CN1267734A - 用棒状细菌经发酵生产l－氨基酸的方法 - Google Patents
用棒状细菌经发酵生产l－氨基酸的方法 Download PDFInfo
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Abstract
本发明涉及用棒状细菌通过发酵生产L－氨基酸的方法,其中所述细菌中mqo基因被扩增。
Description
本发明提供一种用其中mqo基因被扩增的棒状细菌(Coryneformbacteria)经发酵生产L-氨基酸，尤其是L-赖氨酸的方法。
L-氨基酸，尤其是L-赖氨酸用于动物饲料，人的内服药及制药工业。
已知那些氨基酸由棒状细菌，尤其是谷氨酸棒杆菌的发酵菌株生产。由于其非常之重要性，持续进行一些研究工作以改进生产方法。改进的方法可涉及与发酵过程相关的措施，如搅拌及氧补给，或营养基的组分如发酵期糖的浓度，或如经离子交换层析加工产物形式，或微生物自身的内在性质。
为改进那些微生物的性质，可使用诱变，选择及突变体选择方法。在此方式中，获得对抗代谢物如赖氨酸同源物S-[2-氨乙基]-半胱氨酸有抗性的菌株，或在调节方面重要氨基酸的营养缺陷体，并产生L-氨基酸。
几年来，通过扩增氨基酸生物合成的各个基因，并研究对L-氨基酸生产的影响，重组DNA技术已被用于改进谷氨酸棒杆菌的L-氨基酸生产菌株。此方面的文章见于Kinoshita(“Glutamic AcidBacteria”，in Biology of Industrial Microorganisms，Demain andSolomon(eds。)，Benjamin Cummings，London，UK，1985，115-142)，Hilliger(BioTec 2，40-44(1991))，Eggeling(Amino Acids 6，261-272(1994)，Jetten and Sinskey(Critical Reviews in Biotechnology 15，73-103(1995)和Sahm et al.(Annuals of the New York Academy ofScience 782，25-39(1996)。
本发明之目的是寻求适当新措施，以通过发酵改进L-氨基酸尤其是L-赖氨酸的生产。
L-氨基酸尤其L-赖氨酸用于动物的饲料，人内服药及制药工业。因而，寻求适当的改进方法生产那些化合物令人瞩目。
应了解后文提及的任何L-赖氨酸或赖氨酸不只意味着碱，也指盐，如赖氨酸单盐酸盐或赖氨酸硫酸盐。
本发明提供一种用棒状细菌通过发酵生产L-氨基酸，尤其是L-赖氨酸的方法，特别是所述棒状细菌已能生产所需氨基酸，且其中编码苹果酸：醌氧化还原酶的核酸序列被扩增，特别是被过量表达。
优选的实施方案见于权利要求书。
在此，术语“扩增”指通过应用强启动子或应用编码具有高活性的相应酶的基因或任选的这些措施的结合增加基因的拷贝数，从而增加微生物中由相应DNA编码的一或多种酶的细胞内活性。
本发明提供的微生物可从葡萄糖、蔗糖、乳糖、果糖、麦芽糖、糖蜜、淀粉、纤维素中，或从甘油和乙醇中生产L-氨基酸、特别是L-赖氨酸。它们是棒状细菌特别是棒杆菌属的代表。在棒杆菌属中，特别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。
适当的棒杆菌属尤其谷氨酸棒杆菌的菌株是下列的已知野生型菌株：
谷氨酸棒杆菌 ATTC13032
醋谷氨酸棒杆菌 ATTC15806
嗜乙酰乙酸棒杆菌 ATTC13870
Corynebacterium thermoaminogenes FERM BP-1539
黄色短杆菌 ATCC14067
乳发酵短杆菌 ATCC13869和
扩展短杆菌 ATCC14020及产生L-氨基酸特别是产生L-赖氨酸的突变株以及由其产生的菌株如：
谷氨酸棒杆菌 FERM-P 1709
黄色短杆菌 FERM-P1708
乳发酵短杆菌 FERM-P1712
黄色短杆菌 FERM 6463及
黄色短杆菌 FERM-P6464。
本发明人已发现棒状细菌菌在苹果酸：醌氧化还原酶的过量表达后，以一种改进方式生产L-氨基酸特别是L-赖氨酸。mqo基因编码苹果酸：醌氧化还原酶(EC 1.1.99.16)，该酶通过将电子转移至泛醌-1，催化苹果酸为草酰乙酸(Molenaar et al.，European Journal ofBiochemistry 254，395-403(1998))。谷氨酸棒杆菌mqo基因的核苷酸序列已被Molenaar等测定(European Journal of Biochemistry 254，395-403(1998))，且通常可在国立生物技术信息中心(NCBI，Behesda，MD，USA)的数据库中获得，登记号AJ 22 4946。
根据本发明，可使用Molenaar等(European Journal of biochemistry254，395-403(1998))所述的谷氨酸棒杆菌的mqo基因。也可使用得自遗传密码的简并或经功能一中性有义突变产生的mqo基因的等位基因。
为达到过量表达，可提高相应基因的拷贝数，或使位于结构基因前的启动子及调节区突变。插入结构基因前的表达盒具有相同作用。利用可诱导启动子还可在发酵生产L-赖氨酸过程中增加表达。表达也可通过延长m-RNA的寿命的措施加以改进。酶的活性也可通过防止酶蛋白的降解而提高。基因或基因构建体可以不同拷贝数存在于质粒中，或在染色体中整合及扩增。或者，基因的过量表达也可通过改变培养基组分而达到，在此方式中要进行培养。
本领域技术人员将在下列文献中得到指导：Martin等(Bio/Technology 5，137-146(1987))，Guerrero等(基因138，35-41(1994))，Tsuchiya及Mirinaga(Bio/Technology 6，428-430(1998))，Eikmanns等(基因102，93-98(1991))，欧洲专利说明书EP-B 0 472869，美国专利4,601,893，Schwarzer及Puhler(Bio/Technology 9，84-87(1991))，Reinscheid等(Applied and Environmental Microbiology 60，126-132(1994))，LaBarre等(Journal of Bacteriology 175，1001-1007(1993))，专利申请WO96/15246，Malumbers等(基因134，15-24(1993))，Jensen及Hammer(Biotechnology and Bioengineering 58，191-195(1998))，Makrides(Microbiological Reviews 60：512-538(1996))以及已知遗传及分子生物学的教科书。
辅助苹果酸：醌氧化还原酶过量表达的质粒例如pRM17(Molenaar等，1998，欧洲生物化学杂志254，395-403)。质粒pRM17基于穿梭载体pJC1，其见于Cremer等所述(Molecular and GeneralGenetics 220，478-480)。
另外，对于L-氨基酸的生产可能有利的是过量表达相应生物合成途径的一或多种酶以及苹果酸：醌氧化还原酶。因此如在L-赖氨酸的生产中，
编码二氢吡啶二羧酸合酶的dapA基因可同时被过量表达(EP-B0 197 335)，或
·介导S-(2-氨乙基)-半胱氨酸抗性的DNA片段同时被扩增(EP-A 0 088 166)。
对于L-氨基酸的生产也可能有利的是除了苹果酸：醌氧化还原酶的过量表达之外，阻止非所需二级反应(Nakayama：“生产氨基酸的微生物的培育”，微生物产物的过量表达，Krumphanzl，Sikyta，Vanek(eds.)，Academic Press，London，UK，1982)。
根据本发明产生的微生物可在分批工艺中或补料分批或重复补料分批方法中连续或非连续培养，以生产L-氨基酸。熟知的培养法的综述见于Chemiel的教科书(Bioprozesstechnik 1.Einfuhrung in dieBioverfahrenstechnik(Gustav Fischer Verlag，Stuttgart，1991)，或Storhas的教科书(Bioreaktoren and periphere Einrichtungen(Vieweg Verlag，Braunschweig/-Wiesbaden，1994))。
所用培养基必须以适当方式满足所研究菌株的要求。各种微生物的培养基的阐述包含于美国细菌学会的“普通细菌学方法手册”(Washington D.C.，USA，1981)内。用作碳源的糖及碳水化合物如：葡萄糖，蔗糖，乳糖，果糖，麦芽糖，糖蜜，淀粉及纤维素，油及脂肪如大豆油，葵花籽油，花生油，及椰子油，脂肪酸如棕榈酸，硬脂酸及亚油酸，醇如甘油及乙醇，及有机酸如乙酸。这些物质可单独或以混合物形式应用。用作氮源的有机含氮化合物如胨，酵母提取物，肉膏，麦芽提取物，玉米浆，大豆粉及尿素，或无机化合物如硫酸铵，氯化铵，磷酸铵，碳酸铵及硝酸铵。氮源可单独或以混合物方式应用。用作磷源的如磷酸二氢钾或磷酸氢二钾或相应的含钠盐。培养基还必须含有金属盐如硫酸镁或硫酸铁，它们是生长所必须的。最后，除了上述物质外还可应用生长必需氨基酸及维生素。另外，在培养基中可加入适当的预时期。所述物质可以单批形式加入培养基中，或在培养期间以适当方式补料。
为调节培养pH值，可以适当方式应用碱性化合物如氢氧化钠，氢氧化钾，氨，或酸性化合物如磷酸或硫酸。消泡剂，如脂肪酸聚乙二醇酯，可以用来控制起泡。合适的选择性作用物质，如抗生素，可以添加至培养基中，以保持质粒稳定性。氧气或含氧的气体混合物，如空气，引入培养基，以保持需氧条件。培养温度通常从20℃至45℃，优选地为25℃至40℃。培养继续至形成最大量的所需L-氨基酸。这一目标通常在10小时至160小时之内实现。
如Spackman等所述(Analytical Chemistry，30，(1958)，1190)，通过离子交换层析随后进行茚三酮衍生化，进行L-氨基酸分析。
谷氨酸棒杆菌菌株DM22/pRM17已经按照布达佩斯条约保藏在德意志微生物保藏中心(DSMZ，Braunschweig，德国)，保藏号为DSM12711。
本发明的方法通过应用棒状细菌用于发酵生产L-氨基酸，特别是L-天冬氨酸，L-天冬酰胺，L-高丝氨酸，L-苏氨酸，L-异亮氨酸及L-蛋氨酸，尤其是L-赖氨酸的生产。
本发明通过以下实施例得以更详细阐述。
为此，应用生产L-赖氨酸的谷氨酸棒杆菌菌株DSM5715(EP-B-0435132)进行实验，其中权利要求的方法的优点将更清晰。
实施例1
含有扩增的苹果酸：醌氧化还原酶的L-赖氨酸生产菌的产生
将菌株DSM5715用质粒pRM17(Molenaar等，1998，欧洲生物化学杂志254(395-403))，如Liebl等所述(FEMS MicrobiologyLetters 65，299-304(1989))进行转化。在已加入25mg/ml卡那霉素的LBHIS琼脂上进行转化体的选择。LBHIS琼脂是由在LB培养基(Sambrook等，分子克隆实验手册(1989)，冷泉港实验室)中加入37g/l来自Merck(Darmstadt，德国)的脑心肉汤，0.5M山梨醇及15g/l琼脂组成的。以此方式形成菌株DSM5715/pRM17。以相同方式产生菌株DSM5715/pJC1。
实施例2
L-赖氨酸的生产
首先将菌株DSM5715/pRM17及DSM5715/pJC1在已加入卡那霉素(25mg/ml)的脑心琼脂上在33℃温育24小时。为在液体培养基中培养，应用已另加入卡那霉素(25mg/ml)的CgIII培养基(Kase& Nakayama，Agricultural and Biology Chemistry 36(9)1611-1621(1972))。将包含在具有4个缓冲板的100ml Erlenmeyer烧瓶中的10ml培养基用菌株的接种物接种，并将培养物在240rpm，30℃温育16小时，随后此培养物用于进一步预培养。
用于生产或试验的培养基是已另加入卡那霉素(25mg/ml)的MM培养基。
在用菌株DSM5715的方法中，相应的培养基不含卡那霉素。
MM培养基的组成及制备如下：
玉米浆(CSL) 5g/L
3-吗啉代丙磺酸(MOPS) 20g/L
葡萄糖(单独高压灭菌的) 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 0.3mg/l(经过滤灭菌)
盐酸维生素B1 0.2mg/L(经过滤灭菌)
CaCO3 25g/L
亮氨酸 0.1g/L
用氨水将CSL，MOPS及盐溶液调节至pH为7并高压灭菌，然后加入灭菌基质及维生素溶液及干燥高压灭菌的CaCO3。
在已装入10mL上述生产培养基的具有缓冲板的100mlErlenmeyen烧瓶中进行培养。用预培养物如此接种此培养基，以使起始光密度为0.1。在33℃及80％相对湿度进行培养。
在温育72小时后，测定培养物悬浮液的光密度及已生成的L-赖氨酸的浓度。用Dr.Lange的LP2W光度计(Berlin，Germany)在660nm测定波长测定光密度。用Eppendorf-BioTronik(Hamburg，Germany)氨基酸分析仪通过离子交换层析及与茚三酮检测的柱后反应测定L-赖氨酸，试验结果示于表1：
表1：
| 菌株 | OD | L-赖氨酸(g/L) |
| DSM5715 | 10.1 | 16.4 |
| DSM5715/pJC1 | 9.9 | 16.5 |
| DSM5715/pRM17 | 10.2 | 17.8 |
实施例3
含有扩增的苹果酸：醌氧化还原酶的苏氨酸生产菌的产生
将质粒pRM17(Molenaar等，1998，欧洲生物化学杂志254(395-403))用如Tauch等所述的电穿孔方法(FEMS MicrobiologyLetters 123：343-347(1994))电穿孔入谷氨酸棒杆菌菌株DSM5399。菌株DSM5399为苏氨酸生产菌，其描述于EP-B-0358940。将电穿孔沉积物涂布在已加入25mg/ml卡那霉素的LB琼脂上进行转化体的选择(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)。以此方式形成菌株DSM5399/pRM17。
实施例4
苏氨酸的生产
将实施例3中得到的谷氨酸棒杆菌菌株DSM5399/pRM17在适于生产苏氨酸的类似培养基中培养，并随后测定苏氨酸含量。
首先将菌株在已加入相应的抗生素的琼脂平板(含卡那霉素(25mg/ml)的脑心琼脂)上在33℃温育24小时。以琼脂板培养物为预培养接种物起始接种(100ml Erlenmeyer烧瓶中的10ml培养基)。所述预培养的培养基为CgIII培养基(Kase & Nakayama，Agriculturaland Biology Chemistry 36(9)1611-1621(1972))，其中已另加入卡那霉素(25mg/ml)。将预培养物在摇床上于240rpm，33℃温育24小时，随后此预培养物用作主培养接种物接种培养基，以使主培养物起始光密度(660nm)为0.1。主培养所用培养基为MM-苏氨酸培养基。
MM-苏氨酸培养基：
玉米浆(CSL) 5g/L
3-吗啉代丙磺酸(MOPS) 20g/L
葡萄糖(单独高压灭菌的) 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(经过滤灭菌) 0.3mg/l
盐酸维生素B1(经过滤灭菌) 0.2mg/L
CaCO3 25g/L
用氨水将CSL，MOPS及盐溶液调节至pH为7并高压灭菌，然后加入灭菌基质及维生素溶液及干燥高压灭菌的CaCO3。
在已装入10mL上述培养基的具有缓冲板的100ml Erlenmeyen烧瓶中进行培养。加入卡那霉素(25mg/L)。在33℃及80％相对湿度进行培养。
在温育48小时后，用Biomek 1000(Beckman Instruments GmbH，Munich)在660nm测定波长测定光密度。用Eppendorf-BioTronik(Hamburg，Germany)氨基酸分析仪通过离子交换层析及与茚三酮检测的柱后反应测定苏氨酸浓度。
试验结果示于表2。
表2
| 菌株 | OD(660) | L-苏氨酸(g/L) |
| DSM5399/pRM17 | 13.1 | 0.61 |
| DSM5399 | 13.9 | 0.43 |
Claims (10)
1、通过棒状细菌的发酵生产L-氨基酸的方法，其中使用编码苹果酸：醌氧化还原酶的核苷酸序列被扩增、尤其是被过量表达的细菌。
2、权利要求1的方法，其中所用细菌中所需L-氨基酸的生物合成途径的其它基因也被扩增。
3、权利要求1的方法，其中所用细菌中至少一些降低所需L-氨基酸形成的代谢途径被除去。
4、根据前述一或多项权利要求的方法，其中使用了经质粒载体转化的菌株，且质粒载体携带编码苹果酸：醌氧化还原酶的核苷酸序列。
5、如权利要求4的方法，其中使用的是经质粒载体pRM17转化的菌株，所说的质粒载体以在谷氨酸棒杆菌中的形式保藏，保藏号为DSM12711。
6、如前述一或多项权利要求的方法，其中使用生产L-天冬氨酸，L-天冬酰胺，L-高丝氨酸，L-苏氨酸，L-异亮氨酸或L-蛋氨酸的棒状细菌。
7、如权利要求1-5的一或多项权利要求的方法，其中使用生产L-赖氨酸的棒状细菌。
8、如权利要求7的方法，其中编码二氢吡啶二羧酸合酶的dapA基因同时被过量表达。
9、如权利要求7的方法，其中介导S-(2-氨乙基)-半胱氨酸抗性的DNA片段同时被扩增。
10、如前述一或多项权利要求所述的通过发酵生产L-氨基酸的方法，其中进行以下步骤：
a)发酵生产所需L-氨基酸的细菌，所说的细菌中至少苹果酸：醌氧化还原酶基因被扩增，
b)在培养基中或在细菌细胞中积累L-氨基酸，和
c)分离所产生的L-氨基酸。
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| DE19912384A DE19912384A1 (de) | 1999-03-19 | 1999-03-19 | Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
| DE19912384.5 | 1999-03-19 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| US6630332B2 (en) | 2000-07-18 | 2003-10-07 | Degussa Ag | Process for the fermentative preparation of L-threonine |
| EP1303590A1 (en) * | 2000-07-18 | 2003-04-23 | Degussa AG | Process for the fermentative preparation of l-threonine |
| EP1239041B1 (en) * | 2001-02-13 | 2006-04-19 | Ajinomoto Co., Inc. | Method for producing L-amino acid using bacteria belonging to the genus Escherichia |
| US7049106B2 (en) | 2001-04-10 | 2006-05-23 | Degussa Ag | Process for the production of L-amino acids by fermentation using coryneform bacteria with an attenuated mqo gene |
| DE10117816A1 (de) * | 2001-04-10 | 2002-10-17 | Degussa | Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
| WO2003029457A1 (fr) * | 2001-09-28 | 2003-04-10 | Kyowa Hakko Kogyo Co., Ltd. | Procede de production d'acide amine |
| DE10254074A1 (de) * | 2002-11-19 | 2004-06-17 | Forschungszentrum Jülich GmbH | Verfahren zur mikrobiellen Herstellung von Stoffwechselprodukten |
| 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 |
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| JPS58126789A (ja) * | 1981-12-29 | 1983-07-28 | Kyowa Hakko Kogyo Co Ltd | レースレオニンの製造法 |
| JPH0655149B2 (ja) * | 1985-03-12 | 1994-07-27 | 協和醗酵工業株式会社 | Ｌ―リジンの製造法 |
| GB2223754B (en) * | 1988-09-12 | 1992-07-22 | Degussa | Dna encoding phosphoenolpyruvate carboxylase |
| DE3943117A1 (de) * | 1989-12-27 | 1991-07-04 | Forschungszentrum Juelich Gmbh | Verfahren zur fermentativen herstellung von aminosaeure, insbesondere l-lysin, dafuer geeignete mikroorganismen und rekombinante dna |
| AU705550B2 (en) * | 1995-06-07 | 1999-05-27 | Ajinomoto Co., Inc. | Method of producing L-lysine |
| DE19907347A1 (de) * | 1999-02-20 | 2000-08-24 | Degussa | Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
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- 2000-03-16 AU AU22369/00A patent/AU2236900A/en not_active Abandoned
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Cited By (4)
| 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 |
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| Publication number | Publication date |
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| DE19912384A1 (de) | 2000-09-21 |
| ZA200001354B (en) | 2000-10-20 |
| KR20000076897A (ko) | 2000-12-26 |
| EP1038969A3 (de) | 2000-12-27 |
| AU2236900A (en) | 2000-09-21 |
| BR0001342A (pt) | 2001-05-02 |
| HUP0001176A2 (hu) | 2002-04-29 |
| ID29659A (id) | 2001-09-20 |
| EP1038969A2 (de) | 2000-09-27 |
| HU0001176D0 (en) | 2000-05-28 |
| CA2301407A1 (en) | 2000-09-19 |
| JP2000270888A (ja) | 2000-10-03 |
| SK3742000A3 (en) | 2000-10-09 |
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