CN1295131A - 利用棒状细菌发酵生产l-赖氨酸的方法 - Google Patents
利用棒状细菌发酵生产l-赖氨酸的方法 Download PDFInfo
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- CN1295131A CN1295131A CN00130374A CN00130374A CN1295131A CN 1295131 A CN1295131 A CN 1295131A CN 00130374 A CN00130374 A CN 00130374A CN 00130374 A CN00130374 A CN 00130374A CN 1295131 A CN1295131 A CN 1295131A
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Abstract
本发明涉及生产L-氨基酸,特别是L-赖氨酸的方法,其特征在于进行下述的步骤,a)发酵产生所需L-氨基酸的细菌,细菌中至少csp1基因被弱化,b)在培养基中或细菌细胞中积累所需的L-氨基酸,以及c)分离L-氨基酸。任选地所用的细菌中所需L-氨基酸的生物合成途径的基他基因被扩增,或者所用的细菌中降低所需L-氨基酸形成的代谢途径至少部分被抑制。
Description
本发明提供了一种利用弱化了csp1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。
L-氨基酸,特别是L-赖氨酸,被应用在动物营养,人类医药,以及制药工业上。
众所周知,这些氨基酸由棒状细菌菌株,特别是由谷氨酸棒杆菌发酵产生。因为它们的重要性,人们一直在努力改进其生产方法。对这些方法的改进可能涉及与发酵技术有关的措施,例如搅拌和氧气的供应,或者培养基的成分,例如发酵过程中糖的浓度,或者通过例如离子交换层析对产物的处理,或者微生物本身的固有生产特征。
这些微生物本身的固有生产特征,可利用基因诱变,选择和突变选择的方法来提高。这些方法中,获得的菌株对抗代谢物有抗性,例如赖氨酸类似物S-(2-氨乙基)半胱氨酸抗性,或者有重要代谢调节物的营养缺陷,并且能够产生L-氨基酸。
近年来,重组DNA技术同样被用于通过扩增各个生物合成基因以及研究其对L-氨基酸产量的影响来改造产生L-氨基酸的棒杆菌菌株。本主题的相关参考文献可以在Kinoshita(“谷氨酸棒杆菌”,在:工业微生物学,Demam与Solomon(Eds.),Benjamin Cummings,伦敦,UK,1985,115-142),Hilliger(生物技术2,40-44(1991)),Eggeling(氨基酸6:261-272(1994)),Jetten与Sinskey(CriticalReviews in Biotechnology 15,73-103(1995))以及Sahm等(纽约科学院年报782,25-39(1996))中找到。
发明人的目的是提供一种新的用来改进用棒状细菌发酵生产L-氨基酸特别是L-赖氨酸的方法的基础。
L-氨基酸,特别是L-赖氨酸,被用在人用药与医药工业,食品工业以及特别是动物营养中。因此,人们普遍对提供新的改进的方法来生产L-氨基酸特别是L-赖氨酸感兴趣。
任一下面所述的L-赖氨酸或赖氨酸不仅指碱,而且包括盐,例如赖氨酸单盐酸盐或赖氨酸硫酸盐。
本发明提供了一种利用棒状细菌发酵生产L-氨基酸,特别是L-赖氨酸的方法,其中至少细菌中的编码Csp1-基因产物的核苷酸序列被弱化特别是在较低的水平表达,所希望的产物积累在培养基或细胞中并且L-赖氨酸被分离。
所用菌株优选在csp1基因被弱化前已经产生L-氨基酸,特别是L-赖氨酸。
优选的实施方案描述在权利要求中。
在本文中,术语“弱化”是指降低或抑制微生物中一个或多个酶(蛋白)的细胞内活性,其中的酶被相应的DNA(在本申请中为csp1基因)编码,例如通过利用一个弱的启动子或编码相应的具有低活性的酶的基因或等位基因,或使相应的基因(蛋白)失去活性,以及任选地通过这些方法的组合。
本发明所提供的微生物可由葡萄糖,蔗糖,乳糖,果糖,麦芽糖,糖蜜,淀粉,纤维素或从甘油以及乙醇生产氨基酸,特别是赖氨酸。微生物可包括棒状细菌的代表性菌株特别是棒杆菌属,在棒杆菌属中,谷氨酸棒杆菌种可以特别被提及,因为其生产L-氨基酸的能力被专家们所公知。
合适的棒杆菌属的菌株,特别是谷氨酸棒杆菌种的菌株,尤其是已知的野生型菌株
谷氨酸棒杆菌ATCC13032
醋谷棒杆菌ATCC15806
嗜乙酰乙酸棒杆菌ATCC13870
Corynebaeterium melassecola ATCC17965
嗜热产氨棒杆菌FERM BP-1539
黄色短杆菌-ATCC14067
乳发酵短杆菌ATCC13869以及
扩展短杆菌ATCC14020
以及由其产生的生产L-氨基酸的突变体或菌株,
例如生产L-赖氨酸的菌株
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以及
谷氨酸棒杆菌DSM 5714。
人们发现一旦csp1基因被弱化,棒状细菌便以一种改良的方式产生L-氨基酸,特别是L-赖氨酸。
编码PS1蛋白的csp1基因,仍未被证实具有任何酶的活性。csp1基因的核苷酸序列已被Joliff等人描述(分子微生物学1992 Aug;6(16):2349-62)。序列通常可从国家生物技术信息中心(NCBI,Bethesda,MD,USA)的核苷酸序列数据库的注册号g40486得到。相关参考中描述的csp1基因可被用于本发明。来自遗传密码的简并或功能性中性有义突变的csp1基因的等位基因也可被使用。
弱化作用可通过降低或抑制csp1基因的表达或基因产物的催化特性来完成。两种方法可任选地联合使用。
基因表达的降低可通过适当地控制培养或通过基因表达信号结构的遗传修饰(突变)来进行。基因表达的信号结构是,例如,阻遏基因,激活基因,操纵子,启动子,弱化子,核糖体结合位点,起始密码子以及终止子。本领域的技术人员将在下述发现相关信息例如在专利WO 96/15246lBoyd & Murphy(细菌学杂志170:5949(1988))V,oskuil & Chambliss(核酸研究26:3548(1998)),Jensen &Hammer(生物技术与生物工程58:191(1998)),Patek等。(微生物学142:1297(1996))以及公知的遗传学和分子生物学的教材上,例如Knippers的教材(″Molekulare Genetik″,第6版,Georg ThiemeVerlag,Stuttgart,德国,1995)或Winnacker的教材(″基因与克隆″,VCH Verlagsgesellschaft,Weinheim,德国,1990)。
从现有技术中可知产生酶蛋白催化特性的变化或减少的突变;可被提及的例子有Qiu与Goodman的论文(生物化学杂志272:8611-8617(1997)),Sugimoto等(生物科学生物技术与生物化学61:1760-1762(199-7))以及Mockel(″Die Threonindehydratase ausCorynebacterium glutamicum:Aufhebuflg der allosterischen Regulationund Struktur des Enzyms″,Berichte des Forschungszentrums Julichs,Jul-2906,155N09442952,Julich,德国,1994)。摘要说明可以在已知的遗传学和分子生物学教材中找到,例如Hagemann的教材(″AllgemeineGenetik″,Gustav Fischer Verlag,Stuttgart,1986)。
突变可以为碱基转换,易位,***以及删除。依照氨基酸变化对酶活性的影响,突变被分为错义突变或无义突变。基因中至少一个碱基的***或删除产生了移码突变,结果不正确的氨基酸被***或翻译被提前终止。两个或更多密码子的删除结果导致酶活性被完全破坏。产生这种突变的方法为已知的技术并且可在遗传学和分子生物学的教材中找到,例如在Knippers的教材(″Molekulare Genetik″,6th edition,Georg Thieme Verlag,Stuttgart,德国,1995),Winnacker的(″Ge-ne und Klone″,VCH Verlagsgesellschaft,Weinheim,德国,1990)或Hagemann的(″Allgemeine Genetik″,Gustav Fischer Verlag,Stuttgart,1986)。
突变的csp1基因的一个例子为包含在质粒pK18mobsacBAcspl(图1)中的/△csp1等位基因。△csp1等位基因仅含有csp1基因的5′末端与3′末端的序列;编码区1690bp长的一段序列被缺失(删除),此△csp1等位基因可通过整合诱变被掺入到棒状细菌中。上述不能在谷氨酸棒杆菌中复制的质粒pK18mobsacBAcsp1,被用于这种目的。在转化以及通过实现整合的第一次“交换”和实现csp1基因删除的第二次“交换”的同源重组之后,△csp缺失掺入并且在特定的菌株中获得了功能完全的丧失。
与整合诱变相关的方法和说明可以找到,例如在Schwarzer与Puhler(生物技术9,84-87(1991))或Peters-Wendisch等(应用微生物学144,915-927(1998))。
一个携带弱化的csp1基因的棒状细菌氨基酸生产菌株的例子为赖氨酸生产菌谷氨酸棒杆菌R1674△csp1。
除了弱化csp1基因外,扩增一个或多个特定生物合成途径的,糖酵解的,回补代谢的,柠檬酸循环的或氨基酸输出的酶有益于L-氨基酸,特别是L-赖氨酸的生产。
因此,例如,对于L-赖氨酸生产,
·dapA基因(EP-B 0 197 335),其编码二氢-2,6-吡啶二羧酸合酶,可同时被过量表达,和/或
·gap基因,其编码甘油醛-3-磷酸脱氢酶(Eikmanns(1992),细菌学杂志174:6076-6086),可同时被过量表达,或
·pyc基因(Eikmarms(1992),细菌学杂志174:6076-6086),其编码丙酮酸羧化酶,可同时被过量表达,或
·mqo基因(Molenaar等人,欧洲生物化学杂志254,395-403(1998)),其编码苹果酸:醌氧化还原酶,可同时被过量表达,或
·lysE基因(DE-A-195 48 222),其编码赖氨酸输出蛋白,可同时被过量表达。
除了csp1基因外,同时弱化下列基因,有益于L-氨基酸,特别是L-赖氨酸的生产,
·pck基因(DE 199 50 409.1,DSM 13047),其编码磷酸烯醇丙酮酸羧激酶,和/或
·pgi基因(US 09/396,478,DSM 12969),其编码葡萄糖-6-磷酸异构酶。
最后,除了弱化csp1基因外,抑制不需要的次级反应,有益于L-氨基酸,特别是L-赖氨酸的生产,(Nakayama:″氨基酸生产微生物的培养″,在:微生物产物的过量产生,Krumphanzl,Sikyta,Vanek-(eds.),学院出版社,伦敦,英联邦,1982)。
所用的培养基必须充分地满足特定菌株的需求。不同微生物的培养基被描述在美国细菌学协会的“普通细菌学方法手册”中(华盛顿D.C.,美国,1981)。所用的碳源为糖与碳水化合物,例如葡萄糖,蔗糖,乳糖,果糖,麦芽糖,糖蜜,淀粉以及纤维素,油和脂肪,例如大豆油,向日葵油,花生油和椰子油,例如,脂肪酸,如软脂酸,硬脂酸,以及亚油酸,例如醇,如甘油和乙醇,例如有机酸,如乙酸。这些物质可被单独的或混合使用。所用氮源包括含氮的有机物,例如蛋白胨,酵母提取物,肉提取物,麦芽提取物,谷浸液,豆饼粉以及尿素或无机化合物,例如硫酸铵,氯化铵,磷酸铵,碳酸铵以及硝酸铵。氮源可被单独的或混合使用。所用磷源为磷酸,磷酸二氢钾或磷酸氢二钾或相应的含钠的盐。培养基另外还含有金属的盐,例如硫酸镁,或硫酸铁,其是生长所必需的。最后,除了上述的物质外,必要的生长促进物质例如氨基酸和维生素也可使用。此外合适的前体可被加到培养基中。所述的培养物质可被一次性加到培养基中或在培养过程的适当的时候加到培养基中。
碱性化合物,例如氢氧化钠,氢氧化钾,氨或氨水,或酸性化合物,例如磷酸或硫酸,可适当的用于调节培养基的pH。可以通过使用止泡剂例如脂肪酸聚乙二醇酯控制起泡。质粒稳定性可以通过加合适的选择性作用物质到培养基中来维持,例如抗生素。氧或含氧的混合气体例如空气被引到培养物中来维持需氧环境。培养温度通常为从20℃到45℃且优选从25℃到40℃。培养物一直培养到所希望产物的最大量的形成。此目标通常在10到160小时内实现。
检测L-氨基酸的方法是现有技术中已知的。分析可以通过Spackman等描述的阴离子交换层析法然后茚三酮衍生来进行(分析化学,30,(1958),1190),或通过Lindroth等人描述的反相HPLC(分析化学(1979)51:1167-1174)来进行。
下面的微生物已依据布达佩斯协议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S17-1/pK18mobsacB△csp1,保藏号DSM13048
本发明通过下面的实施例被详细的阐明。
实施例1
用于csp1基因的缺失诱变的缺失载体的构建
通过使用Eikmanns等人所述的方法从菌株ATCC 13032中分离染色体DNA(微生物学140:1817 1828(1994))。谷氨酸棒杆菌的csp1基因的核苷酸序列可从国家生物技术信息中心(NCBI,Bethesda,MD,USA)的核苷酸序列数据库的注册号g40486得到。在已知序列的基础上,下面的寡核苷酸被选出用于聚合酶链反应:
csp1-10:
5′GAT CTA G(GA TC)C CGA TGA GCG CGT CCA TGT GT3′
csp1-11:
5′GAT CTA G(GA TC)C TCG ACC TTG CGG TGC TGC TT3′
csp1-del:
5′GGA ATA CGT AGC CAC CTT CGG TCC CGA AAG TTCCCC GCT T3′
所用的引物由MWG生物技术公司(Ebersberg,德国)合成且PCR反应是通过Karreman的标准PCR(生物技术24:736-742,1-998)方法利用来自Boehringer的Pwo聚合酶来进行的。引物csp1-10和csp1-11各含有一个BamHI限制酶的***限制位点,此位点显示在上述的括号中。一个大约0.9kb大小的DNA片段,其携带csp1基因1690bp的缺失,借助于聚合酶链反应被分离。
扩增的DNA片段用限制性酶BamHI酶切并在琼脂糖凝胶(0.8%)上纯化。质粒pK18mobsacB(Jager等,细菌学杂志,1:784-791(1992))也用限制性酶BamHI酶切。质粒pK18mobsacB以及PCR片段被连接。然后将连接混合物电穿孔(Hanahan,DNA克隆应用方法。Vol.1.IRL-出版社,牛津,华盛顿DC,USA,1985)进E.coli菌株SlT-1(Simon等,1993,Bio/Technology 1:784-791)。通过将转化混合物涂布在LB琼脂上,携带质粒细胞被挑选出来(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,N.Y.,1989),LB琼脂用25mg/l的卡那霉素补充。质粒DNA通过来自Qiagen的QlAprep Spin Miniprep试剂盒从转化子中分离并通过限制性BamHI酶限制接着琼脂糖凝胶(0-8%)电泳证实。质粒被命名为pK18mobsacB△csp1。菌株被命名为大肠杆菌S17-1/pK18mobsacB△csp1,其保藏在德意志微生物保藏中心(DSMZ,不伦瑞克,德国),保藏号为DSM13048。
实施例2
csp1基因的缺失诱变导入谷氨酸棒杆菌野生型R167
将实施例1中的命名为pK18mobsacB△csp1的载体通过Tauch等人的电穿孔法(FEMS Microbiological Letters,123:343-347(1994))被电穿孔进入谷氨酸棒杆菌R167(Liebl等。(1989)FEMS MicrobiologicalLetters 65:299-304)。菌株R167是一个限制性缺陷的谷氨酸棒杆菌野生型菌株。载体pK18mobsacB△csp1在谷氨酸棒杆菌中不能独立地复制并且如果被整合到染色体中其保留在细胞中。带有被整合到染色体中的pK18mobsacB△csp1的克隆通过在LB琼脂上涂布电穿孔混合物而筛选(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,N.Y.,1989),其中的LB琼脂补充了15mg/l的卡那霉素。生长的克隆被挑到含25mg/l卡那霉素的LB琼脂上并33℃培养16小时。为了完成质粒及csp1基因的完整染色体拷贝的的切除,克隆然后在含有10%蔗糖的LB琼脂上培养。质粒pK18mobsacB含有sacB基因的一个拷贝,其转化蔗糖为对谷氨酸棒杆菌有毒的果聚糖生成酶。因此,在带有蔗糖的LB琼脂上生长的克隆为那些整合的pK18mobsacB△csp1被切除的克隆。质粒的切除可能伴随着csp1基因的完整染色体拷贝或携带内部缺失的不完整拷贝的切除。为了证实csp1的不完整拷贝遗留在染色体中,质粒pK18mobsacB△csp1片段利用来自Boehringer的Dig杂交试剂盒通过Boehringer Mannheim GmbH(Mannheim,德国,1993)的与″用于滤膜杂交的DIG***用户指南″相应的方法被标记。潜在的缺失突变体的染色体DNA通过Eikmanns等人的(微生物学140:1817-1828(1994))方法分离并用限制性酶EcoRⅠ酶切。生成的片段琼脂糖凝胶电泳分离并在68℃通过Dig-杂交试剂盒杂交。两种大约6500bp和大约4000bp的杂交片段从对照菌株中获得,然而从突变株中获得的杂交片段为大约6500bp和大约3200bp。其因此显示出菌株R167丢失了csp1基因的完整拷贝并且现在仅具有带有大约1690bp缺失的不完整拷贝。此菌株被称为谷氨酸棒杆菌R167△csp1。
实施例3
赖氨酸的生产
实施例2中获得的谷氨酸棒杆菌菌株DSM167△csp1在适于产生赖氨酸的营养培养基上培养且培养物上清液的赖氨酸含量被监测。
为了达到此目的,菌株最初在33℃在琼脂平板上培养33小时。从这种琼脂平板培养物开始接种预培养物(100ml锥形瓶中的10ml培养基)。完全培养基CgⅢ被用作此预培养物的培养基。预培养物33℃在摇床上240rpm振荡培养48小时。主培养基从此预培养物接种,从而主培养基的起始OD(660nm)是0.1OD。MM培养基用作主培养基。
MM培养基
CSL(玉米浸液) 5g/l
MOPS 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(无菌过滤) 0.3mg/l
硫胺素·HCl(无菌过滤) 0.2mg/l
CaCO3 25g/l
CSL,MOPS以及盐溶液用氨水调节到pH 7并高压灭菌。然后加入无菌底物和维生素溶液及干热灭菌的CaCO3。
100ml带有挡流器的锥形瓶中装10ml体积的培养液进行培养。培养在33℃和80%的湿度下进行。
48小时后,利用Biomek 1000(Beckmann Instruments GmbH,慕尼黑)在660nm波长处测定OD值。形成的赖氨酸的量利用Eppendorf-BioTronik(汉堡,德国)的氨基酸分析仪通过离子交换层析和带有茚三酮检测的柱后衍生来测定。
表1显示了实验的结果。
表1
菌株 | OD(660) | 赖氨酸HCl |
R167 | 13.8 | 0.00 |
R167△csp1 | 12.6 | 0.99 |
有如下附图:
图1:质粒pK18mobsacB△csp1图谱
缩写与命名定义如下。所述的长度应被认为是大约的。
sacB:sacB基因
oriV:复制起点V
KmR:卡那霉素抗性
BamHⅠ:限制性酶BamHⅠ的限制位点
csp1′:带有内部1690bp缺失的csp1基因的不完整片段
Claims (9)
1.生产L-氨基酸,特别是L-赖氨酸的方法,其特征在于进行下述的步骤,
a)发酵产生所需L-氨基酸的细菌,细菌中至少csp1基因被弱化,
b)在培养基中或细菌细胞中积累所需的L-氨基酸,以及
c)分离L-氨基酸。
2.如权利要求1所述的方法,特征在于所用细菌中所需L-氨基酸的生物合成途径的其他基因被额外扩增。
3.如权利要求1所述的方法,特征在于所用细菌中降低所需L-氨基酸的形成的代谢途径至少部分地被抑制。
4.如权利要求1所述的方法,特征在于编码csp1基因的多核苷酸的表达被降低。
5.如权利要求1所述的方法,特征在于多核苷酸csp1编码的多肽(酶蛋白)的催化特性被降低。
6.如权利要求1所述的方法,特征在于用通过载体pK18mobsacB△csp1的整合诱变的方法获得弱化,其中载体pK18mobsacB△csp1显示于图1并保藏在编号DSM 13048的大肠杆菌中。
7.如权利要求1所述的方法,特征在于L-赖氨酸通过发酵如下细菌产生,该细菌中选自如下一组的一或多个基因同时过量表达或扩增:
7.1 dapA基因,其编码-二氢-2,6-吡啶二羧酸合成酶,
7.2 DNA片段,其赋予S-(2-氨乙基)半胱氨酸抗性,
7.3 pyc基因,其编码丙酮酸羧化酶,
7.4 dapD基因,其编码四氢-2,6-吡啶二羧酸琥珀酰化酶,
7.5 dapE基因,其编码琥珀酰二氨基庚二酸脱琥珀酰化酶,
7.6 gap基因,其编码甘油醛-3-磷酸脱氢酶,
7.7 mqo基因,其编码苹果酸:醌氧化还原酶,
7.8 lysE基因,其编码赖氨酸输出蛋白。
8.如权利要求1所述的方法,特征在于L-赖氨酸通过发酵如下细菌产生,所述细菌中的选自如下一组的一或多个基因被同时弱化:
8.1 pck基因,其编码磷酸烯醇丙酮酸羧激酶,
8.2 pgi基因,其编码葡萄糖-6-磷酸异构酶。
9.如前述一个或多个权利要求所述的方法,特征在于使用谷氨酸棒杆菌。
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CN111286520A (zh) * | 2018-12-10 | 2020-06-16 | 上海凯赛生物技术股份有限公司 | 用于发酵生产l-赖氨酸的重组dna、菌株及其应用 |
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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 |
JP4152320B2 (ja) * | 2001-09-28 | 2008-09-17 | 協和醗酵工業株式会社 | アミノ酸の製造法 |
KR101565770B1 (ko) | 2013-12-13 | 2015-11-04 | 씨제이제일제당 주식회사 | L-라이신 생산능이 향상된 코리네박테리움 속 미생물 및 이를 이용한 l-라이신을 생산하는 방법 |
JP2015156844A (ja) * | 2014-02-25 | 2015-09-03 | 花王株式会社 | 枯草菌変異株及びそれを用いたジピコリン酸の製造方法 |
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CN111286520A (zh) * | 2018-12-10 | 2020-06-16 | 上海凯赛生物技术股份有限公司 | 用于发酵生产l-赖氨酸的重组dna、菌株及其应用 |
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