CN1304997A - 编码poxB基因的新核苷酸序列 - Google Patents
编码poxB基因的新核苷酸序列 Download PDFInfo
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- CN1304997A CN1304997A CN00133779A CN00133779A CN1304997A CN 1304997 A CN1304997 A CN 1304997A CN 00133779 A CN00133779 A CN 00133779A CN 00133779 A CN00133779 A CN 00133779A CN 1304997 A CN1304997 A CN 1304997A
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Abstract
本发明涉及一种分离的多核苷酸,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。本发明还涉及经弱化poxB基因发酵生产L-氨基酸的方法。
Description
本发明涉及来自棒状细菌的编码poxB基因的核苷酸序列,和通过弱化poxB基因发酵生产氨基酸的方法。
L-氨基酸特别是赖氨酸用于人用药物和制药工业,食品工业,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有抗性或重要的调节代谢物缺陷的并产生氨基酸的菌株。
一段时间以来,重组DNA技术的方法也用于棒杆菌生产L-氨基酸的菌株的改良。
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。
L-氨基酸、尤其是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。
本发明提供了一种分离的多核苷酸,其包括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2的多核苷酸,含有SEQ ID NO:1所示的核苷酸序列,
权利要求2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pCR2.1poxBint,保藏在大肠杆菌DSM 13114中,
以及作为宿主细胞的棒状细菌,其含有pox基因的插入或缺失。
本发明还提供了多核苷酸,其基本上由多核苷酸序列组成,其可通过用具有相应于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,以分离编码丙酮酸氧化酶的全长cDNA,以及分离与丙酮酸氧化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)制备编码丙酮酸氧化酶的基因的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个连续碱基。具有至少40或50个碱基对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,特别是具有丙酮酸氧化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特别是L-赖氨酸的棒状细菌发酵生产氨基酸特别是赖氨酸的方法,在该棒状细菌中编码poxB基因的核苷酸序列是弱化的,尤其是低水平表达的。
文中术语“弱化”是指微生物中由相应DNA编码的一或多种酶(蛋白质)的胞内活性的降低或抑制,例如通过用弱启动子或编码低活性相应酶的基因或等位基因,或失活相应基因或酶(蛋白质),及如果需要组合使用这些方法。
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氨基酸特别是赖氨酸。此微生物可以是棒状细菌的代表菌,尤其是棒杆菌属。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
Corynebacterium melassecola ATCC 17965
嗜热产氨棒杆菌FERM BP-1539
黄色短杆菌ATCC 14067
乳发酵短杆菌ATCC 13869和
扩展短杆菌ATCC 14020
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-P 1709
黄色短杆菌FERM-P 1708
乳发酵短杆菌FERM-P 1712
谷氨酸棒杆菌FERM-P 6463
谷氨酸棒杆菌FERM-P 6464和
谷氨酸棒杆菌DSM5714。
本发明人已成功地分离谷氨酸棒杆菌的编码丙酮酸氧化酶(EC1.2.2.2)的新poxB基因。
为了分离谷氨酸棒杆菌的poxB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ATCC 13032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。O′Donohue(来自谷氨酸棒杆菌的4个常见芳香氨基酸生物合成基因的克隆和分子分析,博士论文,国立爱尔兰大学,Galway,1997)描述了用由Short等(核酸研究16:7583)所述的λZap表达系统克隆谷氨酸棒杆菌的基因。
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DH5α(Jeffrey H.Miller:细菌遗传学简要教程,大肠杆菌及相关细菌的实验室手册,冷泉港实验室出版社,1992)。
借助于粘粒或其他λ载体克隆的长DNA片段随后可亚克隆并用适于测序的通用载体测序。
DNA测序方法例如如Sanger等(美国科学院院报74:5463-5467,1977)所述。
获得的DNA序列然后可用已知的算法或序列分析程序进行研究,然后与公共数据库中的序列进行比较。所述算法或序列分析程序例如Staden程序(核酸研究14,217-132(1986)),Butler的GCG程序(生化分析方法39,74-97(1998)),Pearson & Lipman的FASTA算法(美国科学院院报85,2444-2448(1988))或Altschul等的BLAST算法(自然遗传学6,119-129(1994))。公共数据库例如是欧洲分子生物学实验室数据库(EMBL,Heidelberg,德国)或国家生物技术信息中心数据库(NCBI,Bethesda,MD,USA)。
以此方式可获得编码poxB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poxB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自SEQ ID NO:1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。本发明还提供了用得自SEQ ID NO:1的引物经聚合酶链反应(PCR)产生的DNA序列。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poxB基因弱化后,棒状细菌以改良方式生产L-氨基酸特别是L-赖氨酸。
为获得弱化,可降低或抑制poxB基因的表达或酶蛋白的催化活性。两种措施可任选地组合。
可通过合适控制培养或遗传修饰(突变)用于基因表达的信号结构而实现降低基因表达。用于基因表达的信号结构例如是阻遏基因、激活基因、操纵子、启动子、弱化子、核糖体结合位点、起始密码子和终止子。本领域技术人员可在如下文献中发现此方面的信息,例如专利申请WO96/15246,Boyd & Murphy(细菌学杂志170:5949(1988)),Voskuil & Chambliss(核酸研究26:3548(1998)),Jensen& Hammer(生物技术和生物工程58:191(1998)),Patek等(微生物学142:1297(1996))以及已知的遗传学和分子生物学教科书如Knippers的教科书(分子遗传学,第6版,Georg Thieme Veflag,德国斯图加特,1995),或Winnacker的教科书(基因和克隆,VCHVerlagsgesellschaft,Weinheim,德国,1990)。
导致酶蛋白催化活性的变化或降低的突变是现有技术中已知的,可提及的例子如Qiu and Goodman的论文(生物化学杂志272:8611-8617(1997)),Sugimoto等(生物科学生物技术和生物化学61:1760-1762(1997))以及Mockel(谷氨酸棒杆菌的苏氨酸脱水酶:酶的变构调节和结构,Berichte des Forschungszentrums Julichs,Jul-2906,ISSN09442952,Julich,德国,1994)。综述可参见遗传学和分子生物学的已知教科书,如Hagemann的教科书("Allgemeine Genetik",Gustav Fischer Verlag,Stuttgart,1996)。
可以考虑的突变是转换、颠换、插入和缺失。根据交换氨基酸对酶活性的影响,突变已知为错义突变或无义突变。在基因中插入或缺失至少一个碱基对导致移码突变,其结果是插入不正确的氨基酸或者翻译过早终止。缺失两个或多个密码子典型地导致酶活性的完全丧失。产生此类突变的指导属于现有技术并可在遗传学和分子生物学的已知教科书中找到,如Knippers的教科书(分子遗传学,第6版,Georg Thieme Vedag,德国斯图加特,1995),Winnacker的教科书(基因和克隆,VCH Verlagsgesellschaft,Weinheim,德国,1990)或Hagemann的教科书("Allgemeine Genetik",Gustav FischerVerlag,Stuttgart,1996)。
利用其可对poxB基因进行插入诱变的质粒例如是pCR2.1poxBint(图1)。
质粒pCR2.1poxBint由掺入了SEQ ID NO:3所示的poxB基因的内部片段的质粒pCR2.1-TOPO组成。转化和同源重组进染色体poxB基因(插入)后,这一质粒导致酶功能的完全丧失。例如,以此方式产生其中的丙酮酸氧化酶被抑制的菌株DSM5715∷pCR2.1poxBint。关于插入诱变的进一步指导和解释可见例如Schwarzer and Puhler(生物/技术9,84-87(1991))或Fitzpatrick等(应用微生物学和生物技术42,575-580(1994))。
另外,除了poxB基因的弱化之外,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特别是过表达,对L-氨基酸特别是L-赖氨酸的生产可以是有益的。
因此,例如为生产L-赖氨酸:
·编码编码二氢-2,6-吡啶二羧酸合酶的dapA基因(EP-B-0197335)可以同时过表达,或
·编码四氢-2,6-吡啶二羧酸琥珀酰化酶的dapD基因(Wehrmann等,细菌学杂志180,3159-3165(1998))可以同时过表达,或
·编码琥珀酰二氨基庚二酸脱琥珀酰化酶的dapE基因(Wehrmann等,细菌学杂志177,5991-5993(1995))可以同时过表达,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)可以同时过表达,或
·编码丙酮酸羧化酶的pyc基因(Eikmanns(1992),细菌学杂志174,6076-6086)可以同时过表达,或
·编码苹果酸::醌氧化还原酶的mqo基因(Molenaar等,欧洲生化杂志254,395-403(1998))可以同时过表达,或
·编码赖氨酸输出蛋白的lysE基因(DE-A-195 48 222)可以同时过表达。
除poxB基因的弱化之外,抑制非所需的二级反应对L-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)。
本发明还提供了含有权利要求1的多核苷酸的微生物,其可连续培养或在分批方法,或在补料分批法或重复补料分批法中分批培养以生产L-氨基酸特别是L-赖氨酸。已知的培养法由Chmiel(Bioprozesstechnik l.Einfuhrung in die Bioverfahrenstechnik(GustavFischer Verlag,Stuttgart,1991)所著教材,或由Storhas(Bioreaktorenund periphere Einrichtungen(Vieweg Verlag,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℃,持续培养直至所需产物形成最大量。此目的通常在10~160小时范围达到。
L-氨基酸的分析方法是现有技术中已知的,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,或通过反相HPLC,如Lindroth等(分析化学(1979)51:1167-1174)。
以下微生物根据布达佩斯条约,已保藏在德意志微生物保藏中心(DSMZ,不伦瑞克,德国):
·大肠杆菌菌株DH5α/pCR2.1poxBint,保藏号DSM 13114。
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ATCC 13032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 poxB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)根据厂商指导分离一个菌落的粘粒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Zimmeermann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”。在带有购白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。
得到的原始序列资料然后用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)制备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一1737碱基对的开放读框,其被称为poxB基因。poxB基因编码579个氨基酸的多肽。
实施例3用于poxB基因的整合诱变的整合载体的产生
用Eikmanns等的方法(微生物学140:1817-1828(1994))从菌株ATCC 13032中分离染色体DNA。基于实施例2已知的谷氨酸棒杆菌的poxB基因的序列,选择如下寡核苷酸进行聚合酶链反应:
poxBint1:
5’-TGC GAG ATG GTG AAT GGT GG-3’
poxBint2:
5’-GCA TGA GGC AAC GCA TTA GC-3’。
所述引物由MWG生物技术公司(Ebersberg,德国)合成,根据Innis等的标准PCR方法(PCR方案,方法和应用指南,1990,学术出版社)用来自宝灵格公司的Pwo聚合酶进行PCR反应。从PCR反应中分离出示于SEQ ID NO:3的约0.9kb大小的DNA片段,其携带poxB基因的内部片段。
用来自Invitrogen公司的TOPO TA克隆试剂盒(Carsbad,CA,USA;目录号K4500-01)将扩增的DNA片段连接进载体pCR2.1-TOPO(Mead等,(1991)生物/技术9:657-663)中。这一连接混合物电穿孔进大肠杆菌菌株DH5α(Hanahan,DNA克隆实用方法,卷Ⅰ,IRL出版社,Oxford,华盛顿特区,美国,1985)。通过将转化混合物在补加25mg/l卡那霉素的LB琼脂上铺板选择携带质粒的细胞(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,1989)。用来自Qiagen的QIAprep Spin Miniprep试剂盒从转化子中分离质粒DNA,并用限制酶EcoRⅠ限制消化并琼脂糖凝胶电泳(0.8%)而证实。质粒命名为pCR2.1poxBint。
实施例4 poxB基因在赖氨酸生产菌DSM 5715中的整合诱变
用Tauch等的电穿孔方法(FEMS微生物学通信123:343-347(1994))将实施例3中的载体pCR2.1poxBint电穿孔进谷氨酸棒杆菌DSM 5715中。菌株DSM5715是AEC抗性赖氨酸生产菌。载体pCR2.1poxBint在谷氨酸棒杆菌DSM 5715中不能独立复制,只有在其整合进DSM5715的染色体中后才能在细胞中保持。通过将电穿孔混合物在补加15mg/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,1989)上铺板选择携带整合进染色体的pCR2.1poxBint的克隆。通过用来自宝灵格公司的Dig杂交试剂盒根据宝灵格曼海姆有限公司的“用于滤膜杂交的DIG系统用户指南”(曼海姆,德国,1993)的方法标记poxBint片段以检测整合。用Eikmanns等的方法(微生物学140:1817-1828(1994))从潜在整合子中分离染色体DNA,并分别用限制酶SalⅠ,SacⅠ和HindⅢ切割。经琼脂糖凝胶电泳分离得到的片段并用来自宝灵格公司的Dig杂交试剂盒在68℃杂交。实施例3中的质粒pCR2.1poxBint已插入DSM5715的染色体中的染色体poxB基因内。该菌株命名为DSM5715∷CR2.1poxBint。
实施例5赖氨酸的生产
实施例3中获得的谷氨酸棒杆菌菌株DSM5715∷CR2.1poxBint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(具有卡那霉素(25mg/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,向此培养基中加入卡那霉素(25mg/l)。在摇床上于33℃以240rpm振荡培养预培养物24小时。以此预培养物接种主培养物,从而主培养物的起始光密度(OD,660nm)是0.1。培养基MM用作主培养基。培养基MM:CSL(玉米浆) 5g/lMOPS 20g/l葡萄糖(单独高压灭菌) 50g/l盐:(NH4)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/l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。
48小时后,用Biomek1000(Beckmann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的量。
所得结果示于表1。
表1
菌株 | OD | 赖氨酸HClg/L |
DSM5715 | 13.1 | 9.5 |
DSM5715∷pCR2.1poxBint | 12.5 | 12.9 |
实施例6 poxB基因在缬氨酸生产菌FERM-BP1763中的整合诱变
用Tauch等的电穿孔方法(FEMS微生物学通信123:343-347(1994))将实施例3中的载体pCR2.1poxBint电穿孔进乳发酵短杆菌FERM-BP1763中。菌株FERM-BP1763是霉酚酸抗性缬氨酸生产菌(US-A-5188948)。载体pCR2.1poxBint在FERM-BP1763中不能独立复制,只有在其整合进FERM-BP1763的染色体中后才能在细胞中保持。通过将电穿孔混合物在补加15mg/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,1989)上铺板选择携带整合进染色体的pCR2.1poxBint的克隆。通过用来自宝灵格公司的Dig杂交试剂盒根据宝灵格曼海姆有限公司的“用于滤膜杂交的DIG系统用户指南”(曼海姆,德国,1993)的方法标记poxBint片段以检测整合。用Eikmanns等的方法(微生物学140:1817-1828(1994))从潜在整合子中分离染色体DNA,并分别用限制酶SalⅠ,SacⅠ和HindⅢ切割。经琼脂糖凝胶电泳分离得到的片段并用来自宝灵格公司的Dig杂交试剂盒在68℃杂交。实施例3中的质粒pCR2.1poxBint已插入FERM-BP1763的染色体中的染色体poxB基因内。该菌株命名为FERM-BP1763∷pCR2.1poxBint。
实施例7缬氨酸的生产
实施例6中获得的乳发酵短杆菌菌株FERM-BP1763∷pCR2.1poxBint在适于生产缬氨酸的营养培养基中培养,并确定培养物上清中的缬氨酸含量。
为此,首先在33℃在具有合适抗生素的琼脂板(具有卡那霉素(25mg/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,向此培养基中加入卡那霉素(25mg/l)。在摇床上于33℃以240rpm振荡培养预培养物48小时。以此预培养物接种主培养物,从而主培养物的起始光密度(OD,660nm)是0.1。培养基MM用作主培养基。培养基MM:CSL 5g/lMOPS 20g/l葡萄糖(单独高压灭菌) 50g/l盐:(NH4)2SO4 25g/lKH2PO4 0.1g/lMgSO4·7H2O 1.0g/lCaCl2·2H2O 10mg/lFeSO4·7H2O 10mg/lMnSO4·H2O 5.0mg/l异亮氨酸(过滤灭菌) 0.1g/l甲硫氨酸(过滤灭菌) 0.1g/l硫胺素·HCl(过滤灭菌) 0.2mg/l亮氨酸(过滤灭菌) 0.1g/lCaCO3 25g/l
用氨水将CSL(玉米浆),MOPS(吗啉代丙磺酸)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。
48小时后,用Biomek1000(Beckmann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缬氨酸的量。
所得结果示于表2。
表2
菌株 | OD | 缬氨酸HClg/L |
FERM-BP1763 | 8.6 | 12.1 |
FERM-BP1763∷pCR2.1poxBint | 9.5 | 13.0 |
本发明包括如下附图:
图1:质粒pCR2.1poxBint图。
图中所采用的缩写和符号有如下含义:
ColE1 ori:质粒ColE1的复制源点
lacZ:β-半乳糖苷酶基因的5’末端
f1 ori:噬菌体f1的复制源点
KmR:卡那霉素抗性
ApR:氨苄青霉素抗性
BamHⅠ:限制酶BamHⅠ的酶切位点
EcoRⅠ:限制酶EcoRⅠ的酶切位点
poxBint:poxB基因的内部片段
序列表<110>德古萨-于尔斯股份公司<120>编码poxB基因的新核苷酸序列<130>990159 BT<140><141><160>3<170>PatentIn Ver.2.1<210>1<211>2160<212>DNA<213>谷氨酸棒杆菌<220><221>CDS<222>(327)..(2063)<220><221>-35_信号<222>(227)..(232)<220><221>-10_信号<222>(256)..(261)<400>1ttagaggcga ttctgtgagg tcactttttg tggggtcggg gtctaaattt ggccagtttt 60cgaggcgacc agacaggcgt gcccacgatg tttaaatagg cgatcggtgg gcatctgtgt 120ttggtttcga cgggctgaaa ccaaaccaga ctgcccagca acgacggaaa tcccaaaagt 180gggcatccct gtttggtacc gagtacccac ccgggcctga aactccctgg caggcgggcg 240aagcgtggca acaactggaa tttaagagca caattgaagt cgcaccaagt taggcaacac 300aatagccata acgttgagga gttcag atg gca cac agc tac gca gaa caa tta 353
Met Ala His Ser Tyr Ala Glu Gln Leu
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220 225 230ctg ggt ggt aag cag tac atc cag cat gag aat ccg ttt gag gtc ggc 1073Leu Gly Gly Lys Gln Tyr Ile Gln His Glu Asn Pro Phe Glu Val Gly
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285 290 295ggt cga cgt acc acg gtg aag tat ccg gtg acc ggt gat gtt gct gca 1265Gly Arg Arg Thr Thr Val Lys Tyr Pro Val Thr Gly Asp Val Ala Ala
300 305 310aca atc gaa aat att ttg cct cat gtg aag gaa aaa aca gat cgt tcc 1313Thr Ile Glu Asn Ile Leu Pro His Val Lys Glu Lys Thr Asp Arg Ser
315 320 325ttc ctt gat cgg atg ctc aag gca cac gag cgt aag ttg agc tcg gtg 1361Phe Leu Asp Arg Met Leu Lys Ala His Glu Arg Lys Leu Ser Ser Val330 335 340 345gta gag acg tac aca cat aac gtc gag aag cat gtg cct att cac cct 1409Val Glu Thr Tyr Thr His Asn Val Glu Lys His Val Pro Ile His Pro
350 355 360gaa tac gtt gcc tct att ttg aac gag ctg gcg gat aag gat gcg gtg 1457Glu Tyr Val Ala Ser Ile Leu Asn Glu Leu Ala Asp Lys Asp Ala Val
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380 385 390gag aat ccg gag gga acg cgc gac ttt gtg ggt tca ttc cgc cac ggc 1553Glu Asn Pro Glu Gly Thr Arg Asp Phe Val Gly Ser Phe Arg His Gly
395 400 405acg atg gct aat gcg ttg cct cat gcg att ggt gcg caa agt gtt gat 1601Thr Met Ala Asn Ala Leu Pro His Ala Ile Gly Ala Gln Ser Val Asp410 415 420 425cga aac cgc cag gtg atc gcg atg tgt ggc gat ggt ggt ttg ggc atg 1649Arg Asn Arg Gln Val Ile Ala Met Cys Gly Asp Gly Gly Leu Gly Met
430 435 440ctg ctg ggt gag ctt ctg acc gtt aag ctg cac caa ctt ccg ctg aag 1697Leu Leu Gly Glu Leu Leu Thr Val Lys Leu His Gln Leu Pro Leu Lys
445 450 455gct gtg gtg ttt aac aac agt tct ttg ggc atg gtg aag ttg gag atg 1745Ala Val Val Phe Asn Asn Ser Ser Leu Gly Met Val Lys Leu Glu Met
460 465 470ctc gtg gag gga cag cca gaa ttt ggt act gac cat gag gaa gtg aat 1793Leu Val Glu Gly Gln Pro Glu Phe Gly Thr Asp His Glu Glu Val Asn
475 480 485ttc gca gag att gcg gcg gct gcg ggt atc aaa tcg gta cgc atc acc 1841Phe Ala Glu Ile Ala Ala Ala Ala Gly Ile Lys Ser Val Arg Ile Thr490 495 500 505gat ccg aag aaa gtt cgc gag cag cta gct gag gca ttg gca tat cct 1889Asp Pro Lys Lys Val Arg Glu Gln Leu Ala Glu Ala Leu Ala Tyr Pro
510 515 520gga cct gta ctg atc gat atc gtc acg gat cct aat gcg ctg tcg atc 1937Gly Pro Val Leu Ile Asp Ile Val Thr Asp Pro Asn Ala Leu Ser Ile
525 530 535cca cca acc atc acg tgg gaa cag gtc atg gga ttc agc aag gcg gcc 1985Pro Pro Thr Ile Thr Trp Glu Gln Val Met Gly Phe Ser Lys Ala Ala
540 545 550acc cga acc gtc ttt ggt gga gga gta gga gcg atg atc gat ctg gcc 2033Thr Arg Thr Val Phe Gly Gly Gly Val Gly Ala Met Ile Asp Leu Ala
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20 25 30Val Asp Ala Val Arg Gln Ser Asp Ile Glu Trp Val His Val Arg Asn
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165 170 175Ile Set Ser Gly Thr Pro Val Val Phe Pro Asp Pro Thr Glu Ala Ala
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195 200 205Gly Ala Gly Val Lys Asn Ala Arg Ala Gln Val Leu Glu Leu Ala Glu
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435 440 445Val Lys Leu His Gln Leu Pro Leu Lys Ala Val Val Phe Asn Asn Ser
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Claims (16)
1、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。
2、权利要求1的多核苷酸,其中多核苷酸是能复制的优选重组的DNA。
3、权利要求1的多核苷酸,其中多核苷酸是RNA。
4、权利要求2的多核苷酸,含有SEQ ID NO:1所示的核苷酸序列。
5、权利要求2的多核苷酸序列,其编码含有SEQ ID NO:2所示的氨基酸序列的多肽。
6、权利要求2的能复制的DNA,含有
(ⅰ)如SEQ ID NO:1所示的核苷酸序列,或
(ⅱ)在遗传密码简并区内相应于的(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。
7、含有权利要求1特别是其中d)的多核苷酸的载体,保藏于大肠杆菌DSM 13114中。
8、作为宿主细胞的含有poxB基因的缺失或插入的棒状细菌。
9、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)发酵生产所需氨基酸的细菌,该细菌中至少poxB基因是弱化的,
(b)富集培养基或细菌细胞中的所需L-氨基酸,及
(c)分离L-氨基酸。
10、权利要求9的方法,其特征在于使用如下细菌,该细菌中所需L-氨基酸生物合成途径的其它基因额外被扩增。
11、权利要求9的方法,其特征在于使用如下细菌,该细菌中降低所需L-氨基酸生成的代谢途径至少被部分抑制。
12、权利要求9的方法,其特征在于权利要求1特别是1a至1c的多核苷酸的表达被降低。
13、权利要求9的方法,其特征在于权利要求1特别是1a至1c的多核苷酸编码的多肽(酶蛋白)的催化活性被降低。
14、权利要求9的方法,其特征在于使用如下细菌,该细菌中弱化是通过用质粒pCR2.1poxBint或其组成部分进行整合诱变而实现,所述质粒示于图1并以保藏号DSM13114保藏。
15、权利要求9的方法,其特征在于发酵如下细菌生产L-赖氨酸,该细菌中选自如下一组的一或多个基因同时过表达:
·编码编码二氢-2,6-吡啶二羧酸合酶的dapA基因,
·赋予S-(2-氨基乙基)半胱氨酸抗性的DNA片段,
·编码丙酮酸羧化酶的pyc基因,
·编码琥珀酰二氨基庚二酸脱琥珀酰化酶的dapE基因,
·编码甘油醛-3-磷酸脱氢酶的gap基因,
·编码苹果酸::醌氧化还原酶的mqo基因,
·编码赖氨酸输出蛋白的lysE基因。
16、前述任一项权利要求的方法,其特征在于使用谷氨酸棒杆菌的微生物。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7736880B2 (en) | 2005-11-30 | 2010-06-15 | Cj Cheiljedang Corporation | Microorganism of corynebacterium genus having resistance to kanamycin and enhanced L-lysine productivity and method of producing L-lysine using the same |
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US6797509B1 (en) | 1999-07-09 | 2004-09-28 | Degussa-Huls Ag | Nucleotide sequences which code for the tal gene |
US20060014259A9 (en) * | 1999-07-09 | 2006-01-19 | Kevin Burke | Process for the preparation of L-amino acids with amplification of the zwf gene |
US20050112733A1 (en) * | 2000-03-20 | 2005-05-26 | Degussa Ag | Process for the preparation of L-amino acids with amplification of the zwf gene |
US20030175911A1 (en) * | 2000-03-20 | 2003-09-18 | Stephen Hans | Process for the preparation of L-amino acids with amplification of the zwf gene |
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DE10109690A1 (de) | 2000-09-02 | 2002-03-14 | Degussa | Neue für das metY-Gen kodierende Nukleotidsequenzen |
AU2001293741A1 (en) * | 2000-09-14 | 2002-03-26 | Degussa A.G. | Nucleotide sequences coding for the suga gene |
US6946271B2 (en) * | 2000-09-20 | 2005-09-20 | Degussa Ag | Nucleotide sequences which code for the menE gene |
DE10047865A1 (de) * | 2000-09-27 | 2002-04-18 | Degussa | Neue für das deaD-Gen kodierende Nukleotidsequenzen |
WO2002036797A2 (en) * | 2000-11-04 | 2002-05-10 | Degussa Ag | Process for the fermentative preparation of l-amino acids using strains of the enterobacteriaceae family |
US20030017554A1 (en) * | 2000-11-15 | 2003-01-23 | Mechthild Rieping | Process for the fermentative preparation of L-amino acids using strains of the enterobacteriaceae family |
DE10210527A1 (de) | 2002-03-09 | 2003-09-18 | Degussa | Allele des aceA-Gens aus coryneformen Bakterien |
JP4469568B2 (ja) | 2003-07-09 | 2010-05-26 | 三菱化学株式会社 | 有機酸の製造方法 |
CN100575496C (zh) | 2003-08-28 | 2009-12-30 | 三菱化学株式会社 | 产生琥珀酸的方法 |
CN1852978A (zh) | 2003-09-17 | 2006-10-25 | 三菱化学株式会社 | 制备无-氨基有机酸的方法 |
JP4631706B2 (ja) * | 2003-09-30 | 2011-02-16 | 味の素株式会社 | 発酵液からのコハク酸の精製方法 |
JP5572279B2 (ja) * | 2004-05-20 | 2014-08-13 | 味の素株式会社 | コハク酸生産菌及びコハク酸の製造方法 |
BRPI0510919A (pt) | 2004-05-20 | 2008-05-20 | Ajinomoto Kk | bactéria produtora de ácido succìnico e processo para produzir ácido succìnico |
JP4595506B2 (ja) | 2004-11-25 | 2010-12-08 | 味の素株式会社 | L−アミノ酸生産菌及びl−アミノ酸の製造方法 |
US20070092951A1 (en) * | 2005-03-24 | 2007-04-26 | Degussa Ag | Alleles of the zwf gene from coryneform bacteria |
DE102005048818A1 (de) | 2005-10-10 | 2007-04-12 | Degussa Ag | Mikrobiologische Herstellung von 3-Hydroxypropionsäure |
JP5088136B2 (ja) | 2005-10-18 | 2012-12-05 | 味の素株式会社 | コハク酸の製造方法 |
JP5180060B2 (ja) | 2006-02-24 | 2013-04-10 | 三菱化学株式会社 | 有機酸生産菌及び有機酸の製造法 |
KR101431084B1 (ko) * | 2011-11-18 | 2014-09-23 | 한국생명공학연구원 | PoxB 유전자를 이용한 활성 인클루젼 바디 생산방법 |
CA3007635A1 (en) | 2015-12-07 | 2017-06-15 | Zymergen Inc. | Promoters from corynebacterium glutamicum |
KR20200026881A (ko) | 2017-06-07 | 2020-03-11 | 지머젠 인코포레이티드 | 코리네박테리움 글루타미컴으로부터의 프로모터 및 보조 유전자 발현을 조절하는 데 이의 용도 |
EP3456833A1 (en) * | 2017-09-18 | 2019-03-20 | Evonik Degussa GmbH | Method for the fermentative production of l-amino acids |
RU2019128538A (ru) | 2018-09-26 | 2021-03-11 | Эвоник Оперейшенс ГмбХ | Способ ферментативного получения l-лизина |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20070087093A (ko) * | 1999-06-25 | 2007-08-27 | 바스프 악티엔게젤샤프트 | 탄소 대사 및 에너지 생산과 관련된 단백질을 코딩하는코리네박테리움 글루타미쿰 유전자 |
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1999
- 1999-10-28 DE DE19951975A patent/DE19951975A1/de not_active Withdrawn
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2000
- 2000-10-14 EP EP00122505A patent/EP1096013A3/de not_active Withdrawn
- 2000-10-19 ID IDP20000899A patent/ID27958A/id unknown
- 2000-10-20 SK SK1573-2000A patent/SK15732000A3/sk unknown
- 2000-10-24 MX MXPA00010419A patent/MXPA00010419A/es unknown
- 2000-10-25 CA CA002322553A patent/CA2322553A1/en not_active Abandoned
- 2000-10-25 AU AU68075/00A patent/AU6807500A/en not_active Abandoned
- 2000-10-25 JP JP2000325437A patent/JP2001161386A/ja active Pending
- 2000-10-26 ZA ZA200006039A patent/ZA200006039B/xx unknown
- 2000-10-27 CN CN00133779A patent/CN1304997A/zh active Pending
- 2000-10-27 BR BR0005091-1A patent/BR0005091A/pt not_active IP Right Cessation
- 2000-10-27 KR KR1020000063500A patent/KR20010051289A/ko not_active Application Discontinuation
- 2000-10-27 HU HU0004188A patent/HUP0004188A2/hu unknown
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7736880B2 (en) | 2005-11-30 | 2010-06-15 | Cj Cheiljedang Corporation | Microorganism of corynebacterium genus having resistance to kanamycin and enhanced L-lysine productivity and method of producing L-lysine using the same |
CN101177686B (zh) * | 2006-11-10 | 2011-05-18 | 中国科学院上海生命科学研究院 | 一种丙酮酸氧化酶基因、其重组表达质粒及转化的菌株 |
Also Published As
Publication number | Publication date |
---|---|
ZA200006039B (en) | 2001-05-03 |
HU0004188D0 (zh) | 2001-01-29 |
MXPA00010419A (es) | 2002-07-22 |
DE19951975A1 (de) | 2001-05-03 |
BR0005091A (pt) | 2001-06-19 |
SK15732000A3 (sk) | 2001-11-06 |
AU6807500A (en) | 2001-05-03 |
ID27958A (id) | 2001-05-03 |
US20050196848A1 (en) | 2005-09-08 |
EP1096013A2 (de) | 2001-05-02 |
CA2322553A1 (en) | 2001-04-28 |
JP2001161386A (ja) | 2001-06-19 |
EP1096013A3 (de) | 2005-06-01 |
KR20010051289A (ko) | 2001-06-25 |
HUP0004188A2 (hu) | 2003-03-28 |
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