CN1222622C - 用棒状细菌发酵生产l－氨基酸的方法 - Google Patents
用棒状细菌发酵生产l－氨基酸的方法 Download PDFInfo
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- CN1222622C CN1222622C CNB00102728XA CN00102728A CN1222622C CN 1222622 C CN1222622 C CN 1222622C CN B00102728X A CNB00102728X A CN B00102728XA CN 00102728 A CN00102728 A CN 00102728A CN 1222622 C CN1222622 C CN 1222622C
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Abstract
本发明涉及用其中谷氨酸脱氢酶基因被扩增的棒状细菌发酵生产L-氨基酸的方法。
Description
本发明提供了用其中谷氨酸脱氢酶基因被扩增的棒状细菌发酵生产L-氨基酸的方法。
L-氨基酸被用于动物营养，人类医药以及制药工业中。
L-氨基酸由利用产生L-氨基酸的棒状细菌(特别是利用谷氨酸棒杆菌)菌株经发酵生产。由于这一组产品的重要性，人们坚持不懈地努力改进其生产方法。方法的改进可能与涉及发酵技术的手段有关(例如搅拌和供氧)，或者与营养培养基的组成有关(例如发酵期间的糖浓度)，或者与产物的回收有关(例如，离子交换层析)，或者与微生物自身的内在效能特征有关。
利用诱变，选择和突变体选择这些方法改进这些微生物的效能特征。按这一方式，获得对抗代谢物(例如赖氨酸类似物S-(2-氨乙基)半胱氨酸)具有抗性或者对通常主要的氨基酸营养缺陷并且生产L-氨基酸的菌株。
一些年来，重组体DNA技术的方法也用来改进生产L-氨基酸的谷氨酸棒杆菌的菌株，其是通过扩增个体生物合成基因，并且研究其对L-氨基酸生产的作用。这一课题的回顾性文章在以下文献中可以找到：Kinoshita(″谷氨酸杆菌″，工业微生物学，Demain和Solomon(编者)，Benjamin Cummings，London，UK，1985，115-142)，Hilliger(BioTec2，40-44(1991))，Jetten和Sinskey(生物技术关键性综述15，73-103(1995))和Sahm等(纽约科学院年报782，25-39(1996))。
谷氨酸脱氢酶催化α-氧代戊二酸的还原性氨基化，产生谷氨酸。法国出版的专利申请2 575 492描述了Corynebacterium melassecola801的DNA片段，其携带有谷氨酸脱氢酶基因。它可以用来在Corynebacterium melassecola的发酵中增加谷氨酸的产量。谷氨酸棒杆菌ATCC13032的谷氨酸脱氢酶基因的核苷酸序列已由Brmann等描述(分子微生物学6，317-326(1992))。不解糖消化链球菌的谷氨酸脱氢酶基因的核苷酸序列由Snedecor等说明(细菌学杂志193，6162-6167)。
本发明目的在于提供改进发酵生产其他L-氨基酸的新的手段。
L-氨基酸用于动物营养，人类医药以及制药工业中。因此为L-氨基酸的生产提供改进的方法总是令人有兴趣的。
当以下提到L-氨基酸时，其是指形成蛋白质的氨基酸L-赖氨酸，L-苏氨酸，L-异亮氨酸，L-缬氨酸，L-脯氨酸，L-色氨酸和它们的盐，以及L-高丝氨酸，特别是L-赖氨酸，L-苏氨酸和L-色氨酸。
本发明提供了利用棒状细菌(coryneform bacteria)发酵生产L-氨基酸的方法，特别是所说的细菌已能生产对应的L-氨基酸，且其中编码酶谷氨酸脱氢酶的核苷酸序列被扩增，特别是被超量表达。
优选的的实施方案在权利要求中阐明。
就此而言，术语″扩增″描述了在微生物中的一种或多种酶的胞内活性增加，所说的酶由对应的DNA编码，例如通过增加基因的拷贝数，通过利用强启动子或者编码相应酶(具有提高的活性)的基因，或选择性地组合这些手段来实现活性增加。
本发明提供的微生物能够由葡萄糖，蔗糖，乳糖，果糖，麦芽糖，糖蜜，淀粉，纤维素或者甘油和乙醇产生L-氨基酸。这些微生物可以包括代表性的棒状细菌，特别是棒状杆菌属。在棒状杆菌属内，可以特别提到谷氨酸棒杆菌，本领域技术人员都知道其生产L-氨基酸的能力。
棒状杆菌属，特别是谷氨酸棒杆菌种的合适的菌株是已知的野生型菌株
谷氨酸棒杆菌ATCC13032
醋谷棒状杆菌ATCC15806
嗜乙酰乙酸棒状杆菌ATCC13870
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
和它们的突变体或者来源于它们的菌株，例子是
生产L-赖氨酸的菌株
谷氨酸棒杆菌FERM-P 1709
黄色短杆菌FERM-P1708和
乳发酵短杆菌FERM-P 1712，
生产L-苏氨酸的菌株
谷氨酸棒杆菌FERM-P 5835
黄色短杆菌FERM-P 4164和
乳发酵短杆菌FERM-P 4180，
生产L-异亮氨酸的菌株
谷氨酸棒杆菌FERM-P 756
黄色短杆菌FERM-P 759和
乳发酵短杆菌FERM-P 4192，
生产L-缬氨酸的菌株
黄色短杆菌FERM-P 512和
乳发酵短杆菌FERM-P 1845，
生产L-色氨酸的菌株
谷氨酸棒杆菌FERM-BP 478
黄色短杆菌FERM-BP 475和
乳发酵短杆菌FERM-P 7127。
本发明发明人发现，在超量表达L-谷氨酸脱氢酶后，棒状细菌以一种改进的方式生产L-氨基酸，本文不要求对L-谷氨酸的保护。
谷氨酸棒杆菌的谷氨酸脱氢酶基因由Brmann等描述(分子微生物学6，317-326(1992))，按照本发明可以使用。其他微生物的谷氨酸脱氢酶基因，例如从不解糖消化链球菌获得的(由Snedecor等(细菌学杂志173，6162-6167(1991))描述)也是合适的。由遗传密码简并或由功能性中性突变产生的所述基因的等位基因也可以使用。
可以通过增加相应基因的拷贝数达到超量表达，或者可以突变位于结构基因上游的启动子和调节区。掺入结构基因上游的表达盒以相同的方式起作用。在发酵生产L-氨基酸期间，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。通过阻止酶蛋白质的降解额外地扩增酶活性。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，并被扩增。另外，基因的超量表达也可以通过改变营养培养基的组成与培养条件来完成。
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 472869，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，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。
辅助谷氨酸脱氢酶超量表达的质粒的例子是pEK1.9gdh-1和pEKExpgdh，其存在于菌株ATCC 13032/pEK1.9gdh-1和DH5α/pEKExpgdh中。质粒pEK1.9gdh-1是穿梭载体，其含有谷氨酸棒杆菌的NADP-依赖性谷氨酸脱氢酶基因。质粒pEKExpgdh是穿梭载体，其含有不解糖消化链球菌的NADP-依赖性谷氨酸脱氢酶基因。
为生产相应的L-氨基酸以超量表达一种或多种特殊的氨基酸生物合成途径的酶以及谷氨酸脱氢酶也可能是有利的，这样的例子是：
●编码二氢吡啶二羧酸合酶的dapA基因可被额外地超量表达，以改进生产L-赖氨酸的棒状细菌(EP-B 0197335)，
●编码乙酰羟酸合酶的基因可被额外地超量表达，以改进生产L-缬氨酸的棒状细菌(EP-B 0356739)，
●编码邻氨基苯甲酸转磷酸核糖基酶的基因可被额外地超量表达，以改进生产L-色氨酸的棒状细菌(EP-B 0124048)，
●编码高丝氨酸脱氢酶的基因可被额外地超量表达，以改进生产L-苏氨酸或者L-异亮氨酸的棒状细菌(EP-A 0131171)。
除超量表达谷氨酸脱氢酶之外，为产生相应的L-氨基酸可以更有利地阻断不需要的次级反应(Nakayama：″生产氨基酸的微生物的培育″，在“微生物产物的超量产生”中，Krumphanzl，Sikyta，Vanek(编者)，学院出版社，伦敦，英国，1982)。
为生产L-氨基酸的目的，根据本发明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。合适的前体也可以进一步地添加至培养基中。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自动分析，如Spackman等所描述的(分析化学，30，1190(1958))。
下列微生物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)：
谷氨酸棒杆菌菌株ATCC13032/pEK1.9gdh-1，保藏号为DSM12614。
大肠杆菌K12菌株DH5α/pEKExpgdh，保藏号为DSM 12613。
实施例
本发明由下列实施例更详细地说明。
这里，试验用生产氨基酸的菌株完成，其中显示了所要求的方法的优越性：
a)生产L-赖氨酸的菌株谷氨酸棒杆菌DSM5715，(EP-B-0435132)和
b)生产L-苏氨酸与L-异亮氨酸的菌株黄色短杆菌DSM5399(EP-B-0385 940)和
c)生产L-缬氨酸，需要异亮氨酸的菌株ATCC13032ΔilvA，该菌株已按照依据布达佩斯条约以保藏号DSM12455保藏在德意志微生物保藏中心(德国)。
实施例1
产生具有扩增的谷氨酸脱氢酶的L-氨基酸生产菌
质粒pEK1.9gdh-1相当于由Brmann等(分子微生物学6，317-326(1992))描述的质粒pEK1.9gdh。其从ATCC13032/pEK1.9gdh-1分离而来。以相同的方式从大肠杆菌菌株DH5α/pEKExpgdh分离已知质粒pEKExpgdh(Marx等，代谢工程1，35-48(1999))，其携带有不解糖消化链球菌的谷氨酸脱氢酶基因(Snedecor等.细菌学杂志，173，6162-6167(1991))。
如Liebl等(FEMS微生物学通信65，299-304(1989))描述的方法用质粒pEK1.9gdh-1转化菌株DSM5715，DSM5399和ATCC13032ΔilvA。将转化体在来源于Merck(Darmstadt，德国)的脑/心琼脂上选择，该琼脂已经补充有50mg/l卡那霉素。按这一方式，获得菌株DSM5715/pEK1.9gdh-1，DSM5399/pEK1.9gdh-1和ATCC13032ΔilvA/pEK1.9gdh-1。以相同的方式用质粒pEKExpgdh转化菌株DSM5715，获得菌株DSM5715/pEKExpgdh。
实施例2
生产L-赖氨酸
将菌株DSM5715/pEK1.9gdh-1在复合培养基2TY中预培养，所说的培养基由蛋白胨16g/l，酵母膏10g/l以及NaCl 5g/l组成。为此，用接种环将菌株接种到带有2个阻流片(flow spoilers)的500ml锥形瓶中的60ml 2TY中，在150rpm和30℃下培养12小时。
为了接种在带有2个阻流片的500ml锥形瓶中的60ml生产培养基，将预培养物在Sepatech Minifuge RF(Heraeus，Hanau，德国)离心机中于5000rpm下离心10分钟。弃去上清液，将离心沉淀悬浮在1毫升生产培养基中。将一小份这样的细胞悬液添加至生产培养基中，由此达到OD600约2.0。所使用的生产培养基是具有pH7.0的培养基CGXII(表1)，该培养基由Keilhauer等(细菌学杂志175，5595-5603(1993))描述过，其补充有葡萄糖20g/l，亮氨酸350mg/l以及卡那霉素单硫酸盐50mg/l。将培养物在30℃和150rpm下培养72小时。
表1
| 组分 | 每升浓度 |
| (NH4)2ISO4 | 20g |
| 尿素 | 5g |
| KH2PO4 | 1g |
| K2HPO4 | 1g |
| MgSO4·7H2O | 0.25g |
| 3-吗啉代丙烷磺酸 | 42g |
| FeSO4·7H2O | 10mg |
| MnSO4·H2O | 10mg |
| ZnSO4·7H2O | 1mg |
| CuSO4 | 0.2mg |
| NiCl2·6H2O | 0.02mg |
| CaCl2 | 10mg |
| 原儿茶酸 | 0.03mg |
| 生物素 | 200μg |
然后在600nm测定波长测定光密度(OD)(Biochrom Novaspec4049，LKB Instrument GmbH，Grfelfing，德国)，采用来源于Eppendorf-BioTronik(汉堡，德国)的氨基酸分析仪，经离子交换层析和柱后与茚三酮反应检测所形成的L-赖氨酸的浓度。表2显示试验的结果。
表2
| 菌株 | OD | L-赖氨酸g/l |
| DSM5715 | 16.5 | 4.5 |
| DSM5715/pEK1.9gdh-1 | 19.4 | 6.2 |
实施例3
生产L-苏氨酸和L-异亮氨酸
将菌株DSM5399/pEK1.9gdh-1在具有50μg/ml卡那霉素的完全培养基CgIII(Kase & Nakayama，农业和生物化学36(9)1611-1621(1972))中预培养。为此，用接种环将菌株接种到带有4个阻流片的100ml锥形瓶中的10ml培养基CgIII中，在240rpm和30℃下培养16小时。
为了接种在带有4个阻流片的100ml锥形瓶中的10ml生产培养基，测定预培养物的OD(660nm)。主培养物接种至0.1OD。所使用的生产培养基是由Keilhauer等(细菌学杂志1993，175：5595-5603)描述的培养基CgXII。培养基的组成在实施例2中显示。添加4％葡萄糖和50mg/l卡那霉素硫酸盐。将细胞在33℃，250rpm以及80％大气湿度下培养48小时。
然后在660nm测定波长测定光密度(OD)(Biochrom Novaspec4049，LKB Instrument GmbH，Grfelfing，德国)，如实施例2所述测定形成的L-苏氨酸与L-异亮氨酸的浓度。表3显示试验的结果。
表3
| 菌株 | OD | L-苏氨酸g/l | L-异亮氨酸g/l |
| DSM5399 | 10.5 | 1.77 | 1.05 |
| DSM5399/pEK1.9gdh-1 | 11.0 | 2.26 | 1.44 |
实施例4
产生L-缬氨酸
将菌株ATCC13032ΔilvA/pEK1.9gdh-1在具有50μg/ml卡那霉素的完全培养基CgIII(Kase & Nakayama，农业和生物化学36(9)1611-1621(1972))中预培养。为此，用接种环将菌株接种到带有4个阻流片的500ml锥形瓶中的50ml培养基CgIII中，在140rpm和30℃下培养16小时。
为了接种在带有4个阻流片的500ml锥形瓶中的60ml生产培养基，测定预培养物的OD(660nm)。将主培养物离心，弃去上清液，将离心沉淀悬浮在5毫升生产培养基中，将主培养物接种至OD 0.3。所使用的生产培养基是如实施例3所描述的培养基CgXII(具有4％葡萄糖)(Keilhauer等，细菌学杂志1993，175：5595-5603)。培将细胞在30℃，150rpm下培养48小时。
然后在660nm测定波长测定光密度(OD)(Biochrom Novaspec4049，LKB Instrument GmbH，Grfelfing，德国)，如实施例2所述测定形成的L-缬氨酸的浓度。表4显示试验的结果。
表4
| 菌株 | OD | L-缬氨酸g/l |
| ATCC13032ΔilvA | 18.5 | 0.29 |
| ATCC13032ΔilvA/pEK1.9gdh-1 | 17.6 | 0.45 |
实施例5
生产L-赖氨酸，L-缬氨酸和L-丙氨酸
将菌株DSM5715/pEKExpgdh在复合培养基2TY中预培养，所说的培养基由蛋白胨16g/l，酵母膏10g/l以及NaCl 5g/l组成。为此，用接种环将菌株接种到带有2个阻流片(flow spoilers)的500ml锥形瓶中的60ml 2TY中，在150rpm和30℃下培养12小时。
为了接种在带有2个阻流片的500ml锥形瓶中的60ml生产培养基，将预培养物在Sepatech Minifuge RF(Heraeus，Hanau，德国)离心机中于5000rpm下离心10分钟。弃去上清液，将离心沉淀悬浮在1毫升生产培养基中。将一小份这样的细胞悬液添加至生产培养基中，由此达到OD600约0.4。所使用的生产培养基是培养基CGC(表5)，该培养基由Schrumpf等(细菌学杂志173，4510-4516(1991))描述过，其补充有葡萄糖25g/l，亮氨酸350m/l，3-吗啉代丙烷磺酸42g/l以及卡那霉素单硫酸盐50mg/l(pH7)。将培养物在30℃和150rpm下培养30小时。
表5
| 组分 | 每升浓度 |
| (NH4)2SO4 | 5g |
| 尿素 | 5g |
| KH2PO4 | 0.5g |
| K2HPO4 | 0.5g |
| MgSO4·7H2O | 0.25g |
| FeSO4·7H2O | 10mg |
| MnSO4·H2O | 10mg |
| ZnSO4·7H2O | 1mg |
| CuSO4 | 0.2mg |
| NiCl2·6H2O | 0.02mg |
| CaCl2·2H2O | 10mg |
| 生物素 | 200μg |
然后在600nm测定波长测定光密度(OD)(Biochrom Novaspec4049，LKB Instrument GmbH，Grfelfing，德国)，采用来源于Eppendorf-BioTronik(汉堡，德国)的氨基酸分析仪，经离子交换层析和柱后与茚三酮反应检测所形成的L-丙氨酸，L-赖氨酸和L-缬氨酸的浓度。表6显示试验的结果。
表6
| 菌株 | OD | L-丙氨酸g/l | L-赖氨酸g/l | L-缬氨酸g/l |
| DSM5715 | 29.4 | 微量 | 1.6 | 0.1 |
| DSM5715/PEKExpgdh | 19.4 | 0.6 | 2.1 | 0.6 |
Claims (7)
1.通过棒状细菌发酵生产L-氨基酸的方法，其中所述L-氨基酸选自L-苏氨酸、异亮氨酸、L-缬氨酸和L-丙氨酸中的一种或多种，其特征在于进行以下步骤：
a)将生产L-苏氨酸、异亮氨酸、L-缬氨酸、L-丙氨酸中的一或多种L-氨基酸的细菌进行发酵，所说的细菌中至少编码谷氨酸脱氢酶的核苷酸序列被扩增，
b)在培养基中或在细菌细胞中积累所需的L-氨基酸，和
c)分离所说的L-氨基酸。
2.按照权利要求1的方法，其特征在于，在所使用的细菌中所产生的谷氨酸脱氢酶是NADP依赖性的。
3.按照权利要求1的方法，其特征在于，在所使用的细菌中所产生的谷氨酸脱氢酶是NAD依赖性的。
4.按照权利要求1的方法，其特征在于，使用其中在形成所需L-氨基酸的代谢途径中的其余基因被附加扩增的细菌，所述基因选自编码二氢吡啶二羧酸合酶的dapA基因，编码乙酰羟酸合酶的基因，编码邻氨基苯甲酸转磷酸核糖基酶的基因和编码高丝氨酸脱氢酶的基因。
5.按照权利要求1的方法，其特征在于，使用以质粒载体转化的菌株，所说的质粒载体携带有编码谷氨酸脱氢酶的核苷酸序列。
6.按照权利要求5的方法，其特征在于，使用以质粒载体pEK1.9gdh-1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DSM 12614。
7.按照权利要求5的方法，其特征在于，使用以质粒载体pEKExpgdh转化的菌株，所说的质粒载体以在大肠杆菌中的形式保藏，保藏号DSM 12613。
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| DE19907347A DE19907347A1 (de) | 1999-02-20 | 1999-02-20 | Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien |
| DE19907347.3 | 1999-02-20 | ||
| US09/324,940 US6355454B1 (en) | 1999-02-20 | 1999-06-03 | Process for the fermentative production of L-amino acids using coryneform bacteria |
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| JP3965821B2 (ja) * | 1999-03-09 | 2007-08-29 | 味の素株式会社 | Ｌ−リジンの製造法 |
| WO2002022828A1 (en) * | 2000-09-12 | 2002-03-21 | Degussa Ag | Isolation and sequencing of the pknb gene of c. glutamicum |
| US6939692B2 (en) | 2000-09-12 | 2005-09-06 | Degussa Ag | Nucleotide sequences coding for the pknB gene |
| DE10045496A1 (de) * | 2000-09-14 | 2002-03-28 | Degussa | Neue für das ptsi-Gen kodierende Nukleotidsequenzen |
| DE102004037572A1 (de) * | 2004-08-03 | 2006-02-23 | Degussa Ag | Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von Stämmen der Familie Enterobacteriaceae |
| JP2010017081A (ja) * | 2006-10-10 | 2010-01-28 | Ajinomoto Co Inc | Ｌ−アミノ酸の製造法 |
| DE102006050489A1 (de) * | 2006-10-26 | 2008-04-30 | Evonik Degussa Gmbh | Allele des prpD1-Gens aus coryneformen Bakterien |
| DE102006054202A1 (de) * | 2006-11-17 | 2008-05-21 | Evonik Degussa Gmbh | Allele des oxyR-Gens aus coryneformen Bakterien |
| CN101565723B (zh) * | 2009-05-25 | 2011-06-01 | 河南孟成生物药业股份有限公司 | 一种l-色氨酸的发酵生产工艺 |
| CN102653736A (zh) * | 2012-04-12 | 2012-09-05 | 大连大学 | 微生物发酵生产谷氨酸脱氢酶的方法 |
| CN103184247A (zh) * | 2013-04-08 | 2013-07-03 | 绍兴市安杰生物科技有限公司 | 一种l-赖氨酸联产l-乳酸的方法 |
| PL2811028T3 (pl) * | 2013-06-03 | 2017-07-31 | Evonik Degussa Gmbh | Sposób wytwarzania L-waliny z zastosowaniem zrekombinowanych bakterii maczugowatych zawierających operon ilvBN indukowany propionianem |
| KR101518860B1 (ko) * | 2013-10-11 | 2015-05-12 | 씨제이제일제당 (주) | L-아미노산의 생산 방법 |
| RU2697005C2 (ru) * | 2013-10-11 | 2019-08-08 | СиДжей ЧЕИЛДЗЕДАНГ КОРП. | Способ получения l-аминокислот |
| CN105505894A (zh) * | 2014-09-24 | 2016-04-20 | 中国科学院天津工业生物技术研究所 | 天冬氨酸激酶/高丝氨酸脱氢酶突变体及其应用 |
| CN105506014B (zh) * | 2015-12-23 | 2019-02-12 | 湖南宝利士生物技术有限公司 | 高光学纯l-高丝氨酸及其衍生物的生物合成方法 |
| KR102112240B1 (ko) * | 2018-09-28 | 2020-05-18 | 씨제이제일제당 주식회사 | 알파-글루코시다제의 활성이 강화된 l-아미노산을 생산하는 미생물 및 이를 이용한 l-아미노산 생산 방법 |
| CN109337854B (zh) * | 2018-11-19 | 2020-11-03 | 南京工业大学 | 一株敲除胞外核酸酶ExeR的谷氨酸棒杆菌及其构建方法与应用 |
| CN111197014B (zh) * | 2020-04-01 | 2022-11-08 | 宜昌三峡普诺丁生物制药有限公司 | 谷氨酸棒状杆菌诱变菌株及其应用 |
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| JPS61268185A (ja) * | 1984-12-27 | 1986-11-27 | Asahi Chem Ind Co Ltd | グルタミン酸デヒドロゲナ−ゼ産生遺伝子を含むｄｎａ断片 |
| 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 |
| FI980551A (fi) * | 1998-03-11 | 1999-09-12 | Valtion Teknillinen | Transformoidut mikro-organismit, joilla on parannettuja ominaisuuksia |
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| RU2247778C2 (ru) | 2005-03-10 |
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| ATE263246T1 (de) | 2004-04-15 |
| CA2299105A1 (en) | 2000-08-20 |
| ES2218009T3 (es) | 2004-11-16 |
| HU0000747D0 (en) | 2000-04-28 |
| EP1029919B1 (de) | 2004-03-31 |
| CN1266905A (zh) | 2000-09-20 |
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