CN100387714C - 编码dapF基因的新核苷酸序列 - Google Patents
编码dapF基因的新核苷酸序列 Download PDFInfo
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- CN100387714C CN100387714C CNB001244868A CN00124486A CN100387714C CN 100387714 C CN100387714 C CN 100387714C CN B001244868 A CNB001244868 A CN B001244868A CN 00124486 A CN00124486 A CN 00124486A CN 100387714 C CN100387714 C CN 100387714C
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- polynucleotide
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- methionin
- dapf
- bacterium
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Abstract
本发明涉及分离的多核苷酸，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)与编码由dapF基因表达的多肽的多核苷酸至少70%相同的多核苷酸，所述dapF基因包含在保藏的谷氨酸棒杆菌菌株DSM 12968中的质粒pEC-XT99A-dapF中，c)编码包括与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，d)与a)，b)或c)的多核苷酸互补的多核苷酸，以及e)包括a)，b)，c)或d)的多核苷酸序列的至少15个连续碱基的多核苷酸。本发明还涉及用dapF基因的扩增而发酵制备L-赖氨酸的方法。
Description
本发明涉及编码dapF基因的核苷酸序列，和用棒状细菌生产L-赖氨酸的方法，其中棒状细菌中dapF基因是扩增的。
L-赖氨酸用于人用药物及制药工业，但特别用于动物营养。
已知L-赖氨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L-赖氨酸的极其重要性，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，或营养培养基的组分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-氨乙基)-半胱氨酸有抗性或重要的调节氨基酸缺陷的并产生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L-赖氨酸的菌株的改良，其通过扩增各个赖氨酸生物合成基因，并研究对L-赖氨酸生产的作用而进行改良。此方面的综述文章见Kinoshita(″谷氨酸细菌″，工业微生物的生物学，Demain和Soloman编辑，Benjamin Cummings，英国伦敦，1985，115-142)，Hilliger(生物技术2，40-44(1991))，Eggeling(氨基酸6：261-272(1994))，Jetten和Sinskey(生物技术的关键回顾15，73-103(1995))和Sahm等(纽约科学协会年报782，25-39(1996))。
在原核生物中，已知D，L-二氨基庚二酸或L-赖氨酸的生物合成有三个不同的途径，这些途径的不同在于L-哌啶-2，6-二羧酸酯(四氢-2，6-吡啶二羧酸酯)。
在琥珀酰化酶途径，四氢-2，6-吡啶二羧酸酯经四氢-2，6-吡啶二羧酸琥珀酰化酶酰化，随后的酮基的转氨基作用(N-琥珀酰氨基酮庚二酸转氨酶)，脱琥珀酰化(N-琥珀酰氨基酮庚二酸脱琥珀酰化酶)，然后差向异构化(二氨基庚二酸差向异构酶)而转化成D，L-二氨基庚二酸酯(Gilvarg，1958，生物化学杂志，233：1501-1504)。
在乙酰化酶途径，其在枯草芽胞杆菌和Bacillus megaterium中实现，四氢-2，6-吡啶二羧酸酯的乙酰化通过乙酰基进行(Weinberger & Gilvarg，1970，细菌学杂志101：323-324)。
第三个途径是用于描述B.sphaericus(Misono等，1976，细菌学杂志，137：22-27)。在称作脱氢酶途径的这一生物合成步骤中，发生四氢-2，6-吡啶二羧酸酯的直接还原性转氨基作用，生成D，L-DAP。
借助于遗传学和酶学研究，Schrumpf等(细菌学杂志173，4510-4516(1991))证明在谷氨酸棒杆菌中，赖氨酸生物合成经脱氢酶途径和琥珀酰化酶途径进行。
经Marx等(生物技术及生物工程56，168-180(1997))和Sonntag(欧洲生物化学杂志213：1325-1331(1993))的体内NMR研究，发现在谷氨酸棒杆菌中，琥珀酰化酶途径和脱氢酶途径均导致L-赖氨酸的产生。
谷氨酸棒杆菌的脱琥珀酰化酶基因(dapE)已被Wehrmann等(细菌学杂志177：5991-5993(1995))克隆并测序。还可以克隆和测序来自谷氨酸棒杆菌的琥珀酰化酶基因(dapD)(Wehrmann等，细菌学杂志180：3159-3165(1998))。
本发明人目的在于为改良L-赖氨酸的生产而提供新措施。
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L-赖氨酸的新改良方法总是令人感兴趣的。
当下文提及L-赖氨酸或赖氨酸时，不仅指其本身，也指盐，如赖氨酸单盐酸盐或赖氨酸硫酸盐。
本发明提供了衍生自棒状细菌的分离的多核苷酸，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)与编码由dapF基因表达的多肽的多核苷酸至少70％相同的多核苷酸，所述dapF基因包含在保藏的谷氨酸棒杆菌菌株DSM 12968中的质粒pEC-XT99A-dapF中，
c)编码包括与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，
d)与a)，b)或c)的多核苷酸互补的多核苷酸，以及
e)包括a)，b)，c)或d)的多核苷酸序列的至少15个连续碱基的多核苷酸。
本发明还提供了一种多核苷酸，其优选是能复制的DNA，包括
(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)中有功能的中义突变。
本发明还提供了
一种多核苷酸，包括SEQ ID NO：1所示的核苷酸序列，
一种多核苷酸，其编码包括SEQ ID NO：2所示的氨基酸序列的多肽，
含有本发明的多核苷酸序列的载体，特别是pEC-XT99A-dapF，以DSM 12968保藏，
以及作为宿主细胞的棒状细菌，其含有上述本发明的载体。
本发明还提供了多核苷酸，其基本上包括多核苷酸序列，其可通过用包括所述多核苷酸的序列的相应于SEQ ID NO：1或其片段的探针杂交相应的基因文库，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，以分离编码二氨基庚二酸差向异构酶的全长cDNA，以及分离与二氨基庚二酸差向异构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)制备编码二氨基庚二酸差向异构酶的DNA或基因的引物。
作为探针或引物的这种寡核苷酸包括至少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，特别是具有二氨基庚二酸差向异构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L-赖氨酸的棒状细菌发酵L-赖氨酸的方法，在该棒状细菌中编码dapF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提高，例如通过提高基因的拷贝数，或用强启动子或编码高活性相应酶的基因，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L-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
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-赖氨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DSM5715
本发明人已成功地分离谷氨酸棒杆菌的编码二氨基庚二酸差向异构酶(EC 5.1.1.7)的新dapF基因。
为了分离谷氨酸棒杆菌的dapF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。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dapF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dapF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。另外，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15bp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A(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dapF基因过表达后，棒状细菌以改良方式生产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WO 96/15246，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。
利用其可过表达dapF基因的质粒例如是pEC-XT99A-dapF(图2)，其包含于菌株DSM5715/pEC-XT99A-dapF中。质粒pEC-XT99A-dapF是基于质粒pEC-XT99A(图1)的谷氨酸棒杆菌-E.coli穿梭载体，这一质粒载体含有质粒pGA1(US-B5,175,108)的复制区和质粒pAG1(国家生物技术信息中心的登记号为AF121000，Bethesda，美国马里兰州)的四环素抗性基因。其它可在谷氨酸棒杆菌中复制的质粒载体如pEKEx1(Eikmanns等，基因102：93-98(1991))或pZ8-1(EP-B-0375889)可以同样方式使用。
另外，除了dapF基因之外，赖氨酸生物合成途径的一或多种酶的过表达，对L-赖氨酸的生产可以是有益的，例如：
·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
·赋予S-(2-氨基乙基)-半胱氨酸抗性的DNA片段(EP-A-0088166)可以同时被扩增，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。
除dapF基因的过表达之外，排除非所需的二级反应对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在分批方法(分批培养)，或在补料分批(补料法)或重复补料分批法(重复补料法)中分批培养以生产L-赖氨酸。已知的培养法由Chmiel(Bioprozesstechnik 1.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L-赖氨酸形成最大量。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。
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)：
·谷氨酸棒杆菌DSM5715/pEC-XT99A，保藏号DSM 12967
·谷氨酸棒杆菌DSM5715/pEC-XT99A-dapF，保藏号DSM12968。
本发明方法用于发酵制备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ATCC13032DNA片段混合并用T4DNA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 dapF基因的分离和克隆
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)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一831碱基对的开放读框，其被称为dapF基因。dapF基因编码277个氨基酸的多肽。
实施例3 表达载体pEC-XT99A的构建
用大肠杆菌表达载体pTRC99A(Amann等，1988，基因69：301-315)作为起始载体构建大肠杆菌-谷氨酸棒杆菌穿梭表达载体pEC-XT99A。在BspHI限制酶切(Roche诊断学有限公司，曼海姆，德国，产品描述BspHI，产品号1467123)和随后的Klenow处理(Amersham Pharmacia Biotech，Freiburg，德国，产品描述DNA聚合酶I的Klenow片段，产品号27-0928-01；Sambrook等的方法，1989，分子克隆实验手册，冷泉港)之后，氨苄青霉素抗性基因被谷氨酸棒杆菌质粒pAGl(GenBank登录号AF121000)的四环素抗性基因替换。为此，携带该抗性基因的区域以AluI片段(AmershamPharmacia Biotech，Freiburg，德国，产品描述AluI，产品号27-0884-01)克隆进线性化的大肠杆菌表达载体pTRC99A中。如Sambrook等所述(1989，分子克隆实验手册，冷泉港)进行连接，DNA混合物与T4连接酶(Amersham Pharmacia Biotech，Freiburg，德国，产品描述T4-DNA-连接酶，产品号27-0870-04)保温过夜。然后这一连接混合物电穿孔(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)。所构建的大肠杆菌表达载体称为pXT99A。质粒pGAl(Sonnen等，1991，基因，107：69-74)用作克隆来自谷氨酸棒杆菌的最小复制子的基础。通过BalI/PstI限制酶切(Promega有限公司，曼海姆，德国，产品描述BalI，产品号R6691；AmershamPharmacia Biotech，Freiburg，德国，产品描述PstI，产品号27-0976-01)载体pGAl，可以克隆用SmaI和PstI(Amersham PharmaciaBiotech，Freiburg，德国，产品描述SmaI，产品号27-0942-02，产品描述PstI，产品号27-0976-01)片段化的pK18mob2(Tauch等，1998，微生物学档案169：303-312)中的3484bp片段。通过BamHI/XhoI限制酶切(Amersham Pharmacia Biotech，Freiburg，德国，产品描述BamHI，产品号27-0868-03，产品描述XhoI，产品号27-0950-01)和随后的Klenow处理(Amersham PharmaciaBiotech，Freiburg，德国，产品描述DNA聚合酶I的Klenow片段，产品号27-0928-01；Sambrook等的方法，1989，分子克隆实验手册，冷泉港)，缺失839bp大小的片段。从用T4连接酶(AmershamPharmacia Biotech，Freiburg，德国，产品描述T4-DNA-连接酶，产品号27-0870-04)重连接的构建中，可以将谷氨酸棒杆菌最小复制子作为2645bp片段克隆进大肠杆菌表达载体pXT99A中。为此，携带最小复制子的构建体DNA用限制酶KpnI(Amersham PharmaciaBiotech，Freiburg，德国，产品描述KpnI，产品号27-0908-01)和PstI(Amersham Pharmacia Biotech，Freiburg，德国，产品描述PstI，产品号27-0886-03)酶切，随后用Klenow-聚合酶(AmershamPharmacia Biotech，Freiburg，德国，产品描述DNA聚合酶I的Klenow片段，产品号27-0928-01)进行3′-5′外切核酸酶处理(Sambrook等，1989，分子克隆实验手册，冷泉港)。在平行批次中，用限制酶RsrII(Roche Diagnostics，德国曼海姆，产品描述RsrII，产品号1292587)酶切大肠杆菌表达载体pXT99A，并用Klenow-聚合酶(AmershamPharmacia Biotech，Freiburg，德国，产品描述DNA聚合酶I的Klenow片段，产品号27-0928-01)制备以用于连接。最小复制子与载体构建体pXT99A的连接如Sambrook等(1989，分子克隆实验手册，冷泉港)所述进行，DNA混合物与T4连接酶(Amersham PharmaciaBiotech，Freiburg，德国，产品描述T4-DNA-连接酶，产品号27-0870-04)保温过夜。以此方式构建的大肠杆菌-谷氨酸棒杆菌穿梭表达载体pEC-XT99A通过电穿孔(Liebl等，1989，FEMS微生物学通信，53：299-303)转移进谷氨酸棒杆菌中。通过重新分离的质粒DNA的分析可证实转化子。如此获得的质粒构建体称为pEC-XT99A(图2)。质粒pEC-XT99A转化进大肠杆菌菌株DH5αmcr获得的大肠杆菌菌株称为DH5αmcr/pEC-XT99A。
实施例4 dapF基因的表达
从如SEQ ID NO：1所示的来自谷氨酸棒杆菌ATCC13032的二氨基庚二酸差向异构酶基因dapF的核苷酸序列开始合成PCR引物(ARK Scientific GmbH Biosystems，Darmstadt，德国)。选择这些引物以便扩增的片段含有该基因及其天然的核糖体结合位点，但不含可能的启动子区域。另外，插入了允许克隆进靶载体的合适的限制酶切位点。PCR引物的序列，插入的酶切位点(下划线序列)和扩增的基因(片段大小以bp表示在括号中)列于表1。
用聚合酶链反应(PCR)和表1所述的合成寡核苷酸扩增来自谷氨酸棒杆菌ATCC13032的二氨基庚二酸差向异构酶基因dapF。PCR实验用来自Stratagene公司的Pfu DNA聚合酶(La Jolla，CA，产品描述天然Pfu DNA聚合酶，产品号600250)在一“PCT-100热循环仪”(MJ研究公司，Watertown，Mass.，USA)中进行。94℃3分钟的单个变性步骤后接94℃30秒的变性步骤，在引物依赖性温度T＝(2AT+4GC)-5℃(Suggs等，1981，p.683-693，D.D.Brown，C.F.Fox编辑，使用纯化的基因的发育生物学，学术出版社，纽约，USA)30秒的退火步骤以及72℃持续90秒的延伸步骤。最后3个步骤重复循环30次，反应终止为在72℃5分钟的延伸步骤。用琼脂糖凝胶电泳测试如此制备的产物的大小。
图1所示的大肠杆菌-谷氨酸棒杆菌穿梭表达载体pEC-XT99A(实施例3)用作表达的基本载体。用限制酶BamHI和MunI完全酶切得到的PCR产物，并连接进已用EcoRI和BamHI(AmershamPharmacia Biotech，Freiburg，德国，产品描述EcoRI，产品号27-0854-03，产品描述BamHI，产品号27-0868-03)酶切的表达载体pEC-XT99A中。
以此方式，无启动子的dapF基因被置于在此质粒上所含的trc启动子的控制下。
如Sambrook等所述(1989，分子克隆实验手册，冷泉港)进行dapFex扩增子与表达载体pEC-XT99A的连接，DNA混合物与T4连接酶(Amersham Pharmacia Biotech，Freiburg，德国，产品描述T4-DNA-连接酶，产品号27-0870-04)保温过夜。此连接混合物然后通过电穿孔(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5微克/毫升四环素和40微克/毫升X-Gal(5-溴-4-氯-3-吲哚基β-D-半乳糖苷)的LB琼脂(Lennox，1955，病毒学，1：190)上铺板。
在37℃保温24小时后，借助于α-互补(Sambrook等，1989，分子克隆实验手册，冷泉港)可鉴别具有携带插入子的质粒的菌落。通过Bimboim和Doly(1997，核酸研究7：1513-1523)的“碱裂解法”重新分离质粒DNA，从转化子获得相应的表达构建体的DNA。通过测序插入物检查表达质粒的正确克隆。
如此获得的质粒构建体称为pEC-XT99A-dapF。质粒pEC-XT99A-dapF转化进大肠杆菌菌株DH5αmcr获得的大肠杆菌菌株称为DH5αmcr/pEC-XT99A-dapF。
实施例5 用质粒pEC-XT99A和pEC-XT99A-dapF转化菌株DSM5715
通过电穿孔法(Liebl等，1989，FEMS微生物学通信，53：299-303)将质粒pEC-XT99A(实施例3)和pEC-XT99A-dapF(实施例4)导入菌株DSM5715。
由电穿孔获得的转化子在具有15mg/l卡那霉素的选择琼脂(LBHIS琼脂(18.5g/l脑心浸液，0.5M山梨醇，5g/l Bacto-胰胨，2.5g/lBacto-酵母膏，5g/l NaCl，18g/l Bacto-琼脂))上分离。用常规方法(Peters-Wendich等，1998，微生物学，144，915-927)分离质粒DNA，用合适的限制性内切酶(PstI(Amersham Pharmacia Biotech，Freiburg，德国，产品描述PstI，产品号27-0886-03))切割并检查。获得的菌株称为DSM5715/pEC-XT99A和DSM5715/pEC-XT99A-dapF。
实施例6制备L-赖氨酸
实施例5中制备的谷氨酸棒杆菌菌株DSM5715/pEC-XT99A和DSM5715/pEC-XT99A-dapF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(Bacto-肽胨10g/l，Bacto-酵母膏10g/l，NaCl 5g/l，pH7.4(Eggeling等，1987，应用微生物学和生物技术，25：346-351)。加入四环素(5mg/l)。在摇床上于33℃以240rpm振荡培养预培养物24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0.2。培养基MM用作主培养物。
培养基MM：
CSL 5g/l
MOPS 20g/l
葡萄糖 50g/l(单独高压灭菌)
盐：
(NH4)2SO4 25g/l
KH2PO4 0.1g/l
MgSO4·7H2O 1.0g/l
CaCl2·2H2O 10mg/l
FeSO4·7H2O 10mg/l
MnSO4·H2O 5.0mg/l
L-亮氨酸 0.1g/l
生物素 0.3mg/l(过滤灭菌)
硫胺素·HCl 0.2mg/l(过滤灭菌)
CaCO3 25g/l
缩写：
CSL：玉米浆
MOPS：吗啉代丙磺酸
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，加入四环素(5mg/l)。在33℃和80％大气湿度下进行培养。
为诱导dapF表达，加入1mM IPTG(异丙基硫代-β-半乳糖苷，Gibco BRL生命技术，Eggenstein，德国，货号15529-019)。
72小时后，确定在660nm测量波长的OD以及形成的赖氨酸的浓度。用Lange公司的LD 1W数字式光度计(柏林，德国)确定光密度(OD660)。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赖氨酸。
所得结果示于表2。
表2
| 菌株 | IPTG | OD | L-赖氨酸g/L |
| DSM5715/pEC-XT99A | 8.2 | 11.9 | |
| DSM5715/pEC-XT99A-dapF | 8.5 | 12.6 | |
| DSM5715/pEC-XT99A-dapF | 1mM | 9.1 | 13.5 |
序列表
<110>德古萨-干尔斯股份公司
<120>编码dapF基因的新核苷酸序列
<130>990126 BT
<140>
<141>
<160>2
<170>PatentIn Ver.2.1
<210>1
<211>1084
<212>DNA
<213>谷氨酸棒杆菌
<220>
<221>-35_信号
<222>(117)..(123)
<220>
<221>-10_信号
<222>(138)..(143)
<220>
<221>CDS
<222>(151)..(981)
<400>1
agacgccttc gaacgcacgg tcaccggaac cagacgctat gtcaggcgcc aacgcagctg 60
gttcaacaga gaccaccgcg tgtcctgggt cgacgcctct ggcgatccca ccgcacaagc 120
cttggagatt ttgggtctac aatagcgagg gtg aat ttg acc atc ccc ttt gcc 174
Val Asn Leu Thr Ile Pro Phe Ala
1 5
aaa ggc cac gcc acc gaa aac gac ttc atc atc atc ccc gat gag gat 222
Lys Gly His Ala Thr Glu Asn Asp Phe Ile Ile Ile Pro Asp Glu Asp
10 15 20
gcg cgc cta gat tta act cca gaa atg gtg gtc acg ctg tgt gac cgc 270
Ala Arg Leu Asp Leu Thr Pro Glu Met Val Val Thr Leu Cys Asp Arg
25 30 35 40
cgc gcc ggg atc ggt gct gat ggt atc ctc cgc gtg gtt aaa gct gca 318
Arg Ala Gly Ile Gly Ala Asp Gly Ile Leu Arg Val Val Lys Ala Ala
45 50 55
gac gta gaa ggc tcc acg gtc gac cca tcg ctg tgg ttc atg gat tac 366
Asp Val Glu Gly Ser Thr Val Asp Pro Ser Leu Trp Phe Met Asp Tyr
60 65 70
cgc aac gcc gat gga tct ttg gct gaa atg tgc ggc aat ggt gtg cgc 414
Arg Asn Ala Asp Gly Ser Leu Ala Glu Met Cys Gly Asn Gly Val Arg
75 80 85
ctg ttc gcg cac tgg ctg tac tcc cgc ggt ctt gtt gat aat acg agc 462
Leu Phe Ala His Trp Leu Tyr Ser Arg Gly Leu Val Asp Asn Thr Ser
90 95 100
ttt gat atc ggt acc cgc gcc ggt gtc cgc cac gtt gat att ttg cag 510
Phe Asp Ile Gly Thr Arg Ala Gly Val Arg His Val Asp Ile Leu Gln
105 110 115 120
gca gat caa cat tct gcg cag gtc cgc gtt gat atg ggc atc cct gac 558
Ala Asp Gln His Ser Ala Gln Val Arg Val Asp Met Gly Ile Pro Asp
125 130 135
gtc acg gga tta tcc acc tgc gac atc aac ggc caa gta ttc gct ggc 606
Val Thr Gly Leu Ser Thr Cys Asp Ile Asn Gly Gln Val Phe Ala Gly
140 145 150
ctt ggc gtt gat atg ggt aac cca cac cta gcg tgc gtt gtg ccg ggc 654
Leu Gly Val Asp Met Gly Asn Pro His Leu Ala Cys Val Val Pro Gly
155 160 165
tta agt gcg tcg gct ctt gcc gat atg gaa ctg cgc gca cct acg ttt 702
Leu Ser Ala Ser Ala Leu Ala Asp Met Glu Leu Arg Ala Pro Thr Phe
170 175 180
gat cag gaa ttc ttc ccc cac ggt gtg aac gta gaa atc gtc aca gaa 750
Asp Gln Glu Phe Phe Pro His Gly Val Asn Val Glu Ile Val Thr Glu
185 190 195 200
tta gaa gat gac gca gta tcg atg cgc gtg tgg gaa cgc gga gtg ggc 798
Leu Glu Asp Asp Ala Val Ser Met Arg Val Trp Glu Arg Gly Val Gly
205 210 215
gaa acc cgc tcc tgt ggc acg gga acc gtt gct gca gcg tgt gct gct 846
Glu Thr Arg Ser Cys Gly Thr Gly Thr Val Ala Ala Ala Cys Ala Ala
220 225 230
tta gct gat gct gga ttg gga gaa ggc aca gtt aaa gtg tgc gtt cca 894
Leu Ala Asp Ala Gly Leu Gly Glu Gly Thr Val Lys Val Cys Val Pro
235 240 245
ggt ggg gaa gta gaa gtc cag atc ttt gac gac ggc tcc aca ctc acc 942
Gly Gly Glu Val Glu Val Gln Ile Phe Asp Asp Gly Ser Thr Leu Thr
250 255 260
ggc cca agc gcc atc atc gca ctc ggt gag gtg cag atc taagattcgc 991
Gly Pro Ser Ala Ile Ile Ala Leu Gly Glu Val Gln Ile
265 270 275
gattgtagtt cggcccaagt ttctgggccg ctttacgcgc atccagccac gtttcccgca 1051
gctctagtgc gcgctcgtcc gttactttga gaa 1084
<210>2
<211>277
<212>PRT
<213>谷氨酸棒杆菌
<400>2
Val Asn Leu Thr Ile Pro Phe Ala Lys Gly His Ala Thr Glu Asn Asp
1 5 10 15
Phe Ile Ile Ile Pro Asp Glu Asp Ala Arg Leu Asp Leu Thr Pro Glu
20 25 30
Met Val Val Thr Leu Cys Asp Arg Arg Ala Gly Ile Gly Ala Asp Gly
35 40 45
Ile Leu Arg Val Val Lys Ala Ala Asp Val Glu Gly Ser Thr Val Asp
50 55 60
Pro Ser Leu Trp Phe Met Asp Tyr Arg Asn Ala Asp Gly Ser Leu Ala
65 70 75 80
Glu Met Cys Gly Asn Gly Val Arg Leu Phe Ala His Trp Leu Tyr Ser
85 90 95
Arg Gly Leu Val Asp Asn Thr Ser Phe Asp Ile Gly Thr Arg Ala Gly
100 105 110
Val Arg His Val Asp Ile Leu Gln Ala Asp Gln His Ser Ala Gln Val
115 120 125
Arg Val Asp Met Gly Ile Pro Asp Val Thr Gly Leu Ser Thr Cys Asp
130 135 140
Ile Asn Gly Gln Val Phe Ala Gly Leu Gly Val Asp Met Gly Asn Pro
145 150 155 160
His Leu Ala Cys Val Val Pro Gly Leu Ser Ala Ser Ala Leu Ala Asp
165 170 175
Met Glu Leu Arg Ala Pro Thr Phe Asp Gln Glu Phe Phe Pro His Gly
180 185 190
Val Asn Val Glu Ile Val Thr Glu Leu Glu Asp Asp Ala Val Ser Met
195 200 205
Arg Val Trp Glu Arg Gly Val Gly Glu Thr Arg Ser Cys Gly Thr Gly
210 215 220
Thr Val Ala Ala Ala Cys Ala Ala Leu Ala Asp Ala Gly Leu Gly Glu
225 230 235 240
Gly Thr Val Lys Val Cys Val Pro Gly Gly Glu Val Glu Val Gln Ile
245 250 255
Phe Asp Asp Gly Ser Thr Leu Thr Gly Pro Ser Ala Ile Ile Ala Leu
260 265 270
Gly Glu Val Gln Ile
275
本发明包括如下附图：
图1：质粒pEC-XT99A图。
图2：质粒pEC-XT99A-dapF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
dapF：谷氨酸棒杆菌的dapF基因
oriE：大肠杆菌的质粒编码的复制源点
rep：谷氨酸棒杆菌质粒pGA1的质粒编码的复制源点
per：pGA1的控制拷贝数的基因
EcoRI：限制酶EcoRI的酶切位点
EcoRV：限制酶EcoRV的酶切位点
HincII：限制酶HincII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
SalI：限制酶SalI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
PvuII：限制酶PvuII的酶切位点
BamHI：限制酶BamHI的酶切位点
Claims (17)
1.分离的多核苷酸，其为选自如下一组的多核苷酸：
a)与编码由SEQ ID NO：2的氨基酸序列组成的多肽的多核苷酸相同的多核苷酸，或
b)与a)的多核苷酸互补的多核苷酸。
2.权利要求1的多核苷酸，其中该多核苷酸是能在棒状细菌中复制的重组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的能复制的多核苷酸，其由如SEQ ID NO：1所示的核苷酸序列组成。
5.权利要求2的多核苷酸，其编码由SEQ ID NO：2的氨基酸序列组成的多肽。
6.包含权利要求1的多核苷酸的载体pEC-XT99A-dapF，其以保藏号DSM 12968保藏。
7.含有权利要求1的多核苷酸的作为宿主细胞的棒状细菌。
8.经棒状细菌发酵制备L-赖氨酸的方法，其特征在于使用这样的细菌，所述细菌中权利要求1的多核苷酸被过表达。
9.权利要求8的方法，其特征在于使用这样的细菌，所述细菌中所需的L-氨基酸生物合成途径的其它基因被额外过表达。
10.权利要求8的方法，其特征在于使用这样的细菌，所述细菌中降低L-赖氨酸生成的代谢途径至少被部分关闭。
11.权利要求8的方法，其特征在于使用经质粒载体转化的菌株，所述质粒载体携带编码dapF基因的核苷酸序列。
12.权利要求8的方法，其特征在于使用经质粒载体pEC-XT99A-dapF转化的细菌，该质粒载体以在谷氨酸棒杆菌中的形式保藏，保藏号DSM 12968。
13.权利要求8的方法，其特征在于使用产生L-赖氨酸的谷氨酸棒杆菌物种的细菌。
14.权利要求8的方法，其特征在于编码二氢-2，6-吡啶二羧酸合酶的dapA基因被同时过表达。
15.权利要求8的方法，其特征在于赋予S-(2-氨基乙基)-半胱氨酸抗性的DNA片段被同时过表达。
16.权利要求8的发酵制备L-赖氨酸的方法，其特征在于进行以下步骤：
(a)发酵产生L-赖氨酸的棒状细菌，其中至少dapF基因是过表达的，
(b)富集培养基或细菌细胞中的L-赖氨酸，及
(c)分离L-赖氨酸。
17.由SEQ ID NO：1的至少15个连续核苷酸组成的探针或引物。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19943587.1 | 1999-09-11 | ||
| DE19943587A DE19943587A1 (de) | 1999-09-11 | 1999-09-11 | Neue für das dapF-Gen codierende Nukleotidsequenzen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1288055A CN1288055A (zh) | 2001-03-21 |
| CN100387714C true CN100387714C (zh) | 2008-05-14 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB001244868A Expired - Fee Related CN100387714C (zh) | 1999-09-11 | 2000-09-11 | 编码dapF基因的新核苷酸序列 |
Country Status (17)
| Country | Link |
|---|---|
| US (1) | US6670156B1 (zh) |
| EP (1) | EP1085094B2 (zh) |
| JP (1) | JP2001095593A (zh) |
| KR (1) | KR20010050423A (zh) |
| CN (1) | CN100387714C (zh) |
| AT (1) | ATE280232T1 (zh) |
| AU (1) | AU5507400A (zh) |
| BR (1) | BR0004059A (zh) |
| CA (1) | CA2317058A1 (zh) |
| DE (2) | DE19943587A1 (zh) |
| DK (1) | DK1085094T3 (zh) |
| ES (1) | ES2231089T5 (zh) |
| HU (1) | HUP0003553A2 (zh) |
| ID (1) | ID27239A (zh) |
| MX (1) | MXPA00008678A (zh) |
| SK (1) | SK13282000A3 (zh) |
| ZA (1) | ZA200004723B (zh) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4075087B2 (ja) | 1996-12-05 | 2008-04-16 | 味の素株式会社 | Ｌ−リジンの製造法 |
| US20040109600A1 (en) * | 2002-07-18 | 2004-06-10 | Cory Watkins | Inspection tool with partial framing camrea |
| US20040096095A1 (en) * | 2002-07-18 | 2004-05-20 | August Technology Corp. | Inspection tool with partial framing/windowing camera |
| EP1761487B1 (en) * | 2004-06-10 | 2013-08-07 | Board of Trustees of Michigan State University | Synthesis of caprolactam from lysine |
| WO2008103366A2 (en) * | 2007-02-20 | 2008-08-28 | Board Of Trustees Of Michigan State University | Catalytic deamination for carprolactam production |
| BRPI0911733A8 (pt) * | 2008-07-24 | 2017-10-03 | Amyris Inc | Método para produzir uma amida cíclica, método para produzir uma poliamida, método para produzir a-amino-e-caprolactama, método para produzir e-caprolactama, método para produzir policaprolactama, processo para a síntese de a-amino-e-caprolactama, processo para a síntese da e-caprolactama e método para produzir nylon 6 |
| EP2479279A1 (de) | 2011-01-20 | 2012-07-25 | Evonik Degussa GmbH | Verfahren zur fermentativen Herstellung schwefelhaltiger Aminosäuren |
| BR112016004367B1 (pt) | 2013-08-30 | 2022-06-14 | Evonik Operations Gmbh | Microrganismo recombinante e método para a produção fermentativa de metionina e/ou seus derivados |
| EP3204405B1 (en) * | 2014-10-09 | 2020-12-02 | Cathay Biotech Inc. | Expression of recombinant tetracycline efflux pumps for the production of lysine or lysine-derived products, and methods and applications thereof |
| EP3467099A1 (en) | 2017-10-05 | 2019-04-10 | 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 |
| WO2023222505A1 (en) | 2022-05-18 | 2023-11-23 | Evonik Operations Gmbh | Biotechnological production of monomers of bisucaberins, desferrioxamines and analogs thereof |
| WO2023222510A1 (en) | 2022-05-18 | 2023-11-23 | Evonik Operations Gmbh | Biotechnological production of desferrioxamines and analogs thereof |
| WO2023222515A1 (en) | 2022-05-18 | 2023-11-23 | Evonik Operations Gmbh | Biotechnological production of bisucaberins, desferrioxamines and analogs thereof |
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| US4560654A (en) * | 1980-05-16 | 1985-12-24 | Ajinomoto Company Incorporated | Method for producing L-lysine by fermentation |
| US4954441A (en) * | 1985-03-12 | 1990-09-04 | Kyowa Hakko Kogyo Co. Ltd. | Process for producing L-lysine |
| EP0854189A2 (en) * | 1996-12-05 | 1998-07-22 | Ajinomoto Co., Inc. | Method for producing L-lysine |
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|---|---|---|---|---|
| JPH07155184A (ja) * | 1993-12-08 | 1995-06-20 | Ajinomoto Co Inc | 発酵法によるｌ−リジンの製造法 |
| JP2003135066A (ja) * | 1999-03-19 | 2003-05-13 | Ajinomoto Co Inc | Ｌ−リジンの製造法 |
| US20030049804A1 (en) | 1999-06-25 | 2003-03-13 | Markus Pompejus | Corynebacterium glutamicum genes encoding metabolic pathway proteins |
| KR20070087034A (ko) | 1999-06-25 | 2007-08-27 | 바스프 악티엔게젤샤프트 | 대사 경로 단백질을 코딩하는 코리네박테리움 글루타미쿰유전자 |
| JP4623825B2 (ja) | 1999-12-16 | 2011-02-02 | 協和発酵バイオ株式会社 | 新規ポリヌクレオチド |
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- 1999-10-27 US US09/427,548 patent/US6670156B1/en not_active Expired - Lifetime
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- 2000-09-06 SK SK1328-2000A patent/SK13282000A3/sk unknown
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- 2000-09-08 JP JP2000273705A patent/JP2001095593A/ja active Pending
- 2000-09-08 BR BR0004059-2A patent/BR0004059A/pt not_active Application Discontinuation
- 2000-09-08 HU HU0003553A patent/HUP0003553A2/hu unknown
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4560654A (en) * | 1980-05-16 | 1985-12-24 | Ajinomoto Company Incorporated | Method for producing L-lysine by fermentation |
| US4954441A (en) * | 1985-03-12 | 1990-09-04 | Kyowa Hakko Kogyo Co. Ltd. | Process for producing L-lysine |
| EP0854189A2 (en) * | 1996-12-05 | 1998-07-22 | Ajinomoto Co., Inc. | Method for producing L-lysine |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200004723B (en) | 2001-03-13 |
| DE60015042D1 (de) | 2004-11-25 |
| JP2001095593A (ja) | 2001-04-10 |
| EP1085094B1 (en) | 2004-10-20 |
| ES2231089T3 (es) | 2005-05-16 |
| AU5507400A (en) | 2001-03-15 |
| MXPA00008678A (es) | 2002-05-23 |
| CA2317058A1 (en) | 2001-03-11 |
| SK13282000A3 (sk) | 2002-02-05 |
| ES2231089T5 (es) | 2008-05-01 |
| HUP0003553A2 (hu) | 2003-03-28 |
| EP1085094A2 (en) | 2001-03-21 |
| ID27239A (id) | 2001-03-15 |
| DK1085094T3 (da) | 2005-02-21 |
| DE60015042T3 (de) | 2008-03-06 |
| CN1288055A (zh) | 2001-03-21 |
| ATE280232T1 (de) | 2004-11-15 |
| KR20010050423A (ko) | 2001-06-15 |
| EP1085094A3 (en) | 2003-01-15 |
| EP1085094B2 (en) | 2007-11-07 |
| DE19943587A1 (de) | 2001-03-15 |
| US6670156B1 (en) | 2003-12-30 |
| BR0004059A (pt) | 2001-10-02 |
| DE60015042T2 (de) | 2006-02-09 |
| HU0003553D0 (en) | 2000-09-08 |
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