CN1501979A - 通过发酵棒状细菌生产l-氨基酸的方法 - Google Patents
通过发酵棒状细菌生产l-氨基酸的方法 Download PDFInfo
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Abstract
本发明涉及一种生产L-氨基酸的方法,其中进行以下步骤:(a)发酵生产所需L-氨基酸的棒状细菌,该细菌中至少mqo基因是弱化的,(b)富集培养基或细菌细胞中的所需产物,及(c)分离所需L-氨基酸,及任选地,所用细菌中所需L-氨基酸生物合成途径的其它基因被额外增强,或者所用细菌中降低所需L-氨基酸形成的至少一些代谢途径被排除。
Description
发明领域
本发明提供了通过发酵棒状细菌(Coryneform bacteria)生产L-氨基酸,尤其L-赖氨酸的方法,其中所用棒状细菌中编码苹果酸醌氧化还原酶(malate quinone oxidoreductase)的mqo基因已经弱化。
现有技术
L-氨基酸特别是L-赖氨酸用于人用药物和制药工业,食品工业,特别是动物饲养。
已知氨基酸可通过棒状细菌菌株,尤其谷氨酸棒杆菌(Corynebacterium glutamicum)的发酵而生产。由于生产方法具有的极其重要性,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,以此方法所获得的菌株对抗代谢物例如赖氨酸类似物S-(2-氨乙基)-半胱氨酸有抗性,或者对具有重要调节作用的代谢物产生营养缺陷,且所述的菌株产生L-氨基酸。
多年以来,重组DNA技术的方法也用于对产生L-氨基酸的谷氨酸棒杆菌菌株的改良,通过扩增各个氨基酸生物合成基因和研究对L-氨基酸生产的作用而进行。
发明目的
在EP-A-1038969中,阐述了通过mqo基因的增强、特别是过表达而增加通过发酵方法的L-氨基酸的产量。
本发明人的目的在于为用于通过发酵棒状细菌生产L-氨基酸尤其是L-赖氨酸的改良方法而提供新的基础。
发明阐述
当下文提及L-氨基酸时,应理解是指选自以下的一或多种氨基酸,包括其盐:L-天冬酰胺,L-苏氨酸,L-丝氨酸,L-谷氨酸,L-甘氨酸,L-丙氨酸,L-半胱氨酸,L-缬氨酸,L-甲硫氨酸,L-异亮氨酸,L-亮氨酸,L-酪氨酸,L-苯丙氨酸,L-组氨酸,L-赖氨酸,L-色氨酸,和L-精氨酸。特别优选L-赖氨酸。
当下文提及L-赖氨酸或赖氨酸时,应理解不仅是指碱还指盐,例如赖氨酸盐酸盐或赖氨酸硫酸盐。
本发明提供了一种通过发酵棒状细菌生产L-赖氨酸的方法,其中至少编码苹果酸醌氧化还原酶的核苷酸序列(mqo基因)是弱化的,尤其是排除的(excluded)或在低水平表达。
本发明还提供了一种通过发酵生产L-氨基酸的方法,其中进行以下步骤:
a)发酵生产L-氨基酸的棒状细菌,所述棒状细菌中至少编码苹果酸醌氧化还原酶的核苷酸序列是弱化的,尤其是排除的或低水平表达的;
b)浓缩培养基中或细菌细胞中的L-氨基酸;及
c)分离所需的L-氨基酸,其中任选地在终产物中保留部分或全部发酵液成分和/或生物量。
所用菌株优选甚至在mqo基因弱化之前就产生L-氨基酸、尤其是产生L-赖氨酸。
优选的实施方案见于权利要求书。
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排除,例如通过用弱启动子或编码低水平活性的相应酶的基因或等位基因,或失活相应基因或酶(蛋白质),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氨基酸。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。
适当的棒杆菌菌株,尤其谷氨酸棒杆菌菌种,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(Corynebacterium thermoaminogenes)FERMBP-1539
黄色短杆菌ATCC 14067
乳发酵短杆菌ATCC 13869和
扩展短杆菌ATCC 14020
和从中获得的产生L-氨基酸的突变体或菌株如生产L-赖氨酸的菌株:
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
谷氨酸棒杆菌ATCC 21513
谷氨酸棒杆菌ATCC 21544
谷氨酸棒杆菌ATCC 21543
谷氨酸棒杆菌DSM 4697和
谷氨酸棒杆菌DSM 5715。
与现有技术(EP-A-1038969)相反,发现在mqo基因弱化后可使棒状细菌的L-氨基酸产生增加。
谷氨酸棒杆菌的mqo基因的核苷酸序列已经由Molenar等公布(欧洲生物化学杂志254,395-403(1998)),也可以取自基因文库,登记号AJ22 4946。该核苷酸序列也示于SEQ ID NO:1,蛋白质的氨基酸序列示于SEQ ID NO:2。
根据本发明可以使用在提及的参考文献中所述的编码苹果酸醌氧化还原酶的序列。还可以使用苹果酸醌氧化还原酶的等位基因,其通过遗传密码的简并或通过中性功能的有义突变形成。
为获得弱化,可降低或排除mqo基因的表达或基因产物的催化性质。两种措施可任选地组合。
可通过以适当方式培养或遗传修饰(突变)用于基因表达的信号结构而降低基因表达。用于基因表达的信号结构例如是阻抑物(repressor)基因、激活子基因、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和Murphy(细菌学杂志170:5949(1988)),Voskuil和Chambliss(核酸研究26:3548(1998)),Jensen和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 ThiemeVerlag,德国斯图加特,1995),或Winnacker的教科书(基因和克隆,VCH Verlagsgesellschaft,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和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(transition)、颠换(transversion)、插入和缺失。氨基酸取代对酶活性的影响,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。如果终止密码子由于突变而在编码区内形成,则很可能通常导致翻译的过早终止。
缺失一些密码子典型地导致酶活性完全丧失。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 Verlag,德国斯图加特,1995),Winnacker的教科书(基因和克隆,VCHVerlagsgesellschaft,Weinheim,德国,1990)或Hagemann的教科书("Allgemeine Genetik",Gustav Fischer Verlag,Stuttgart,1986)。
本发明提供了mqo基因的等位基因672,示于SEQ ID NO:3,该等位基因在DNA序列(见SEQ ID NO:1)的第672位是腺嘌呤代替鸟嘌呤,其导致编码色氨酸-224(见SEQ ID NO:2)的TGG密码子由乳白(TGA)终止密码子取代。
本发明还提供了mqo基因的等位基因1230,示于SEQ ID NO:4,该等位基因在DNA序列(见SEQ ID NO:1)的第672位是腺嘌呤代替鸟嘌呤,其导致编码色氨酸-224(见 SEQ ID NO:2)的tgg密码子由乳白终止密码子取代,该等位基因另外在第1230位的胞嘧啶由胸腺嘧啶取代。
对谷氨酸棒杆菌基因进行诱变的一种常用方法是基因破坏和基因置换方法,如Schwarzer和Puhler所述(生物/技术学9,84-87(1991))。
在基因破坏方法中,将所述基因编码区的中心部分克隆入一个质粒载体中,该载体在宿主(典型为大肠杆菌)中能复制,但在谷氨酸棒杆菌中不能复制。适当的载体例如是pSUP301(Simon等,生物/技术学1,784-791(1983)),pK18mob或pK19mob(Schafer等,基因145,69-73(1994)),pK18mobsacB或pK19mobsacB(Jager等,细菌学杂志174:5462-5465(1992)),pGEM-T(Promega公司,Madison,WI,美国),pCR2.1-TOPO(Shuman(1994),生物化学杂志269:32678-32684;美国专利5487993),pCRBlunt(Invitrogen,Groningen,Netherlands;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)。然后将含有所述基因编码区中心部分的质粒载体通过缀合或转化移至所需的谷氨酸棒杆菌菌株中。缀合方法例如Schafer等所述(应用和环境微生物学60,756-759(1994))。转化方法例如Thierbach等所述(应用微生物和生物技术学29,356-362(1988)),Dunican和Shivnan(生物/技术学7,1067-1070(1989))和Tauch等(FEMS微生物学通讯123,343-347(1994))。在通过交叉同源重组后,所述基因的编码区被载体序列终端,获得分别缺失3’和5’末端的两个不完整的等位基因。这种方法例如由Fitzpatrick等使用(应用微生物学和生物技术学42,575-580(1994),以排除谷氨酸棒杆菌的recA基因。
在基因置换方法中,在体外在所述基因中产生突变,例如缺失,插入或碱基取代。将产生的等位基因依次克隆入在谷氨酸棒杆菌中不复制的载体中,然后将该载体通过转化或接合移至所需谷氨酸棒杆菌宿主中。在通过首次实现整合的交换和实现靶基因或靶序列切除的第二次交换同源重组后,实现掺入突变或等位基因。
这种方法例如由Peters-Wendisch等使用(微生物学144,915-927(1998)),以通过缺失排除谷氨酸棒杆菌的pyc基因。这种方法由Schafer等使用(基因145:69-73(1994)),以在hom-thrB基因区域中掺入缺失。同样,Schafer等(细菌学杂志176:7309-7319(1994))在谷氨酸棒杆菌的cgl基因区域中导入一个缺失。
因此在mqo基因中可掺入缺失、插入或碱基取代。
另外,除了mqo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、戊糖磷酸循环、氨基酸输出的一或多种酶,及任选调节蛋白的增强尤其过表达,对L-氨基酸的生产可以是有益的。
文中术语“增加”或“增强”是指微生物中由相应的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,及任选地组合使用这些方法
因此,例如为生产L-赖氨酸,除了弱化mqo基因之外,可以(同时)增强、尤其是过表达选自以下一组的一或多个基因:
·编码反馈抗性天冬氨酸激酶的lysC基因(登记号No.p26512,EP-B-0387527;EP-A-0699759;WO00/63388)
·编码二氢-2,6-吡啶二羧酸合酶的dapA基因(EP-B-0197335)
·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)
·编码丙酮酸羧化酶的pyc基因(DE-A-19831609)(同时)
·编码葡萄糖-6-磷酸脱氢酶的zwf基因(JP-A-09224661)
·编码赖氨酸输出蛋白的lysE基因(DE-A-19548222)(同时)
·编码Zwa1蛋白的zwa1基因(DE:19959328.0,DSM 13115)
·编码丙糖磷酸异构酶的tpi基因(Eikmanns(1992),细菌学杂志174:6076-6086),及
·编码3-磷酸甘油酸激酶的pgk基因(Eikmanns(1992),细菌学杂志174:6076-6086)。
除了弱化mqo基因之外,同时弱化选自以下一组的一或多个基因、尤其减少其表达对氨基酸的生产也是有益的:
·编码烯醇丙酮酸磷酸羧激酶的pck基因(DE19950409.1,DSM13047)
·编码葡糖6-磷酸异构酶的pgi基因(U.S.Ser.No.09/396478;DSM12969),
·编码编码丙酮酸氧化酶的poxB基因(DE:19951975.7;DSM13114),
·编码Zwa2蛋白的zwa2基因(DE:19959327.2;DSM13113)。
最后,除mqo基因的弱化之外,排除非所需的二级反应对氨基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,英国伦敦,1982)。
根据本发明产生的微生物也形成了本发明的一部分,并为了生产L-氨基酸可以连续或不连续地通过分批培养,或者通过补料分批法或重复补料分批法培养。已知的培养法见Chmiel(Bioprozesstechnik1.Einfuhrung in die Bioverfahrenstechnik(Gustav Fischer Verlag,Stuttgart,1991)所著教材,或由Storhas(Bioreaktoren und periphereEinrichtungen(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、酵母膏、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。也可以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。
为调节培养物的pH值,可以适当加入碱性化合物如NaOH、KOH、氨或氨水,或酸性化合物如磷酸或硫酸。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。
现有技术已知测定L-氨基酸的方法。分析可以如Speckman等(分析化学,30,(1958),1190)所述,通过阴离子交换层析,随后经茚三酮衍生化作用进行,或通过反相HPLC进行,如Lindroth等(分析化学(1979)51:1167-1174)所述。
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:制备用于在谷氨酸棒杆菌中IPTG诱导的mqo基因表达的表达载体pxk99Emobmqo
1.1克隆mqo基因
用Eikmanns等所述方法(微生物学140:1817-1828(1994))从菌株ATCC 13032中分离染色体DNA。基于已知的谷氨酸棒杆菌的mqo基因的序列,选择如下寡核苷酸进行聚合酶链反应(见SEQ IDNO:5和SEQ ID NO:6):
mqo-oPl:
5′-GA
GGA TCC GCA GAG AAC TCG CGG AGA TA-3′
mqo-hind:
5′-CT
AAG CTT CGT AGC GAG CCT TGA TGT AT-3′。
对引物加以选择以便扩增的片段含有从不具有启动子区域的天然核糖体结合位点开始的不完整基因,和mqo基因的前方区域。另外引物mqo-oP1含有限制性内切酶BamHI的切割位点序列,引物mqo-hind含有限制性内切酶HindIII的切割位点序列,在上述核苷酸序列中下划线处所示。
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Roche Diagnostics GmbH(德国曼海姆)的Pwo聚合酶进行PCR反应。借助于聚合酶链反应,该引物可以扩增大小为468bp的一个DNA片段,其携带不完整的mqo基因,包括天然核糖体结合位点。
将大小为468bp的mqo片段用限制性内切酶BamHI和HindIII切割,然后用QiaExII凝胶提取试剂盒(产品号No.20021,Qiagen,Hilden,德国)从琼脂糖凝胶中分离。
1.2构建表达载体pXK99Emob
根据现有技术构建IPTG可诱导的表达载体pXK99Emob。该载体基于大肠杆菌表达载体pTRC99A(Amann等,基因69:301-315(1988)),并含有可以通过加入乳糖衍生的IPTG(异丙基β-D-硫代半乳糖吡喃糖苷)而诱导trc启动子,终止区域T1和T2,大肠杆菌的复制起点ColE1,lacIq基因(大肠杆菌lac操纵子阻抑基因),多克隆位点(mcs)(Norrander,J.M.等,基因26,101-106(1983)),大肠杆菌卡那霉素抗性基因aph(3’)-Iia(Beck等(1982),基因19:327-336)和克隆载体pK18mobsacB的RP4-活动位点(Schaefer等,基因14:69-73(1994))。
已经发现载体pXK99Emob非常特异性适于调节基因的表达,尤其在棒状细菌中产生弱化表达。载体pXK99Emob是一种大肠杆菌表达载体,而且可以用于大肠杆菌中以增强基因的表达。
由于该载体不能在棒状细菌中独立复制,只有在将其整合入染色体中才可以保留在细胞中。该载体的这种特性在此用于调节基因的表达,在从载体中的相应基因的前方区域中克隆一个含有起始密码子和天然核糖体结合位点的基因节段后,随后将载体整合入棒状细菌中,尤其是谷氨酸棒杆菌中。基因表达通过在营养培养基中加入计量的IPTG而调节。0.5μM直至10μM数量的IPTG使相应基因非常弱地表达,10μM直至100μM使相应基因的正常表达略减弱。
将构建的大肠杆菌表达载体pXK99Emob通过电穿孔(Tauch等,1994,FEMS微生物学通讯123:343-347)移至大肠杆菌DH5αmcr中(Grant,1990,美国科学院院报87:4645-4649)。在补加50mg/ml卡那霉素的LB琼脂上(Sambrook等,分子克隆实验指导,第二版,冷泉港实验室出版社,冷泉港,N.Y.,1989)选择转化体。
质粒DNA通过常规方法从转化体中分离(Peters-Wendisch等,1998,微生物学144,915-927),用限制性内切酶NcoI切割,并随后通过琼脂糖凝胶电泳检测质粒。
以此方式获得的质粒构建体称为pXK99Emob(图1)。通过将质粒pXK99Emob电穿孔入大肠杆菌菌株DH5αmcr中获得的菌株称为大肠杆菌DH5alphamcr/pXK99Emob。
1.3在大肠杆菌表达载体pXK99Emob中克隆mqo片段
实施例1.2中阐述的大肠杆菌表达载体pXK99Emob用作载体。将这个质粒的DNA用限制性内切酶BamHI和HindIII完全切割,然后用虾碱性磷酸酶去磷酸化(Roche Diagnostics GmbH,德国曼海姆,产品描述SAP,产品号No.1758250)。
将通过PCR及用限制性内切酶BamHI和HindIII切割获得的实施例1.1所述大小为大约458bp的mqo片段与制备的载体pXK99Emob混合,然后将此混合物用T4 DNA连接酶处理(AmershamPharmacia,Freiburg,德国,T4-DNA-连接酶产品说明书,编码号No.27-0870-04)。然后将连接混合物转化入大肠杆菌菌株DH5αmcr中(Hanahan,DNA克隆实用方案,第1卷,IRL出版社,Oxford,美国华盛顿)。将转化混合物铺板于含有50mg/l卡那霉素的LB琼脂(Lennox,1955,病毒学,1:190)上选择携带质粒的细胞。在37℃温育过夜后,选择重组的各个克隆。用Qiaprep Spin Miniprep试剂盒(产品号No.27106,Qiagen,Hilden,德国)根据厂商说明书从转化体中分离质粒DNA,并用限制性内切酶BamHI和HindIII切割,以通过随后的琼脂糖凝胶电泳检测该质粒。所得质粒称为pXK99Emobmqo。示于图2。
以下微生物根据布达佩斯条约以纯培养物形式于2002年2月15日保藏在德意志微生物和细胞培养物保藏中心(DSMZ)(Braunschweig,德国):
大肠杆菌DH5alphamcr/pXK99Emobmqo(=DH5αmcr/pXK99Emobmqo),保藏号DSM14815。
实施例2:将载体pXK99Emobmqo整合入谷氨酸棒杆菌菌株DSM5715的基因组中
将实施例1所述载体pXK99Emobmqo通过Tauch等所述电穿孔方法(1989,FEMS微生物学通讯123:343-347)电穿孔入谷氨酸棒杆菌菌株DSM5715中。该载体在谷氨酸棒杆菌中不能独立复制,而且只有在整合入染色体中才能在细胞中保留。将电穿孔混合物铺板于补加了15mg/l卡那霉素和IPTG(1mM)的LB琼脂(Sambrook等,分子克隆实验指导,第二版,冷泉港,纽约,1989)上,选择具有整合的pXK99Emobmqo的克隆。
一种选择的卡那霉素抗性克隆,其中实施例1所述质粒pXK99Emobmqo插入DSM5715的染色体mqo基因中,将其称为DSM5715∷pXK99Emobmqo。
实施例3:赖氨酸的制备
将实施例2中获得的谷氨酸棒杆菌菌株DSM5715∷pXK99Emobmqo在适于生产赖氨酸的营养培养基中培养,并确定培养物上清液中的赖氨酸含量。加入IPTG,通过trc启动子的调节,mqo基因表达弱化。
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)和IPTG(10μM)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。
培养基Cg III
NaCl 2.5g/l
Bacto-蛋白胨 10g/l
Bacto-酵母膏 10g/l
葡萄糖(单独高压灭菌) 2%(w/v)
将pH调节为7.4。
向此培养基中加入卡那霉素(25mg/l)和IPTG(10μM)。在摇床上于33℃以240rpm振荡培养预培养物16小时。该预培养物的光密度(OD,660nm)是0.5。将500μl该预培养物接种主培养物。通过从预培养物中转移含有IPTG的培养基,主培养物中IPTG的浓度为大约0.5μM。培养基MM用作主培养基。
培养基MM:
CSL(玉米浆) 5g/l
MOPS(吗啉代丙烷磺酸) 20g/l
葡萄糖(单独高压灭菌) 50g/l
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
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/l
CaCO3 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。
72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(660nm) | 赖氨酸HCl(g/L) |
DSM5715 | 6.8 | 12.82 |
DSM5715∷pXK99Emobmqo | 6.4 | 14.85 |
附图简述:
图1:质粒pXK99Emob图。
图2:质粒pXK99Emobmqo图。
图中所采用的缩写和符号有如下含义:
Kan:大肠杆菌的卡那霉素抗性基因aph(3’)-IIa
BamHI:限制酶BamHI的切割位点
HindIII:限制酶HindIII的切割位点
NcoI:限制酶NcoI的切割位点
Ptrc:trc启动子
T1:终止区域T1
T2:终止区域T2
LacIq:大肠杆菌lac操纵子的lacIq阻抑基因
oriV:大肠杆菌ColE1的复制源点
mob:RP4-活动位点
mqo:mqo基因的克隆区域
序列表
<110>德古萨股份公司
<120>通过发酵棒状细菌生产L-氨基酸的方法
<130>010145 BT
<160>6
<170>PatentIn version 3.1
<210>1
<211>1503
<212>DNA
<213>谷氨酸棒杆菌
<220>
<221>CDS
<222>(1)..(1500)
<223>mqo-基因
<400>1
atg tca gat tcc ccg aag aac gca ccg agg att acc gat gag gca gat 48
Met Ser Asp Ser Pro Lys Asn Ala Pro Arg Ile Thr Asp Glu Ala Asp
1 5 10 15
gta gtt ctc att ggt gcc ggt atc atg agc tcc acg ctg ggt gca atg 96
Val Val Leu Ile Gly Ala Gly Ile Met Ser Ser Thr Leu Gly Ala Met
20 25 30
ctg cgt cag ctg gag cca agc tgg act cag atc gtc ttc gag cgt ttg 144
Leu Arg Gln Leu Glu Pro Ser Trp Thr Gln Ile Val Phe Glu Arg Leu
35 40 45
gat gga ccg gca caa gag tcg tcc tcc ccg tgg aac aat gca gga acc 192
Asp Gly Pro Ala Gln Glu Ser Ser Ser Pro Trp Asn Asn Ala Gly Thr
50 55 60
ggc cac tct gct cta tgc gag ctg aac tac acc cca gag gtt aag ggc 240
Gly His Ser Ala Leu Cys Glu Leu Asn Tyr Thr Pro Glu Val Lys Gly
65 70 75 80
aag gtt gaa att gcc aag gct gta gga atc aac gag aag ttc cag gtt 288
Lys Val Glu Ile Ala Lys Ala Val Gly Ile Asn Glu Lys Phe Gln Val
85 90 95
tcc cgt cag ttc tgg tct cac ctc gtt gaa gag gga gtg ctg tct gat 336
Ser Arg Gln Phe Trp Ser His Leu Val Glu Glu Gly Val Leu Ser Asp
100 105 110
cct aag gaa ttc atc aac cct gtt cct cac gta tct ttc ggc cag ggc 384
Pro Lys Glu Phe Ile Asn Pro Val Pro His Val Ser Phe Gly Gln Gly
115 120 125
gca gat cag gtt gca tac atc aag gct cgc tac gaa gct ttg aag gat 432
Ala Asp Gln Val Ala Tyr Ile Lys Ala Arg Tyr Glu Ala Leu Lys Asp
130 135 140
cac cca ctc ttc cag ggc atg acc tac gct gac gat gaa gct acc ttc 480
His Pro Leu Phe Gln Gly Met Thr Tyr Ala Asp Asp Glu Ala Thr Phe
145 150 155 160
acc gag aag ctg cct ttg atg gca aag ggc cgt gac ttc tct gat cca 528
Thr Glu Lys Leu Pro Leu Met Ala Lys Gly Arg Asp Phe Ser Asp Pro
165 170 175
gta gca atc tct tgg atc gat gaa ggc acc gac atc aac tac ggt gct 576
Val Ala Ile Ser Trp Ile Asp Glu Gly Thr Asp Ile Asn Tyr Gly Ala
180 185 190
cag acc aag cag tac ctg gat gca gct gaa gtt gaa ggc act gaa atc 624
Gln Thr Lys Gln Tyr Leu Asp Ala Ala Glu Val Glu Gly Thr Glu Ile
195 200 205
cgc tat ggc cac gaa gtc aag agc atc aag gct gat ggc gca aag tgg 672
Arg Tyr Gly His Glu Val Lys Ser Ile Lys Ala Asp Gly Ala Lys Trp
210 215 220
atc gtg acc gtc aag aac gta cac act ggc gac acc aag acc atc aag 720
Ile Val Thr Val Lys Asn Val His Thr Gly Asp Thr Lys Thr Ile Lys
225 230 235 240
gca aac ttc gtg ttc gtc ggc gca ggc gga tac gca ctg gat ctg ctt 768
Ala Asn Phe Val Phe Val Gly Ala Gly Gly Tyr Ala Leu Asp Leu Leu
245 250 255
cgc agc gca ggc atc cca cag gtc aag ggc ttc gct gga ttc cca gta 816
Arg Ser Ala Gly Ile Pro Gln Val Lys Gly Phe Ala Gly Phe Pro Val
260 265 270
tcc ggc ctg tgg ctt cgt tgc acc aac gag gaa ctg atc gag cag cac 864
Ser Gly Leu Trp Leu Arg Cys Thr Asn Glu Glu Leu Ile Glu Gln His
275 280 285
gca gcc aag gta tat ggc aag gca tct gtt ggc gct cct cca atg tct 912
Ala Ala Lys Val Tyr Gly Lys Ala Ser Val Gly Ala Pro Pro Met Ser
290 295 300
gtt cct cac ctt gac acc cgc gtt atc gag ggt gaa aag ggt ctg ctc 960
Val Pro His Leu Asp Thr Arg Val Ile Glu Gly Glu Lys Gly Leu Leu
305 310 315 320
ttt gga cct tac ggt ggc tgg acc cct aag ttc ttg aag gaa ggc tcc 1008
Phe Gly Pro Tyr Gly Gly Trp Thr Pro Lys Phe Leu Lys Glu Gly Ser
325 330 335
tac ctg gac ctg ttc aag tcc atc cgc cca gac aac att cct tcc tac 1056
Tyr Leu Asp Leu Phe Lys Ser Ile Arg Pro Asp Asn Ile Pro Ser Tyr
340 345 350
ctt ggc gtt gct gct cag gaa ttt gat ctg acc aag tac ctt gtc act 1104
Leu Gly Val Ala Ala Gln Glu Phe Asp Leu Thr Lys Tyr Leu Val Thr
355 360 365
gaa gtt ctc aag gac cag gac aag cgt atg gat gct ctt cgc gag tac 1152
Glu Val Leu Lys Asp Gln Asp Lys Arg Met Asp Ala Leu Arg Glu Tyr
370 375 380
atg cca gag gca caa aac ggc gat tgg gag acc atc gtt gcc gga cag 1200
Met Pro Glu Ala Gln Asn Gly Asp Trp Glu Thr Ile Val Ala Gly Gln
385 390 395 400
cgt gtt cag gtt att aag cct gca gga ttc cct aag ttc ggt tcc ctg 1248
Arg Val Gln Val Ile Lys Pro Ala Gly Phe Pro Lys Phe Gly Ser Leu
405 410 415
gaa ttc ggc acc acc ttg atc aac aac tcc gaa ggc acc atc gcc gga 1296
Glu Phe Gly Thr Thr Leu Ile Asn Asn Ser Glu Gly Thr Ile Ala Gly
420 425 430
ttg ctc ggt gct tcc cct gga gca tcc atc gca cct tcc gca atg atc 1344
Leu Leu Gly Ala Ser Pro Gly Ala Ser Ile Ala Pro Ser Ala Met Ile
435 440 445
gag ctg ctt gag cgt tgc ttc ggt gac cgc atg atc gag tgg ggc gac 1392
Glu Leu Leu Glu Arg Cys Phe Gly Asp Arg Met Ile Glu Trp Gly Asp
450 455 460
aag ctg aag gac atg atc cct tcc tac ggc aag aag ctt gct tcc gag 1440
Lys Leu Lys Asp Met Ile Pro Ser Tyr Gly Lys Lys Leu Ala Ser Glu
465 470 475 480
cca gca ctg ttt gag cag cag tgg gca cgc acc cag aag acc ctg aag 1488
Pro Ala Leu Phe Glu Gln Gln Trp Ala Arg Thr Gln Lys Thr Leu Lys
485 490 495
ctt gag gaa gcc taa 1503
Leu Glu Glu Ala
500
<210>2
<211>500
<212>PRT
<213>谷氨酸棒杆菌
<400>2
Met Ser Asp Ser Pro Lys Asn Ala Pro Arg Ile Thr Asp Glu Ala Asp
1 5 10 15
Val Val Leu Ile Gly Ala Gly Ile Met Ser Ser Thr Leu Gly Ala Met
20 25 30
Leu Arg Gln Leu Glu Pro Ser Trp Thr Gln Ile Val Phe Glu Arg Leu
35 40 45
Asp Gly Pro Ala Gln Glu Ser Ser Ser Pro Trp Asn Asn Ala Gly Thr
50 55 60
Gly His Ser Ala Leu Cys Glu Leu Asn Tyr Thr Pro Glu Val Lys Gly
65 70 75 80
Lys Val Glu Ile Ala Lys Ala Val Gly Ile Asn Glu Lys Phe Gln Val
85 90 95
Ser Arg Gln Phe Trp Ser His Leu Val Glu Glu Gly Val Leu Ser Asp
100 105 110
Pro Lys Glu Phe Ile Asn Pro Val Pro His Val Ser Phe Gly Gln Gly
115 120 125
Ala Asp Gln Val Ala Tyr Ile Lys Ala Arg Tyr Glu Ala Leu Lys Asp
130 135 140
His Pro Leu Phe Gln Gly Met Thr Tyr Ala Asp Asp Glu Ala Thr Phe
145 150 155 160
Thr Glu Lys Leu Pro Leu Met Ala Lys Gly Arg Asp Phe Ser Asp Pro
165 170 175
Val Ala Ile Ser Trp Ile Asp Glu Gly Thr Asp Ile Asn Tyr Gly Ala
180 185 190
Gln Thr Lys Gln Tyr Leu Asp Ala Ala Glu Val Glu Gly Thr Glu Ile
195 200 205
Arg Tyr Gly His Glu Val Lys Ser Ile Lys Ala Asp Gly Ala Lys Trp
210 215 220
Ile Val Thr Val Lys Asn Val His Thr Gly Asp Thr Lys Thr Ile Lys
225 230 235 240
Ala Asn Phe Val Phe Val Gly Ala Gly Gly Tyr Ala Leu Asp Leu Leu
245 250 255
Arg Ser Ala Gly Ile Pro Gln Val Lys Gly Phe Ala Gly Phe Pro Val
260 265 270
Ser Gly Leu Trp Leu Arg Cys Thr Asn Glu Glu Leu Ile Glu Gln His
275 280 285
Ala Ala Lys Val Tyr Gly Lys Ala Ser Val Gly Ala Pro Pro Met Ser
290 295 300
Val Pro His Leu Asp Thr Arg Val Ile Glu Gly Glu Lys Gly Leu Leu
305 310 315 320
Phe Gly Pro Tyr Gly Gly Trp Thr Pro Lys Phe Leu Lys Glu Gly Ser
325 330 335
Tyr Leu Asp Leu Phe Lys Ser Ile Arg Pro Asp Asn Ile Pro Ser Tyr
340 345 350
Leu Gly Val Ala Ala Gln Glu Phe Asp Leu Thr Lys Tyr Leu Val Thr
355 360 365
Glu Val Leu Lys Asp Gln Asp Lys Arg Met Asp Ala Leu Arg Glu Tyr
370 375 380
Met Pro Glu Ala Gln Asn Gly Asp Trp Glu Thr Ile Val Ala Gly Gln
385 390 395 400
Arg Val Gln Val Ile Lys Pro Ala Gly Phe Pro Lys Phe Gly Ser Leu
405 410 415
Glu Phe Gly Thr Thr Leu Ile Asn Asn Ser Glu Gly Thr Ile Ala Gly
420 425 430
Leu Leu Gly Ala Ser Pro Gly Ala Ser Ile Ala Pro Ser Ala Met Ile
435 440 445
Glu Leu Leu Glu Arg Cys Phe Gly Asp Arg Met Ile Glu Trp Gly Asp
450 455 460
Lys Leu Lys Asp Met Ile Pro Ser Tyr Gly Lys Lys Leu Ala Ser Glu
465 470 475 480
Pro Ala Leu Phe Glu Gln Gln Trp Ala Arg Thr Gln Lys Thr Leu Lys
485 490 495
Leu Glu Glu Ala
500
<210>3
<211>1503
<212>DNA
<213>谷氨酸棒杆菌
<220>
<221>-
<222>(1)..(1500)
<223>mqo-等位基因672
<220>
<221>其他特征
<222>(1)..(3)
<223>起始密码子
<220>
<221>其他特征
<222>(670)..(672)
<223>opal终止密码子
<400>3
atgtcagatt ccccgaagaa cgcaccgagg attaccgatg aggcagatgt agttctcatt 60
ggtgccggta tcatgagctc cacgctgggt gcaatgctgc gtcagctgga gccaagctgg 120
actcagatcg tcttcgagcg tttggatgga ccggcacaag agtcgtcctc cccgtggaac 180
aatgcaggaa ccggccactc tgctctatgc gagctgaact acaccccaga ggttaagggc 240
aaggttgaaa ttgccaaggc tgtaggaatc aacgagaagt tccaggtttc ccgtcagttc 300
tggtctcacc tcgttgaaga gggagtgctg tctgatccta aggaattcat caaccctgtt 360
cctcacgtat ctttcggcca gggcgcagat caggttgcat acatcaaggc tcgctacgaa 420
gctttgaagg atcacccact cttccagggc atgacctacg ctgacgatga agctaccttc 480
accgagaagc tgcctttgat ggcaaagggc cgtgacttct ctgatccagt agcaatctct 540
tggatcgatg aaggcaccga catcaactac ggtgctcaga ccaagcagta cctggatgca 600
gctgaagttg aaggcactga aatccgctat ggccacgaag tcaagagcat caaggctgat 660
ggcgcaaagt gaatcgtgac cgtcaagaac gtacacactg gcgacaccaa gaccatcaag 720
gcaaacttcg tgttcgtcgg cgcaggcgga tacgcactgg atctgcttcg cagcgcaggc 780
atcccacagg tcaagggctt cgctggattc ccagtatccg gcctgtggct tcgttgcacc 840
aacgaggaac tgatcgagca gcacgcagcc aaggtatatg gcaaggcatc tgttggcgct 900
cctccaatgt ctgttcctca ccttgacacc cgcgttatcg agggtgaaaa gggtctgctc 960
tttggacctt acggtggctg gacccctaag ttcttgaagg aaggctccta cctggacctg 1020
ttcaagtcca tccgcccaga caacattcct tcctaccttg gcgttgctgc tcaggaattt 1080
gatctgacca agtaccttgt cactgaagtt ctcaaggacc aggacaagcg tatggatgct 1140
cttcgcgagt acatgccaga ggcacaaaac ggcgattggg agaccatcgt tgccggacag 1200
cgtgttcagg ttattaagcc tgcaggattc cctaagttcg gttccctgga attcggcacc 1260
accttgatca acaactccga aggcaccatc gccggattgc tcggtgcttc ccctggagca 1320
tccatcgcac cttccgcaat gatcgagctg cttgagcgtt gcttcggtga ccgcatgatc 1380
gagtggggcg acaagctgaa ggacatgatc ccttcctacg gcaagaagct tgcttccgag 1440
ccagcactgt ttgagcagca gtgggcacgc acccagaaga ccctgaagct tgaggaagcc 1500
taa 1503
<210>4
<211>1503
<212>DNA
<213>谷氨酸棒杆菌
<220>
<221>-
<222>(1)..(1500)
<223>mqo-等位基因1230
<220>
<221>其他特征
<222>(1)..(3)
<223>起始密码子
<220>
<221>其他特征
<222>(670)..(672)
<223>opal终止密码子
<220>
<221>其他特征
<222>(1230)..(672)
<223>C-T转换
<400>4
atgtcagatt ccccgaagaa cgcaccgagg attaccgatg aggcagatgt agttctcatt 60
ggtgccggta tcatgagctc cacgctgggt gcaatgctgc gtcagctgga gccaagctgg 120
actcagatcg tcttcgagcg tttggatgga ccggcacaag agtcgtcctc cccgtggaac 180
aatgcaggaa ccggccactc tgctctatgc gagctgaact acaccccaga ggttaagggc 240
aaggttgaaa ttgccaaggc tgtaggaatc aacgagaagt tccaggtttc ccgtcagttc 300
tggtctcacc tcgttgaaga gggagtgctg tctgatccta aggaattcat caaccctgtt 360
cctcacgtat ctttcggcca gggcgcagat caggttgcat acatcaaggc tcgctacgaa 420
gctttgaagg atcacccact cttccagggc atgacctacg ctgacgatga agctaccttc 480
accgagaagc tgcctttgat ggcaaagggc cgtgacttct ctgatccagt agcaatctct 540
tggatcgatg aaggcaccga catcaactac ggtgctcaga ccaagcagta cctggatgca 600
gctgaagttg aaggcactga aatccgctat ggccacgaag tcaagagcat caaggctgat 660
ggcgcaaagt gaatcgtgac cgtcaagaac gtacacactg gcgacaccaa gaccatcaag 720
gcaaacttcg tgttcgtcgg cgcaggcgga tacgcactgg atctgcttcg cagcgcaggc 780
atcccacagg tcaagggctt cgctggattc ccagtatccg gcctgtggct tcgttgcacc 840
aacgaggaac tgatcgagca gcacgcagcc aaggtatatg gcaaggcatc tgttggcgct 900
cctccaatgt ctgttcctca ccttgacacc cgcgttatcg agggtgaaaa gggtctgctc 960
tttggacctt acggtggctg gacccctaag ttcttgaagg aaggctccta cctggacctg 1020
ttcaagtcca tccgcccaga caacattcct tcctaccttg gcgttgctgc tcaggaattt 1080
gatctgacca agtaccttgt cactgaagtt ctcaaggacc aggacaagcg tatggatgct 1140
cttcgcgagt acatgccaga ggcacaaaac ggcgattggg agaccatcgt tgccggacag 1200
cgtgttcagg ttattaagcc tgcaggattt cctaagttcg gttccctgga attcggcacc 1260
accttgatca acaactccga aggcaccatc gccggattgc tcggtgcttc ccctggagca 1320
tccatcgcac cttccgcaat gatcgagctg cttgagcgtt gcttcggtga ccgcatgatc 1380
gagtggggcg acaagctgaa ggacatgatc ccttcctacg gcaagaagct tgcttccgag 1440
ccagcactgt ttgagcagca gtgggcacgc acccagaaga ccctgaagct tgaggaagcc 1500
taa 1503
<210>5
<211>28
<212>DNA
<213>人工序列
<220>
<223>含有限制性酶切位点的引物
<220>
<221>其他特征
<222>(1)..(28)
<223>引物mqo_oP1
<400>5
gaggatccgc agagaactcg cggagata 28
<210>6
<211>28
<212>DNA
<213>人工序列
<220>
<223>含有限制性酶切位点的引物
<220>
<221>其他特征
<222>(1)..(28)
<223>引物mqo_hind
<400>6
ctaagcttcg tagcgagcct tgatgtat 28
Claims (15)
1、通过发酵生产L-氨基酸特别是L-赖氨酸的方法,所述方法包括进行以下步骤:
(a)发酵生产所需L-氨基酸的棒状细菌,该细菌中至少mqo基因是弱化的,
(b)富集培养基或细菌细胞中的所需产物,及
(c)分离所需L-氨基酸,其中任选地在终产物中保留部分或全部发酵液和/或生物量。
2、权利要求1的方法,其中所用的细菌中所需L-氨基酸生物合成途径的其它基因额外被扩增。
3、权利要求1的方法,其中所用的细菌中减少所需L-氨基酸生成的至少一些代谢途径被排除。
4、权利要求1的方法,其中编码mqo基因的多核苷酸的表达被降低。
5、权利要求1的方法,其中多核苷酸mqo编码的多肽(酶蛋白)的催化活性被降低。
6、权利要求1的方法,其中为生产L-赖氨酸,所发酵的棒状微生物中选自以下一组的一或多种基因同时被增强、尤其是被过表达:
6.1编码反馈抗性天冬氨酸激酶的lysC基因,
6.2编码二氢-2,6-吡啶二羧酸合酶的dapA基因,
6.3编码甘油醛-3-磷酸脱氢酶的gap基因,
6.4编码丙酮酸羧化酶的pyc基因,
6.5编码葡萄糖-6-磷酸脱氢酶的zwf基因,
6.6编码赖氨酸输出的lysE基因(同时)
6.7编码Zwa1蛋白的zwa1基因,
6.8编码丙糖磷酸异构酶的tpi基因,及
6.9编码3-磷酸甘油酸激酶的pgk基因。
7、权利要求1的方法,其中为生产L-氨基酸,所发酵的棒状微生物中选自以下一组的一或多种基因同时被弱化:
7.1编码烯醇丙酮酸磷酸羧激酶的pck基因,
7.2编码葡糖6-磷酸异构酶的pgi基因,
7.3编码丙酮酸氧化酶的poxB基因,或
7.4编码Zwa2蛋白的zwa2基因。
8、前述权利要求中任一项的方法,其中使用谷氨酸棒杆菌菌种的微生物。
9、棒状细菌,其中至少mqo基因以弱化形式存在。
10、权利要求9的棒状细菌,其中弱化通过缺失、插入、转换、或颠换突变而实现。
11、权利要求9或10的棒状细菌,其中mqo基因含有一个终止密码子。
12、权利要求11的棒状细菌,其中终止密码子、尤其是乳白密码子,位于SEQ ID NO:2的氨基酸序列的第224位。
13、权利要求9-12中任一项的棒状细菌,其含有SEQ ID NO:3的mqo等位基因。
14、权利要求9-12中任一项的棒状细菌,其含有SEQ ID NO:4的mqo等位基因。
15、大肠杆菌菌株DH5alphamcr/pXK99Emobmqo(=DH5αmcr/pXK99Emobmqo),保藏在德意志微生物和细胞培养物保藏中心(DSMZ,Brunswick,德国),保藏号DSM14815。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10117816.6 | 2001-04-10 | ||
DE10117816A DE10117816A1 (de) | 2001-04-10 | 2001-04-10 | Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
Publications (2)
Publication Number | Publication Date |
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CN1501979A true CN1501979A (zh) | 2004-06-02 |
CN1231591C CN1231591C (zh) | 2005-12-14 |
Family
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Application Number | Title | Priority Date | Filing Date |
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CNB028081048A Expired - Fee Related CN1231591C (zh) | 2001-04-10 | 2002-04-04 | 通过发酵棒状细菌生产l-氨基酸的方法 |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP1377674A2 (zh) |
KR (1) | KR20040014489A (zh) |
CN (1) | CN1231591C (zh) |
AU (1) | AU2002316834A1 (zh) |
DE (1) | DE10117816A1 (zh) |
HU (1) | HUP0303926A3 (zh) |
WO (1) | WO2002086137A2 (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102317433A (zh) * | 2008-12-17 | 2012-01-11 | Cj第一制糖株式会社 | 具有提高的5’-黄苷一磷酸生产力的棒状菌菌株和用其生产5’-黄苷一磷酸的方法 |
CN102300982B (zh) * | 2008-12-17 | 2014-10-29 | Cj第一制糖株式会社 | 用于提高5’-鸟苷一磷酸生产力的棒状菌菌株和用其生产5’-鸟苷一磷酸的方法 |
CN109957537A (zh) * | 2017-12-14 | 2019-07-02 | 赢创德固赛有限公司 | 发酵生产l-赖氨酸的方法 |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100786987B1 (ko) * | 2004-01-30 | 2007-12-18 | 아지노모토 가부시키가이샤 | L-아미노산 생산 미생물 및 l-아미노산 생산 방법 |
DE102004011248A1 (de) * | 2004-03-09 | 2005-09-22 | Degussa Ag | Verfahren zur Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
DE102005032429A1 (de) * | 2005-01-19 | 2006-07-20 | Degussa Ag | Allele des mqo-Gens aus coryneformen Bakterien |
US7214526B2 (en) | 2005-01-19 | 2007-05-08 | Degussa Ag | Alleles of the mqo gene from coryneform bacteria |
KR100789270B1 (ko) * | 2005-11-30 | 2008-01-02 | 씨제이 주식회사 | L-라이신 생산능이 향상된 코리네박테리움 속 미생물 및그를 이용하여 l-라이신을 생산하는 방법 |
KR100733928B1 (ko) * | 2005-11-30 | 2007-07-02 | 씨제이 주식회사 | 카나마이신 내성을 갖고 l-라이신 생산능이 향상된코리네형 미생물 및 그를 이용하여 l-라이신을 생산하는방법 |
DE102006026328A1 (de) * | 2006-06-02 | 2008-01-03 | Evonik Degussa Gmbh | Verfahren zur Herstellung eines L-Lysin enthaltenden Futtermitteladditivs |
DE102006050489A1 (de) | 2006-10-26 | 2008-04-30 | Evonik Degussa Gmbh | Allele des prpD1-Gens aus coryneformen Bakterien |
KR102572849B1 (ko) * | 2022-07-11 | 2023-08-31 | 대상 주식회사 | L-글루탐산을 생산하는 코리네박테리움 속 변이 미생물 및 이를 이용한 l-글루탐산의 생산 방법 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19912384A1 (de) * | 1999-03-19 | 2000-09-21 | Degussa | Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
DE19941478A1 (de) * | 1999-09-01 | 2001-03-08 | Degussa | Neue für das thrE-Gen codierende Nukleotidsequenzen und Verfahren zur fermentativen Herstellung von L-Threonin mit coryneformen Bakterien |
-
2001
- 2001-04-10 DE DE10117816A patent/DE10117816A1/de not_active Withdrawn
-
2002
- 2002-04-04 AU AU2002316834A patent/AU2002316834A1/en not_active Abandoned
- 2002-04-04 HU HU0303926A patent/HUP0303926A3/hu unknown
- 2002-04-04 CN CNB028081048A patent/CN1231591C/zh not_active Expired - Fee Related
- 2002-04-04 WO PCT/EP2002/003728 patent/WO2002086137A2/en not_active Application Discontinuation
- 2002-04-04 KR KR20037013212A patent/KR20040014489A/ko not_active Application Discontinuation
- 2002-04-04 EP EP02745213A patent/EP1377674A2/en not_active Withdrawn
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102317433A (zh) * | 2008-12-17 | 2012-01-11 | Cj第一制糖株式会社 | 具有提高的5’-黄苷一磷酸生产力的棒状菌菌株和用其生产5’-黄苷一磷酸的方法 |
CN102300982B (zh) * | 2008-12-17 | 2014-10-29 | Cj第一制糖株式会社 | 用于提高5’-鸟苷一磷酸生产力的棒状菌菌株和用其生产5’-鸟苷一磷酸的方法 |
CN104130966A (zh) * | 2008-12-17 | 2014-11-05 | Cj第一制糖株式会社 | 具有提高的5’-黄苷一磷酸生产力的棒状菌菌株和用其生产5’-黄苷一磷酸的方法 |
CN102317433B (zh) * | 2008-12-17 | 2016-09-07 | Cj第一制糖株式会社 | 具有提高的5’-黄苷一磷酸生产力的棒状菌菌株和用其生产5’-黄苷一磷酸的方法 |
CN109957537A (zh) * | 2017-12-14 | 2019-07-02 | 赢创德固赛有限公司 | 发酵生产l-赖氨酸的方法 |
Also Published As
Publication number | Publication date |
---|---|
HUP0303926A3 (en) | 2005-12-28 |
HUP0303926A2 (hu) | 2004-03-01 |
AU2002316834A1 (en) | 2002-11-05 |
KR20040014489A (ko) | 2004-02-14 |
AU2002316834A8 (en) | 2005-10-13 |
DE10117816A1 (de) | 2002-10-17 |
CN1231591C (zh) | 2005-12-14 |
EP1377674A2 (en) | 2004-01-07 |
WO2002086137A3 (en) | 2003-08-28 |
WO2002086137A2 (en) | 2002-10-31 |
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