184-LH载体中，得到pKP228质粒。
一个合成的多重克隆位点被引入pKP228质粒中，步骤如下：pKP228被BglII酶酶切，切点再由klenow酶补平(依建议方法操作)，及用碱性磷酸酶进行去磷酸作用。在由此制备的载体中插入一个合成的具有下述序列的双链DNA片段，所述的序列为
5’-GAAGATCTAGGAGGCCTAGCATATGTGAATTCCCGGGCTGCTGCAGCAGCTG-3’(SEQ ID NO：9)。产生的pKP504质粒，在GAPDH启动子的下游含有多重克隆位点。
将pKP504用PvuII酶切，去磷酸化，并使用T4-DNA连接酶将其与含有大鼠肝的S-腺苷甲硫氨酸合成酶的基因(基因序列为SEQ ID NO：10；蛋白质序列为SEQ ID NO：11)的磷酸化的聚合酶链反应产物进行连接(依说明操作)。用该连接混合物，依照本领域人员已知的方式，通过电穿孔法转化大肠杆菌菌株DH5α的细胞。将转化混合物倒至含氨苄青霉素的LB-琼脂培养基(10克/升胰蛋白胨；5克/升酵母提取物；5克/升氯化纳；15克/升琼脂；20毫克/升四环素)上，在37℃下隔夜培养。
通过QlAprep Spin Miniprep试剂盒(Qiagen，希尔登，德国)进行质粒的分离，通过限制酶分析及核苷酸序列分析，得到目标转化子。
在如上所述得到的质粒pMSRLSSK中，RLSS基因(其编码大鼠肝的S-腺苷甲硫氨酸合成酶)被置于大肠杆菌gapA基因的组成型GAPDH启动子的调控下。
实验例5：制备第二种S-腺苷甲硫氨酸生产菌
实验例4中所描述的pMSRLSSK质粒，通过氯化钙法转入大肠杆菌W3110(ATCC 27325)，然后，通过含有20毫克/升四环素的LB固态琼脂养基进行筛选，自转化子中再次进行分离，并用限制核酸内切酶酶切及检查。此菌株称为W3110/pMSRLSSK，其适合用于S-腺苷甲硫氨酸的生产。此菌株依布远佩斯条约，保藏于德国微生物及细胞培养物保藏中心，编号为DSM 16133。
实验例6：S-腺苷甲硫氨酸的发酵生产
A.S-腺苷甲硫氨酸的生产
W3110/pMSRLSSK菌株用于发酵生产S-腺苷甲硫氨酸，而不具有质粒的W3110起始菌株(ATCC 27325)则培养在相同条件下，作为比较用。
以下为所用的培养基：1升培养基中含有0.0147克的二水氯化钙，0.3克七水硫酸镁，0.15毫克二水钼酸钠，2.5毫克硼酸，0.7毫克六水氯化钴，0.25毫克五水硫酸铜，1.6毫克四水氯化锰，0.3毫克七水硫酸锌，3克磷酸二氢钾，12克磷酸氢二钾，5克硫酸铵，0.6克氯化钠，0.002克七水硫酸亚铁，1克二水柠檬酸钠，15克葡萄糖，1克胰蛋白胨，及0.5克酵母提取物，培养W3110/pMSRLSSK则需再加入20微克/毫升的四环素至培养基中。在表二中有标示处为额外再加入0.5克/升L-甲硫氨酸或1克/升D，L-甲硫氨酸至培养基中。
首先，将10毫升的培养基放入100毫升的三角瓶中，植入适当的菌株，在37℃，于160转的震荡器上培养上16小时，得到用于培养生产菌的预培养物，由此制备的细胞最终接种于50毫升相同的培养基(用300毫升的三角瓶)中培养，直到OD600吸光值(在600nm波长吸光值)达0.1，再将生产菌在37℃，于160转的震荡器中培养48小时。在24及48小时分别取样，通过离心将细胞从培养基中除去。
B.定量所生产的S-腺苷甲硫氨酸
可通过高效液相层析法(HPLC)定量在培养物上清液中所含有的S-腺苷甲硫氨酸的量(Develosil RP-Aqueous C 30柱，5微米，250×4.6毫米)(购自普亨奥孟克斯公司，阿赫亚芬贝格，德国)，加入10微升的培养物上清液，在等度洗脱下进行分离，洗脱液为1升水中含3毫升85％的磷酸，常温，0.5毫升/分钟流速，在260nm波长处用真空二极管探测器定量S-腺苷甲硫氨酸。表二列出在特定的培养物上清液中S-腺苷甲硫氨酸的含量。
表二：
| 菌株 | S-腺苷甲硫氨酸(毫克/升) | |||||
| 无甲硫氨酸的培养 | 含0.5克/升的L-甲硫氨酸的培养 | 含1克/升的D，L-甲硫氨酸的培养 | ||||
| 24小时 | 48小时 | 24小时 | 48小时 | 24小时 | 48小时 | |
| W3110 | 5 | 0 | 0 | 0 | 0 | 0 |
| W3110/pMSRLSSK | 9 | 46 | 78 | 82 | 63 | 68 |
序列表
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gaagcggtaa tggaagagat catcaagcca attctgcccg ctgaatggct gacttctgcc 660
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Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr Gly
325 330 335
Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys Asn
340 345 350
Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys Lys
355 360 365
Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser Glu
370 375 380
Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe
385 390 395
Claims (13)
1.一种发酵生产S-腺苷甲硫氨酸的方法，包括在一种培养基中培养可得自一种起始菌株、且与该起始菌株相比具有增强的S-腺苷甲硫氨酸合成酶活性的大肠杆菌菌株，该大肠杆菌菌株将S-腺苷甲硫氨酸分泌到该培养基中，以及自该培养基中收集该S-腺苷甲硫氨酸，其中所使用的S-腺苷甲硫氨酸合成酶是一种含有SEQ ID NO：1的序列的蛋白质或者是该蛋白质的一种功能性变体，该功能性变体的序列与SEQ ID NO：1的相似性大于40％且具有S-腺苷甲硫氨酸合成酶活性。
2.权利要求1的方法，其中的相似性大于60％。
3.权利要求1的方法，其中的相似性大于80％。
4.权利要求1-3中任一项的方法，其中的大肠杆菌菌株在最基本盐类培养基中培养。
5权利要求1-3中任一项的方法，其中所使用的碳源是葡萄糖或甘油。
6.权利要求1-3中任一项的方法，其中所使用的氮源是尿素、氨或其盐或硝酸盐。
7.权利要求1-3中任一项的方法，其中所述的大肠杆菌菌株在有氧培养条件下培养16至150小时且对于所述大肠杆菌菌株而言培养温度在其最适生长温度范围内。
8.权利要求1-3中任一项的方法，其中L-甲硫氨酸被添加到所述的最基本盐类培养基中，添加浓度为0.05至25克/升。
9.权利要求8的方法，其中添加浓度为1至5克/升。
10.权利要求1-3中任一项的方法，其中D，L-甲硫氨酸被添加到所述的最基本盐类培养基中，添加浓度为0.05至25克/升。
11.权利要求10的方法，其中添加浓度为1至5克/升。
12.权利要求1-3中任一项的方法，其中通过对培养基进行离心自该培养基中回收S-腺苷甲硫氨酸，然后进行S-腺苷甲硫氨酸的层析纯化、络合、过滤、或沉淀。
13.权利要求12的方法，其中所述过滤为交叉流过滤。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10309856A DE10309856A1 (de) | 2003-03-06 | 2003-03-06 | Verfahren zur fermentativen Herstellung von S-Adenosylmethionin |
| DE10309856.9 | 2003-03-06 |
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| Publication Number | Publication Date |
|---|---|
| CN1570126A CN1570126A (zh) | 2005-01-26 |
| CN1308457C true CN1308457C (zh) | 2007-04-04 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB2004100074938A Expired - Fee Related CN1308457C (zh) | 2003-03-06 | 2004-03-05 | 发酵生产s－腺苷甲硫氨酸的方法 |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US20040175805A1 (zh) |
| EP (1) | EP1457569A1 (zh) |
| JP (1) | JP2004267209A (zh) |
| CN (1) | CN1308457C (zh) |
| CA (1) | CA2457423A1 (zh) |
| DE (1) | DE10309856A1 (zh) |
| RU (1) | RU2004106512A (zh) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102005009751A1 (de) * | 2005-03-03 | 2006-09-07 | Consortium für elektrochemische Industrie GmbH | Verfahren zur fermentativen Herstellung von S-Adenosyl-Methionin |
| US20090012036A1 (en) * | 2005-05-24 | 2009-01-08 | Hebert Rolland F | Stable S-adenosyl-L-methionine |
| CN100363499C (zh) * | 2006-02-27 | 2008-01-23 | 上海国佳生化工程技术研究中心有限公司 | 一种新的腺苷甲硫氨酸的体外酶促转化方法 |
| CN101932694A (zh) * | 2007-11-30 | 2010-12-29 | 加利福尼亚大学董事会 | 使用表达卤代甲烷转移酶的重组生物进行有机化合物的工业生产 |
| CN101392230B (zh) * | 2008-01-07 | 2012-05-23 | 北京凯因科技股份有限公司 | 一株表达腺苷蛋氨酸合成酶的重组大肠埃希氏菌 |
| CN101285085B (zh) * | 2008-01-22 | 2011-04-27 | 西北工业大学 | 一种利用整细胞催化合成腺苷甲硫氨酸的方法 |
| CA2805041A1 (en) | 2009-07-22 | 2011-01-27 | The Regents Of The University Of California | Cell-based systems for production of methyl formate |
| BR112012012915B1 (pt) | 2009-11-30 | 2020-12-01 | Ajinomoto Co., Inc. | método para produção de l-cisteína, l-cistina, um derivado das mesmas, ou uma mistura das mesmas |
| RU2460793C2 (ru) | 2010-01-15 | 2012-09-10 | Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) | Способ получения l-аминокислот с использованием бактерий семейства enterobacteriaceae |
| CN101870964B (zh) * | 2010-05-25 | 2012-07-04 | 北京凯因科技股份有限公司 | 一种提高sam合成酶表达量的方法 |
| CN103119154B (zh) | 2010-09-14 | 2015-11-25 | 味之素株式会社 | 含硫氨基酸生产菌以及含硫氨基酸的制造方法 |
| JP2014087259A (ja) | 2011-02-22 | 2014-05-15 | Ajinomoto Co Inc | Ｌ−システイン生産菌及びｌ−システインの製造法 |
| BR112013023465B1 (pt) | 2011-04-01 | 2020-10-27 | Ajinomoto Co., Inc | método para produzir l-cisteína |
| WO2018228247A1 (zh) * | 2017-06-15 | 2018-12-20 | 安徽古特生物科技有限公司 | 一种利用腺苷代替atp进行酶促反应的生产方法 |
| WO2019110101A1 (de) | 2017-12-06 | 2019-06-13 | Wacker Chemie Ag | Mikroorganismen-stamm und verfahren zur fermentativen herstellung von methylanthranilat |
| CN111394384B (zh) * | 2020-04-07 | 2022-07-01 | 河南科技大学 | 一种用于检测s-腺苷甲硫氨酸的生物传感器及其制备方法 |
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| EP0647712A1 (en) * | 1993-10-07 | 1995-04-12 | Boehringer Ingelheim Espana S.A. | Production of S-adenosyl-methionine (SAM) by fermentation of transformed bacteria |
| CN1357630A (zh) * | 2001-11-30 | 2002-07-10 | 中国科学院上海生物化学研究所 | 一种生产腺苷甲硫氨酸的方法 |
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| GB2116172B (en) * | 1982-02-25 | 1986-07-09 | Nippon Zeon Co | Microbial cells containing s-adenosyl methionine in high concentrations and process for production of s adenosyl methionine |
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| KR100526770B1 (ko) * | 2001-07-16 | 2005-11-08 | 서주원 | 스트렙토마이세스 속 균주 유래의 아데노실메치요닌 생합성 효소, 이를 코딩하는 유전자 염기서열 및 항생제를 포함하는 유용한 이차대사산물의 대량 생산방법 |
-
2003
- 2003-03-06 DE DE10309856A patent/DE10309856A1/de not_active Withdrawn
-
2004
- 2004-02-27 US US10/789,493 patent/US20040175805A1/en not_active Abandoned
- 2004-03-03 CA CA002457423A patent/CA2457423A1/en not_active Abandoned
- 2004-03-04 EP EP04005193A patent/EP1457569A1/de not_active Withdrawn
- 2004-03-05 JP JP2004062766A patent/JP2004267209A/ja active Pending
- 2004-03-05 CN CNB2004100074938A patent/CN1308457C/zh not_active Expired - Fee Related
- 2004-03-05 RU RU2004106512/13A patent/RU2004106512A/ru unknown
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| JPS63279798A (ja) * | 1987-05-12 | 1988-11-16 | Kyowa Hakko Kogyo Co Ltd | Ｓ−アデノシルメチオニンの製造法 |
| EP0647712A1 (en) * | 1993-10-07 | 1995-04-12 | Boehringer Ingelheim Espana S.A. | Production of S-adenosyl-methionine (SAM) by fermentation of transformed bacteria |
| CN1357630A (zh) * | 2001-11-30 | 2002-07-10 | 中国科学院上海生物化学研究所 | 一种生产腺苷甲硫氨酸的方法 |
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Also Published As
| Publication number | Publication date |
|---|---|
| JP2004267209A (ja) | 2004-09-30 |
| EP1457569A1 (de) | 2004-09-15 |
| CN1570126A (zh) | 2005-01-26 |
| DE10309856A1 (de) | 2004-09-23 |
| US20040175805A1 (en) | 2004-09-09 |
| CA2457423A1 (en) | 2004-09-06 |
| RU2004106512A (ru) | 2005-08-10 |
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