CN1297054A - 编码pfkA基因的新核苷酸序列 - Google Patents
编码pfkA基因的新核苷酸序列 Download PDFInfo
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- CN1297054A CN1297054A CN00132480A CN00132480A CN1297054A CN 1297054 A CN1297054 A CN 1297054A CN 00132480 A CN00132480 A CN 00132480A CN 00132480 A CN00132480 A CN 00132480A CN 1297054 A CN1297054 A CN 1297054A
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Abstract
本发明涉及分离的多核苷酸,其含有选自如下一组的多核苷酸序列:a)与编码包括SEQ ID NO:2的氨基酸序列的多肽的多核苷酸至少70%相同的多核苷酸,b)编码含有与SEQ ID NO:2的氨基酸序列至少70%相同的氨基酸序列的多肽的多核苷酸,c)与a)或b)的多核苷酸互补的多核苷酸,以及d)含有a),b)或c)的多核苷酸序列的至少15个连续碱基的多核苷酸。本发明还提供了经扩增pfkA基因发酵生产L-氨基酸的方法,以及本发明多核苷酸作为引物或杂交探针的应用。
Description
本发明提供了编码pfkA基因的核苷酸序列,和通过具有扩增的pfkA基因的棒状细菌发酵生产L-氨基酸特别是L-赖氨酸的方法。
氨基酸特别是L-赖氨酸用于人用药物和制药工业,特别是动物营养。
已知氨基酸可通过棒状细菌菌株,尤其谷氨酸棒杆菌的发酵而生产。由于其极其重要性,已持续进行改良生产方法的尝试。生产方法的改良可涉及发酵措施,如搅拌和供氧,或营养培养基的组成如发酵期间的糖浓度,或产物形式的加工方法,例如通过离子交换层析,或微生物本身的固有生产性质。
为改良这些微生物的生产性质,可使用诱变,选择及突变体选择等方法,以此方法可获得对抗代谢物如赖氨酸类似物S-(2-氨基乙基)半胱氨酸有抗性或重要的调节代谢物营养缺陷的并产生L-氨基酸如L-赖氨酸的菌株。
一段时间以来,重组DNA技术的方法也用于棒杆菌生产氨基酸的菌株的改良,其是通过扩增各个生物合成基因并研究对氨基酸生产的作用而进行。这一主题的综述文章可在Kinoshita(“谷氨酸细菌”,工业微生物的生物学,Demain和Soloman编辑,BenjaminCummings,英国伦敦,1985,115-142),Hilliger(BioTec 2,40-44(1991)),Eggeling(氨基酸6:261-272(1994)),Jetten和Sinskey(生物技术学关键综述15,73-103(1995))和Sahm等(纽约科学学会年报782,25-39(1996))。
本发明人目的在于为改良氨基酸尤其是L-赖氨酸的发酵生产而提供新措施。
氨基酸、尤其是L-赖氨酸用于人用药物及制药工业,特别是动物营养,因此提供生产氨基酸特别是L-赖氨酸的新改良方法总是令人感兴趣的。
当下文提到L-赖氨酸或赖氨酸时,不仅是指碱,也指盐,如赖氨酸单盐酸盐或赖氨酸硫酸盐。
本发明提供了一种来自棒状细菌的分离的多核苷酸,其含有选自如下一组的多核苷酸序列:
a)与编码含有SEQ ID NO:2的氨基酸序列的多肽的多核苷酸至少70%相同的多核苷酸,
b)编码含有与SEQ ID NO:2的氨基酸序列至少70%相同的氨基酸序列的多肽的多核苷酸,
c)与a)或b)的多核苷酸互补的多核苷酸,以及
d)含有a),b)或c)的多核苷酸序列的至少15个连续碱基的多核苷酸。
本发明还提供了根据权利要求1的多核苷酸,其优选包括能复制的DNA,含有
(ⅰ)SEQ ID NO:1的核苷酸序列,或
(ⅱ)在遗传密码简并范围内相应于(ⅰ)序列的至少一个序列,或
(ⅲ)与互补于(ⅰ)或(ⅱ)序列的序列杂交的至少一个序列,和任选地,
(ⅳ)(ⅰ)中有功能的中性有义突变。
本发明还提供了
权利要求4的多核苷酸,含有SEQ ID NO:1所示的核苷酸序列,
权利要求6的多核苷酸,其编码含有SEQ ID NO:2所示的氨基酸序列的多肽,
含有权利要求1的多核苷酸序列的载体,特别是穿梭载体或质粒载体,
以及作为宿主细胞的含有载体的棒状细菌。
本发明还提供了基本上由多核苷酸序列组成的多核苷酸,其可通过用含有相应于SEQ ID NO:1所述多核苷酸或其片段的序列的探针杂交合适的基因文库,并分离所述的DNA序列来筛选获得,所述的文库包括具有相应于SEQ ID NO:1的所述多核苷酸序列的完整基因。
本发明的多核苷酸序列适用作RNA、cDNA和DNA的杂交探针,以分离编码果糖磷酸激酶的全长cDNA,以及分离与果糖磷酸激酶基因具有高度序列相似性的cDNA或基因。
本发明的多核苷酸序列还适用作通过聚合酶链反应(PCR)制备编码磷酸果糖激酶的基因的DNA的引物。
作为探针或引物的这种寡核苷酸含有至少30个、优选至少20个、特别优选至少15个连续核苷酸。具有至少40或50个核苷酸的寡核苷酸也是合适的。
“分离的”是指从其天然环境中分离出来。
“多核苷酸”一般地涉及多聚核糖核苷酸和多聚脱氧核糖核苷酸,其可以是非修饰的RNA或DNA,或修饰的RNA或DNA。
“多肽”应理解为包括经肽键结合的两个或多个氨基酸的肽或蛋白质。
本发明的多肽包括SEQ ID NO:2的多肽,特别是具有果糖磷酸激酶生物学活性的多肽,以及与SEQ ID NO:2的多肽至少70%相同的多肽,优选地与SEQ ID NO:2的多肽至少80%、特别是90-95%相同并具有所述活性的多肽。
本发明另外还提供了用尤其已生产氨基酸的棒状细菌发酵生产氨基酸特别是L-赖氨酸的方法,在该棒状细菌中编码pfkA基因的核苷酸序列是扩增的,尤其是过表达的。
文中术语“扩增”是指微生物中由相应DNA编码的一或多种酶的胞内活性的增加,例如通过增加一或多个基因的拷贝数,通过用强启动子或编码具有升高的活性的相应酶的基因或等位基因,及任选地组合使用这些方法。
本发明的微生物可从葡萄糖、蔗糖、乳糖、果糖、麦芽糖、糖蜜、淀粉、纤维素或从甘油和乙醇中生产L-氨基酸特别是L-赖氨酸。此微生物可以是棒状细菌尤其是棒杆菌属的代表菌。在棒杆菌属中尤其应提及的是谷氨酸棒杆菌,本领域技术人员已知其生产L-氨基酸的能力。
适当的棒杆菌菌属,尤其谷氨酸棒杆菌菌株,是例如已知的野生型菌株:
谷氨酸棒杆菌ATCC 13032
醋谷棒杆菌ATCC 15806
嗜乙酰乙酸棒杆菌ATCC 13870
嗜热产氨棒杆菌FERM BP-1539
Corynebacterium melassecola ATCC 17965
黄色短杆菌ATCC14067
乳发酵短杆菌ATCC13869和
扩展短杆菌ATCC14020
和从中获得的生产L-氨基酸的突变体或菌株如:
谷氨酸棒杆菌FERM-P1709
黄色短杆菌FERM-P1708
乳发酵短杆菌FERM-P1712
谷氨酸棒杆菌FERM-P6463
谷氨酸棒杆菌FERM-P6464和
谷氨酸棒杆菌DSM5715。
本发明人已成功地分离谷氨酸棒杆菌的编码果糖磷酸激酶(EC2.7.1.11)的新pfkA基因。
为了分离谷氨酸棒杆菌的pfkA基因或其他基因,首先在大肠杆菌中建立这一微生物的基因文库。可根据通常已知的教材和手册建立基因文库。例如由Winnacker所著教材:基因与克隆,基因工程入门(Verlag Chamie,Weinheim,德国,1990),或由Sambrook等所著手册:分子克隆实验手册(Cold Spring Harbor Laboratory Press,1989)。一非常熟知的基因文库是已由Kohara等(细胞50,495-508(1987))在λ载体中建立的E.coli K-12菌株W3110的基因文库。Bathe等(分子及普通遗传学,252:255-265,1996)阐述了借助于粘粒载体SuperCos I(Wahl等,1987,Proceeding of the NationalAcademy of Sciences USA,84:2160-2164),在E.coli K-12菌株NM554(Raleigh等,1988,核酸研究16:1563-1575)中建立的谷氨酸棒杆菌ATCC13032的基因文库。Bormann等(分子微生物学6(3),317-326)描述了用粘粒pHC79(Hohn和Collins,基因11,291-298(1980))制备谷氨酸棒杆菌ATCC13032的基因文库。为在大肠杆菌中制备谷氨酸棒杆菌的基因文库,也可使用质粒如pBR322(Bolivar,生命科学25,807-818(1979))或pUC9(Viera等,1982,基因19:259-268)。合适的宿主尤其是限制和重组缺陷的大肠杆菌菌株,一个例子是菌株DH5αmcr,如Grant等(美国科学院院报7(1990)4645-4649)所述。借助于粘粒克隆的长DNA片段随后可亚克隆并用适于测序的通用载体测序,如Sanger等(美国科学院院报74:5463-5467,1977)所述。
以此方式可获得编码pfkA基因的谷氨酸棒杆菌的新DNA序列,示作SEQ ID NO:1,是本发明的一部分,用前述方法从此DNA序列中也已衍生出相应蛋白质的氨基酸序列。所得pfkA基因产物的氨基酸序列以SEQ ID NO:2表示。
通过遗传密码的简并性产生的编码DNA序列也是本发明的一部分。同样,与SEQ ID NO:1或SEQ ID NO:1的部分杂交的DNA序列也是本发明的一部分。另外,蛋白质中的保守氨基酸置换,如用丙氨酸置换蛋白质中甘氨酸,或用谷氨酸置换蛋白质中天冬氨酸,本领域已知是“有义突变”,其不引起蛋白质任何功能的改变,即是中性的。另外已知蛋白质N和/或C-末端的改变基本不影响其功能,或者甚至可以稳定其功能,本领域技术人员可在以下文献中发现对此的论述,参见Ben-Bassat等(细菌学杂志169:751-757(1987))O’Regan等(基因77:237-251.(1989)),Sahin-Toth等(蛋白质科学3:240-247(1994)),Hochuli等(生物/技术6:1321-1325(1988)),及已知关于遗传和分子生物学的教材。以相应方式产生自SEQ ID NO:2的氨基酸序列也是本发明的一部分。
同样,与SEQ ID NO:1或SEQ ID NO:1的部分杂交的DNA序列也是本发明的组成部分。最后,用产生自SEQ ID NO:1的引物经聚合酶链反应(PCR)制备的DNA序列也是本发明的组成部分。这种寡核苷酸典型地具有至少15个核苷酸的长度。
通过杂交鉴别DNA序列的指导可参见宝灵格曼海姆有限公司的手册“用于滤膜杂交的DIG系统用户指南”(曼海姆,德国,1993)以及Liebl等(系统细菌学国际杂志(1991)41:255-260)。用聚合酶链反应(PCR)扩增DNA序列的指导参见Gait的手册:寡核苷酸合成实用方法(IRL出版社,英国牛津,1984)及Newton和Graham:PCR(Spektrum Akademischer Verlag,Heidelberg,德国,1994)。
本发明人发现,在pfkA过表达后,棒状细菌以改良方式生产L-氨基酸尤其是L-赖氨酸。
为获得过表达,可提高相应基因的拷贝数,或可使位于结构基因上游的启动子和调节区或核糖体结合位点突变。掺入结构基因上游的表达盒以同样方式工作。通过可诱导启动子,在L-赖氨酸发酵生产期间增强表达也是可能的。通过目的在于延长mRNA寿命的措施,也可改良表达。通过防止酶蛋白的分解也可提高酶促活性。基因或基因构建体可以不同拷贝数存在于质粒中,或可在染色体中整合与扩增。或者,通过改变培养基的组分和培养条件,也可获得相关基因的过表达。
本领域熟练技术人员从以下文献中可发现对此的详述,参见Martin等(生物/技术5,137-146(1987)),Guerrero等(基因138,35-41(1994)),Tsuchiya和Morinaga(生物/技术6,428-430(1988)),Eikmanns等(基因102,93-98(1991)),欧洲专利说明书EPS0472869,美国专利4,601,893,Schwarzer和Puhler(生物/技术9,84-87(1991)),Reinscheid等(应用及环境微生物学60,126-132(1994)),LaBarre等(细菌学杂志175,1001-1007(1993)),专利申请WO96/15246,Malumbres等(基因134,15-24(1993)),日本特许公开JP-A-10-229891,Jensen和Hammer(生物技术及生物工程58,191-195(1998)),Makrides(微生物学综述60:512-538(1996))及已知关于遗传及分子生物学的教材。
例如,本发明的pfkA基因可借助于质粒过表达。
合适的质粒是在棒状细菌中复制的质粒。各种已知的质粒载体,如pZ1(Menkel等,应用及环境微生物学(1989)64:549-554),pEKEx1(Eikmanns等,基因102:93-98(1991))或pHS2-1(Sonnen等,基因107:69-74(1991))是基于隐蔽质粒pHM1519,pBL1或pGA1。其它可质粒载体如基于pCG4(US-A4,489,160),或pNG2(Serwold-Davis等,FEMS微生物学通信66,119-124(1990))或pAG1(US-A5,158,891)可以同样方式使用。
其他合适的质粒载体是那些可通过整合进染色体而进行基因扩增的质粒载体,例如,由Reinscheid等(应用及环境微生物学60:126-132(1994))所述的用于复制或扩增hom-thrB操纵子的质粒载体。在这一方法中,将完整基因克隆进能在宿主(典型地是大肠杆菌)中复制而在谷氨酸棒杆菌中不能复制的质粒载体中。可考虑的载体例如是pSUP301(Simon等,生物/技术1,784-791(1983)),pK18mob或pK19mob(Schafer等,基因145,69-73(1994)),pGEM-T(Promega公司,Madison,WI,USA),pCR2.1-TOPO(Shuman(1994),生物化学杂志269:32678-84;US-A 5,487,993),pCRBlunt(Invitrogen,Groningen,荷兰;Bernard等,分子生物学杂志,234:534-541(1993))或pEM1(Schrumpf等,1991,细菌学杂志173:4510-4516)。含有待扩增基因的质粒载体然后可经接合或转化转移进希望的谷氨酸棒杆菌菌株。接合方法例如如Schafer等(应用及环境微生物学60,756-759(1994))所述。转化方法例如如Thierbach等(应用微生物学及细菌学29,356-362(1988)),Dunican和Shivnan(生物/技术7,1067-1070(1989))和Tauch等(FEMS微生物学通信123,343-347(1994))所述。经“交换”而同源重组后,得到的菌株含有至少两个拷贝的所需基因。
另外,不仅是pfkA基因,而且特定生物合成途径、糖酵解、回补代谢、柠檬酸循环或氨基酸输出的一或多种酶的扩增或过表达,对氨基酸特别是L-赖氨酸的生产可以是有益的。
因此,为生产L-赖氨酸,例如可同时过表达一或多种选自如下一组的基因:
·编码二氢-2,6-吡啶二羧酸合酶的dapA基因(EP-B-0197335),或
·编码甘油醛-3-磷酸脱氢酶的gap基因(Eikmanns(1992),细菌学杂志174,6076-6086),或
·编码三糖磷酸异构酶的tpi基因(Eikmanns(1992),细菌学杂志174,6076-6086),或
·编码3-磷酸甘油激酶的pgk基因(Eikmanns(1992),细菌学杂志174,6076-6086),或
·编码丙酮酸羧化酶的pyc基因(Eikmanns(1992),细菌学杂志174,6076-6086),或
·编码赖氨酸输出蛋白的lysE基因(DE-A-19548222)。
对于氨基酸特别是L-赖氨酸的生产,除了扩增pfkA基因外,还有利的是同时弱化
·编码烯醇丙酮酸磷酸羧激酶的pck基因(DE19950409.1,DSM13047)和/或
·编码葡萄糖-6-磷酸异构酶的pgi基因(US09/396,478,DSM12969)。
除pfkA基因的过表达之外,抑制非所需的二级反应对氨基酸特别是L-赖氨酸的生产也是有益的(Nakayama:“生产氨基酸的微生物的育种”,微生物产物的过量产生,Krumphanzl,Sikyta,Vanek(编辑),学术出版社,伦敦,英国,1982)。
根据本发明产生的微生物可连续培养或用分批方法,补料分批方法或重复补料分批法分批培养以生产氨基酸特别是L-赖氨酸。已知的培养法由Chmiel(Bioprozesstechnik 1.Einfuhrung in dieBioverfahrenstechnik(Gustav Fischer Verlag,Stuttgart,1991)所著教材,或由Storhas(Bioreaktoren und periphere Einrichtungen(ViewegVerlag,Brunswick/Wiesbaden,1994))所著教材中提供。
所用培养基必须以适当方式符合各菌株的需求,关于各种微生物培养基的阐述见于,美国细菌学会的“细菌学通用方法手册”(华盛顿D.C.,USA,1981)。可使用的碳源包括糖及碳水化合物,例如葡萄糖,蔗糖,乳糖,果糖,麦芽糖,糖蜜,淀粉和纤维素,油和脂肪如豆油,葵花油,落花生油和椰子油,脂肪酸如棕榈酸,硬脂酸和亚油酸,醇如甘油和乙醇,及有机酸如乙酸,这些物质可单独或混合使用,可使用的氮源包括含氮的有机化合物如胨,酵母提取物,肉膏,麦芽提取物,玉米浸液、大豆粉和尿素,或无机化合物如硫酸铵,氯化铵,磷酸铵,碳酸铵和硝酸铵。氮源可单独或混合使用,可使用的磷源包括磷酸,磷酸二氢钾或磷酸氢二钾,或相应钠盐培养基另外还必须含有为生长所需的金属盐如硫酸镁或硫酸铁。最后,除了上述物质之外,可使用生长必需物质如氨基酸和维生素,此外,可将适当前体加入培养基中上述物质可以单批形式或在培养期间以适当方式加入培养物中。
可以适当方式加入碱性化合物如NaOH,KOH,氨或氨水,或酸性化合物如磷酸或硫酸,以调节培养物的PH值,抗泡沫剂例如脂肪酸聚乙二醇酯可用于控制泡沫产生。适当的选择性作用物质例如抗生素,可加入培养基中以保持质粒的稳定性。氧气或含氧混合气,例如空气,可充入培养物中以保持有氧条件。培养温度通常在20℃~45℃,优选25℃~40℃,持续培养直至赖氨基酸形成最大量。此目的通常在10~160小时范围达到。
L-赖氨酸的分析可通过阴离子交换层析,随后经如Speckman等(分析化学,30,(1958),1190)所述茚三酮衍生化作用进行。
本发明方法用于发酵制备氨基酸尤其是L-赖氨酸。
本发明借助于以下提供的实施例得以更详述阐述。
实施例1 制备谷氨酸棒杆菌ATCC13032的基因组粘粒基因文库
如Tauch等(1995,质粒33:168-179)所述分离谷氨酸棒杆菌ATCC13032的染色体DNA并用限制性内切酶Sau3AI(AmershamPharmacia,Freiburg,德国,产品描述Sau3AI,编码27-0913-02)部分酶切。用虾碱性磷酸酶(Roche Molecular Biochemicals,德国曼海姆,产品描述SAP,编码1758250)将DNA片段去磷酸化。得自Stratagene公司(La Jolla,USA,产品描述SuperCosl粘粒载体试剂盒,编码251301)的粘粒载体SuperCosl(Wahl等(1987)美国科学院院报84:2160-2164)的DNA用限制性内切酶XbaI(AmershamPharmacia,Freiburg,德国,产品描述XbaI,编码27-0948-02)酶切并类似地用虾碱性磷酸酶去磷酸化。粘粒DNA然后用限制性内切酶BamHI(Amersham Pharmacia,Freiburg,德国,产品描述BamHI,编码27-0868-04)酶切。以此方式处理的粘粒DNA与处理的ATCC13032 DNA片段混合并用T4 DNA连接酶(AmershamPharmacia,Freiburg,德国,产品描述T4-DNA-连接酶,编码27-0870-04)处理。连接混合物然后用Gigapack Ⅱ XL包装提取物(Stratagene,La Jolla,USA,产品描述Gigapack Ⅱ XL包装提取物,编码200217)包装进噬菌体中。为感染大肠杆菌菌株NM554(Raleigh等,1988,核酸研究16:1563-1575),将细胞置于10mM MgSO4并与噬菌体悬液混合。如Sambrook等(1989,分子克隆实验手册,冷泉港)所述进行粘粒文库的感染和滴定,细胞在含100微克/毫升氨苄青霉素的LB琼脂(Lennox,1955,病毒学,1:190)上铺板。在37℃保温过夜后,选择重组克隆。
实施例2 pfkA基因的分离和测序
用Qiaprep Spin微量制备试剂盒(产品号27106,Qiagen,Hilden,德国)根据厂商指导分离各个菌落的粘粒DNA,并用限制性内切酶Sau3AI(Amersham Pharmacia,Freiburg,德国,产品描述Sau3AI,编码27-0913-02)部分酶切。用虾碱性磷酸酶(Roche MolecularBiochemicals,德国曼海姆,产品描述SAP,编码1758250)将DNA片段去磷酸化。凝胶电泳分离后,用QiaExⅡ凝胶提取试剂盒(产品号20021,Qiagen,Hilden,德国)分离大小范围为1500-2000bp的粘粒片段。得自Invitrogen公司(Groningen,荷兰,产品描述Zero背景克隆试剂盒,产品号K2500-01)的测序载体pZero-1的DNA用限制性内切酶BamHI(Amersham Pharmacia,Freiburg,德国,产品描述BamHI,产品号27-0868-04)酶切。如Sambrook等(1989,分子克隆实验手册,冷泉港)所述进行粘粒片段在测序载体pZero-1中的连接,DNA混合物与T4连接酶(Pharmacia Biotech,Freiburg,德国)保温过夜。然后将连接混合物电穿孔(Tauch等,1994,FEMS微生物学通信,123:343-7)进大肠杆菌菌株DH5αMCR(Grant,1990,美国科学院院报87:4645-4649)并在含有50微克/毫升zeocin的LB琼脂(Lennox,1955,病毒学,1:190)上铺板。重组克隆的质粒制备用Biorobot 9600(产品号900200,Qiagen,Hilden,德国)进行。测序用Zimmermann等(1990,核酸研究18:1067)改良的Sanger等(1977,美国科学院院报74:5463-5467)的双脱氧链终止法进行。使用得自PE应用生物系统公司(产品号403044,Weiterstadt,德国)的“RR罗丹明终止循环测序试剂盒”。在带有购自PE应用生物系统公司(Weiterstadt,德国)的“ABI Prism 377”测序仪的“Rotiphoresis NF”丙烯酰胺/双丙烯酰胺凝胶(29∶1)(产品号A124.1,Roth,Karlsruhe,德国)中进行凝胶电泳分离和序列分析。
原始数据资料然后用Staden程序包(1986,核酸研究,14:217-231)版本97-0处理。pZero-1衍生物的各个序列组装成连续重叠群。用XNIP程序(Staden,1986,核酸研究,14:217-231)进行计算机辅助编码区分析。同源性分析用“BLAST搜索程序’(Altschul等,1997,核酸研究,25:3389-3402)对“国家生物技术信息中心”(NCBI,Bethesda,MD,USA)的非冗余数据库进行。
获得的核苷酸序列如SEQ ID NO:1所示。对该核苷酸序列的分析显示一1029碱基对的开放读框,其被称为pfkA基因。pfkA基因编码343个氨基酸的多肽。
实施例3 用于在谷氨酸棒杆菌中过量表达pfkA的质粒的制备
3.1将pfkA克隆进pCR-Blunt载体
如Tauch等(1995,质粒33:168-179)所述分离谷氨酸棒杆菌ATCC13032的染色体DNA。基于实施例2已知的谷氨酸棒杆菌pfkA基因的序列,选择下述寡核苷酸用于聚合酶链反应:
pfkA-exp
5′-AAC TGC AGC TCT GGC GAT TA-3′
pfkA-ex2
5′-AAC TAT CCA AAC ATT GCC TG-3′
所述引物由MWG生物技术公司(Ebersberg,德国)合成,并根据Innis等(PCR方案,方法和应用指南,1990,学术出版社)的标准PCR方法用来自Roche诊断学有限公司(德国曼海姆)的Pwo聚合酶进行PCR反应。用聚合酶链反应分离携带pfkA基因的约1160bp的DNA片段。
用Invitrogen公司(Carlsbad,CA,USA;目录号K2700-20)的Zero Blunt PCR克隆试剂盒将扩增的DNA片段连接进载体pCR2Blunt载体(Bernard等,(1983),分子生物学杂志234:534-541)中。用连接混合物转化大肠杆菌菌株Top10F(Grand等,(1990),美国科学院院报87:4645-4649)。通过将转化混合物在补加50mg/l卡那霉素的LB琼脂(Sambrook等,分子克隆实验手册,第2版,冷泉港实验室出版社,冷泉港,纽约)上铺板而选择携带质粒的细胞。用来自Qiagen的QIAprep Spin Miniprep试剂盒从转化子分离质粒DNA,并用限制酶EcoRI限制随后琼脂糖凝胶电泳(0.8%)证实。质粒命名为pCRB1-pfkAexp1。
3.2 穿梭载体pEC-T18mob2的制备
根据现有技术构建大肠杆菌-谷氨酸棒杆菌穿梭载体。该载体含有质粒pGA1的包括复制效应子per(US-A5,175,108;Nesvera等,细菌学杂志179,1525-1532(1997))的复制区rep,质粒pAG1的赋予四环素抗性的tetA(Z)基因(US-A5,158,891;国家生物技术信息中心(NCBI,Bethesda,MD,USA)基因文库登录号AF121000),质粒pMB1(Sutcliffe,定量生物学冷泉港研讨会43,77-90(1979))的复制起点oriV,包括1ac启动子和多克隆位点(mcs)的1acZα基因片段(Norrander等,基因26,101-106(1983)),以及质粒RP4(Simon等,(1983)生物/技术1:784-791)的mob区。然后将构建的载体转化进大肠杆菌菌株DH5α(Hanahan,DNA克隆实用方案,第Ⅰ卷,IRL出版社,Oxford,华盛顿特区,美国)。通过将转化混合物在补加5mg/l四环素的LB琼脂(Sambrook等,分子克隆实验手册,第2版,冷泉港实验室出版社,冷泉港,纽约)上铺板而选择携带质粒的细胞。用来自Qiagen的QIAprep Spin Miniprep试剂盒从转化子分离质粒DNA,并用限制酶EcoRI和HindⅢ限制随后琼脂糖凝胶电泳(0.8%)证实。质粒命名为pEC-T18mob2并如图1所示。
3.3将pfkA克隆进穿梭载体pEC-T18mob2中
所用载体是实施例3.2所述的大肠杆菌-谷氨酸棒杆菌穿梭载体pEC-T18mob2。来自这一质粒的DNA用限制酶EcoRI完全酶切,然后用虾碱性磷酸酶(Roche Molecular Biochemicals,德国曼海姆,产品描述SAP,编码1758250)去磷酸化。
通过用限制酶EcoRI完全酶切从实施例3.1所述的质粒pCRB1-pfkAexp1中分离pfkA基因。用QiaExⅡ凝胶提取试剂盒(产品号20021,Qiagen,Hilden,德国)从琼脂糖凝胶中分离约1160bp pfkA片段。
以此方式获得的pfkA片段与制备的pEC-T18mob2载体混合,并用T4 DNA连接酶(Amersham Pharmacia,Freiburg,德国,产品描述T4-DNA-连接酶,编码27-0870-04)处理。连接混合物然后转化进大肠杆菌菌株DH5α(Hanahan,DNA克隆实用方案,第Ⅰ卷,IRL出版社,Oxford,华盛顿特区,美国)。通过将转化混合物在含有5mg/l四环素的LB琼脂(Lennox,1955,病毒学1:90)上铺板而选择携带质粒的细胞。37℃保温过夜后,选择各个重组克隆。根据厂商指导用QIAprep Spin Miniprep试剂盒(产品号27106,Qiagen,Hilden,德国)从转化子分离质粒DNA,并用限制酶EcoRI酶切以经随后的琼脂糖凝胶电泳检查质粒。得到的质粒命名为pT-pfkAexp,其示于图2。
实施例4 用质粒pT-pfkAexp转化菌株DSM5715
通过Liebl等所述的电穿孔法(FEMS微生物学通信,53:299-303(1989))用质粒pT-pfkAexp转化菌株DSM5715(EP-B-0435132)。在补加5mg/l四环素的LBHIS琼脂(18.5g/l脑心浸液,0.5M山梨醇,5g/l Bacto-胰胨,2.5g/l Bacto-酵母膏,5g/l NaCl,18g/l Bacto-琼脂))上筛选转化子。在33℃保温2天。
用常规方法(Peters-Wendich等,1998,微生物学,144,915-927)从转化子分离质粒DNA,用限制性内切酶EcoRI切割并经随后的琼脂糖凝胶电泳检查质粒。获得的菌株称为DSM5715/pT-pfkAexp。
实施例5 生产赖氨酸
实施例4中制备的谷氨酸棒杆菌菌株DSM5715/pT-pfkAexp在适于生产赖氨酸的营养培养基中培养,并确定培养物上清中的赖氨酸含量。
为此,首先在33℃在具有合适抗生素的琼脂板(具有四环素(5mg/l)的脑心琼脂)上培养菌株24小时。从这些琼脂板培养物开始,接种预培养物(10ml培养基于100ml锥形瓶)。用于预培养的培养基是完全培养基CgⅢ(NaCl 2.5g/l,Bacto-肽胨10g/l,Bacto-酵母膏10g/l,葡萄糖20g/l,pH7.4)。加入四环素(5mg/l)。在摇床上于33℃以240rpm振荡培养预培养物16小时。从此预培养物接种主培养物,从而主培养物的起始OD(测量波长660nm)是0.1。培养基MM用作主培养物。培养基MM:CSL(玉米浆) 5g/lMOPS(吗啉代丙磺酸) 20g/l葡萄糖(单独高压灭菌) 50g/lNH4)2SO4 25g/lKH2PO4 0.1g/lMgSO4·7H2O 1.0g/lCaCl2·2H2O 10mg/lFeSO4·7H2O 10mg/lMnSO4·H2O 5.0mg/l生物素(过滤灭菌) 0.3mg/l硫胺素·HCl(过滤灭菌) 0.2mg/lL-亮氨酸(过滤灭菌) 0.1g/lCaCO3 25g/l
用氨水将CSL,MOPS和盐溶液调至pH7并高压灭菌。然后加入无菌的底物和维生素溶液,并加入干态高压灭菌的CaCO3。
以在具有档板的100ml锥形瓶中的10ml体积进行培养,加入卡那霉素(25mg/l)。在33℃和80%大气湿度下进行培养。
24小时后,用Biomek(Beckman仪器有限公司,慕尼黑)确定在660nm测量波长的OD。用Eppendorf-BioTronik公司的氨基酸分析仪(汉堡,德国)经离子交换层析和用茚三酮检测柱后衍生化而确定形成的赖氨酸量。
所得结果示于表1。表1
菌株 | OD(660) | 赖氨酸HClg/L |
DSM5715/pT-pfkAexp | 14.6 | 10.1 |
DSM5715 | 15.2 | 8.1 |
本发明包括如下附图:
图1:质粒pEC-T18mob2图。
图2:质粒pT-pfkAexp图。
图中所采用的缩写和符号有如下含义:
Tet:四环素抗性基因
oriV:大肠杆菌的质粒编码的复制起点
RP4mob:质粒迁移的mob区
rep:谷氨酸棒杆菌质粒pGA1的质粒编码的复制起点
per:pGA1的控制拷贝数的基因
lacZ-alpha:β-半乳糖苷酶基因的lacZα基因片段(N末端)
′lacZa′:lacZα基因片段的5’末端
lacZ-alpha′:lacZα基因片段的3’末端
pfkA:谷氨酸棒杆菌ATCC13032的pfkA基因
BamHI:限制酶BamHI的酶切位点
EcoRI:限制酶EcoRI的酶切位点
HindⅢ:限制酶HindⅢ的酶切位点
KpnI:限制酶KpnI的酶切位点
PstI:限制酶PstI的酶切位点
PvuI:限制酶PvuI的酶切位点
SalI:限制酶SalI的酶切位点
SacI:限制酶SacI的酶切位点
SmaI:限制酶SmaI的酶切位点
SphI:限制酶SphI的酶切位点
XbaI:限制酶XbaI的酶切位点
XhoI:限制酶XhoI的酶切位点
序列表<110>德古萨-于尔斯股份公司<120>编码pfkA基因的新核苷酸序列<130>990169 BT<140><141><160>2<170>PatentIn Ver.2.1<210>1<211>1274<212>DNA<213>谷氨酸棒杆菌<220><221> CDS<222> (143)..(1171)<400>1gtcgatttgt taatgaaact gcagctctgg cgattaaata agatggtcag agacagtttt 60ttggcctgtc aacccctgtg attctcttat ttttgggtga ttgttccggc gcgggtgttg 120tgatgggttt aatatggaag ac atg cga att gct act ctc acg tca ggc ggc 172
Met Arg Ile Ala Thr Leu Thr Ser Gly Gly
1 5 10gac tgc ccc gga cta aac gcc gtc atc cga gga atc gtc cgc aca gcc 220Asp Cys Pro Gly Leu Asn Ala Val Ile Arg Gly Ile Val Arg Thr Ala
15 20 25agc aat gaa ttt ggc tcc acc gtc gtt ggt tat caa gac ggt tgg gaa 268Ser Asn Glu Phe Gly Ser Thr Val Val Gly Tyr Gln Asp Gly Trp Glu
30 35 40gga ctg tta ggc gat cgt cgc gta cag ctg tat gac gat gaa gat att 316Gly Leu Leu Gly Asp Arg Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile
45 50 55gac cga atc ctc ctt cga ggc ggc acc att ttg ggc act ggt cgc ctc 364Asp Arg Ile Leu Leu Arg Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu
60 65 70cat ccg gac aag ttt aag gcc gga att gat cag att aag gcc aac tta 412His Pro Asp Lys Phe Lys Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu75 80 85 90gaa gac gcc ggc atc gat gcc ctt atc cca atc ggt ggc gaa gga acc 460Glu Asp Ala Gly Ile Asp Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr
95 100 105ctg aag ggt gcc aag tgg ctg tct gat aac ggt atc cct gtt gtc ggt 508Leu Lys Gly Ala Lys Trp Leu Ser Asp Asn Gly Ile Pro Val Val Gly
110 115 120gtc cca aag acc att gac aat gac gtg aat ggc act gac ttc acc ttc 556Val Pro Lys Thr Ile Asp Asn Asp Val Asn Gly Thr Asp Phe Thr Phe
125 130 135ggt ttc gat act gct gtg gca gtg gct acc gac gct gtt gac cgc ctg 604Gly Phe Asp Thr Ala Val Ala Val Ala Thr Asp Ala Val Asp Arg Leu
140 145 150cac acc acc gct gaa tct cac aac cgt gtg atg atc gtg gag gtc atg 652His Thr Thr Ala Glu Ser His Asn Arg Val Met Ile Val Glu Val Met155 160 165 170ggc cgc cac gtg ggt tgg att gct ctg cac gca ggt atg gcc ggc ggt 700Gly Arg His Val Gly TrP Ile Ala Leu His Ala Gly Met Ala Gly Gly
175 180 185gct cac tac acc gtt att cca gaa gta cct ttc gat att gca gag atc 748Ala His Tyr Thr Val Ile Pro Glu Val Pro Phe Asp Ile Ala Glu Ile
190 195 200tgc aag gcg atg gaa cgt cgc ttc cag atg ggc gag aag tac ggc att 796Cys Lys Ala Met Glu Arg Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile
205 210 215atc gtc gtt gcg gaa ggt gcg ttg cca cgc gaa ggc acc atg gag ctt 844Ile Val Val Ala Glu Gly Ala Leu Pro Arg Glu Gly Thr Met Glu Leu
220 225 230cgt gaa ggc cac att gac cag ttc ggt cac aag acc ttc acg gga att 892Arg Glu Gly His Ile Asp Gln Phe Gly His Lys Thr Phe Thr GIy Ile235 240 245 250gga cag cag atc gct gat gag atc cac gtg cgc ctc ggc cac gat gtt 940Gly Gln Gln Ile Ala Asp Glu Ile His Val Arg Leu Gly His Asp Val
255 260 265cgt acg acc gtt ctt ggc cac att caa cgt ggt gga acc cca act gct 988Arg Thr Thr Val Leu Gly His Ile Gln Arg Gly Gly Thr Pro Thr Ala
270 275 280ttc gac cgt gtt ctg gcc act cgt tat ggt gtt cgt gca gct cgt gcg 1036Phe Asp Arg Val Leu Ala Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala
285 290 295tgc cat gag gga agc ttt gac aag gtt gtt gct ttg aag ggt gag agc 1084Cys His Glu Gly Ser Phe Asp Lys Val Val Ala Leu Lys Gly Glu Ser
300 305 310att gag atg atc acc ttt gaa gaa gca gtc gga acc ttg aag gaa gtt 1132Ile Glu Met Ile Thr Phe Glu Glu Ala Val Gly Thr Leu Lys Glu Val315 320 325 330cca ttc gaa cgc tgg gtt act gcc cag gca atg ttt gga tagtttttcg 1181Pro Phe Glu Arg Trp Val Thr Ala Gln Ala Met Phe Gly
335 340ggcttttatc aacagccaat aacagctctt tcgcccattg aggtggaggg gctgtttttt 1241catgccgtaa ggaaagtgca agtaagtgaa atc 1274<210>2<211>343<212>PRT<213>谷氨酸棒杆菌<400>2Met Arg Ile Ala Thr Leu Thr Ser Gly Gly Asp Cys Pro Gly Leu Asn1 5 10 15Ala Val Ile Arg Gly Ile Val Arg Thr Ala Ser Asn Glu Phe Gly Ser
20 25 30Thr Val Val Gly Tyr Gln Asp Gly Trp Glu Gly Leu Leu Gly Asp Arg
35 40 45Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile Asp Arg Ile Leu Leu Arg
50 55 60Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu His Pro Asp Lys Phe Lys65 70 75 80Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu Glu Asp Ala Gly Ile Asp
85 90 95Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr Leu Lys Gly Ala Lys Trp
100 105 110Leu Ser Asp Asn Gly Ile Pro Val yal Gly Val Pro Lys Thr Ile Asp
115 120 125Asn Asp Val Asn Gly Thr Asp Phe Thr Phe Gly Phe Asp Thr Ala Val
130 135 140Ala Val Ala Thr Asp Ala Val Asp Arg Leu His Thr Thr Ala Glu Ser145 150 155 160His Asn Arg Val Met Ile Val Glu Val Met Gly Arg His Val Gly Trp
165 170 175Ile Ala Leu His Ala Gly Met Ala Gly Gly Ala His Tyr Thr Val Ile
180 185 190Pro Glu Val Pro Phe Asp Ile Ala Glu Ile Cys Lys Ala Met Glu Arg
195 200 205Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile Ile Val Val Ala Glu Gly
210 215 220Ala Leu Pro Arg Glu Gly Thr Met Glu Leu Arg Glu Gly His Ile Asp225 230 235 240Gln Phe Gly His Lys Thr Phe Thr Gly Ile Gly Gln Gln Ile Ala Asp
245 250 255Glu Ile His Val Arg Leu Gly His Asp Val Arg Thr Thr Val Leu Gly
260 265 270His Ile Gln Arg Gly Gly Thr Pro Thr Ala Phe Asp Arg Val Leu Ala
275 280 285Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala Cys His Glu Gly Ser Phe
290 295 300Asp Lys Val Val Ala Leu Lys Gly Glu Ser Ile Glu Met Ile Thr Phe305 310 315 320Glu Glu Ala Val Gly Thr Leu Lys Glu Val Pro Phe Glu Arg Trp Val
325 330 335Thr Ala Gln Ala Met Phe Gly
340
Claims (16)
1、来自棒状细菌的分离的多核苷酸,其含有选自如下一组的多核苷酸序列:
a)与编码包括SEQ ID NO:2的氨基酸序列的多肽的多核苷酸至少70%相同的多核苷酸,
b)编码含有与SEQ ID NO:2的氨基酸序列至少70%相同的氨基酸序列的多肽的多核苷酸,
c)与a)或b)的多核苷酸互补的多核苷酸,以及
d)含有a),b)或c)的多核苷酸序列的至少15个连续碱基的多核苷酸。
2、权利要求1的多核苷酸,其中多核苷酸是能在棒状细菌中复制的优选地是重组的DNA。
3、权利要求1的多核苷酸,其中该多核苷酸是RNA。
4、权利要求2的多核苷酸,含有如SEQ ID NO:1所示的核酸序列。
5、权利要求2的可复制的DNA,含有
(ⅰ)如SEQ ID NO:1所示的核苷酸序列,或
(ⅱ)在遗传密码简并范围内相应于的(ⅰ)序列的至少一个序列,或
(ⅲ)与互补于(ⅰ)或(ⅱ)序列的序列杂交的至少一个序列,和任选地
(ⅳ)(ⅰ)中有功能的中性有义突变。
6、权利要求2的多核苷酸序列,其编码含有示于SEQ ID NO:2的氨基酸序列的多肽。
7、发酵生产L-氨基酸特别是L-赖氨酸的方法,其特征在于进行以下步骤:
(a)发酵生产L-氨基酸的棒状细菌,该细菌中至少pfkA基因或其编码核苷酸序列是扩增的,特别是过表达的,
(b)在培养基或细菌细胞中积累L-氨基酸,及
(c)分离L-氨基酸。
8、权利要求7的方法,特征在于所用细菌中所需的L-氨基酸生物合成途径的其它基因是额外扩增的。
9、权利要求7的方法,其特征在于所用细菌中降低L-赖氨酸生成的代谢途径至少被部分抑制。
10、权利要求7的方法,其特征在于使用经质粒载体转化的菌株,且此质粒载体携带编码pfkA基因的核苷酸序列。
11、权利要求7-10任一项的方法,其特征在于使用产生L-赖氨酸的棒状细菌。
12、权利要求6的方法,其特征在于发酵这样的细菌生产赖氨酸,所述细菌中选自如下一组的一或多个基因同时扩增,特别是过表达:
12.1编码二氢-2,6-吡啶二羧酸合酶的dapA基因,
12.2编码丙酮酸羧化酶的pyc基因,
12.3编码三糖磷酸异构酶的tpi基因,
12.4编码琥珀酰二氨基庚二酸脱琥珀酰化酶的dapE基因,
12.5编码甘油醛-3-磷酸脱氢酶的gap基因,
12.6编码3-磷酸甘油激酶的pgk基因,
12.7编码赖氨酸输出蛋白的lysE基因。
13、权利要求9的方法,其特征在于发酵这样的细菌生产赖氨酸,所述细菌中选自如下一组的一或多个基因同时弱化:
13.1编码烯醇丙酮酸磷酸羧激酶的pck基因,
13.2编码葡萄糖-6-磷酸异构酶的pgi基因。
14、前述一或多条权利要求的方法,其特征在于使用谷氨酸棒杆菌的微生物。
15、权利要求1的多核苷酸序列作为经聚合酶链反应产生编码果糖磷酸激酶的基因的DNA的引物的应用。
16、权利要求1的多核苷酸序列作为杂交探针的应用。
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DE19956133.8 | 1999-11-23 | ||
DE19956133 | 1999-11-23 | ||
DE10011922.0 | 2000-03-11 | ||
DE10011922A DE10011922A1 (de) | 1999-11-23 | 2000-03-11 | Neue für das pfkA-Gen codierende Nukleotidsequenzen |
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JP (1) | JP2001186896A (zh) |
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CN (1) | CN1297054A (zh) |
BR (1) | BR0005531A (zh) |
CA (1) | CA2324485A1 (zh) |
HU (1) | HUP0004675A2 (zh) |
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DE10030702A1 (de) | 2000-06-23 | 2002-01-03 | Degussa | Verfahren zur fermentativen Herstellung von D-Pantothensäure unter Verwendung coryneformer Bakterien |
DE10112992A1 (de) * | 2001-03-17 | 2002-09-26 | Degussa | Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
DE102007044789A1 (de) | 2007-09-19 | 2009-04-02 | Wacker Chemie Ag | Selbsthaftende additionsvernetzende Siliconzusammensetzung |
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MXPA01012842A (es) * | 1999-06-25 | 2002-07-09 | Basf Ag | Genes de corynebacterium glutamicum que codifican proteinas involucradas en el metabolismo del carbono y la produccion de energia. |
TWI289604B (en) * | 1999-10-04 | 2007-11-11 | Ajinomoto Kk | neform bacteria Genes for a heat resistant enzymes of amino acid biosynthetic pathway derived from thermophilic cory |
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DE10030702A1 (de) * | 2000-06-23 | 2002-01-03 | Degussa | Verfahren zur fermentativen Herstellung von D-Pantothensäure unter Verwendung coryneformer Bakterien |
DE10112992A1 (de) * | 2001-03-17 | 2002-09-26 | Degussa | Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
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- 2000-10-19 EP EP00122746A patent/EP1106622A3/de not_active Withdrawn
- 2000-11-15 ID IDP20000984A patent/ID28423A/id unknown
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PL344073A1 (en) | 2001-06-04 |
CA2324485A1 (en) | 2001-05-23 |
EP1106622A2 (de) | 2001-06-13 |
SK17372000A3 (sk) | 2001-12-03 |
HU0004675D0 (zh) | 2001-02-28 |
JP2001186896A (ja) | 2001-07-10 |
EP1106622A3 (de) | 2004-01-02 |
MXPA00011412A (es) | 2002-05-23 |
KR20010051876A (ko) | 2001-06-25 |
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