CN1275622A - 用棒状细菌发酵制备l－氨基酸的方法 - Google Patents
用棒状细菌发酵制备l－氨基酸的方法 Download PDFInfo
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- CN1275622A CN1275622A CN00109311A CN00109311A CN1275622A CN 1275622 A CN1275622 A CN 1275622A CN 00109311 A CN00109311 A CN 00109311A CN 00109311 A CN00109311 A CN 00109311A CN 1275622 A CN1275622 A CN 1275622A
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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Abstract
本发明提供了一种利用棒状细菌发酵制备L－氨基酸的方法,在所使用的棒状细菌中,编码乙酰辅酶A羧化酶的携带生物素—羧基载体蛋白结构域和生物素—羧化酶结构域的亚基的核苷酸序列(accBC基因)被扩增,特别是过量表达。
Description
本发明提供了通过用其中accBC基因被扩增的棒状细菌发酵制备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菌株发酵生产。由于这些产物非常之重要性，一直尝试改进制备方法。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，或营养培养基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。
为改进这些微生物的性质，可使用诱变，选择及突变体选择方法。以这种方式可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(“谷氨酸细菌”，见《工业微生物学》，Demain和Solomon编辑，Benjamin Cummings，英国伦敦，1985，115-142)，Hilliger(BioTec 2，40-44(1991))，Eggeling(氨基酸6，261-272(1994))，Jetten和Sinskey(Critical Reviews inBiotechnology 15，73-103(1995))和Sahm et al.(纽约科学院年报782，25-39(1996))。
本发明的目的是提供一种新的通过发酵制备L-氨基酸，尤其是L-赖氨酸的改良方法。
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。因而提供新改进的制备这些化合物的方法通常是令人感兴趣的。
应了解后文提及的任何L-赖氨酸或赖氨酸不只意味着其本体，也指盐，如赖氨酸单盐酸盐或赖氨酸硫酸盐。
本发明提供了用棒状细菌经发酵制备L-氨基酸特别是L-赖氨酸的方法，所述棒状细菌尤其是已产生所需L-氨基酸，且其中编码乙酰辅酶A羧化酶的携带生物素—羧基载体蛋白结构域和生物素羧化酶结构域的亚基的核苷酸序列(accBC基因)是扩增的，尤其是过量表达的。
优选的实施方案见权利要求书。
在此，术语“扩增”指通过增加基因的拷贝数、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，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。这些微生物是棒状细菌成员、特别是棒杆菌属。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 ATTC 13032
醋谷氨酸棒杆菌 ATTC 15806
嗜乙酰乙酸棒杆菌 ATTC 13870
Corynebacterium thermoaminogenes FERM BP-1539
黄色短杆菌 ATCC 14067
乳发酵短杆菌 ATCC13869和
扩展短杆菌 ATCC 14020及产生L-氨基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-P 1708
乳发酵短杆菌 FERM-P 1712
黄色短杆菌 FERM-P 6463及
黄色短杆菌 FERM-P 6464。
accBC基因编码乙酰辅酶A羧化酶的携带生物素—羧基载体蛋白结构域和生物素—羧化酶结构域的亚基。谷氨酸棒杆菌的accBC基因的核苷酸序列已由Jager等测定(Archives of Microbiology166，76-82(1996))，且通常可从欧洲分子生物学实验室(EMBL，Heidelberg，德国)的数据库获得，登记号为U35023。
Jager等(Archives of Microbiology 166，76-82(1996))所述的谷氨酸棒杆菌的accBC基因可用于本发明。另外由于遗传密码的简并性或功能—中性有义突变产生的accBC基因的等位基因也可使用。
为达到过量表达，可以增加相应基因的拷贝数，或者可以突变位于编码序列上游的启动子和调节区或核糖体结合位点。掺入编码序列上游的表达盒以相同的方式起作用。在发酵生产L-赖氨酸期间，通过以诱导型启动子的方式增加表达也是可能的。也可以通过延长mRNA寿命的方法改善表达。另外也可通过阻止酶蛋白质的降解增加酶活性。基因或者基因构建体可以以可变拷贝数存在于质粒中或者整合进染色体，并扩增。另外，基因的过量表达也可以通过改变营养培养基的组成与培养技术来完成。
在这一点上，本领域技术人员将会从以下文献中得到指导：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EPS O 472 869，美国专利4,601,893，Schwarzer和Piihler(生物/技术9，84-87(1991)，Reinscheid等(应用和环境微生物学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(生物技术和生物工程58，191-195(1998))，Makrides(微生物学回顾60：512-538(1996))和已知的遗传学与分子生物学教科书。
能过量表达accBC基因的质粒的一实例是pZlaccBC(图1)，其包含在菌株MH20-22B/pZlaccBC中。质粒pZlaccBC是E.coli-谷氨酸棒杆菌穿梭载体，其携带accBC基因并基于质粒pZl(Menkel etal.，应用和环境微生物学55(3)，684-688(1989))。
另外，对L-氨基酸生产有利的是不仅accBC基因而且一或多种生物合成途径的酶的过量表达，这样，例如对于L-赖氨酸的制备，·可以同时过量表达编码二氢-2，6-吡啶二羧酸合酶的dapA基因(EP-B 0 197 335)，或·可以同时扩增赋予S-(2-氨乙基)—半胱氨酸抗性的DNA片段(EP-A 0 088 166)。
另外，为产生L-氨基酸，除了过量表达accBC基因，切断非所需副反应也是有利的(Nakayama：＂生产氨基酸的微生物的培育＂，见《微生物产物的过量产生》，Krumphanzl，Sikyta，Vanek(编者)，学院出版社，伦敦，英国，1982)。
为生产L-氨基酸，根据本发明制备的微生物可采用分批或流加方法或重复流加方法连续培养或不连续培养。已知培养方法总结在以下教科书中：Chmiel(Bioprozesstechnik 1.Einfhrung in dieBioverfahrenstechnik(Gustav Fischer Verlag，Stuttgart，1991))或Storhas(Bioreaktoren und periphere Einrichtungen(Vieweg Verlag，Braunschweig/Wiesbaden，1994))。
所使用的培养基必须完全满足特定菌株的需要。各种微生物的培养基在美国细菌学会的 “普通细菌学方法手册” (华盛顿D.C.，美国)中有描述。可以利用的碳源包括糖和碳水化合物，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，如大豆油，向日葵油，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，以及有机酸，如乙酸。这些物质可以单独使用或者混合使用。可以利用的氮源包括含氮有机化合物，如蛋白胨，酵母膏，肉膏，麦芽汁，玉米浆，大豆粉和尿素；或者无机化合物，如硫酸铵，氯化铵，磷酸铵，碳酸铵和硝酸铵。氮源可以单独使用或者混合使用。可以利用的磷源是磷酸二氢钾或者磷酸氢二钾或者相应的含钠盐。培养基必须还含有生长必需的金属盐，如硫酸镁或者硫酸铁。最后，除了以上所述的物质外，对促生长必需的物质如氨基酸和维生素也可以使用。合适的前体也可以添加至培养基中。所述的营养物质可以以单批或者在培养期间适当流加至培养基中。
碱性化合物，如氢氧化钠，氢氧化钾，氨或氨水，或者酸性化合物，如磷酸或者硫酸，用来适当地控制培养物的pH值。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。
L-氨基酸可以采用阴离子交换层析和随后的茚三酮衍生化分析，如Spackman等所描述的(分析化学，30，1190(1958))。
谷氨酸棒杆菌菌株DSM5715/pZlaccBC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DSM 12786。
本发明的方法可用于经发酵棒状细菌制备L-氨基酸，尤其是L-天冬氨酸，L-天冬酰胺、L-高丝氨酸，L-苏氨酸、L-异亮氨酸和L-甲硫氨酸。其尤其用于制备L-赖氨酸。
实施例
通过以下实施例，对本发明进一步详加例证。
为此，用L-赖氨酸生产菌株DSM5715(EP-B-0 435 132)进行实验，由此证实了本发明方法的优越性。
实施例1
构建表达质粒pZlaccBC和菌株DSM5715/pZlaccBC
为了构建表达质粒pZlaccBC，包含accBC基因的pWJ71质粒(Jager等，微生物学报(1996)166：76-82)用限制性内切酶PvuI和Nae I消化，Klenow聚合酶补平，碱性磷酸酶处理，琼脂糖凝胶法回收2.1kb的accBC基因，上述操作方法参照Sambrook等(分子克隆实验手册(1989)，冷泉港实验室)。在制备accBC基因的同时，将质粒pZ1(Menkel等，应用与环境微生物学55(3)，684-688(1989))用Sac I酶切，Klenow聚合酶补平，碱性磷酸酶处理。然后将上述方法处理的所制备的accBC基因和载体pZl连接，并用连接混合物转化菌株DSM5715，连接方法参见Libel等(FEMS微生物学通讯65，299-304(1989))。转化子使用Merck公司(Darmstadt，德国)的脑/心琼脂筛选，琼脂内预先加有50mg/l的卡那霉素，将鉴定为阳性的一个转化子命名为DSM5715/pZlaccBC。表达质粒pZlaccBC的限制性酶切图谱见图1。
实施例2
L-赖氨酸的制备
菌株DSM5715/pZlaccBC首先在含有50μg/ml卡那霉素的CgIII完全培养基(Kase和Nakayama，农业和生物化学36(9)1611-1621(1972))上预培养。为达到此目的，将10mlCgIII完全培养基加入100ml的有4个挡板的锥形瓶，接种种菌，30℃，每分钟240转培养16小时。
测定预培养物660nm下的OD值(光密度)后用以接种含10ml生产培养基的有4个挡板的100ml锥形瓶，接种后含有生产培养基的主培养物的OD值为0.1。Keilhauer等(微生物杂志175：5595-5603(1993))描述的CgXII培养基被用作生产培养基。培养基中需加入4％的葡萄糖和50mg/l的卡那霉素硫酸盐。接种后细胞的培养条件是33℃，80％的湿度，每分钟250转培养48小时。
在仅仅使用菌株DSM5715的方法中，培养基中不含卡那霉素。
最后，在660nm下测定光密度，使用Eppendorf-BioTronik公司(Hamburg，德国)的氨基酸分析仪通过离子交换层析，过柱后用水合茚三酮检测的方法来确定产生的L-赖氨酸的浓度。实验结果见表1。
表1
| 菌株 | OD | L-赖氨酸(g/l) |
| DSM 5715 | 31.4 | 7.2 |
| DSM 5715/pZlaccBC | 27.6 | 9.6 |
本说明书有以下附图：图1：质粒pZlaccBC的图谱。
Claims (10)
1.一种利用棒状细菌发酵制备L-氨基酸的方法，其中
所使用的细菌中编码accBC基因的核苷酸序列或携带该基因的核苷酸序列被扩增，特别是过量表达。
2.如权利要求1所述的方法，其中
所使用的细菌中所需L-氨基酸生物合成途径中的其它基因也被扩增。
3.如权利要求1所述的方法，其中
所使用的细菌中降低所需L-氨基酸的产生的代谢途径至少被部分关闭。
4.如前述权利要求的一项或几项所述的方法，其中
使用用质粒载体转化的菌株，该质粒载体携带编码accBC基因的核苷酸序列。
5.如权利要求4所述的方法，其中
使用用图1所示的质粒载体pZlaccBC转化的、保藏于德意志微生物保藏中心的保藏号为DSM12786的谷氨酸棒杆菌。
6.如前述权利要求的一项或几项所述的方法，其中
使用产生L-天冬氨酸，L-天冬酰胺，L-高丝氨酸，L-苏氨酸，L-异亮氨酸和L-甲硫氨酸的棒状细菌。
7.如权利要求1至5的一或多项所述的方法，其中
使用产生L-赖氨酸的棒状细菌。
8.如权利要求7所述的方法，其中
编码二氢-2，6-吡啶二羧酸合酶的dapA基因被同时过量表达。
9.如权利要求7所述的方法，其中
赋予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发酵制备L-氨基酸的方法，其中进行以下步骤：
a)发酵产生所需L-氨基酸的细菌，该细菌中至少accBC基因被扩增，
b)在培养基中或在细菌的细胞中富集所需L-氨基酸，
c)分离所产生的氨基酸。
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| US20110012309A1 (en) * | 2009-07-15 | 2011-01-20 | David Schreff | Aerodynamic sports toy, game and method of play |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56160997A (en) | 1980-05-16 | 1981-12-11 | Ajinomoto Co Inc | Preparation of l-lysine by fermenaition method |
| JPS58126789A (ja) * | 1981-12-29 | 1983-07-28 | Kyowa Hakko Kogyo Co Ltd | レースレオニンの製造法 |
| DE3823451C2 (de) | 1988-07-11 | 1997-02-20 | Forschungszentrum Juelich Gmbh | Rekombinante DNA, damit transformierte Mikrooganismen und Verwendung dieser Mikroorganismen zur Herstellung von L-Lysin mit Hilfe dieser Mikroorganismen |
| GB2223754B (en) | 1988-09-12 | 1992-07-22 | Degussa | Dna encoding phosphoenolpyruvate carboxylase |
| DE3908201A1 (de) | 1989-03-14 | 1990-09-27 | Degussa | Verfahren zur fermentativen herstellung von l-lysin |
| 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 |
-
1999
- 1999-05-27 DE DE19924364A patent/DE19924364A1/de not_active Withdrawn
- 1999-07-29 US US09/363,389 patent/US6379934B1/en not_active Expired - Lifetime
-
2000
- 2000-05-10 AT AT00109843T patent/ATE214736T1/de not_active IP Right Cessation
- 2000-05-10 ES ES00109843T patent/ES2173838T3/es not_active Expired - Lifetime
- 2000-05-10 DE DE50000123T patent/DE50000123D1/de not_active Expired - Lifetime
- 2000-05-10 EP EP00109843A patent/EP1055730B1/de not_active Expired - Lifetime
- 2000-05-19 CN CN00109311A patent/CN1275622A/zh active Pending
- 2000-05-19 ID IDP20000421D patent/ID26177A/id unknown
- 2000-05-22 SK SK766-2000A patent/SK7662000A3/sk unknown
- 2000-05-24 JP JP2000153549A patent/JP2000350580A/ja active Pending
- 2000-05-24 BR BR0001906-2A patent/BR0001906A/pt not_active Application Discontinuation
- 2000-05-25 KR KR1020000028238A patent/KR20010049405A/ko not_active Application Discontinuation
- 2000-05-26 AU AU36446/00A patent/AU3644600A/en not_active Abandoned
- 2000-05-26 ZA ZA200002659A patent/ZA200002659B/xx unknown
- 2000-05-26 CA CA002309481A patent/CA2309481A1/en not_active Abandoned
- 2000-05-26 HU HU0002020A patent/HUP0002020A2/hu unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP1055730B1 (de) | 2002-03-20 |
| CA2309481A1 (en) | 2000-11-27 |
| ZA200002659B (en) | 2001-11-26 |
| DE19924364A1 (de) | 2000-11-30 |
| ES2173838T3 (es) | 2002-11-01 |
| EP1055730A1 (de) | 2000-11-29 |
| HUP0002020A2 (en) | 2002-09-28 |
| ID26177A (id) | 2000-12-07 |
| ATE214736T1 (de) | 2002-04-15 |
| DE50000123D1 (de) | 2002-04-25 |
| HU0002020D0 (en) | 2000-08-28 |
| AU3644600A (en) | 2000-11-30 |
| JP2000350580A (ja) | 2000-12-19 |
| US6379934B1 (en) | 2002-04-30 |
| SK7662000A3 (en) | 2000-12-11 |
| KR20010049405A (ko) | 2001-06-15 |
| BR0001906A (pt) | 2001-08-21 |
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