CN101563453B - L-氨基酸的生产方法 - Google Patents
L-氨基酸的生产方法 Download PDFInfo
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Abstract
在培养基中培养具有L-氨基酸生产能力并且经过修饰使得琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性降低的微生物,在该培养基中或菌体内生成并蓄积L-氨基酸,并且从该培养基或菌体收集L-氨基酸,从而生产L-氨基酸。
Description
技术领域
本发明涉及使用微生物的L-谷氨酸等L-氨基酸的生产方法。L-谷氨酸作为调味品原料等,其它L-氨基酸作为动物饲料用添加剂、保健食品成分或氨基酸输液等,在产业上是有用的。
背景技术
为了通过使用微生物的发酵方法来产生目标物质,例如L-氨基酸等,存在下述方法:使用野生型细菌(野生型菌株)的方法、使用从野生型菌株衍生的营养缺陷菌株的方法、使用从野生型菌株作为对各种药物的抗性突变体菌株而衍生的代谢调节突变体菌株的方法、使用同时具有营养缺陷菌株和代谢调节突变菌株特征的菌株等。
例如,L-谷氨酸主要通过使用属于短杆菌属(Brevibacterium)、棒杆菌属(Corynebacterium)、微杆菌属(Microbacterium)的称作棒状杆菌型细菌(coryneform bacteria)的L-谷氨酸生产菌或者它们的突变菌株的发酵方法来生产(参照例如非专利文献1)。作为通过使用其它菌株的发酵方法来生产L-谷氨酸的方法,已知有下述方法:使用芽孢杆菌属(Bacillus)、链霉菌属(Streptomyces)或青霉属(Penicillium)属等微生物的方法(参照例如专利文献1),使用假单胞菌属(Pseudomonas)、节杆菌属(Arthrobacter)、沙雷氏菌属(Serratia)或假丝酵母属(Candida)等微生物的方法(参照例如专利文献2),使用芽孢杆菌属、假单胞菌属、沙雷氏菌属或产气气杆菌(Aerobacteraerogenes)(现在名为产气肠杆菌(Enterobacter aerogenes))等微生物的方法(例如参照专利文献3),使用大肠杆菌的突变菌株的方法(参照例如专利文献4),等等。此外,还公开了使用属于克雷伯氏菌属(Klebsiella)、欧文氏菌属(Erwinia)、泛菌属(Pantotea)或肠杆菌属(Enterobacter)的微生物的L-谷氨酸生产方法(参照例如专利文献5~7)。
近年来,在目的物质的发酵生产中运用了重组DNA技术。例如,通过增强编码L-氨基酸生物合成体系酶的基因的表达(专利文献8、专利文献9)、 或者增强碳源向L-氨基酸生物合成体系的流入(专利文献10),可以改善细菌的L-氨基酸生产力。
例如,已经有报道称,对于L-谷氨酸的生产,在棒杆菌属或短杆菌属的细菌中引入源自大肠杆菌或谷氨酸棒杆菌(Corynebacterium glutamicum)的编码柠檬酸合酶的基因,对于棒状杆菌型细菌的L-谷氨基酸生产能力的增强是有效的(参照例如专利文献11)。另外,已经有报道称,将源自棒状杆菌型细菌的柠檬酸合酶基因引入属于肠杆菌属、克雷伯氏菌属、沙雷氏菌属、欧文氏菌属或埃希氏菌属的肠细菌(enterobacterium)中,有效地增强了L-谷氨酸产生能力(参照例如专利文献12)。
此外,已知通过缺损微生物的α-酮戊二酸脱氢酶(α-KGDH)来生产显著量的L-谷氨酸(专利文献13、专利文献14、专利文献15)。
琥珀酸脱氢酶(SDH)是催化由琥珀酸到富马酸的反应的酶,已知在棒状杆菌型细菌中通过缺损该酶的基因,生成微量的L-谷氨酸(专利文献16)。
另一方面,已知属于肠内细菌群的大肠杆菌(Esherichia coli)中也存在琥珀酸脱氢酶缺损株(非专利文献2),但尚不清楚其与L-谷氨酸生产之间的关联。
此外,已知α-酮戊二酸脱氢酶缺损的大肠杆菌呈琥珀酸营养缺陷性,但若与SDH一起双重缺损,则琥珀酸营养缺陷性可以恢复(非专利文献2)。然而,α-酮戊二酸脱氢酶与琥珀酸脱氢酶的双重缺损对L-谷氨酸等L-氨基酸生产的影响尚属未知。
专利文献1美国专利第3,220,929号说明书
专利文献2美国专利第3,563,857号说明书
专利文献3特公昭32-9393号公报
专利文献4特开平5-244970号公报
专利文献5特开2000-106869号公报
专利文献6特开2000-189169号公报
专利文献7特开2000-189175号公报
专利文献8美国专利第5,168,056号说明书
专利文献9美国专利第5,776,736号说明书
专利文献10美国专利第5,906,925号说明书
专利文献11特公平7-121228号公报
专利文献12特开2000-189175号公报
专利文献13欧州专利申请公开771879号公报
专利文献14欧州专利申请公开0952221号公报
专利文献15欧州专利申请公开1078989号公报
专利文献16欧州专利申请公开1106684号公报
非专利文献1明石邦彦等著,氨基酸发酵,学会出版中心,195~215页,1986年
非专利文献2J Gen Microbiol.1978Jul;107(1):1-13
发明内容
发明所要解决的问题
本发明的课题是:提供能够高效地生产L-谷氨酸等L-氨基酸的细菌,和使用该细菌高效地生产L-氨基酸的方法。
解决问题的手段
本发明人等为解决上述问题进行了深入研究,结果发现通过对细菌进行修饰使得琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性降低,可以提高L-谷氨酸等L-氨基酸的生产力,从而完成了本发明。
即,本发明如下所述。
(1).一种生产L-氨基酸的方法,包括在培养基中培养具有L-氨基酸生产能力且经过修饰使得琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性降低的微生物,使L-氨基酸在该培养基中或菌体内生成并蓄积,并且从该培养基或菌体收集L-氨基酸。
(2).上述方法,其中通过降低琥珀酸脱氢酶编码基因或α-酮戊二酸脱氢酶编码基因的表达量,或者破坏所述基因,来降低琥珀酸脱氢酶活性或α-酮戊二酸脱氢酶活性。
(3).上述方法,其中,所述琥珀酸脱氢酶编码基因是选自sdhA基因、sdhB基因、sdhC基因和sdhD基因中的1种或2种以上的基因。
(4).上述方法,其中,α-酮戊二酸脱氢酶编码基因是选自sucA基因、odhA基因和sucB基因中的1种或2种以上的基因。
(5).上述方法,其中,所述微生物为属于肠杆菌科(Enterobacteriacae)的细菌或者棒状杆菌型细菌。
(6).上述方法,其中,所述氨基酸为L-谷氨酸或以L-谷氨酸为前体生物合成的L-氨基酸。
(7).上述方法,其中,所述L-氨基酸选自L-精氨酸、L-脯氨酸、L-鸟氨酸、L-瓜氨酸和L-谷氨酰胺。
附图说明
图1显示了辅助质粒RSF-Red-TER的结构。
图2显示了辅助质粒RSF-Red-TER的构建。
实施发明的最佳模式
以下,对本发明进行详细说明。
<1>用于本发明的微生物
本发明的方法是使用具有L-氨基酸生产能力、且经过修饰使得琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性降低的微生物的L-氨基酸生产方法。
用于本发明的微生物可以这样获得:以具有L-氨基酸生产能力的微生物为亲本菌株,通过修饰使其琥珀酸脱氢酶和α-酮戊二酸脱氢酶活性降低。此外,用于本发明的微生物还可以这样获得:以经过修饰而使得琥珀酸脱氢酶和α-酮戊二酸脱氢酶活性降低的微生物为亲本菌株,赋予或增强L-氨基酸生产能力。
用于本发明的微生物可以是本来就具有L-氨基酸生产能力的微生物,也可以是通过利用突变法、重组DNA技术等的选育而被赋予了L-氨基酸生产能力的微生物。
此处,“L-氨基酸生产能力”是指:当在培养基中培养用于本发明的微生物时,其具有以可从细胞或培养基中回收的水平生产L-氨基酸的能力。优选具有与在相同条件下培养的野生型菌株或非修饰菌株相比具有生产更多量的L-氨基酸的能力。
作为L-氨基酸,可以列举出:L-赖氨酸、L-谷氨酸、L-苏氨酸、L-缬氨酸、L-亮氨酸、L-异亮氨酸、L-丝氨酸、L-天冬氨酸、L-天冬酰胺、L-谷氨酰胺、L-精氨酸、L-半胱氨酸(胱氨酸)、L-甲硫氨酸、L-苯丙氨酸、L-色氨酸、L-酪氨酸、L-甘氨酸、L-丙氨酸、L-脯氨酸、L-鸟氨酸、L-瓜氨酸、L-高丝氨酸;优选L-谷氨酸或以L-谷氨酸为前体的L-氨基酸,特别优选L- 谷氨酸、L-谷氨酰胺、L-脯氨酸、L-精氨酸、L-鸟氨酸、L-瓜氨酸。
作为在本发明的生产方法中使用的微生物,可以列举出细菌,例如埃希氏菌属、泛菌属、肠杆菌属等属于肠杆菌科(Enterobacteriaceae family)的微生物;谷氨酸棒杆菌和乳发酵短杆菌(Brevibacterium lactofermentum)等棒状杆菌型细菌;和枯草芽孢杆菌(Bacillus subtilis)等属于芽孢杆菌属的细菌。
在本发明中,“棒状杆菌型细菌”还包括原来归类于短杆菌属、但现在归类于棒杆菌属的细菌(Int.J.Syst.Bacteriol.,41,255(1991)),以及与棒杆菌属亲缘关系非常近的短杆菌属的细菌。这样的棒状杆菌型细菌的实例可列举下述:
嗜乙酰乙酸棒杆菌(Corynebacterium acetoacidophilum)
醋谷棒杆菌(Corynebacterium acetoglutamicum)
烷醇棒杆菌(Corynebacterium alkanolyticum)
美棒杆菌(Corynebacterium callunae)
谷氨酸棒杆菌
百合花棒杆菌(Corynebacterium lilium)
栖糖蜜棒杆菌(Corynebacterium melassecola)
嗜热产氨棒杆菌(Corynebacterium thermoaminogenes)(Corynebacteriumefficiens)
力士棒杆菌(Corynebacterium herculis)
二歧短杆菌(Brevibacterium divaricatum)
黄色短杆菌(Brevibacterium flavum)
Brevibacterium immariophilum
乳发酵短杆菌(Brevibacterium lactofermentum)(谷氨酸棒杆菌)
玫瑰色短杆菌(Brevibacterium roseum)
解糖短杆菌(Brevibacterium saccharolyicum)
生硫短杆菌(Brevibacterium thiogenitalis)
产氨棒杆菌(Brevibacterium ammoniagenes)
白色短杆菌(Brevibacterium album)
Brevibacterium Cerinum
嗜氨微杆菌(Microbacterium ammoniaphilum)
具体地,可以下述菌株为例:
嗜乙酰乙酸棒杆菌ATCC13870
醋谷棒杆菌ATCC15806
烷醇棒杆菌ATCC21511
美棒杆菌ATCC15991
谷氨酸棒杆菌ATCC13020、ATCC13032、ATCC13060
百合花棒杆菌ATCC15990
栖糖蜜棒杆菌ATCC17965
嗜热产氨棒杆菌AJ12340(FERM BP-1539)
力士棒杆菌ATCC13868
二歧短杆菌ATCC14020
黄色短杆菌ATCC13826、ATCC14067
Brevibacterium immariophilum ATCC14068
乳发酵短杆菌ATCC13869(谷氨酸棒杆菌ATCC13869)
玫瑰色短杆菌ATCC13825
解糖短杆菌ATCC14066
生硫短杆菌ATCC19240
产氨棒杆菌ATCC6871、ATCC6872
白色短杆菌ATCC15111
Brevibacterium selinum ATCC15112
嗜氨微杆菌ATCC15354
这些菌株可从例如美国典型培养物保藏中心(American Type CultureCollection,ATCC:地址:P.O.Box 1549,Manassas,VA 20108,1,UnitedStates of America)通过分售获得。即,对每种菌株都给予了登录号,可以使用这些号码通过分售获得菌株(http://www.atcc.org/)。美国典型培养物保藏中心的目录中列出了每种菌株的对应登录号。根据布达佩斯条约的规定,将AJ12340菌株于1987年10月27日保藏在通商产业省工业技术院生命工学工业技术研究所(National Institute of Bioscience andHuman-Technology,Agency of Industrial Science and Technology),(目前为,独立行政法人产业技术综合研究所专利微生物保藏中心(InternationalPatent Organism Depositary,National Institute of Advanced Industrial Science and Technology))(305-5466日本国茨城县筑波市东1丁目1番地1中央第6),登录号为FERM BP-1539。
对于用于本发明的属于肠杆菌科的微生物没有特殊限制,只要它们属于埃希氏菌属、肠杆菌属、泛菌属、克雷伯氏菌属、沙雷氏菌属、欧文氏菌属、沙门氏菌属、摩根氏菌属(Morganella)等属,具有L-氨基酸生产能力即可。具体地说,可以使用按照NCBI(美国国家生物技术信息中心,National Center for Biotechnology Information)数据库中记载的分类属于肠杆菌科的细菌(http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Tree&id=1236&lvl=3&keep=1&srchmode=1&unlock)。作为肠杆菌科的亲本菌株,尤其优选使用埃希氏菌属细菌、肠杆菌属细菌或泛菌属细菌。
对于埃希氏菌属细菌的亲本菌株,没有特别限制,具体而言,可以使用Neidhardt等的著作(Neidhardt et al.Escherichia coli and SalmonellaTyphimurium,American Society for Microbiology,Washington D.C.,1029表1)列出的细菌。在这些细菌中,例如可列举大肠杆菌。作为大肠杆菌,具体地可列举源自原型野生型K12菌株的大肠杆菌W3110(ATCC 27325)和大肠杆菌MG1655(ATCC 47076)。
特别地,泛菌属细菌、欧文氏菌属细菌和肠杆菌属细菌是分类为γ-变形细菌(γ-Proteobacteria)的细菌,它们在分类学上的亲缘关系非常近(J GenAppl Microbiol 1997 Dec;43(6)355-361、International Journal of SystematicBacteriology,Oct.1997,p1061-1067)。近年来,依据DNA-DNA杂交实验等,属于肠杆菌属的细菌中,有些被重新分类为成团泛菌(Pantoeaagglomerans)或Pantoea dispersa等(International Journal of SystematicBacteriology,July 1989;39(3).p.337-345)。此外,属于欧文氏菌属的细菌中,有些被重新分类为Pantoea ananas、斯氏泛菌(Pantoea stewartii)(参照International Journal of Systematic Bacteriology,Jan 1993;43(1).p.162-173)。
肠杆菌属细菌的实例包括成团肠杆菌(Enterobacter agglomerans)和产气肠杆菌(Enterobacter aerogenes)。具体地,可以使用欧州专利申请公开952221号说明书示例的菌株。作为肠杆菌属的代表性菌株,可以列举出成团肠杆菌ATCC12287株。
作为泛菌属细菌的代表性菌株,可以列举出Pantoea ananatis、斯氏泛菌、成团泛菌、Pantoea citrea。具体地,可以列举下述菌株。
Pantoea ananatis AJ13355株(FERM BP-6614)(欧洲专利申请公开0952221号说明书)
Pantoea ananatis AJ13356株(FERM BP-6615)(欧洲专利申请公开0952221号说明书)
而且,这些菌株在欧洲专利申请公开0952221号说明书中记载为成团肠杆菌,但现在,如上文所述,依据16S rRNA核苷酸序列分析将其重新分类为Pantoea ananatis。
作为欧文氏菌属细菌,可以列举出解淀粉欧文氏菌(Erwinia amylovora)、胡萝卜软腐欧文氏菌(Erwinia carotovora);作为克雷伯氏菌属细菌,可以列举出植生克雷伯氏菌(Klebsiella planticola)。具体地,可以列举出下述菌株。
解淀粉欧文氏菌ATCC15580株
胡萝卜软腐欧文氏菌ATCC15713株
植生克雷伯氏菌(Klebsiella planticola)AJ13399株(FERM BP-6600)(欧洲专利申请公开955368号说明书)
植生克雷伯氏菌(Klebsiella planticola)AJ13410株(FERM BP-6617)(欧洲专利申请公开955368号说明书)
<1-1>L-氨基酸生产能力的赋予或增强
以下,说明对如上所述的微生物赋予L-氨基酸生产能力的方法,或增强如上所述的微生物的L-氨基酸生产能力的方法。
为了赋予L-氨基酸生产能力,可以使用在棒状杆菌型细菌或埃希氏菌属细菌等氨基酸生产菌的选育中沿用至今的方法,如获取营养缺陷突变体、L-氨基酸类似物抗性菌株或代谢调节突变菌株,创建L-氨基酸生物合成系统酶表达增强的重组菌株等等(参见《氨基酸发酵》,(株)学会出版中心,1986年5月30日初版发行,第77-100页)。这里,在L-氨基酸生产菌的选育中,被赋予的营养缺陷性、类似物抗性或代谢调节突变等性质可以是单独一种,也可以是两种或者三种以上。此外,表达被增强的L-氨基酸生物合成系统酶可以是单独一种,也可以是两种或三种以上。另外,可以将营养缺陷突变、类似物抗性或代谢调节突变等性质的赋予与生物合成系统酶的增强组合进行。
具有L-氨基酸生产能力的营养缺陷突变体菌株、类似物抗性菌株或代谢调节突变菌株可如下获得:对亲本菌株或野生型菌株施以常规突变处 理,即X-射线或UV照射或用诱变剂例如N-甲基-N’-硝基-N-亚硝基胍等处理;其后,从所得的突变菌株中选择显示营养缺陷性、类似物抗性或代谢调节突变并且还具有L-氨基酸生产能力的菌株。此外,L-氨基酸产生菌还可以通过基因重组增加L-氨基酸生物合成系统酶的活性来进行。
以下,对赋予L-氨基酸生产能力的方法和赋予了L-氨基酸生产能力的微生物进行示例。
作为通过选育来赋予或者增强L-谷氨酸生产能力的方法,可以列举例如通过修饰使得编码L-谷氨酸生物合成相关酶的基因的表达增强的方法。L-谷氨酸生物合成相关酶的实例包括谷氨酸脱氢酶(gdhA)、谷氨酰胺合成酶(glnA)、谷氨酸合酶(gltAB)、异柠檬酸脱氢酶(icdA)、乌头酸水合酶(acnA,acnB)、柠檬酸合酶(citrate synthase;gltA)、磷酸烯醇丙酮酸羧化酶(ppc)、丙酮酸羧化酶、丙酮酸脱氢酶(aceEF,lpdA)、丙酮酸激酶(pykA,pykF)、磷酸烯醇丙酮酸合酶(ppsA)、烯醇化酶(eno)、磷酸甘油变位酶(pgmA,pgmI)、磷酸甘油酸激酶(pgk)、甘油醛-3-磷酸脱氢酶(gapA)、丙糖磷酸异构酶(tpiA)、果糖二磷酸醛缩酶(fbp)、磷酸果糖激酶(pfkA,pfkB)、葡糖磷酸异构酶(pgi)和甲基柠檬酸合酶(prpC)等等。此外,酶名称后的括号内为基因名称(以下同)。
作为增强这些基因的表达的方法,可以这样来实现:导入扩增质粒,所述扩增质粒是将包含这些基因的DNA片段导入适当的质粒,例如至少包含负责质粒在微生物内的复制增殖功能的基因的质粒载体而形成的;或者,通过接合、基因转移等使这些基因在染色体上多拷贝化;抑或在这些基因的启动子区域导入突变(参照国际公开小册子WO95/34672号)。
当向上述扩增质粒或者染色体上导入目的基因时,用于表达这些基因的启动子可以是能够在棒状杆菌型细菌中发挥功能的任何启动子,可以是所使用的基因自身的启动子,也可以是经修饰的启动子。也可以通过适宜地选择在棒状杆菌型细菌中强力发挥功能的启动子,或者通过使启动子的-35、-10区域与共有序列接近,来进行基因表达量的调节。作为通过如上所述的方法进行修饰从而增强了柠檬酸合酶基因、异柠檬酸脱氢酶、丙酮酸脱氢酶基因和/或谷氨酸脱氢酶基因的表达的微生物,可以例举WO00/18935号小册子、欧洲专利申请公开1010755号说明书等中记载的微生物。
为了赋予L-谷氨酸生产能力的修饰可以通过降低或缺损下述酶的活性 来进行,所述酶催化从L-谷氨酸生物合成途径分支出来而产生其它化合物的反应。作为催化从L-谷氨酸生物合成途径中分支出来而生成L-谷氨酸以外的化合物的反应的酶,可以列举出:异柠檬酸裂合酶、乙酰羟酸合酶、乙酰乳酸合酶、甲酸乙酰转移酶、乳酸脱氢酶、谷氨酸脱羧酶、1-吡咯啉-5-羧酸脱氢酶(1-ピロリン-5-カルボキシレ一トデヒドロゲナ一ゼ)、乙酰CoA水化酶(WO2006/057450)等。
降低或者缺损上述酶的活性,可以按照与后述的降低琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性相似的方法来进行。
而且,作为在棒状杆菌型细菌中赋予L-谷氨酸生产能力的方法,还可以采用下述方法:扩增yggB基因(NCgl 1221;NP_600492.Reportssmall-conductance...[gi:19552490])的方法,导入在编码区内导入突变而得到的突变型yggB基因的方法(WO2006/070944)。
此外,作为提高L-谷氨酸生产能力的方法,可以列举导入编码D-木酮糖-5-磷酸磷酸酮醇酶(D-xylulose-5-phosphate phosphoketolase)和/或果糖-6-磷酸磷酸酮醇酶(fructose-6-phosphate phosphoketolase)(这里合称磷酸酮醇酶)的基因的方法。例如,作为磷酸酮醇酶活性提高的微生物,可以列举以下微生物(WO2006/016705)。
乳发酵短杆菌ATCC13869ΔsucA(pVK9-xfp)、
乳发酵短杆菌ATCC13869ΔsucA(pVK9-PS2_xpkA)
L-谷氨酸生产能力可以通过增强6-磷酸葡糖酸脱水酶活性、或2-酮-3-脱氧-6-磷酸葡糖酸醛缩酶活性、或者这两者的活性来赋予。作为6-磷酸葡糖酸脱水酶活性、2-酮-3-脱氧-6-磷酸葡糖酸醛缩酶活性提高的微生物,可以列举出特开2003-274988公开的微生物。此外,L-谷氨酸生产能力还可以通过扩增作为L-谷氨酸分泌基因的yhfK基因来赋予(WO2005/085419)。
此外,作为用于本发明的L-谷氨酸生产微生物,可以使用具有于酸性条件下培养时在液体培养基中蓄积超过L-谷氨酸饱和浓度的量的L-谷氨酸之能力(以下也称为酸性条件下的L-谷氨酸蓄积能力)的微生物。例如,按照欧洲公开公报1078989号记载的方法,通过获取在低pH环境下对L-谷氨酸的抗性提高的菌株,能够赋予蓄积超过饱和浓度的量的L-谷氨酸的能力。
作为本来具有酸性条件下的L-谷氨酸蓄积能力的微生物,具体地可以列举出:Pantoea ananatis AJ13355株(FERM BP-6614)、AJ13356株(FERM BP-6615)、AJ13601株(FERM BP-7207)(以上,参照欧洲专利申请公开0952221号说明书)、SC17sucA株、SC17sucA/RSFCPG+pSTVCB株、NP106株和NA1株等。
Pantoea ananatis AJ13355株是作为低pH下能够在含L-谷氨酸和碳源的培养基中增殖的菌株从静冈县磐田市的土壤中分离出来的菌株。AJ13356为AJ13355株的αKGDH-E1亚基基因(sucA)缺损而得到的菌株。
Pantoea ananatis AJ13355株和AJ13356株已于平成10年(1998年)2月19日保藏于通产省工业技术院生命工学工业技术研究所(现名称为产业技术综合研究所专利生物保藏中心,地址:邮政编码305-8566茨城县筑波市东1丁目1番地1中央第6),保藏号分别为FERM P-16644和FERM P-16645,并于平成11年(1999年)1月11日转为基于布达佩斯条约的国际保藏,并给予保藏号FERM BP-6614和FERM BP-6615。而且,这些菌株在分离时被鉴定为成团肠杆菌(Enterobacter agglomerans),并当作成团肠杆菌AJ13354、AJ13355进行了保藏。但是,近年来,基于16S rRNA核苷酸序列分析等,它们被重新分类为Pantoea ananatis(参照后述实施例)。而下文所述的AJ13601同样也是在上述保藏机构中作为成团肠杆菌保藏的,而在本说明书中表述为Pantoea ananatis。AJ13601已于1999年8月18日保藏于工业技术院生命工学工业技术研究所(现名称为产业技术综合研究所专利生物保藏中心,地址:邮政编码305-8566茨城县筑波市东1丁目1番地1中央第6),保藏号为FERM P-17516,并且于2000年7月6日转为基于布达佩斯条约的国际保藏,并给与保藏号FERM BP-7207。
此外,作为Pantoea ananatis的L-谷氨酸生产菌,可以列举出α-酮戊二酸脱氢酶(αKGDH)活性缺损、或者αKGDH活性降低的属于泛菌属的细菌。作为这样的菌株有:前述的AJ13356株,以及从AJ13355株中作为粘液质低生产突变菌株选择出来的SC17株衍生的sucA基因缺损株SC17sucA(美国专利第6,596,517号)。SC17sucA株的内部编号为AJ417株,已于2004年2月26日保藏于产业技术综合研究所专利生物保藏中心(地址:邮政编码305-8566茨城县筑波市东1丁目1番地1中央第6),保藏号FERMBP-08646。
此外,上述SC17sucA/RSFCPG+pSTVCB株是在SC17sucA株中导入包含大肠杆菌来源的柠檬酸合酶基因(gltA)、磷酸烯醇丙酮酸羧化酶基因(ppsA) 和谷氨酸脱氢酶基因(gdhA)的质粒RSFCPG,以及包含乳发酵短杆菌来源的柠檬酸合酶基因(gltA)的质粒pSTVCB而得到的菌株。AJ13601株是从该SC17sucA/RSFCPG+pSTVCB株中作为低pH下显示出对高浓度L-谷氨酸的抗性的菌株而选择出的菌株。此外,NP106株是如实施例所述那样,质粒RSFCPG+pSTVCB从AJ13601株脱落而得到的菌株。
作为赋予或者增强L-谷氨酸生产能力的其它方法,还可以列举:赋予对有机酸类似物、呼吸链抑制剂等的抗性的方法,和赋予对细胞壁合成抑制剂的敏感性的方法。例如:赋予单氟乙酸抗性的方法(特开昭50-113209)、赋予腺嘌呤或胸腺嘧啶抗性的方法(特开昭57-065198)、削弱脲酶的方法(特开昭52-038088)、赋予丙二酸抗性的方法(特开昭52-038088)、赋予苯并吡喃酮或萘醌抗性的方法(特开昭56-1889)、赋予HOQNO抗性的方法(特开昭56-140895)、赋予α-酮丙二酸抗性的方法(特开昭57-2689)、赋予胍抗性的方法(特开昭56-35981)和赋予青霉素敏感性的方法(特开平4-88994),等等。
作为这样的抗性菌的具体例子,可以列举出下述菌株:
黄色短杆菌AJ3949(FERM BP-2632;参见特开昭50-113209)
谷氨酸棒杆菌AJ11628(FERM P-5736;参见特开昭57-065198)
黄色短杆菌AJ11355(FERM P-5007;参见特开昭56-1889号公报)
谷氨酸棒杆菌AJ11368(FERM P-5020;参见特开昭56-1889号公报)
黄色短杆菌AJ11217(FERM P-4318;参见特开昭57-2689号公报)
谷氨酸棒杆菌AJ11218(FERM P-4319;参见特开昭57-2689号公报)
黄色短杆菌AJ11564(FERM P-5472;参见特开昭56-140895号公报)
黄色短杆菌AJ11439(FERM P-5136;参见特开昭56-35981号公报)
谷氨酸棒杆菌H7684(FERM BP-3004;参见特开昭04-88994号公报)
乳发酵短杆菌AJ11426(FERM P-5123;参见特开昭56-048890号公报)
谷氨酸棒杆菌AJ11440(FERM P-5137;参见特开昭56-048890号公报)
乳发酵短杆菌AJ11796(FERM P-6402;参见特开昭58-158192号公报)。
具有L-谷氨酰胺生产能力的微生物的优选例子是:谷氨酸脱氢酶活性增强的细菌,谷氨酰胺合成酶(glnA)活性增强的细菌,和谷氨酰胺酶基因被破坏的细菌(欧洲专利申请公开第1229121号和欧洲专利申请公开1424398号)。谷氨酰胺合成酶的活性增强也可以通过谷氨酰胺腺苷酰转移酶基因(glnE)的破坏、PII调控蛋白(glnB)的破坏来实现。另外,作为优选 的L-谷氨酰胺生产菌的例子,可列举具有如下所述的突变型谷氨酰胺合成酶的、属于埃希氏菌属的细菌,所述突变型谷氨酰胺合成酶中谷氨酰胺合成酶第397位的酪氨酸残基被其它氨基酸残基取代(美国专利申请公开号2003-0148474)。
赋予或增强L-谷氨酰胺生产能力的其它方法包括:赋予6-重氮-5-氧-正亮氨酸抗性的方法(特开平3-232497),赋予嘌呤类似物和甲硫氨酸亚砜抗性的方法(特开昭61-202694),和赋予α-酮马来酸抗性(特开昭56-151495),等等。作为具有L-谷氨酰胺生产能力的棒状杆菌型细菌的具体实例,可列举下列微生物:
黄色短杆菌AJ11573(FERM P-5492;特开昭56-161495)
黄色短杆菌AJ11576(FERM BP-10381;特开昭56-161495)
黄色短杆菌AJ12212(FERM P-8123;特开昭61-202694)
作为具有L-脯氨酸生产能力的微生物,可列举例如携带解除了L-脯氨酸反馈抑制的γ-谷氨酰激酶的细菌;和削弱了L-脯氨酸分解系统的细菌。使用编码解除了L-脯氨酸反馈抑制的γ-谷氨酰激酶的DNA来修饰细菌的方法公开在Dandekar,A.M.,Uratsu,S.L.,J.Bacteriol.,170,12,5943-5(1988)中。此外,作为获得削弱了L-脯氨酸分解系统的细菌的方法,可列举通过向脯氨酸脱氢酶基因中引入导致酶活性降低的突变的方法。作为具有L-脯氨酸生产能力的细菌的具体实例,可列举大肠杆菌NRRL B-12403株、NRRL B-12404株(英国专利2075056)、VKPM B-8012株(美国专利公开2002-0058315),以及携带下述质粒突变体的菌株:德国专利第3127361号中公开的质粒突变体,或由Bloom等的文献(The 15th Miami WinterSymposium,1983,p.34)公开的质粒突变体。
此外,优选的具有L-脯氨酸生产能力的微生物包括大肠杆菌702株(VKPM B-8011),其对3,4-脱羟脯氨酸(3,4-dehydroxyproline)和氮杂环丁烷-2-羧酸具有抗性;702ilvA株(VKPM B-8012),其为702株的ilvA缺陷株;和由b2682基因、b2683基因、b1242基因或b3434基因编码的蛋白活性增强的大肠杆菌(特开2002-300874号公报),等等。
作为棒状杆菌型细菌中的L-脯氨酸生产菌,可以列举:DL-3,4-脱氢脯氨酸抗性株(FERM BP-1219.美国专利4224409号公报)、柠檬酸合成酶活性比亲本菌株提高至1.4倍以上的菌株(FERM P-5332、FERM P-5333、FERM P-5342、FERMP-5343日本专利1426823号)、赋予了乙酸营养缺陷性的菌株(FERM P-5931)。
作为具有L-亮氨酸生产能力的细菌,例如可列举表现出对4-氮杂亮氨酸或5,5,5-三氟亮氨酸的抗性的大肠杆菌H-9068株(ATCC 21530)、H-9070株(FERM BP-4704)和H-9072株(FERM BP-4706)(美国专利5,744,331);以及携带解除了L-亮氨酸反馈抑制的异丙基苹果酸合酶(isopropyl malatesynthase)的大肠杆菌(欧洲专利1067191);对β-2-噻吩丙氨酸和β-羟亮氨酸显示抗性的大肠杆菌AJ11478株(美国专利5,763,231)、大肠杆菌57株(VKPM B-7386,俄罗斯专利2140450)等。
作为棒状杆菌型细菌中的L-亮氨酸生产菌,可以列举:2-噻唑丙氨酸和β-羟基亮氨酸抗性菌株(特开平8-266295),缬氨酸类似物抗性菌株(特开昭63-248392),缬氨酸营养缺陷菌株(特公昭38-4395),S-(2-氨基乙基)-L-半胱氨酸(AEC)抗性菌株(特公昭51-37347),苯丙氨酸、缬氨酸和异亮氨酸营养缺陷菌株(特公昭54-36233)等。
作为具有L-半胱氨酸生产能力的微生物,可列举:用编码解除了反馈抑制的丝氨酸乙酰转移酶的cysE基因等位基因转化的大肠杆菌JM15株(美国专利6,218,168);负责细胞毒素物质分泌的蛋白质的编码基因过量表达的大肠杆菌W3110株(美国专利5,972,663);半胱氨酸脱巯基酶活性降低的大肠杆菌(特开平11-155571 A);和其中由cysB基因编码的半胱氨酸调节子的转录激活因子已被扩增的大肠杆菌W3110株(WO 01/27307)等。
作为具有L-异亮氨酸生产能力的微生物,可以列举:显示6-二甲基氨基嘌呤抗性的埃希氏菌属突变菌株(特开平5-304969),对L-异亮氨酸异羟肟酸(L-isoleucine hydroxamate)、硫代异亮氨酸(thiaisoleucine)、DL-乙硫氨酸或精氨酸异羟肟酸显示抗性的突变菌株(特开平5-130882),和具有经扩增的苏氨酸脱氨酶基因和乙酰羟基化物合酶(acetohydroxylate synthase)基因的重组菌株(特开平2-458、特开平2-42988和特开平8-47397)等。
作为棒状杆菌型细菌中的L-异亮氨酸生产菌,可以列举:扩增了编码分枝氨基酸分泌蛋白的brnE基因的棒状杆菌型细菌(特开2001-169788),通过与L-赖氨酸生产菌进行原生质体融合而赋予了L-异亮氨酸生产能力的棒状杆菌型细菌(特开昭62-74293),强化了高丝氨酸脱氢酶的棒状杆菌型细菌(特开昭62-91193),苏氨酸异羟肟酸(スレォ二ンハィドロキサメ一 ト)抗性菌株(特开昭62-195293),α-酮丙二酸抗性菌株(特开昭61-15695),和甲基赖氨酸抗性菌株(特开昭61-15696)。
可以通过例如下述方法赋予L-缬氨酸生产能力:增加由ilvGMEDA操纵子编码的L-缬氨酸合成酶,特别是由ilvG基因编码的乙酰羟基化物合酶的活性(特公平02-748418)。L-缬氨酸合成酶也可以是解除了L-缬氨酸的反馈抑制的L-缬氨酸合成酶。还可以通过降低乙酰乳酸合酶III基因(ilvIH基因)的表达来赋予L-缬氨酸生产能力。
此外,可以通过赋予细菌对氨基酸类似物的抗性来赋予L-缬氨酸生产能力。作为这样的细菌的实例,例如,可列举对L-异亮氨酸和L-甲硫氨酸为营养缺陷性,而且对D-核糖、嘌呤核苷或嘧啶核糖核苷显示抗性的突变株(FERM P-1841、FERM P-5556;特开昭53-025034);和对聚酮化合物(polyketide)显示抗性的突变株(FERM P-9325;特许第1934507号)。
作为L-缬氨酸生产菌的例子,还可以列举出氨酰t-RNA合成酶具有突变的突变菌株(美国专利5,658,766)。例如,可以使用大肠杆菌VL1970,该菌株的编码异亮氨酸tRNA合成酶的ileS基因具有突变。大肠杆菌VL1970于1988年6月24日保藏于俄罗斯国立工业微生物保藏中心(Russian NationalCollection of Industrial Microorganisms)(VKPM)(1Dorozhny proezd.,1Moscow 117545,Russia),保藏号为VKPM B-4411。
而且,生长需要硫辛酸和/或缺失H+-ATPase的突变株可以用作亲本株(WO96/06926)。
作为属于棒状杆菌型细菌的L-缬氨酸生产菌,例如有通过修饰而增强了编码L-缬氨酸生物合成相关酶的基因的表达的菌株。作为L-缬氨酸生物合成相关酶,例如,可列举由ilvBNC操纵子,即由ilvBN编码的乙酰羟酸合成酶和由ilvC编码的异构还原酶(isomero-reductase)(国际公布小册子WO00/50624)。而且,ilvBNC操纵子受到L-缬氨酸和/或L-异亮氨酸和/或L-亮氨酸所致的操纵子的表达调控,所以最好解除弱化作用(releaseattenuation),以消除生成的L-缬氨酸所致的表达抑制。
作为具有L-丙氨酸生产能力的微生物,例如,可列举H+-ATP酶活性缺陷的棒状杆菌型细菌(Appl Microbiol Biotechnol.2001 Nov;57(4):534-40);和天冬氨酸β-脱羧酶的基因经过扩增的棒状杆菌型细菌(特开平07-163383)等。
作为具有L-精氨酸生产能力的微生物,可列举对α-甲基甲硫氨酸、对氟苯丙氨酸、D-精氨酸、精氨酸异羟肟酸、AEC(S-(2-氨乙基)-半胱氨酸)、α-甲基丝氨酸、β-2-噻吩丙氨酸或磺胺胍具有抗性的大肠杆菌突变菌株(特开昭56-106598号公报)。此外,具有对L-精氨酸的反馈抑制的抗性突变,并携带具有高活性的N-乙酰谷氨酸合酶的L-精氨酸生产菌大肠杆菌237株(俄罗斯专利申请第2000117677号)也是合适的L-精氨酸生产菌株。该菌株在2000年4月10日保藏在俄罗斯国立工业微生物保藏中心(RussianNational Collection of Industrial Microorganisms(VKPM),GNII Genetika),并给出了登录号VKPM B-7925,按照布达佩斯条约的规定,于2001年5月18日转为国际保藏。也可以使用大肠杆菌382株,它是237菌株的衍生菌株,是改善了同化乙酸的能力的L-精氨酸生产菌(特开2002-017342号公报)。大肠杆菌382株在2000年4月10日保藏在俄罗斯国立工业微生物保藏中心(VKPM),并给出了登录号VKPM B-7926。
此外,作为具有L-精氨酸生产能力的微生物,可以使用编码L-精氨酸生物合成相关酶的基因的表达量得到提高的微生物。例如,作为L-精氨酸生物合成系统酶,可以列举出选自N-乙酰谷氨酸合酶(argA)、N-乙酰谷氨酰-磷酸还原酶(argC)、鸟氨酸乙酰转移酶(argJ)、N-乙酰谷氨酸激酶(argB)、乙酰鸟氨酸转氨酶(argD)、乙酰鸟氨酸脱乙酰酶(argE)、鸟氨酸氨基甲酰转移酶(argF)、精氨琥珀酸合酶(argG)、精氨琥珀酸裂合酶(argH)和氨甲酰磷酸合酶(carAB)中的1种或2种以上。对于N-乙酰谷氨酸合酶(argA),使用这样的突变型的基因是更优选的:所述突变型对应于野生型第15至19位的氨基酸序列被取代,L-精氨酸所致的反馈抑制得以解除(欧洲申请公开1170361号说明书)。
就属于棒状杆菌型细菌的L-精氨酸生产菌而言,没有特殊限制,只要具有L-精氨酸生产能力即可,可以列举下列:棒状杆菌型细菌野生型菌株;对磺胺类药、2-噻唑丙氨酸或α-氨基-β-羟基戊酸等药剂具有抗性的棒状杆菌型细菌;除2-噻唑丙氨酸抗性外,还具有L-组氨酸、L-脯氨酸、L-苏氨酸、L-异亮氨酸、L-甲硫氨酸或L-色氨酸营养缺陷性的棒状杆菌型细菌(特开昭54-4409号);对酮丙二酸、氟丙二酸(fluoromalonic acid)或单氟乙酸有抗性的棒状杆菌型细菌(特开昭57-18989号);对精氨醇(argininol)有抗性的棒状杆菌型细菌(特开昭62-24075号);或者对x-胍有抗性的棒状杆菌型 细菌(x是脂肪酸或脂肪链衍生物)(特开平2-186995号)等。
此外,具有L-精氨酸生产能力的棒状杆菌型细菌可以作为下述菌株来选育:对5-氮尿嘧啶、6-氮尿嘧啶、2-硫尿嘧啶、5-氟尿嘧啶、5-溴尿嘧啶、5-氮胞嘧啶、6-氮胞嘧啶等具有抗性的突变菌株;对精氨酸异羟肟酸或2-硫尿嘧啶具有抗性的突变菌株,对精氨酸异羟肟酸和6-氮尿嘧啶具有抗性的突变菌株(特开昭49-126819号);对组氨酸类似物或色氨酸类似物具有抗性的突变菌株(特开昭52-114092号);对甲硫氨酸(メチ二ォン)、组氨酸、苏氨酸、脯氨酸、异亮氨酸(ィソロィシィン)、赖氨酸、腺嘌呤、鸟嘌呤或者尿嘧啶(或者尿嘧啶前体)中的至少一种具有营养缺陷性的突变菌株(参见特开昭52-99289号);对精氨酸异羟肟酸具有抗性的突变菌株(特公昭51-6754号);琥珀酸营养缺陷性或对核酸碱基类似物具有抗性的突变菌株(特开昭58-9692号);精氨酸分解能力缺损、对精氨酸拮抗物和刀豆氨酸具有抗性、赖氨酸营养缺陷的突变菌株(特开昭52-8729号);精氨酸、精氨酸异羟肟酸、高精氨酸、D-精氨酸、刀豆氨酸抗性、精氨酸异羟肟酸和6-氮尿嘧啶抗性的突变菌株(特开昭53-143288号);以及刀豆氨酸抗性的突变菌株(特开昭53-3586号),等等。
作为具有L-精氨酸生产能力的棒状杆菌型细菌的具体例子,可以列举出下述菌株。
黄色短杆菌(Brevibacterium flavum)AJ11169(FERM P-4161)
乳发酵短杆菌AJ12092(FERM P-7273)
黄色短杆菌AJ11336(FERM P-4939)
黄色短杆菌AJ11345(FERM P-4948)
乳发酵短杆菌AJ12430(FERM BP-2228)
而且,还可以使用精氨酸阻遏物ArgR缺损的菌株(美国专利申请公开2002-0045223号)、细胞内谷氨酰胺合成酶活性提高的菌株(美国专利申请公开2005-0014236号公报)。
L-瓜氨酸和L-鸟氨酸的生物合成途径与L-精氨酸的生物合成途径也是共通的,可以通过增加下述酶的酶活性来赋予这些氨基酸的生产能力:N-乙酰谷氨酸合酶(argA)、N-乙酰谷氨酰磷酸还原酶(argC)、鸟氨酸乙酰转移酶(argJ)、N-乙酰谷氨酸激酶(argB)、乙酰鸟氨酸转氨酶(argD)和乙酰鸟氨酸脱乙酰酶(argE)(国际公开2006-35831号小册子)。
作为具有L-赖氨酸生产能力的微生物,例如可列举具有L-赖氨酸生产能力的L-赖氨酸类似物抗性菌株或代谢调控突变菌株。其具体可列举:大肠杆菌AJ11442株(FERM BP-1543,NRRL B-12185;参考特开昭56-18596号公报和美国专利4,346,170号说明书)和大肠杆菌VL611株(特开2000-189180号公报)等。此外,作为大肠杆菌L-赖氨酸生产菌,也可使用WC196株(参考国际公布第96/17930号小册子)。WC196菌株是通过赋予源自大肠杆菌K-12菌株的W3110菌株以对AEC(S-(2-氨乙基)-半胱氨酸)的抗性而选育的。WC196菌株被命名为大肠杆菌AJ13069菌株,并于平成6年(1994年)12月6日保藏在工业技术院生命工学工业技术研究所(现称独立行政法人产业技术综合研究所专利生物保藏中心,305-8566日本国茨城县筑波市东1丁目1番地1中央第6),保藏号FERM P-14690,其后根据布达佩斯条约的规定于平成7年(1995年)9月29日转为国际保藏,给出登录号FERM BP-5252。
就属于棒状杆菌型细菌的L-赖氨酸生产菌而言,作为具有L-赖氨酸生产能力的棒状杆菌型细菌,可以列举:S-(2-氨基乙基)-半胱氨酸(以下简写为“AEC”)抗性突变菌株(乳发酵短杆菌AJ11082(NRRL B-11470)株等:参照特公昭56-1914号公报、特公昭56-1915号公报、特公昭57-14157号公报、特公昭57-14158号公报、特公昭57-30474号公报、特公昭58-10075号公报、特公昭59-4993号公报、特公昭61-35840号公报、特公昭62-24074号公报、特公昭62-36673号公报、特公平5-11958号公报、特公平7-112437号公报、特公平7-112438号公报);生长需要L-高丝氨酸等氨基酸的突变菌株(参照特公昭48-28078号公报、特公昭56-6499号公报);显示AEC抗性,且需要L-亮氨酸、L-高丝氨酸、L-脯氨酸、L-丝氨酸、L-精氨酸、L-丙氨酸、L-缬氨酸等氨基酸的突变菌株(参照美国专利第3708395号和第3825472号说明书);对DL-α-氨基-ε-己内酰胺、α-氨基-十二内酰胺、天冬氨酸-类似物、磺胺类药、醌型化合物(キノィド)、N-月桂酰亮氨酸显示抗性的L-赖氨酸生产突变菌株;对草酰乙酸脱碳酸酶(脱羧酶)或者呼吸体系酶抑制剂显示抗性的L-赖氨酸生产突变菌株(特开昭50-53588号公报、特开昭50-31093号公报、特开昭52-102498号公报、特开昭53-9394号公报、特开昭53-86089号公报、特开昭55-9783号公报、特开昭55-9759号公报、特开昭56-32995号公报、特开昭56-39778号公报、特公昭53-43591号公报、特公昭53-1833 号公报);需要肌醇或者乙酸的L-赖氨酸生产突变菌株(特开昭55-9784号公报、特开昭56-8692号公报);对氟丙酮酸或者34℃以上的温度呈敏感性的L-赖氨酸生产突变菌株(特开昭55-9783号公报、特开昭53-86090号公报);对乙二醇呈抗性、且产生L-赖氨酸的短杆菌属或者棒杆菌属的生产突变菌株(美国专利第4411997号说明书);等等。
还可以通过增加L-赖氨酸生物合成系统的酶活性而获得赋予了L-赖氨酸生产能力的微生物。这些酶活性可以通过下述方法增加:增加细胞内编码所述酶的基因的拷贝数,或者修饰表达调控序列。
作为编码L-赖氨酸生物合成系统酶的基因,可以列举:编码二氨基庚二酸途径的酶的基因,如二氢吡啶二羧酸合酶基因(dapA)、天冬氨酸激酶基因(lysC)、二氢吡啶二羧酸还原酶基因(dapB)、二氨基庚二酸脱羧酶基因(lysA)、二氨基庚二酸脱氢酶基因(ddh)(以上,国际公布第96/40934号小册子)、磷酸烯醇丙酮酸羧化酶基因(ppc)(特开昭60-87788号公报)、天冬氨酸氨基转移酶基因(aspC)(特公平6-102028号公报)、二氨基庚二酸差向异构酶基因(dapF)(特开2003-135066号公报)和天冬氨酸半醛脱氢酶基因(asd)(国际公布第00/61723号小册子)等;和编码氨基己二酸途径的酶如高乌头酸水合酶(特开2000-157276号公报)等的基因。
此外,作为天冬氨酸激酶III基因(lysC),优选使用经过修饰从而不受L-赖氨酸所致的反馈抑制的基因。这种经过修饰而不受反馈抑制的lysC基因可以通过美国专利5,932,453号说明书中描述的方法获得。
此外,在具有L-赖氨酸生产能力的微生物中,催化产生除L-赖氨酸之外的产物的反应的酶的活性或者对L-赖氨酸产生起负作用的酶的活性可以是降低的或者缺损的。作为这样的酶,可列举高丝氨酸脱氢酶、赖氨酸脱羧酶(cadA、ldcC)和苹果酸酶,国际公布第WO 95/23864号、第WO96/17930号小册子、第WO 2005/010175号小册子等中描述了这些酶的活性降低或缺失的菌株。
作为具有L-色氨酸生产能力的微生物,优选下述酶活性中的一种或两种以上的活性增强的细菌:邻氨基苯甲酸合成酶活性、磷酸甘油酸脱氢酶活性或色氨酸合酶活性。邻氨基苯甲酸合成酶和磷酸甘油酸脱氢酶分别受L-色氨酸和L-丝氨酸反馈抑制,因此,可以通过使其携带脱敏型的突变酶来强化酶活性。具体地说,例如,使邻氨基苯甲酸合成酶基因(trpE)和/或 磷酸甘油酸脱氢酶基因(serA)突变,使它们不受反馈抑制,并且将所得突变型基因引入细菌,这样可以获得携带脱敏型酶的细菌。作为这样的细菌,更具体地可以列举通过用质粒pGH5(参见国际公布第94/08031号小册子)转化大肠杆菌SV164而获得的转化菌株,其中所述大肠杆菌SV164携带有脱敏型邻氨基苯甲酸合成酶,而质粒pGH5包含编码脱敏型磷酸甘油酸脱氢酶的突变型serA基因。
此外,还可以通过引入包含色氨酸操纵子的重组DNA而赋予L-色氨酸生产能力。具体地说,可列举导入了包含编码脱敏型邻氨基苯甲酸合成酶的基因的色氨酸操纵子的大肠杆菌(特开昭57-71397号公报,特开昭62-244382号公报和美国专利4,371,614说明书)。此外,通过强化色氨酸操纵子中编码色氨酸合酶(trpBA)的基因的表达,也可以增强或赋予L-色氨酸生产能力。色氨酸合酶由α和β亚基组成,其分别由trpA和trpB编码。
此外,还可以通过缺损编码色氨酸操纵子阻遏物的trpR,或者向trpR导入可降低阻遏物活性的突变,从而赋予L-色氨酸生产能力(美国专利第4,371,614号公报、国际公布第WO2005/056776号小册子)。
此外,这样的细菌也是适宜的L-色氨酸生产菌:所述细菌中苹果酸合成酶、异柠檬酸裂合酶、异柠檬酸脱氢酶激酶/磷酸酶操纵子(ace操纵子)为组成型表达,或者该操纵子的表达得到增强。具体地说,优选ace操纵子的启动子不受阻遏物iclR的抑制,或者抑制被解除,这样的细菌可以通过破坏iclR基因来实现。
可以通过下述方法实现ace操纵子的表达增强的细菌:将包含ace操纵子的DNA与强启动子连接,并且通过质粒或同源重组将其导入细菌内,或者利用转座子使上述DNA以多拷贝存在。
而且,作为具有L-色氨酸生产能力的微生物,可以列举出:大肠杆菌AGX17(pGX44)菌株[NRRL B-122363],其表现为L-苯丙氨酸和L-酪氨酸-营养缺陷表型;和AGX6(pGX50)aroP[NRRL B-12264]株,其携带包含色氨酸操纵子的质粒pGX50(均可参见美国专利第4,371,614号说明书)。
作为具有L-色氨酸生产能力的棒状杆菌型细菌,可以使用:磺胺脒(サルファグァ二ジン)抗性菌株谷氨酸棒杆菌AJ12118(FERM BP-478特许01681002号),导入了色氨酸操纵子的棒状杆菌型细菌(特开S63240794号公报),导入了棒状杆菌型细菌来源的莽草酸激酶的编码基因的棒状杆菌型 细菌(特开01994749号公报)。
L-色氨酸、L-苯丙氨酸和L-酪氨酸都是芳香族氨基酸,生物合成系统是共通的。编码芳香族氨基酸生物合成体系酶的基因包括例如脱氧阿拉伯-庚酮糖酸磷酸合酶(deoxyarabino-heptulosonate phosphate synthase)(aroG)、3-脱氢奎宁酸合酶(aroB)、莽草酸脱氢酶(aroE)、莽草酸激酶(aroL)、5-烯醇丙酮酰莽草酸3-磷酸合成酶(5-enolpyruvylshikimate 3-phosphate synthase)(aroA)和分支酸合成酶(aroC)。(欧洲专利申请公布763127号说明书)。因此,可以通过使编码这些酶的基因在质粒中或染色体上多拷贝化,来增强芳香族氨基酸产生能力。此外,已知这些基因受酪氨酸阻抑物(tyrR)的调控,因此也可以通过缺损tyrR基因来增加芳香族氨基酸的生物合成系统酶活性(参见欧洲专利763127号说明书)。
作为具有L-苯丙氨酸生产能力的微生物,可以列举出下述属于埃希氏菌属的菌株,但不限于此:tyrA、tyrR缺损的大肠杆菌AJ12739(tyrA::Tn10,tyrR)(VKPM B-8197),携带突变型pheA34基因的大肠杆菌HW1089(ATCC55371)(美国专利第5,354,672号)、大肠杆菌MWEC101-b(KR8903681)、大肠杆菌NRRL B-12141,NRRL B-12145,NRRL B-12146和NRRL B-12147(美国专利第4,407,952号)等。此外,还可以使用大肠杆菌K-12[W3110(tyrA)/pPHAB](FERM BP-3566)、大肠杆菌K-12[W3110(tyrA)/pPHAD](FERM BP-12659)、大肠杆菌K-12[W3110(tyrA)/pPHATerm](FERMBP-12662)以及命名为AJ 12604的大肠杆菌K-12[W3110(tyrA)/pBR-aroG4,pACMAB](FERM BP-3579)(EP 488424 B1)。此外,还可以列举出苯丙氨酸分泌基因yddG、yedA扩增株(国际公开第03/044192号小册子、美国专利申请公开2003/0148473 A1和2003/0157667 A1)。
作为属于棒状杆菌型细菌的苯丙氨酸生产菌,可以使用磷酸烯醇丙酮酸羧化酶或者丙酮酸激酶活性降低的谷氨酸棒杆菌BPS-13株(FERMBP-1777,K77(FERM BP-2062)和K78(FERM BP-2063)(欧洲专利公开公报331145号JP 02303495),酪氨酸营养缺陷性菌株(JP 05049489)等。
具有L-苏氨酸生产能力的微生物的优选实例可列举L-苏氨酸生物合成系统酶活性增强的属于肠杆菌科的微生物。作为编码L-苏氨酸生物合成系统酶的基因,可列举天冬氨酸激酶III基因(lysC)、天冬氨酸半醛脱氢酶基因(asd)、由thr操纵子编码的天冬氨酸激酶I基因(thrA)、高丝氨酸激酶基因 (thrB)、苏氨酸合酶基因(thrC)。括号中为该基因的简写符号。可以导入这些基因中的两种或更多种。可以将L-苏氨酸生物合成系统基因导入L-苏氨酸分解受到抑制的埃希氏菌属细菌。作为L-苏氨酸降解受到抑制的埃希氏菌属细菌,例如,可列举苏氨酸脱氢酶活性缺陷的TDH6菌株(特开2001-346578)。
L-苏氨酸生物合成系统酶的酶活性受终产物L-苏氨酸的抑制。因此,为了构建L-苏氨酸生产菌,优选修饰L-苏氨酸生物合成系统基因,使之不受L-苏氨酸的反馈抑制。此外,上述thrA、thrB和thrC基因组成苏氨酸操纵子,苏氨酸操纵子形成弱化子结构,苏氨酸操纵子的表达受培养液中异亮氨酸、苏氨酸的抑制,在弱化作用下表达被抑制。这样的修饰可以通过去除弱化区域中的前导序列或弱化子来实现(Lynn,S.P.,Burton,W.S.,Donohue,T.J.,Gould,R.M.,Gumport,R.I.,and Gardner,J.F.J.Mol.Biol.194:59-69(1987);国际公布第02/26993号和国际公布第2005/049808号)。
苏氨酸操纵子的上游存在固有的启动子,但也可以替换成非天然启动子(参见WO 98/04715号小册子),或者,可以构建这样的苏氨酸操纵子,使得苏氨酸生物合成相关基因的表达受λ噬菌体的阻遏物和启动子的控制(参照欧洲专利第0593792号说明书)。此外,为了修饰埃希氏菌属细菌以不受L-苏氨酸反馈抑制,可以选择对α-氨基-β-羟基戊酸(AHV)具有抗性的菌株。
对于经修饰而不受L-苏氨酸反馈抑制的苏氨酸操纵子,优选的是其在宿主内的拷贝数增加,或者其与强启动子连接而提高表达量。拷贝数的增加除了用质粒进行扩增外,还可以通过利用转座子、Mu-噬菌体等将苏氨酸操纵子转移至基因组上来实现。
除L-苏氨酸生物合成系统酶以外,还优选增强下述基因:糖酵解途径、TCA循环和呼吸链相关基因或调控基因表达的基因,和糖摄取基因。作为这些对L-苏氨酸生产有效的基因,可列举转氢酶(pntAB)基因(欧洲专利733712号说明书)、磷酸烯醇丙酮酸羧化酶基因(pepC)(国际公布第95/06114号小册子)、磷酸烯醇丙酮酸合酶基因(pps)(欧洲专利877090号说明书),和棒状杆菌型细菌或芽孢杆菌属细菌的丙酮酸羧化酶基因(国际公布第99/18228号小册子和欧洲申请公开1092776号说明书)。
此外,还优选增强赋予L-苏氨酸抗性的基因、赋予L-高丝氨酸抗性的 基因的表达,或者对宿主赋予L-苏氨酸、L-高丝氨酸抗性。作为赋予抗性的基因,可列举rhtA基因(Res.Microbiol.154:123-135(2003))、rhtB基因(欧洲专利申请公开第0994190号说明书)、rhtC基因(欧洲专利申请公开第1013765号说明书)、yfiK基因和yeaS基因(欧洲专利申请公开第1016710号说明书)。此外,赋予宿主L-苏氨酸抗性的方法还可以参照欧洲专利申请公开第0994190号说明书或国际公布第90/04636号小册子中所述的方法。
作为具有L-苏氨酸生产能力的微生物,还可以大肠杆菌VKPM B-3996株(参考美国专利第5,175,107号说明书)为例来说明。该VKPM B-3996菌株在1987年11月19日保藏在俄罗斯国立工业微生物保藏中心(VKPM),GNII Genetika,给出的登录号为VKPM B-3996。VKPM B-3996株携带质粒pVIC40(国际公布第90/04636号小册子),该质粒是通过向pAYC32中插入苏氨酸生物合成系统基因(苏氨酸操纵子:thrABC)而获得,pAYC32是一种具有链霉素抗性标记的广泛宿主范围的质粒载体(Chistorerdov,A.Y.,and Tsygankov,Y.D.Plasmid,16,161-167(1986))。在该pVIC40中,苏氨酸操纵子中的thrA所编码的天冬氨酸激酶L-高丝氨酸脱氢酶I解除了L-苏氨酸的反馈抑制。
此外,大肠杆菌B-5318菌株(参照欧洲专利第0593792号说明书)也可作为被赋予了优选的L-苏氨酸生产能力的细菌的一个实例。B-5318菌株在1987年11月19日保藏在俄罗斯国立工业微生物保藏中心(VKPM),GNIIGenetika,保藏号为VKPM B-5318。该B-5318菌株是异亮氨酸原养型,并且携带如下构建的重组质粒DNA:其中,缺失了其原本具有的转录调节区域——弱化子区的苏氨酸操纵子(即苏氨酸生物合成的相关基因)位于λ噬菌体的温度敏感型C1阻遏物、PR启动子和Cro蛋白N末端部分的下游,从而使苏氨酸生物合成基因表达受λ噬菌体的阻遏物和启动子的调控。
在本发明中使用的L-氨基酸生产菌中,除编码天然生物合成系统酶的基因之外,还可扩增糖摄取、糖代谢(糖酵解系统)和能量代谢的相关基因。
作为糖代谢的相关基因,可列举编码糖酵解系统酶的基因或糖摄取基因,如葡糖-6-磷酸异构酶基因(pgi;国际公布第01/02542号小册子)、磷酸烯醇丙酮酸合酶基因(pps,欧洲申请公开877090号说明书)、磷酸葡糖变位酶基因(pgm;国际公布第03/04598号小册子)、果糖二磷酸醛缩酶基因 (fbp;国际公布第03/04664号小册子)、丙酮酸激酶基因(pykF;国际公布03/008609号小册子)、转醛醇酶(transaldolase)基因(talB;国际公布03/008611号小册子)、延胡索酸酶基因(fum;国际公布01/02545号小册子)、磷酸烯醇丙酮酸合酶基因(pps;欧洲申请公开877090)、非-PTS蔗糖摄取基因(csc;欧洲申请公开149911)和蔗糖同化基因(scrAB操纵子;国际公布第90/04636号小册子)。
能量代谢中涉及的基因的实例包括转氢酶基因(pntAB;美国专利第5,830,716号说明书)和细胞色素bo型氧化酶(cytochrome bo type oxidase)基因(cyoB;欧洲专利申请公开1070376号说明书)。
用于本发明的微生物是具有如上述的L-氨基酸生产能力的微生物,并且该微生物已被修饰使得琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性降低。
<1-2>琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性的降低
在本发明中,“酶活性降低”是指:与非修饰菌株、例如亲本菌株或野生型菌株相比,琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性低,也包括酶活性完全消失。
琥珀酸脱氢酶(以下也记作“SDH”)是可逆地催化以下反应的EC:1.3.99.1的酶。在本发明中,SDH活性是指催化该反应的活性。
根据微生物物种的不同,SDH由3或4个亚基的结构所构成,这其中至少1种蛋白经过修饰而不正常发挥功能。具体地,SDH由以下亚基(括号内为编码亚基的基因名)构成,而因物种而异,膜锚定蛋白(membrane anchorprotein)可以由sdhC单独编码,或者由sdhC和sdhD编码。
SDHA:黄素蛋白亚基(sdhA)
SDHB:Fe-S蛋白亚基(sdhB)
SDHC:膜锚定蛋白(sdhC)
SDHD:膜锚定蛋白(sdhD)
此外,有些情况下,SDH亚基复合体具有SDH和富马酸还原酶这两者的活性。例如,棒状杆菌型细菌的SDH亚基复合体具有SDH与富马酸还原酶这两者的活性(WO2005/021770)。
SDH活性的确认可以通过以2,6-二氯靛酚(2,6-dichloroindophenol)(DCIP) 的还原为指标进行测定来进行。具体方法记载于Tatsuki Kurokawa andJunshi Sakamoto,Arch Microbiol(2005)183:317-324。
在本发明中,将编码各种SDH亚基的基因、以及包含这些基因的操纵子统称为“编码SDH的基因”。
作为编码肠杆菌SDH的基因,Pantoea ananatis基因的核苷酸序列和各亚基的氨基酸序列如SEQ ID NO:1~6所示。
作为编码棒状杆菌型细菌的SDH的基因,例如,谷氨酸棒杆菌的sdh操纵子(GenBank登录号.NCgl0359(sdhC)NCgl0360(sdhA)NCgl0361(sdhB))和黄色短杆菌的sdh操纵子(特开2005-095169号、EP1672077A1)的序列已公开。
作为编码棒状杆菌型细菌的SDH的基因,谷氨酸棒杆菌ATCC13032的基因的核苷酸序列和各亚基的氨基酸序列示于SEQ ID NO:73~76,乳发酵短杆菌(谷氨酸棒杆菌)ATCC13869的基因的核苷酸序列和各亚基示于SEQID NO:77~80。
在本发明中,α-酮戊二酸脱氢酶(以下也称“α-KGDH”)活性是指催化α-酮戊二酸(2-氧代戊二酸)氧化脱碳酸、生成琥珀酰-CoA(succinyl-CoA)的反应的活性。上述反应由α-KGDH(E1o:α-酮戊二酸脱氢酶,EC:1.2.4.2)、二氢硫辛酰胺S-琥珀酰转移酶(E2o:dihydrolipoamide-S-succinyltransferase;EC:2.3.1.61)、二氢硫辛酰胺脱氢酶(E3:dihydrolipoamide dehydrogenase;EC:1.8.1.4)这3种酶来催化。即,这3种亚基分别催化以下反应,而将催化这3个反应合起来的反应的活性称为α-KGDH活性。α-KGDH活性的确认可以通过Shiio等的方法(Isamu Shiio and Kyoko Ujigawa-Takeda,Agric.Biol.Chem.,44(8),1897-1904,1980)来进行测定。
E1o:2-氧代戊二酸(2-oxoglutarate)+[二氢硫辛酰赖氨酸残基琥珀酰转移酶(dihydrolipoyllysine-residue succinyltransferase)]硫辛酰赖氨酸(lipoyllysine)=[二氢硫辛酰赖氨酸残基琥珀酰转移酶]S-琥珀酰二氢硫辛酰赖氨酸(S-succinyldihydrolipoyllysine)+CO2
E2o:CoA+酶N6-(S-琥珀酰二氢硫辛酰)赖氨酸=琥珀酰-CoA+酶N6-(二氢硫辛酰)赖氨酸
E3:蛋白N6-(二氢硫辛酰)赖氨酸+NAD+=蛋白N6-(硫辛酰)赖氨酸+NADH+H+
而且,α-KGDH也称为氧代戊二酸脱氢酶(oxoglutarate dehydrogenase)或2-氧代戊二酸脱氢酶(2-oxoglutarate dehydrogenase)。
在肠杆菌科例如Pantoea ananatis中,分别具有这3种酶活性的亚基蛋白形成复合体。而且,各亚基分别由各个sucA、sucB和lpd编码,sucA、sucB基因存在于琥珀酸脱氢酶铁-硫蛋白基因(sdhB)的下游(美国专利第6,331,419号)。而且,上述专利中将这些基因描述为成团肠杆菌AJ13355的基因,而该菌株后来被重新分类为Pantoea ananatis。
作为编码肠杆菌α-KGDH的基因,Pantoea ananatis的sucA、sucB以及存在于下游的sucC基因的核苷酸序列和各亚基的氨基酸序列如SEQ IDNO:7~11所示。此外,编码大肠杆菌α-KGDH的sucA、sucB和sucC基因分另公开于GenBank NP_415254,NP_415255。
此外,在棒状杆菌型细菌中,E1o亚基由odhA基因(也称sucA基因,作为GenBank登录号.NC_003450的NCgl1084登录)编码,E3亚基由lpd基因(GenBank登录号.Y16642)编码。另一方面,据推测,E2o亚基与E1o亚基一起以双功能性蛋白的形式由odhA基因所编码(参照Usuda et al.,Microbiology 1996 142,3347-3354),或者由与odhA基因不同的、以GenBank登录号NC_003450的NCgl2126登录的基因所编码。因此,在本发明中,odhA基因是编码E1o亚基的基因,但其也可以同时编码E2o。
乳发酵短杆菌odhA基因的核苷酸序列和E1o亚基的氨基酸序列(GenBank登录号.NC_003450的NCgl1084、WO2006/028298)示于SEQ IDNO:12、13。此外,上述GenBank登录号.NC_003450的NCgl2126的核苷酸序列和由该序列编码的E2o亚基的氨基酸序列示于SEQ ID NO:14、15。
在本发明中,编码各α-KGDH亚基的基因、以及包含这些基因的基因簇统称为“编码α-KGDH的基因”。
可以通过例如以下方法来降低各种酶活性。
(1)通过基因重组来破坏编码各酶的基因的方法
通过基因重组对编码SDH或α-KGDH(以下也简称为“酶”)的基因进行修饰,这样可以相对于亲本菌株或野生型菌株减少平均每个细胞的这些基因所编码的酶蛋白分子的数目,或者完全不生成酶蛋白分子。此外,也可以降低或消除酶蛋白平均每个分子的酶活性。酶蛋白分子的数目可以通过降低编码该酶的基因的表达量来减少。基因表达量的降低包括由该基因转 录的mRNA的转录量的降低、以及该mRNA的翻译量的降低。此外,完全不生成酶蛋白分子或者降低或消除酶蛋白平均每个分子的活性,可以通过破坏编码该酶的基因来实现。就作为比较对象的野生型菌株而言,可以列举:Pantoea ananatis AJ13355株、植生克雷伯氏菌(Klebsiella planticola)AJ13399株、谷氨酸棒杆菌ATCC13032、乳发酵短杆菌ATCC13869、黄色短杆菌ATCC14067等。
作为要修饰的编码SDH的基因,可以是编码SDH的各亚基的基因中的1个或1个以上,此外,也可以是整个操纵子全体,可以向编码各个亚基(SDHA、SDHB、SDHC、SDHD)的基因中导入突变。
此外,作为要修饰的编码α-KGDH的基因,可以是编码α-KGDH的各亚基的基因中的1个或1个以上,此外,也可以是整个基因簇,优选编码E1o亚基的基因(sucA或odhA)或者编码E2o亚基的基因(sucB)。
根据微生物所属的种或菌株不同,有些情况下编码SDH或α-KGDH的各亚基的基因的核苷酸序列存在差异,因此,编码它们的基因也可以是SEQID NO:1、7、12、14、73、77、或81的核苷酸序列的变体。各基因的变体可以以SEQ ID NO:1、7、12、14、73、77或81的核苷酸序列作为参考,通过BLAST等来检索(http://blast.genome.jp/)。此外,各基因的变体还包括能够以各基因同源物例如肠杆菌科、棒状杆菌型细菌等微生物的染色体为模板,使用基于例如SEQ ID NO:1、7、12、14、73或81的核苷酸序列制备的合成寡核苷酸采用PCR扩增而得到的基因。
作为SDH的亚基,可以列举出具有SEQ ID NO:2~4、6、74~76、78~80中的任一氨基酸序列的蛋白;作为α-KGDH的亚基,可以列举出具有SEQ IDNO:8~11、13、15和81中的任一氨基酸序列的蛋白。因细菌的种或菌株的不同,使用的密码子不同,各基因的核苷酸序列存在差异,因此,只要编码的蛋白的功能不变,有些情况下也编码在上述氨基酸序列中包含一个或数个氨基酸的取代、缺失、插入或者增加的氨基酸序列。这里,数个是指例如1~20个、优选1~10个、更优选1~5个。上述一个或数个氨基酸的取代、缺失、插入或者增加是能够正常地维持各蛋白功能的保守突变。关于保守突变,当取代位点是芳香族氨基酸时为Phe、Trp和Tyr之间、当取代位点是疏水氨基酸时为Leu、Ile和Val之间、当是极性氨基酸时为Gln和Asn之间、当是碱性氨基酸时为Lys、Arg和His之间、当是酸性氨基酸时 为Asp和Glu之间、当是具有羟基的氨基酸时为Ser和Thr之间的相互取代的突变。保守突变的代表是保守取代,可视为保守取代的取代可列举:用Ser或Thr取代Ala;用Gln、His或Lys取代Arg;用Glu、Gln、Lys、His或Asp取代Asn;用Asn、Glu或Gln取代Asp;用Ser或Ala取代Cys;用Asn、Glu、Lys、His、Asp或Arg取代Gln;用Asn、Gln、Lys或Asp取代Glu;用Pro取代Gly;用Asn、Lys、Gln、Arg或Tyr取代His;用Leu、Met、Val或Phe取代Ile;用Ile、Met、Val或Phe取代Leu;用Asn、Glu、Gln、His或Arg取代Lys;用Ile、Leu、Val或Phe取代Met;用Trp、Tyr、Met、Ile或Leu取代Phe;用Thr或Ala取代Ser;用Ser或Ala取代Thr;用Phe或Tyr取代Trp;用His、Phe或Trp取代Tyr;和用Met、Ile或Leu取代Val。
各基因的变体不仅可以是具有SEQ ID NO:1、7、12、14、73、77或81的核苷酸序列中的各编码区域的核苷酸序列的基因,也可以是与这些核苷酸序列或编码区域的核苷酸序列互补的序列、或者是与可由这样的核苷酸序列制备的探针在严格条件下杂交的DNA。“严格条件”包括形成所谓特异性杂交体而不形成非特异性杂交体的条件。举一实例,有这样的条件:在该条件下,具有高同源性的DNA,例如具有80%以上,优选90%以上,更优选95%以上,还更优选97%以上,特别优选具有99%以上同源性的DNA相互杂交,而同源性较之为低的DNA则不互相杂交;或者是这样的条件:在与常规Southern杂交的洗涤条件即60℃,1×SSC、0.1%SDS,优选60℃,0.1×SSC、0.1%SDS,更优选68℃,0.1×SSC、0.1%SDS相当的盐浓度和温度下,洗涤一次,更优选两次或三次的条件。探针的长度可以根据杂交条件适宜地选择,通常为100bp~1Kbp。
基因的修饰具体地可以通过缺损染色体上基因编码区域的一部分或全部、或者修饰启动子或shine-dalgarno(SD)序列等表达调节序列等方法来实现。此外,表达调节序列以外的非翻译区域的修饰也可以降低基因表达量。而且,还可以缺失整个基因,包括染色体上的基因的前后的序列。此外,所述修饰还可以这样完成:通过基因重组在染色体上的酶编码区域引入氨基酸取代(错义突变)、或者导入终止密码子(无义突变)、或者导入添加或缺失一~两个核苷酸的移码突变(Journal of biological Chemistry 272:8611-8617(1997),Proceedings of the National Academy of Sciences,USA 955511-5515(1998),Journal of Biological Chemistry 266,20833-20839(1991))。
此外,在肠杆菌中,sucA、sucB基因存在于sdh操纵子的上游,可以认为通过导入降低sdh操纵子表达量的突变,sucA、sucB基因的表达量也会降低,这样的突变也包含在本发明中。
本发明中,“通过基因重组进行修饰”是指:通过利用同源重组缺损染色体上的基因表达调节序列例如启动子区域、或编码区域、或者非编码区域的一部分或全部,或者在这些区域内插入其它序列,来降低细胞内的酶活性。在本发明中基因修饰优选达到这样的程度,即基因的失活无法通过自然突变而复原。
然而,在本发明中,只要是降低SDH活性和α-KGDH活性的修饰即可,也可以是通过X射线或紫外线照射、或者N-甲基-N’-硝基-N-亚硝基胍等突变剂进行的常规突变处理引起的修饰。
表达调节序列的修饰优选为1个核苷酸以上,更优选为2个核苷酸以上,特别优选为3个核苷酸以上。此外,在缺失编码区域时,只要产生的酶蛋白的功能降低或缺失,缺失的区域可以是N末端区域、内部区域、C末端区域中的任何区域,也可以是整个编码区域。通常,缺失的区域较长能够切实地使基因失活。此外,优选的是缺失区域的上游与下游的阅读框不一致。
当在编码区域中插入其它序列时,可以是基因的任何区域,但插入的序列较长能够切实地使编码酶的基因失活。插入部位的前后的序列优选阅读框不一致。对于其它序列没有特殊限制,只要能够降低或缺损酶蛋白的功能即可,可以列举例如携带抗生素抗性基因或对L-谷氨酸生产有用的基因的转座子等。
为了如上述地对染色体上的基因进行修饰,例如可以通过下述方法来实现:制备经过修饰而缺失基因的部分序列并不产生正常发挥功能的酶蛋白的缺失型基因,用包含该基因的DNA转化细菌,使缺失型基因与染色体上的基因发生同源重组,从而用缺失型基因取代染色体上的基因。缺失型基因所编码的酶蛋白即便能够产生,其具有的立体结构也与野生型酶蛋白不同,其功能降低或消失。这种基于利用同源重组的基因取代的基因破坏是已经确立的,包括下列方法:使用线性DNA的方法,例如称为“Red 驱动整合”的方法(Datsenko和Wanner,Proc.Natl.Acad.Sci.USA,2000,Vol.97,No.12,pp.6640-6645),组合使用Red驱动整合法与λ噬菌体来源的切出系统(Cho,E.H.,Gumport,R.I.,Gardner,J.F.J.Bacteriol.184:5200-5203(2002))的方法(参考WO2005/010175号);或者使用含有温度敏感复制起点的质粒、可接合转移的质粒的方法,利用在宿主内不具有复制起点的自杀载体的方法,等等(美国专利第6303383号说明书、或者特开平05-007491号公报)。
而且,在Red驱动整合中,如参考例1所示,可合适地使用对λRed基因产物具有抗性的菌株、例如Pantoea ananatis SC17(0)株。SC17(0)株已于2005年9月21日保藏在俄罗斯国立工业微生物保藏中心(RussianNational Collection of Industrial Microorganisms(VKPM),GNII Genetika),保藏号为VKPM B-9246。
基因转录量降低的确认可以通过比较由该基因转录的mRNA的量与野生型菌株、或非修饰菌株中的量来进行。作为评价mRNA量的方法,可以列举出Northern杂交、RT-PCR等(Molecular cloning(Cold spring HarborLaboratory Press,Cold spring Harbor(USA),2001))。转录量的降低只要是与野生型菌株或非修饰菌株相比降低即可,可以是任意的,优选例如与野生型菌株、非修饰菌株相比降低至至少75%以下、50%以下、25%以下或10%以下,特别优选完全不表达。
基因编码的蛋白量的降低的确认可以使用抗体通过Western印迹来进行(Molecular cloning(Cold spring Harbor Laboratory Press,Cold spring Harbor(USA),2001))。蛋白量的降低只要是与野生型菌株或非修饰菌株相比降低即可,可以是任意的,优选例如与野生型菌株、非修饰菌株相比,与野生型菌株或非修饰菌株相比,减少至至少75%以下、50%以下、25%以下或10%以下,特别优选完全不产生蛋白(活性完全消失)。
(2)利用营养缺陷性获取α-KGDH活性降低株
α-KGDH活性降低的菌株可以采用上述(1)的方法获得,也可以利用营养缺陷性,采用以下方法获得。
例如,用N-甲基-N’-硝基-N-亚硝基胍对微生物菌体实施常规突变处理(例如250μg/ml、30℃、20分钟)后,在固体培养基上进行培养,来形成菌落。从中采用影印法分离出在以L-谷氨酸为唯一碳源和唯一氮源的培养基 上不能生长的突变菌株,这样可以分离出α-KGDH活性降低的菌株。
作为α-KGDH活性降低的菌株,可以列举出例如以下菌株。
乳发酵短杆菌AJ12821(FERM BP-4172;参照美国专利第5,492,818号)
黄色短杆菌AJ12822(FERM BP-4173;参照美国专利第5,492,818号)
谷氨酸棒杆菌AJ12823(FERM BP-4174;参照美国专利第5,492,818号)
乳发酵短杆菌ΔS株(国际公布95/34672号小册子)
谷氨酸棒杆菌OAGN、OA2-2、OAGN2-2(WO2006/028298)
Pantoea ananatis AJ 13601(FERM BP-7207)
植生克雷伯氏菌(Klebsiella planticola)AJ13410株(FERM BP-6617)
Pantoea ananatis AJ13355(FERM BP-6614)
(3)以琥珀酸营养缺陷性为指标获得SDH活性降低株
由于琥珀酰-CoA的供给减少,α-KGDH活性降低株的生长必需琥珀酸。另一方面,已知在α-KGDH与SDH的双重缺损株中琥珀酸营养缺陷性得以恢复(J Gen Microbiol.1978Jul;107(1):1-13)。因此,通过从α-KGDH活性降低的菌株中选择琥珀酸营养缺陷性恢复的菌株,可以获得α-KGDH和SDH活性降低的菌株。具体地,在不含琥珀酸的极限培养基上用α-KGDH活性降低的菌株铺板,使之形成菌落。能够在不含琥珀酸的极限培养基上生长的α-KGDH活性降低菌株中,编码SDH的基因上具有突变的频率高。
<3>本发明的L-氨基酸生产方法
在培养基中培养如上所述的微生物,使L-氨基酸生成并蓄积在该培养基中或菌体内,并且从该培养基或菌体收集L-氨基酸,这样可以生产L-氨基酸。
用于培养的培养基可以使用含有碳源、氮源、无机盐类以及视需要的氨基酸、维生素等有机微量营养元素的常规培养基。可以使用合成培养基或者天然培养基。培养基中使用的碳源和氮源可以是任意种类的,只要所培养的菌株能够利用即可。
碳源可以使用糖类,如葡萄糖、甘油、果糖、蔗糖、麦芽糖、甘露糖、半乳糖、淀粉水解物和糖蜜,此外也可以单独使用或者与其它碳源组合使用有机酸,如乙酸和柠檬酸,以及醇,如乙醇。作为氮源,可以使用氨、铵盐如硫酸铵、碳酸铵、氯化铵、磷酸铵或乙酸铵,或者使用硝酸盐。作为有机痕量营养物,可以使用氨基酸、维生素、脂肪酸和核酸,以及包含 这些营养物的蛋白胨、酪蛋白氨基酸、酵母提取物、大豆蛋白水解物,并且在使用生长需要氨基酸等营养物的营养缺陷型突变菌株时,优选补充所需要的营养物。无机盐类可以使用磷酸盐、镁盐、钙盐、铁盐、锰盐等。
培养优选控制在发酵温度20~45℃、pH 3-9的条件下进行通气培养。而且,pH调整可以使用无机或有机酸性或碱性物质,以及氨气等。在这样的条件下,进行优选约10~120小时左右的培养,在培养液中或菌体内蓄积L-氨基酸。
此外,当目的L-氨基酸为L-谷氨酸时,也可以使用调整至能够使L-谷氨酸析出的条件的液体培养基来进行培养,使得培养基中L-谷氨酸一边生成和蓄积,一边析出。作为使L-谷氨酸析出的条件,可以列举例如:pH5.0~4.0,优选pH4.5~4.0,更优选pH4.3~4.0,特别优选pH4.0。而且,为了兼顾酸性条件下生长的提高和L-谷氨酸的高效析出,pH优选5.0~4.0,更优选4.5~4.0,更优选4.3~4.0。而且,上述pH下的培养可以是整个培养期间,也可以是培养期间的一部分。
作为培养结束后从培养液收集L-氨基酸的方法,可以按照公知的回收方法进行。例如,在从培养液中除去菌体后,通过浓缩晶析方法或离子交换色谱等来收集。当在使L-谷氨酸析出到培养基中的条件下培养时,析出到培养液中的L-谷氨酸可以通过离心分离或过滤等来收集。此时,可以在使溶解在培养基中的L-谷氨酸晶析后,一并进行分离。
而且,可以在培养基中添加海藻糖来进行培养。培养基中所含海藻糖的浓度优选添加0.1g/L以上,更优选0.2g/L以上,更优选0.5g/L以上。这其中,在使用棒状杆菌型细菌时,优选添加0.5g/L以上,更优选0.75g/L以上,更优选2g/L以上。添加在培养基中的海藻糖,可以使用溶解的结晶海藻糖,也可以使用从发酵过程产生的发酵溶液中回收目的物质后的母液中包含的海藻糖。此外,本发明中,培养基中的海藻糖还可以为发酵液中作为副产物产生的海藻糖。
而且,在本发明中,除海藻糖外,还可以通过添加甜菜碱(N-甲基甘氨酸、N-N-二甲基甘氨酸、N-N-N-三甲基甘氨酸、〔2-羟基乙基〕三甲基铵)来进一步改善目的物质的生产力。优选添加0.1g/L以上,优选0.25g/L以上,更优选0.5g/L以上。
实施例
以下,通过实施例对本发明进行更具体的说明。
〔参考例1〕对λRed基因产物具有抗性的Pantoea ananatis菌株的构建
为了在Pantoea ananatis中破坏sdhA基因,构建了用于高效率地进行称作“Red驱动整合”或“Red介导整合”的方法(Proc.Natl.Acad.Sci.USA.97.6640-6645(2000))的受体菌。
首先,构建了表达λ的gam、bet和exo各基因(以下称“λRed基因”)的新辅助质粒RSF-Red-TER(图1)。详细描述见参考例2。
该质粒可以用于具有不同基因背景的广域宿主。理由是:1)其具有在许多革兰氏阴性菌和革兰氏阳性菌甚至植物中都能够稳定保持的RSF1010广宿主域质粒的复制子(Scholz,et al.,1989;Buchanan-Wollaston et al.,1987),2)λRed基因、gam、bet和exo基因受能够被许多细菌的RNA聚合酶识别的PlacUV5启动子的调节(例如、Brunschwig,E.and Darzins,A.,Gene,111,1,35-41(1992);Dehio,M.et al,Gene,215,2,223-229(1998)),3)自调节因子PlacUV5-lacI、以及大肠杆菌rrnB操纵子的ρ非依赖性转录终止子(TrrnB)降低λRed基因的本底表达水平(Skorokhodova,A.Yu et al,Biotekhnologiya(Rus),5,3-21(2004))。而且,RSF-Red-TER质粒包含果聚糖蔗糖酶(levansucrase)基因(sacB),利用该基因,可以在含蔗糖的培养基中从细胞回收质粒。
在大肠杆菌中,PCR反应生成的DNA片段与由RSF-Red-TER质粒提供的短侧翼区域一起发生整合的频率与使用pKD46辅助质粒(Datsenko,K.A.,Wanner,B.L.,Proc.Natl.Acad.Sci.USA,97,6640-6645,(2000))时一样高。然而,λRed基因的表达对Pantoea ananatis显示毒性。用RSF-Red-TER辅助质粒转化的细胞在含IPTG(异丙基-β-D-硫代半乳糖苷,1mM)和适当抗生素(氯霉素25μg/ml或卡那霉素40μg/ml)的LB培养基上显示非常低的生长速度,λRed介导的重组(λRed-mediated recombination)的效率即使能够观察到,也是非常低的(10-8)。
选择了对λRed基因的全部3个基因的表达均具有抗性的Pantoeaananatis突变菌株。为此,通过电穿孔将RSF-Red-TER质粒导入Pantoeaananatis SC17株(美国专利第6,596,517号)。培养18小时后,得到了约106个转化株,有10个克隆的菌落尺寸大,其余的菌落都非常小。培养18小时后,大菌落为约2mm,小菌落为约0.2mm。将培养延长至24小时,小菌 落不再继续生长,而大菌落继续生长。将对λRed基因的全部3个基因(gam、bet和exo)的表达均具有抗性的大菌落Pantoea ananatis突变菌株中的一个用于进一步分析。
从1个大菌落克隆和数个小菌落克隆中分离出RSF-Red-TER质粒DNA,用其再次转化DNA大肠杆菌MG1655,考察了质粒合成Red基因的活性产物的能力。由所得转化体中Red依赖性整合的对照实验可知,只有从大菌落克隆分离的质粒可引起Red依赖性整合所必需的λRed基因的表达。为了考察所选的大菌落克隆中是否发生Red介导的整合,使用PCR反应生成的下述线性DNA片段进行了电穿孔,所述DNA片段设计为包含KmR标记和与hisD基因同源的40bp侧翼区域,并且整合到Pantoea ananatis的hisD基因的SmaI识别位点。将2个小菌落的克隆用作对照。Pantoea ananatis的hisD基因的核苷酸序列示于SEQ ID NO:16。PCR中使用SEQ ID NO:17和18的寡核苷酸作为引物,使用pMW118-(λatt-Kmr-λatt)质粒作为模板。使用对λRed基因不具有抗性的2个小菌落的克隆作为对照。pMW118-(λattL-Kmr-λattR)质粒的构建在参考例3中详细描述。
RSF-Red-TER质粒能够通过该质粒上的lacI基因诱导Red基因的表达。对两种诱导条件进行了考察。在第1组中,电穿孔1小时前添加IPTG(1mM);在第2组中,在用于制备可电穿孔细胞的培养开始时添加IPTG。携带来自大菌落克隆的RSF-Red-TER的细胞的后代的生长速度并不显著地低于不带有SC17质粒的菌株。添加IPTG仅使这些培养物的生长速度有些许降低。另一方面,小菌落克隆的后代在未添加IPTG的条件下生长非常缓慢,而加以诱导后,生长基本上停止。对大菌落克隆的后代的细胞进行电穿孔后,有许多KmR克隆(短诱导时间为18个克隆,延长诱导时间后为约100个克隆)生长。考察的全部100个克隆均具有His-表现型,通过PCR对20个克隆进行了确认,结果确认了这些细胞的染色体结构与预想的相同。另一方面,对小菌落克隆的后代的细胞即使进行电穿孔也未能得到整合的菌株。
使所得大菌落克隆在含7%蔗糖的平板上生长,并使质粒脱落,用RSF-Red-TER再次进行转化。将不携带质粒的菌株命名为SC17(0)。该菌株已于2005年9月21保藏在俄罗斯国立工业微生物保藏中心(RussianNational Collection of Industrial Microorganisms(VKPM),GNII Genetika)(地址:Russia,117545Moscow,1 Dorozhny proezd.1),保藏号VKPM B-9246。
上述再次转化后生长出的全部克隆均与亲本菌株克隆SC17(0)一样具有较大的菌落尺寸。在用RSF-Red-TER质粒再次转化的SC17(0)株中进行了Red介导的整合实验。对于所得的3个独立转化株,使用与前述实验所用的相同的DNA片段进行了考察。采用了短诱导时间(电穿孔1小时前)。在各实验中,生长出超过10个的KmR克隆。测试的全部克隆均具有His-表现型。这样,选择出突变菌株,将其命名为对λRed基因的表达具有抗性的SC17(0)。该菌株可以作为用于对Pantoea ananatis染色体的Red依赖性整合的合适受体菌。
〔参考例2〕辅助质粒RSF-Red-TER的构建
辅助质粒RSF-Red-TER的构建方案如图2所示。
作为构建的最初步骤,设计了RSFsacBPlacMCS载体。为此,分别使用SEQ ID NO:19、20、21、22的寡核苷酸,通过PCR扩增了包含pACYC184质粒的cat基因和枯草芽孢杆菌的sacB基因的结构部分的DNA片段。这些寡核苷酸的5′末端分别包含对进一步克隆而言必需且便利的BglII、SacI、XbaI、和BamHI限制酶位点。将所得1.5kb的sacB片段克隆到此前获得的pMW119-PlaclacI载体的XbaI-BamHI位点。该载体是依照与pMW118-PlaclacI载体的相关描述(Skorokhodova,A.Yu et al,Biotekhnologiya(Rus),5,3-21(2004))相同的方法构建的。只是该载体包含来自pMW219而不是pMW218质粒的多接头位点。
接下来,将上述的1.0kb的cat片段用BglII和SacI处理,并克隆到在先步骤中所得的RSF-PlaclacIsacB质粒的BamHI-SacI位点。所得质粒pMW-PlaclacIsacBcat包含PlacUV5-lacI-sacB-cat片段。为了将该片段亚克隆到RSF1010载体,将pMW-PlaclacIsacBcat用BglII消化,并用DNA聚合酶I克列诺片段处理使末端平滑化,然后用SacI切割。从1%琼脂糖凝胶中洗提出pMWPlaclacIsacBcat质粒的3.8kb的BglII-SacI片段,将其与用PstI和SacI处理的RSF1010载体连接。用连接混合液转化大肠杆菌TG1,将其在含氯霉素(50mg/L)的LB培养基上铺板。对从生长出的克隆中分离的质粒进行制限酶分析,得到了RSFsacB质粒。为了构建RSFsacBPlacMCS载体,以SEQ ID NO:23和24的寡核苷酸为引物,以pMW119-PlaclacI质粒为模板,通过PCR扩增了包含PlacUV5启动子的DNA片段。将所得的146bp片段用SacI和NotI消化,将其与RSFsacB质粒的SacI-NotI大片段连接。然后,以 SEQ ID NO:25和26的寡核苷酸为引物,以pKD46质粒(Datsenko,K.A.,Wanner,B.L.,Proc.Natl.Acad.Sci.USA,97,6640-6645,(2000))为模板,通过PCR扩增了包含λRedαβγ基因和转录终止子tL3的2.3kb的DNA片段。将所得片段克隆到RSFsacBPlacMCS载体的PvuI-NotI位点。这样,设计了RSFRed质粒。
为了排除Red基因的通读转录,将大肠杆菌rrnB操纵子的ρ-依赖性转录终止子插入到cat基因与PlacUV5启动子之间。为此,以SEQ ID NO:27和24的寡核苷酸为引物,以大肠杆菌BW3350的染色体为模板,通过PCR扩增了包含PlacUV5启动子与TrrnB终止子的DNA片段。将所得的这些片段用KpnI处理,并进行连接。然后,以SEQ ID NO:24和28的寡核苷酸为引物,通过PCR扩增了包含PlacUV5和TrrnB这两者的0.5kb片段。将所得DNA片段用EcoRI消化,并用DNA聚合酶I克列诺片段处理使末端平滑化,然后用BamHI切割,将其与RSFsacBPlacMCS载体的Ecl136II-BamHI大片段连接。将所得质粒命名为RSF-Red-TER。
〔参考例3〕pMW118-(λattL-Kmr-λattR)质粒的构建
pMW118-(λattL-Kmr-λattR)质粒是这样构建的:在pMW118-attL-Tc-attR(WO2005/010175)质粒中,用pUC4K质粒的卡那霉素抗性基因取代四环素抗性标记基因。为此,将pMW118-attL-Tc-attR质粒的EcoRI-HindIII大片段与pUC4K质粒的HindIII-PstI(676bp)和EcoRI-HindIII(585bp)这2个片段连接。作为基础的pMW118-attL-Tc-attR是通过连接以下4个片段而得到的。
1)使用引物P1(SEQ ID NO:29)和P2(SEQ ID NO:30)从大肠杆菌W3350(包含λ原噬菌体)染色体的相当于attL的区域通过PCR扩增得到的具有attL(SEQ ID NO:31)的BglII-EcoRI片段(114bp)。这些引物包含BglII和EcoRI的附加识别位点。
2)使用引物P3(SEQ ID NO:32)和P2(SEQ ID NO:33)从大肠杆菌W3350(包含λ原噬菌体)染色体的相当于attR的区域通过PCR扩增得到的具有attR(SEQ ID NO:34)的PstI-HindIII片段(182bp)。这些引物包含PstI和HindIII的附加识别位点。
3)pMW118-ter_rrnB的BglII-HindIII大片段(3916 bp)。质粒pMW118-ter_rrnB是通过连接以下3个DNA片段而得到的。
·pMW118的具有AatII-EcoRI片段的大片段(2359bp)。该片段是通过用 EcoRI消化pMW118、并用DNA聚合酶I克列诺片段进行处理、再用AatII进行消化而得到的。
·具有氨苄青霉素抗性(ApR)的基因bla的pUC19的AatII-BglII小片段(1194bp)。该片段是使用引物P5和P6(SEQ ID NO:35和36)对pUC19质粒的相应区域进行PCR扩增而得到的。这些引物中包含PstI、AatII和BglII的附加识别位点。
·转录终止子ter rrnB的BglII-PstI小片段(363bp)。该片段是通过使用引物P7和P8(SEQ ID NO:37和38)对大肠杆菌MG1655染色体的相应区域进行PCR扩增而得到的。这些引物包含PstI、BglII和PstI的附加识别位点。
4)具有四环素抗性基因和ter_thrL转录终止子的pML-Tc-ter_thrL的EcoRI-PstI小片段(1388bp)(SEQ ID NO:39)。pML-Tc-ter_thrL质粒是通过如下的2个步骤获得的。
·将pML-MCS质粒(Mashko,S.V.et al.,Biotekhnologiya(俄语),2001,no.5,3-20)用XbaI和BamHI消化,然后将大片段(3342bp)与包含ter_thrL终止子的XbaI-BamHI片段(68bp)连接。该包含ter_thrL终止子的片段是使用引物P9和P10(SEQ ID NO:40和41)对大肠杆菌MG1655染色体的相应区域进行PCR而得到的。这样,得到了pML-ter_thrL质粒。这些引物包含PstI、XbaI和BamHI的附加识别位点。
·将pML-ter_thrL质粒用KpnI和XbaI消化,然后用DNA聚合酶I克列诺片段处理,再与具有四环素抗性基因的pBR322的EcoRI-Van91I小片段(1317bp)连接,从而得到了pML-Tc-ter_thrL质粒。而且,将pBR322用EcoRI和Van91I消化,然后再用DNA聚合酶I克列诺片段进行了处理。
〔实施例1〕Pantoea ananatis中sdhA基因和sucA基因的破坏效果
(1)谷氨酸生产质粒RSFPPG的构建
构建了L-谷氨酸生物合成系统基因,prpC基因(国际公布2006/051660号小册子)、ppc基因、gdh基因(欧洲申请公布0999282号说明书)被扩增的质粒RSFPPG。
设计了引物1(SEQ ID NO:42)和引物2(SEQ ID NO:43),用于扩增RSFCPG(欧洲申请公开1233068号说明书)的gltA基因的ORF以外的部分。使用该引物,以RSFCPG为模板进行PCR,获得了约14.9kb的片段。另一方面,对于prpC,使用引物3(SEQ ID NO:44)和引物4(SEQ ID NO:45),以 大肠杆菌W3110株的染色体DNA为模板进行PCR,获得了约1.2kb的片段。将两PCR产物分别用BglII和KpnI处理并进行连接,然后转化大肠杆菌JM109株。收集全部出现的菌落,以混合物的形式提取质粒。用该质粒混合物转化柠檬酸合酶(CS)缺损株大肠杆菌ME8330株,将其涂布于含50mg/L尿嘧啶、5mg/L硫胺-HCl的M9极限培养基(1L纯水中含葡萄糖5g、硫酸镁2mM、磷酸二氢钾3g、氯化钠0.5g、氯化铵1g磷酸氢二钠6g的培养基)。从出现的菌株中提取质粒,命名为RSFPPG。在L-谷氨酸生产菌Pantoeaananatis NP106株中导入上述质粒RSFPPG,构建了L-谷氨酸生产菌NP106/RSFPPG(本菌株称为“NA1株”)。
NP106株是如下述地获得的。将前文例示的Pantoea ananatis AJ13601株在LBGM9液体培养基中于34℃振荡培养一夜,稀释到每个平板为100~200个菌落,将其涂布在含四环素12.5mg/L的LBGM9平板上。对于出现的菌落,将其转印到含四环素12.5mg/L和氯霉素25mg/L的LBGM9平板上,选择氯霉素敏感性的菌株,从而获得了pSTVCB脱落的菌株,将其命名为G106S。而且,将G106S株在LBGM9液体培养基中于34℃振荡培养一夜,稀释到每个平板为100~200个菌落,将其涂布在不含药物的LBGM9平板上。对于出现的菌落,将其转印在含四环素12.5mg/L的LBGM9平板和不含药物的LBGM9平板上,选择四环素敏感性菌株,从而获得了RSFCPG脱落的菌株,将其命名为NP106。这样获得的NP106株不再具有AJ13601株所携带的RSFCPG和pSTVCB这2个质粒。
(2)sdhA基因破坏株的构建
以pMW-attL-Kmr-attR为模板,使用SEQ ID NO:46、47的引物进行PCR,扩增出下述基因片段,所述基因片段中在卡那霉素抗性基因的两端分别增加λ噬菌体的attL和attR序列,并且进一步在它们的外侧分别增加sdhA基因的上游50bp和下游50bp的序列。使用Wizard PCR Prep DNA PurificationSystem(Promega公司生产)对该片段进行了纯化。
接下来,用RSF-Red-TER转化SC17(0),得到了SC17(0)/RSF-Red-TER株。将该菌株在含有25mg/L氯霉素的L培养基(纯水1L中含细菌用胰蛋白胨10g、酵母抽提物5g、NaCl 5g的培养基,pH7.0)中培养过夜,将该过夜培养液以1/100的量接种于含有25mg/L氯霉素和1mM异丙基-β-D-硫代半乳糖苷的L培养基100mL中,于34℃培养3小时。收集这样制备的菌体, 用冰冷却的10%甘油清洗菌体3次,最后将其悬浮在0.5mL的10%甘油中作为感受态细胞,使用前项中制备的PCR片段100ng,利用GENE PULSERII(BioRad公司生产),在电场强度18kV/cm、电容容量25μF、电阻值200Ω的条件进行导入。在细胞悬浮液中添加冰冷放置的SOC培养基(细菌用胰蛋白胨20g/L、酵母抽提物5g/L、NaCl 0.5g/L、葡萄糖10g/L),于34℃振荡培养2小时,将其涂于在L培养基(纯水1L中含细菌用胰蛋白胨10g、酵母抽提物5g、NaCl 5g、琼脂15g的培养基,pH7.0)中添加极限培养基成分(每1L中葡萄糖5g、硫酸镁2mM、磷酸二氢钾3g、氯化钠0.5g、氯化铵1g、磷酸氢二钠6g)和40mg/L的卡那霉素而得到的培养基上。将出现的菌落用同样的培养基纯化后,通过PCR确认了sdhA基因被取代为卡那霉素抗性基因。
使用Edge Biosystems公司生产的Bacterial Genomic DNA PurificationKit从该sdhA基因缺损株中提取染色体。另一方面,将NAI株于在L培养基中添加了上述极限培养基成分和12.5mg/L四环素而得到的琼脂培养基中培养过夜。用接种环刮取菌体,用冰冷放置的10%甘油清洗菌体3次,在菌体中加入10%甘油至终体积为500μL悬浮,作为感受态细胞。对于该感受态细胞,利用GENE PULSER II(BioRad公司生产),在电场强度17.5kV/cm、电容容量25μF、电阻值200Ω的条件下导入前述的染色体DNA600ng。在细胞悬浮液中添加冰冷放置的SOC培养基,于34℃振荡培养2小时,然后将其涂布于在L培养基中添加上述极限培养基成分和12.5mg/L四环素、40mg/L卡那霉素而得到的琼脂培养基上。将出现的菌落用同样的培养基纯化后,通过PCR确认了sdhA基因被取代为卡那霉素抗性基因。
NA1株缺损编码α-KGDH的E1亚基的sucA基因。另一方面,SC17(0)/RSF-Red-TER株的sucA基因中未引入突变。sdhA基因与sucA基因位置非常接近,在用sdhA基因缺损株的染色体DNA进行转化时,野生型sucA基因也以一定比例与sdhA的突变同时转移。因此,在所获得的NA1的sdhA缺损株中,包括sucA基因缺损的菌株和恢复为野生型的菌株二者。因而,通过PCR对NA1中与sucA基因突变位置相应的区域进行扩增,以其是否能被制限酶BglII切割为指标,确认是sucA缺损还是sucA基因恢复为野生型,从而得到了sdhA单独缺损株以及sucAsdhA双重缺损株。
(3)sdhA缺损株、sucAsdhA双重缺损株的L-谷氨酸生产能力的评价
接下来,为了对前项中获得的sdhA缺损株和sucAsdhA双重缺损株的L-谷氨酸生产能力进行评价,将这些菌株接种于注入了具有下述组成的培养基5mL的试管中,培养18小时。sucA单独缺损株NA1株在该培养基中基本不生长,L-谷氨酸蓄积也仅为3.3g/L左右,与此相对,sdhA单独缺损株获得了13.2g/L的L-谷氨酸蓄积,该结果与sucA单独缺损株相比有很大提高。另一方面,sucAsdhA双重缺损株获得了14.7g/L的L-谷氨酸蓄积,可以确认与分别单独缺损sucA、sdhA时相比,具有更高的L-谷氨酸生产能力,生长也得到了大幅改善。
(L-谷氨酸生产评价培养基组成)
(A区)
蔗糖 30g/L
MgSO4·7H2O 0.5g/L
(B区)
(NH4)2SO4 20g/L
KH2PO4 42g/L
FeSO4·7H2O 20mg/L
MnSO4·5H2O 20mg/L
酵母抽提物(Difco) 2g/L
泛酸钙 18mg/L
(C区)
碳酸钙 20g/L
对于A区、B区,115℃,10分钟,高压灭菌器灭菌;对于C区,180℃,3小时,干热灭菌。
冷却至室温后,将3者混合来使用。
表1
RS:残余糖
〔实施例2〕棒状杆菌型细菌中odhA基因和sdhA基因的破坏效果
(1)odhA基因破坏株的构建
由黄色短杆菌(Brevibacterium flavum)ATCC14067株(现在分类为谷氨酸棒杆菌(Corynebacterium glutamicum))构建了下述菌株,所述菌株中odhA基因缺损、并且yggB基因(SEQ ID NO:56)第837位的G被替换成A、gdh基因的启动子被修饰。此外,在本实施例中使用黄色短杆菌ATCC14067株作为亲本菌株,但也可以使用谷氨酸棒杆菌ATCC13032株、乳发酵短杆菌ATCC13869株作为亲本菌株构建具有相同性质的菌株。
首先,构建了用于缺损odhA的质粒pBS3ΔsucA47。以SEQ ID NO:48所示的合成DNA和SEQ ID NO:49所示的合成DNA为引物,以黄色短杆菌ATCC14067株的染色体DNA为模板进行PCR,制备了N末端侧片段。相似地,以SEQ ID NO:50和SEQ ID NO:51的合成DNA为引物,制备了C末端侧片段。然后,将N末端侧片段和C末端侧片段等量混合作为模板,以SEQ ID NO:52和SEQ ID NO:53的合成DNA(附加了BamHI序列)为引物进行PCR,获得了odhA基因的编码区域缺失的片段。将所得突变型odhA片段用BamHI处理,插入pBS3(国际公布2006/070944号小册子)的BamHI位点,将所得质粒命名为pBS3ΔsucA47。
采用电脉冲法将pBS3ΔsucA47导入黄色短杆菌ATCC14067,将其涂布在含卡那霉素25μg/ml的CM-Dex琼脂培养基(葡萄糖5g/l、多蛋白胨10g/l、酵母提取物10g/l、KH2PO4 1g/l、MgSO4·7H2O 0.4g/l、FeSO4·7H2O 0.01g/l、MnSO4·4-5H2O 0.01g/l、尿素3g/l、大豆蛋白水解液1.2g/l、琼脂20g/l,用NaOH调整至pH7.5,高压灭菌器灭菌120℃ 20分钟)上。31.5℃培养2日后,将生长出的菌株作为pBS3ΔsucA47插入到染色体上的菌株ATCC14067-pBS3ΔsucA。然后,将ATCC14067-pBS3ΔsucA株在CM-Dex液体培养基中培养一夜,将所得悬浮液涂布在S10琼脂培养基(蔗糖100g/l、多蛋白胨10g/l、酵母提取物10g/l、KH2PO4 1g/l、MgSO4·7H2O 0.4g/l、FeSO4·7H2O 0.01g/l、MnSO4·4-5H2O 0.01g/l、尿素3g/l、大豆蛋白水解液1.2g/l、琼脂20g/l,用NaOH调整至pH7.5,高压灭菌器灭菌120℃20分钟)上,31.5℃进行培养。从出现的菌落中选择显示卡那霉素敏感性的菌株,进一步在CM-Dex琼脂培养基上进行纯化。从这些菌株制备染色体DNA,以SEQID NO:52和SEQ ID NO:53所示的合成DNA为引物进行PCR,将确认了约 1.9Kb的扩增片段的菌株命名为8L3株。
以8L3株的染色体DNA为模板,以SEQ ID NO:54和SEQ ID NO:55的合成DNA(附加了SacI序列)为引物进行PCR,测定所得扩增片段的序列,结果显示SEQ ID NO:57的第111位的丙氨酸被取代为苏氨酸。也就是说,8L3是在导入odhA缺损时偶然性地导入了该突变而成的双重突变菌株。作为与8L3一样具有yggB突变的菌株,已知有ATCC14067yggB8株(国际公布2006/070944号小册子)。向ATCC14067yggB株导入pBS3ΔsucA47,然后通过上述步骤,可以构建与8L3一样具有yggB突变的odhA缺损株。
接下来,构建了用于向gdh的启动子区域导入突变的质粒pBS4gdh3。以SEQ ID NO:60所示的合成DNA和SEQ ID NO:61所示合成DNA为引物,以乳发酵短杆菌ATCC13869株的染色体DNA为模板进行PCR,制备了N末端侧片段。相似地,以SEQ ID NO:62和SEQ ID NO:63的合成DNA为引物,制备了C末端侧片段。然后,将N末端侧片段和C末端侧片段等量混合作为模板,以SEQ ID NO:64和SEQ ID NO:65的合成DNA(附加了SmaI序列)作为引物进行PCR,获得了在启动子区域中导入了突变的gdh基因片段。将所得突变型gdh片段用SmaI处理,然后插入pBS4S(国际公布2006/070944号小册子)的SmaI位点,将所得质粒命名为pBS4gdh3。
采用电脉冲法将pBS4gdh3导入8L3株,将其涂布在含卡那霉素25μg/ml的CM-Dex琼脂培养基上。31.5℃培养3日后,将生长出的菌株分离作为pBS4gdh3插入到染色体上的菌株8L3-pBS4gdh3。然后,将8L3-pBS4gdh3株在CM-Dex液体培养基中培养一夜,将所得悬浮液涂布在S10琼脂培养基上,31.5℃进行培养。从出现的菌落中选择显示卡那霉素敏感性的菌株,进一步在CM-Dex琼脂培养基上进行了纯化。从这些菌株制备染色体DNA,测定了gdh编码区域上游的序列。将具有SEQ ID NO:66所示序列的菌株命名为8L3G株。
(2)SDH缺损株的构建
构建了用于缺损sdhA的质粒pBS3Δsdh47。以SEQ ID NO:67所示合成DNA和SEQ ID NO:68所示合成DNA为引物,以黄色短杆菌ATCC 14067株的染色体DNA为模板进行PCR,制备了N末端侧片段。相似地,以SEQID NO:69和SEQ ID NO:70的合成DNA为引物,制备了C末端侧片段。然后,将N末端侧片段和C末端侧片段等量混合作为模板,以SEQ ID NO:71 和SEQ ID NO:72的合成DNA为引物进行PCR,获得了缺失编码区域的sdhA基因片段。将所得突变型sdhA片段用BamHI处理,然后将其插入pBS3(国际公布2006/070944号小册子)的BamHI位点,将所得质粒命名为pBS3ΔsdhA47。
和SEQ ID NO:72的合成DNA为引物进行PCR,获得了缺失编码区域的sdhA基因片段。将所得突变型sdhA片段用BamHI处理,然后将其插入pBS3(国际公布2006/070944号小册子)的BamHI位点,将所得质粒命名为pBS3ΔsdhA47。
采用电脉冲法将pBS3ΔsdhA47导入8L3G株,将其涂布在含卡那霉素25μg/ml的CM-Dex琼脂培养基上。31.5℃培养2日后,将生长出的菌株分离作为pBS3ΔsdhA47插入到染色体上的菌株8L3G-pBS3ΔsdhA。然后,将8L3G-pBS3ΔsdhA株在CM-Dex液体培养基中培养一夜,将所得悬浮液涂布在S10琼脂培养基上,31.5℃进行培养。从出现的菌落中选择显示卡那霉素敏感性的菌株,进一步在CM-Dex琼脂培养基上进行纯化。从这些菌株制备染色体DNA,以SEQ ID NO:71和SEQ ID NO:72所示的合成DNA(附加了BamHI序列)为引物进行PCR,将确认到约1Kb的扩增片段的株命名为8L3GΔSDH株。
(6)sdhA缺损株、sucAsdhA双重缺损株的L-谷氨酸生产能力的评价
接下来,为了对前项中获得的8L3G株、及其SDH缺损株即8L3GΔSDH株的L-谷氨酸生产能力进行评价,将这些菌株分别在1个CM-Dex平板培养基上培养一夜,然后从平板上刮取下全部量的菌体,将其接种到装有300mL具有下述组成的培养基的罐(jar)中,31.5℃进行培养。使用氨气将培养中的pH控制在7.2,控制通气搅拌使溶氧浓度保持在5%以上。如表2所示,与sucA单独缺损株即8L3G株相比,sucAsdhA双重缺损株即8L3GΔSDH的谷氨酸生产速度更高,培养第12.5小时的L-谷氨酸蓄积8L3GΔSDH株为19g/L,与sucA单独缺损株即8L3G株所得的16.5g/L相比有大幅提高。这些结果显示:在棒状杆菌型细菌中,同时缺损sucA和sdhA对于L-谷氨酸生产也是有效的。
(L-谷氨酸生产评价培养基组成)
葡萄糖 60g/L
MgSO4·7H2O 0.9g/L
H3PO4 1.54g/L
KOH 1.45g/L
FeSO4·7H2O 10mg/L
大豆水解物 1.54g/L(以氮计)
生物素 3.2mg/L
VB1 0.67mg/L
DL-甲硫氨酸 0.28g/L
用氨水调整至pH4.0后,120℃灭菌15分钟,即将开始培养前用氨气调整至pH7.2,然后进行培养。
表2
〔序列表的说明〕
SEQ ID NO:1:Pantoea ananatis的sdh操纵子的核苷酸序列(SDHD、SDHA、SDHB的氨基酸序列一并给出)
sdhC:527~913
sdhD:910~1254
sdhA:1258~3021
sdhB:3039~3752
SEQ ID NO:2:SDHD的氨基酸序列
SEQ ID NO:3:SDHA的氨基酸序列
SEQ ID NO:4:SDHB的氨基酸序列
SEQ ID NO:5:Pantoea ananatis的sdh操纵子的核苷酸序列(SDHC的氨基酸序列一并给出)
SEQ ID NO:6:SDHC的氨基酸序列
SEQ ID NO:7:Pantoea ananatis的α-KGDH亚基基因与其附近基因的核苷酸序列
sdhB:2~121
sucA:322~3129
sucB:3145~4368
sucC:4437~4556
SEQ ID NO:8:琥珀酸脱氢酶铁-硫蛋白的氨基酸序列(部分)
SEQ ID NO:9:α-KGDH E1o亚基的氨基酸序列
SEQ ID NO:10:α-KGDH E2o亚基的氨基酸序列
SEQ ID NO:11:琥珀酰-CoA合成酶β亚基的一部分
SEQ ID NO:12:乳发酵短杆菌odhA基因的核苷酸序列
SEQ ID NO:13:odhA编码的E1o亚基的氨基酸序列
SEQ ID NO:14:编码乳发酵短杆菌的E2o亚基的基因(GenBank登录号.NC_003450的NCgl2126)的核苷酸序列
SEQ ID NO:15:NCgl2126编码的E2o亚基的氨基酸序列
SEQ ID NO:16:Pantoea ananatis的hisD基因的核苷酸序列
SEQ ID NO:17:用于在hisD基因中整合Kmr基因的片段的扩增用引物
SEQ ID NO:18:用于在hisD基因中整合Kmr基因的片段的扩增用引物
SEQ ID NO:19:cat基因扩增用引物
SEQ ID NO:20:cat基因扩增用引物
SEQ ID NO:21:sacB基因扩增用引物
SEQ ID NO:22:sacB基因扩增用引物
SEQ ID NO:23:含PlacUV5启动子的DNA片段的扩增用引物
SEQ ID NO:24:含PlacUV5启动子的DNA片段的扩增用引物
SEQ ID NO:25:含λRedαβγ基因和tL3的DNA片段的扩增用引物
SEQ ID NO:26:含λRedαβγ基因和tL3的DNA片段的扩增用引物
SEQ ID NO:27:含PlacUV5启动子和TrrnB的DNA片段的扩增用引物
SEQ ID NO:28:含PlacUV5启动子和TrrnB的DNA片段的扩增用引物
SEQ ID NO:29:attL的扩增用引物
SEQ ID NO:30:attL的扩增用引物
SEQ ID NO:31:attL的核苷酸序列
SEQ ID NO:32:attR的扩增用引物
SEQ ID NO:33:attR的扩增用引物
SEQ ID NO:34:attR的核苷酸序列
SEQ ID NO:35:含bla基因的DNA片段的扩增用引物
SEQ ID NO:36:含bla基因的DNA片段的扩增用引物
SEQ ID NO:37:含ter_rrnB的DNA片段的扩增用引物
SEQ ID NO:38:含ter_rrnB的DNA片段的扩增用引物
SEQ ID NO:39:含ter_thrL终止子的DNA片段的核苷酸序列
SEQ ID NO:40:含ter_thrL终止子的DNA片段的扩增用引物
SEQ ID NO:41:含ter_thrL终止子的DNA片段的扩增用引物
SEQ ID NO:42:用于扩增gltA基因的ORF以外部分的引物
SEQ ID NO:43:用于扩增gltA基因的ORF以外部分的引物
SEQ ID NO:44:prpC基因的扩增用引物
SEQ ID NO:45:prpC基因的扩增用引物
SEQ ID NO:46:用于sdhA破坏的DNA片段的扩增用引物
SEQ ID NO:47:用于sdhA破坏的DNA片段的扩增用引物
SEQ ID NO:48:sucA基因上游片段的扩增用引物
SEQ ID NO:49:sucA基因上游片段的扩增用引物
SEQ ID NO:50:sucA基因下游片段的扩增用引物
SEQ ID NO:51:sucA基因下游片段的扩增用引物
SEQ ID NO:52:sucA基因的扩增用引物
SEQ ID NO:53:sucA基因的扩增用引物
SEQ ID NO:54:yggB基因的扩增用引物
SEQ ID NO:55:yggB基因的扩增用引物
SEQ ID NO:56:yggB基因的核苷酸序列
SEQ ID NO:57:YggB的氨基酸序列
SEQ ID NO:58:突变型yggB基因的核苷酸序列
SEQ ID NO:59:突变型YggB的氨基酸序列
SEQ ID NO:60:gdh基因上游的扩增用引物(导入了突变)
SEQ ID NO:61:gdh基因上游的扩增用引物
SEQ ID NO:62:gdh基因下游的扩增用引物
SEQ ID NO:63:gdh基因下游的扩增用引物(导入了突变)
SEQ ID NO:64:gdh基因的扩增用引物
SEQ ID NO:65:gdh基因的扩增用引物
SEQ ID NO:66:突变型gdh基因编码区域上游的核苷酸序列
SEQ ID NO:67:sdhA基因上游的扩增用引物
SEQ ID NO:68:sdhA基因上游的扩增用引物(导入了突变)
SEQ ID NO:69:sdhA基因下游的扩增用引物
SEQ ID NO:70:sdhA基因下游的扩增用引物(导入了突变)
SEQ ID NO:71:sdhA基因的扩增用引物
SEQ ID NO:72:sdhA基因的扩增用引物
SEQ ID NO:73:谷氨酸棒杆菌ATCC13032的sdh操纵子的核苷酸序列(SDHC、SDHA、SDHB的氨基酸序列一并给出)
sdhC:449~1219
sdhA:1239~3257
sdhB:3260~4006
SEQ ID NO:74:SDHC的氨基酸序列
SEQ ID NO:75:SDHA的氨基酸序列
SEQ ID NO:76:SDAB的氨基酸序列
SEQ ID NO:77:乳发酵短杆菌ATCC13869的sdh操纵子的核苷酸序列(SDHC、SDHA、SDHB的氨基酸序列一并给出)
sdhC:449~1219
sdhA:1239~3257
sdhB:3260~4006
SEQ ID NO:78:SDHC的氨基酸序列
SEQ ID NO:79:SDHA的氨基酸序列
SEQ ID NO:80:SDHB的氨基酸序列
工业实用性
通过采用本发明的方法,能够高效率地发酵生产L-谷氨酸等L-氨基酸。
序列表
<110>味之素株式会社(Ajinomoto Co.,Inc.)
<120>L-氨基酸的生产方法
<130>C-C7220
<150>JP2006-341019
<151>2006-12-19
<160>80
<170>PatentIn version 3.3
<210>1
<211>3944
<212>DNA
<213>Pantoea ananatis
<220>
<221>CDS
<222>(910)..(1254)
<220>
<221>CDS
<222>(1258)..(3021)
<220>
<221>CDS
<222>(3039)..(3752)
<400>1
tataagcagt gaagggtaca gagtgagagg cgcgttccac aggaaatgta aagccgtgac 60
atgtgcatat cgcggttgtt aaagatccgg tcaggacata acttctttgt aaccaataac 120
ctgatggttt taaaagctgt tagtggcaat gttacataac ttgagcttag gagaaagtgt 180
atccccataa cttttgtgca tcataggatt ttatcgggat agtttgtaac tgaattgttg 240
aactattgtc aaatcagata attaaatttg tatgaattgt gaaaaacgtg agaatgatca 300
ctgttccacc caaaattctc caaagagttt gtaggggaat tgtaatcaga atgtgatcgt 360
cctatacttc cggccaggtc tccggaatac cctgacacca ggagccaccc agcgtttgta 420
acacgtcatt taacactgga tgttgctatt ttggtgacgg ataaagtctg aacacgttta 480
gggttcaggt cttatgtccg gaggaagcaa aataaaaaag agctgtgtgg gcaaaaccgt 540
gaaaaaacaa agacctgtca acttggatct ctcgacgatc cggtttcccg ttactgcaat 600
atcgtccatt cttcaccgcg tctccggcgt catcaccctg gttgctctcg gcatcctgtt 660
atggcttctc ggtctttccc tctcttctcc tgaaggtttc cagcacgctg catctgtcat 720
gaatggcttc tttgcgaagt tcatcatgtg gggcattttg actgcgctgg cctatcatgc 780
agtgggcggg atacgtcata tgttaatgga ttttggttat ctggccgaaa cgctcgaagc 840
aggtaagcgt tctgcgcagg tgacgtttgg tattactgtc gtgctttcaa ttttagcagg 900
agtcctcgt atg gta agc aat gca tcc gcg ttg ggt cgc aat ggc att cag 951
Met Val Ser Asn Ala Ser Ala Leu Gly Arg Asn Gly Ile Gln
1 5 10
gac tgg ctg ctg ctg cgc gcg acg gca atc ctc atc acg ctt tac atc 999
Asp Trp Leu Leu Leu Arg Ala Thr Ala Ile Leu Ile Thr Leu Tyr Ile
15 20 25 30
atc tat ctc ctc ggt ttc gtg gtg atg aca gac acg ctg acg tat gac 1047
Ile Tyr Leu Leu Gly Phe Val Val Met Thr Asp Thr Leu Thr Tyr Asp
35 40 45
atc tgg cgt ggc ttc ttc gct tct gca ttt aca aaa gtg ttc acg ctc 1095
Ile Trp Arg Gly Phe Phe Ala Ser Ala Phe Thr Lys Val Phe Thr Leu
50 55 60
ctg acg ctg ttt tcc att ctg atc cac ggc tgg atc ggt atg tgg cag 1143
Leu Thr Leu Phe Ser Ile Leu Ile His Gly Trp Ile Gly Met Trp Gln
65 70 75
gtc tta acg gac tac gtt aaa ccg ctg gca acg cgt ctg ctg ttg cag 1191
Val Leu Thr Asp Tyr Val Lys Pro Leu Ala Thr Arg Leu Leu Leu Gln
80 85 90
ttg gtc atc gta gtg gcg ctg ttg tca tat gca att tat gga ttt gtt 1239
Leu Val Ile Val Val Ala Leu Leu Ser Tyr Ala Ile Tyr Gly Phe Val
95 100 105 110
gtg gtg tgg ggt gtg taa atg agt tta cca gta aga gaa ttt gat gcc 1287
Val Val Trp Gly Val Met Ser Leu Pro Val Arg Glu Phe Asp Ala
115 120 125
gtg gtg atc ggc gca ggc ggc gcg ggt atg cgc gcg gcg ttg caa atc 1335
Val Val Ile Gly Ala Gly Gly Ala Gly Met Arg Ala Ala Leu Gln Ile
130 135 140
tcc cag tcg ggc cag acc tgt gcc ctg ttg tcc aaa gtc ttc ccg acc 1383
Ser Gln Ser Gly Gln Thr Cys Ala Leu Leu Ser Lys Val Phe Pro Thr
145 150 155
cgt tcc cat acg gtc tcc gcg cag ggc gga atc acc gtt gcg ctg ggt 1431
Arg Ser His Thr Val Ser Ala Gln Gly Gly Ile Thr Val Ala Leu Gly
160 165 170
aac acc cat gac gat aac tgg gaa tgg cat atg tat gac acc gtc aaa 1479
Asn Thr His Asp Asp Asn Trp Glu Trp His Met Tyr Asp Thr Val Lys
175 180 185
ggt tcc gac tac atc ggt gac cag gac gcg atc gaa tac atg tgt cac 1527
Gly Ser Asp Tyr Ile Gly Asp Gln Asp Ala Ile Glu Tyr Met Cys His
190 195 200 205
gtc ggt ccg gaa gcg att ctg gaa ctg gag cac atg ggc ttg ccc ttc 1575
Val Gly Pro Glu Ala Ile Leu Glu Leu Glu His Met Gly Leu Pro Phe
210 215 220
tcc cgt ctt gat gac ggc cgc gtt tat cag cgt ccg ttt ggt ggt cag 1623
Ser Arg Leu Asp Asp Gly Arg Val Tyr Gln Arg Pro Phe Gly Gly Gln
225 230 235
tcg aaa aac ttc ggc ggt gag cag gcg gcg cgt act gcc gca gcg gcc 1671
Ser Lys Asn Phe Gly Gly Glu Gln Ala Ala Arg Thr Ala Ala Ala Ala
240 245 250
gac cgt acc ggc cac gca ctg ctg cat acg ctg tat cag cag aac ctg 1719
Asp Arg Thr Gly His Ala Leu Leu His Thr Leu Tyr Gln Gln Asn Leu
255 260 265
aaa aat aaa acc act atc ttc tcc gaa tgg tat gca ctg gat ctg gtc 1767
Lys Asn Lys Thr Thr Ile Phe Ser Glu Trp Tyr Ala Leu Asp Leu Val
270 275 280 285
aaa aat gac gac ggt gcc atc gta ggc tgt acg gca atc tgc atg gaa 1815
Lys Asn Asp Asp Gly Ala Ile Val Gly Cys Thr Ala Ile Cys Met Glu
290 295 300
acc ggc gaa acg gtt tac ttc aaa gcc aag gcc acc att ctg gcg acg 1863
Thr Gly Glu Thr Val Tyr Phe Lys Ala Lys Ala Thr Ile Leu Ala Thr
305 310 315
ggc ggt gca gga cgt att tat cag tcc acg acg aac gcc cac atc aat 1911
Gly Gly Ala Gly Arg Ile Tyr Gln Ser Thr Thr Asn Ala His Ile Asn
320 325 330
acc ggc gac ggt gtt ggc atg gcg ctg cgc gcg ggc gtg cct gtg cag 1959
Thr Gly Asp Gly Val Gly Met Ala Leu Arg Ala Gly Val Pro Val Gln
335 340 345
gat atg gaa atg tgg cag ttc cac cca acc ggt atc gcc ggt gct ggt 2007
Asp Met Glu Met Trp Gln Phe His Pro Thr Gly Ile Ala Gly Ala Gly
350 355 360 365
gta ctg gtc acc gaa ggc tgt cgt ggt gaa ggc ggt tat ctg ctg aac 2055
Val Leu Val Thr Glu Gly Cys Arg Gly Glu Gly Gly Tyr Leu Leu Asn
370 375 380
aaa cac ggt gag cgt ttc atg gag cgc tat gcg cct aac gcc aaa gac 2103
Lys His Gly Glu Arg Phe Met Glu Arg Tyr Ala Pro Asn Ala Lys Asp
385 390 395
ctt gcc ggt cgt gac gtt gtg gcc cgt tcg atg atg gtg gag atc cgt 2151
Leu Ala Gly Arg Asp Val Val Ala Arg Ser Met Met Val Glu Ile Arg
400 405 410
gaa ggt cgt ggt tgc gac ggt cca tgg ggc ccg cac atc aag ctg aaa 2199
Glu Gly Arg Gly Cys Asp Gly Pro Trp Gly Pro His Ile Lys Leu Lys
415 420 425
ctc gat cac ctg ggt gcg gaa gtg ctg gaa tcg cgc ctg ccg ggc atc 2247
Leu Asp His Leu Gly Ala Glu Val Leu Glu Ser Arg Leu Pro Gly Ile
430 435 440 445
ctt gag ctg tcc cgt acc ttt gct cac gtt gac ccg att aaa gag ccg 2295
Leu Glu Leu Ser Arg Thr Phe Ala His Val Asp Pro Ile Lys Glu Pro
450 455 460
att ccg gtt atc cca acc tgt cac tac atg atg ggc ggc gtg cca acc 2343
Ile Pro Val Ile Pro Thr Cys His Tyr Met Met Gly Gly Val Pro Thr
465 470 475
aaa gtc acc ggt cag gcg ctg cgt gtg aat gag cag ggc gaa gat gaa 2391
Lys Val Thr Gly Gln Ala Leu Arg Val Asn Glu Gln Gly Glu Asp Glu
480 485 490
gtg att cct ggc ctg ttc gcg gtg ggt gaa atc gcc tgc gta tcg gta 2439
Val Ile Pro Gly Leu Phe Ala Val Gly Glu Ile Ala Cys Val Ser Val
495 500 505
cac ggg gcg aac cgt ctg ggt ggc aac tcg ctg ctg gac ctg gtg gtc 2487
His Gly Ala Asn Arg Leu Gly Gly Asn Ser Leu Leu Asp Leu Val Val
510 515 520 525
ttc ggt cgc gcg gct ggc gtg cat ctg ctt gaa tgt ctg gaa gag cag 2535
Phe Gly Arg Ala Ala Gly Val His Leu Leu Glu Cys Leu Glu Glu Gln
530 535 540
ggt gaa ctg cgt gaa gcc agc cag gaa aac att gat gcc gcg atg gcg 2583
Gly Glu Leu Arg Glu Ala Ser Gln Glu Asn Ile Asp Ala Ala Met Ala
545 550 555
cgt ttc aac cgc tgg gaa aat aac acc acg ggt gaa gat ccg gtt gaa 2631
Arg Phe Asn Arg Trp Glu Asn Asn Thr Thr Gly Glu Asp Pro Val Glu
560 565 570
atc cgc aag gcg ttg caa cgc tgc atg cag aac aac ttc tcg gta ttc 2679
Ile Arg Lys Ala Leu Gln Arg Cys Met Gln Asn Asn Phe Ser Val Phe
575 580 585
cgt gaa ggc gat gcg atg cgt gaa ggg ctt gct gaa ctg aaa gag atc 2727
Arg Glu Gly Asp Ala Met Arg Glu Gly Leu Ala Glu Leu Lys Glu Ile
590 595 600 605
cgt gag cgt ctg aag tcc gcg cgc ctg gat gac cgc tca cct gac ttc 2775
Arg Glu Arg Leu Lys Ser Ala Arg Leu Asp Asp Arg Ser Pro Asp Phe
610 615 620
aat aca cag cgt att gag tgc ctt gag ctg gat aac ctg atg gaa acc 2823
Asn Thr Gln Arg Ile Glu Cys Leu Glu Leu Asp Asn Leu Met Glu Thr
625 630 635
gct tat gcc acc gca gtg gcg gcc aac tac cgc act gag agc cgt ggc 2871
Ala Tyr Ala Thr Ala Val Ala Ala Asn Tyr Arg Thr Glu Ser Arg Gly
640 645 650
gca cac agt cgc ttc gac tat ccg gaa cgt gat gat gcc aac tgg ctg 2919
Ala His Ser Arg Phe Asp Tyr Pro Glu Arg Asp Asp Ala Asn Trp Leu
655 660 665
tgc cat agc ctc tat gtt ccg caa acg gaa agc atg acg cgc cgt gag 2967
Cys His Ser Leu Tyr Val Pro Gln Thr Glu Ser Met Thr Arg Arg Glu
670 675 680 685
gtg aac atg caa ccg aaa ctg cgt gcg gcc ttc ccg ccg aaa gcg cgt 3015
Val Asn Met Gln Pro Lys Leu Arg Ala Ala Phe Pro Pro Lys Ala Arg
690 695 700
acc tac taattgcgga gatcatc atg aga ctt gaa ttt tcg atc tat cgt 3065
Thr Tyr Met Arg Leu Glu Phe Ser Ile Tyr Arg
705 710
tac aac ccg gac gtc gat gac aaa ccg cgc atg cag gac tat acc ctg 3113
Tyr Asn Pro Asp Val Asp Asp Lys Pro Arg Met Gln Asp Tyr Thr Leu
715 720 725
gaa gcg gaa gac ggc cgt gac atg atg ctg ctg gat gcg ctg atc cgt 3161
Glu Ala Glu Asp Gly Arg Asp Met Met Leu Leu Asp Ala Leu Ile Arg
730 735 740
ctg aaa gag aag gat cct acc ctg gcg ttt cgt cgc tca tgc cgt gaa 3209
Leu Lys Glu Lys Asp Pro Thr Leu Ala Phe Arg Arg Ser Cys Arg Glu
745 750 755 760
ggc gtg tgc ggt tca gac ggc ctg aac atg aac ggg aaa aac ggc ctg 3257
Gly Val Cys Gly Ser Asp Gly Leu Asn Met Asn Gly Lys Asn Gly Leu
765 770 775
gcc tgt atc acg ccg gtc tcg gcg tta ggc aac ggc aag cag aaa atc 3305
Ala Cys Ile Thr Pro Val Ser Ala Leu Gly Asn Gly Lys Gln Lys Ile
780 785 790
gtt atc cgt cct cta ccg ggc ctg ccg gtc gtg cgc gac ctg gtg gtg 3353
Val Ile Arg Pro Leu Pro Gly Leu Pro Val Val Arg Asp Leu Val Val
795 800 805
gac atg ggc cag ttt tat gcc cag tat gag aaa att aag cct ttc tta 3401
Asp Met Gly Gln Phe Tyr Ala Gln Tyr Glu Lys Ile Lys Pro Phe Leu
810 815 820
ttg aat aac ggt gaa aac ccg cca gcg cgt gag cat ctg cag tcg ccg 3449
Leu Asn Asn Gly Glu Asn Pro Pro Ala Arg Glu His Leu Gln Ser Pro
825 830 835 840
ggc gag cgt gag cat ctg gac gga ttg tat gag tgt att ctc tgc gcc 3497
Gly Glu Arg Glu His Leu Asp Gly Leu Tyr Glu Cys Ile Leu Cys Ala
845 850 855
tgt tgc tca acc tct tgc ccg tcg ttc tgg tgg aac ccg gac aag ttt 3545
Cys Cys Ser Thr Ser Cys Pro Ser Phe Trp Trp Asn Pro Asp Lys Phe
860 865 870
att ggt cct gca ggc ctg ctg gcc gct tac cgc ttc ctg att gac agc 3593
Ile Gly Pro Ala Gly Leu Leu Ala Ala Tyr Arg Phe Leu Ile Asp Ser
875 880 885
cgt gat acg gaa acg gat gcg cgt ctg gat aat ctg agc gat gca ttc 3641
Arg Asp Thr Glu Thr Asp Ala Arg Leu Asp Asn Leu Ser Asp Ala Phe
890 895 900
agc gtt ttc cgc tgt cac agc atc atg aac tgt gta agt gtt tgt cct 3689
Ser Val Phe Arg Cys His Ser Ile Met Asn Cys Val Ser Val Cys Pro
905 910 915 920
aaa ggg cta aac ccg acg cgc gct atc ggc cac att aag tcg atg ctg 3737
Lys Gly Leu Asn Pro Thr Arg Ala Ile Gly His Ile Lys Ser Met Leu
925 930 935
ctg caa cgc agc gcg tagttatacc accgggaacc tcaggttccc ggtattttac 3792
Leu Gln Arg Ser Ala
940
ggaagcctct gtaaacgcgg tcccaaccac gtttacaaag gttcccttac gggccgggcg 3852
cgcgctgcgc acagtgctcg tatcgctgaa ctcactacgg caaaccgcga aagcggcaac 3912
aaatgaaacc tcaaaaaagc ataacattgc tt 3944
<210>2
<211>115
<212>PRT
<213>Pantoea ananatis
<400>2
Met Val Ser Asn Ala Ser Ala Leu Gly Arg Asn Gly Ile Gln Asp Trp
1 5 10 15
Leu Leu Leu Arg Ala Thr Ala Ile Leu Ile Thr Leu Tyr Ile Ile Tyr
20 25 30
Leu Leu Gly Phe Val Val Met Thr Asp Thr Leu Thr Tyr Asp Ile Trp
35 40 45
Arg Gly Phe Phe Ala Ser Ala Phe Thr Lys Val Phe Thr Leu Leu Thr
50 55 60
Leu Phe Ser Ile Leu Ile His Gly Trp Ile Gly Met Trp Gln Val Leu
65 70 75 80
Thr Asp Tyr Val Lys Pro Leu Ala Thr Arg Leu Leu Leu Gln Leu Val
85 90 95
Ile Val Val Ala Leu Leu Ser Tyr Ala Ile Tyr Gly Phe Val Val Val
100 105 110
Trp Gly Val
115
<210>3
<211>588
<212>PRT
<213>Pantoea ananatis
<400>3
Met Ser Leu Pro Val Arg Glu Phe Asp Ala Val Val Ile Gly Ala Gly
1 5 10 15
Gly Ala Gly Met Arg Ala Ala Leu Gln Ile Ser Gln Ser Gly Gln Thr
20 25 30
Cys Ala Leu Leu Ser Lys Val Phe Pro Thr Arg Ser His Thr Val Ser
35 40 45
Ala Gln Gly Gly Ile Thr Val Ala Leu Gly Asn Thr His Asp Asp Asn
50 55 60
Trp Glu Trp His Met Tyr Asp Thr Val Lys Gly Ser Asp Tyr Ile Gly
65 70 75 80
Asp Gln Asp Ala Ile Glu Tyr Met Cys His Val Gly Pro Glu Ala Ile
85 90 95
Leu Glu Leu Glu His Met Gly Leu Pro Phe Ser Arg Leu Asp Asp Gly
100 105 110
Arg Val Tyr Gln Arg Pro Phe Gly Gly Gln Ser Lys Asn Phe Gly Gly
115 120 125
Glu Gln Ala Ala Arg Thr Ala Ala Ala Ala Asp Arg Thr Gly His Ala
130 135 140
Leu Leu His Thr Leu Tyr Gln Gln Asn Leu Lys Asn Lys Thr Thr Ile
145 150 155 160
Phe Ser Glu Trp Tyr Ala Leu Asp Leu Val Lys Asn Asp Asp Gly Ala
165 170 175
Ile Val Gly Cys Thr Ala Ile Cys Met Glu Thr Gly Glu Thr Val Tyr
180 185 190
Phe Lys Ala Lys Ala Thr Ile Leu Ala Thr Gly Gly Ala Gly Arg Ile
195 200 205
Tyr Gln Ser Thr Thr Asn Ala His Ile Asn Thr Gly Asp Gly Val Gly
210 215 220
Met Ala Leu Arg Ala Gly Val Pro Val Gln Asp Met Glu Met Trp Gln
225 230 235 240
Phe His Pro Thr Gly Ile Ala Gly Ala Gly Val Leu Val Thr Glu Gly
245 250 255
Cys Arg Gly Glu Gly Gly Tyr Leu Leu Asn Lys His Gly Glu Arg Phe
260 265 270
Met Glu Arg Tyr Ala Pro Asn Ala Lys Asp Leu Ala Gly Arg Asp Val
275 280 285
Val Ala Arg Ser Met Met Val Glu Ile Arg Glu Gly Arg Gly Cys Asp
290 295 300
Gly Pro Trp Gly Pro His Ile Lys Leu Lys Leu Asp His Leu Gly Ala
305 310 315 320
Glu Val Leu Glu Ser Arg Leu Pro Gly Ile Leu Glu Leu Ser Arg Thr
325 330 335
Phe Ala His Val Asp Pro Ile Lys Glu Pro Ile Pro Val Ile Pro Thr
340 345 350
Cys His Tyr Met Met Gly Gly Val Pro Thr Lys Val Thr Gly Gln Ala
355 360 365
Leu Arg Val Asn Glu Gln Gly Glu Asp Glu Val Ile Pro Gly Leu Phe
370 375 380
Ala Val Gly Glu Ile Ala Cys Val Ser Val His Gly Ala Asn Arg Leu
385 390 395 400
Gly Gly Asn Ser Leu Leu Asp Leu Val Val Phe Gly Arg Ala Ala Gly
405 410 415
Val His Leu Leu Glu Cys Leu Glu Glu Gln Gly Glu Leu Arg Glu Ala
420 425 430
Ser Gln Glu Asn Ile Asp Ala Ala Met Ala Arg Phe Asn Arg Trp Glu
435 440 445
Asn Asn Thr Thr Gly Glu Asp Pro Val Glu Ile Arg Lys Ala Leu Gln
450 455 460
Arg Cys Met Gln Asn Asn Phe Ser Val Phe Arg Glu Gly Asp Ala Met
465 470 475 480
Arg Glu Gly Leu Ala Glu Leu Lys Glu Ile Arg Glu Arg Leu Lys Ser
485 490 495
Ala Arg Leu Asp Asp Arg Ser Pro Asp Phe Asn Thr Gln Arg Ile Glu
500 505 510
Cys Leu Glu Leu Asp Asn Leu Met Glu Thr Ala Tyr Ala Thr Ala Val
515 520 525
Ala Ala Asn Tyr Arg Thr Glu Ser Arg Gly Ala His Ser Arg Phe Asp
530 535 540
Tyr Pro Glu Arg Asp Asp Ala Asn Trp Leu Cys His Ser Leu Tyr Val
545 550 555 560
Pro Gln Thr Glu Ser Met Thr Arg Arg Glu Val Asn Met Gln Pro Lys
565 570 575
Leu Arg Ala Ala Phe Pro Pro Lys Ala Arg Thr Tyr
580 585
<210>4
<211>238
<212>PRT
<213>Pantoea ananatis
<400>4
Met Arg Leu Glu Phe Ser Ile Tyr Arg Tyr Asn Pro Asp Val Asp Asp
1 5 10 15
Lys Pro Arg Met Gln Asp Tyr Thr Leu Glu Ala Glu Asp Gly Arg Asp
20 25 30
Met Met Leu Leu Asp Ala Leu Ile Arg Leu Lys Glu Lys Asp Pro Thr
35 40 45
Leu Ala Phe Arg Arg Ser Cys Arg Glu Gly Val Cys Gly Ser Asp Gly
50 55 60
Leu Asn Met Asn Gly Lys Asn Gly Leu Ala Cys Ile Thr Pro Val Ser
65 70 75 80
Ala Leu Gly Asn Gly Lys Gln Lys Ile Val Ile Arg Pro Leu Pro Gly
85 90 95
Leu Pro Val Val Arg Asp Leu Val Val Asp Met Gly Gln Phe Tyr Ala
100 105 110
Gln Tyr Glu Lys Ile Lys Pro Phe Leu Leu Asn Asn Gly Glu Asn Pro
115 120 125
Pro Ala Arg Glu His Leu Gln Ser Pro Gly Glu Arg Glu His Leu Asp
130 135 140
Gly Leu Tyr Glu Cys Ile Leu Cys Ala Cys Cys Ser Thr Ser Cys Pro
145 150 155 160
Ser Phe Trp Trp Asn Pro Asp Lys Phe Ile Gly Pro Ala Gly Leu Leu
165 170 175
Ala Ala Tyr Arg Phe Leu Ile Asp Ser Arg Asp Thr Glu Thr Asp Ala
180 185 190
Arg Leu Asp Asn Leu Ser Asp Ala Phe Ser Val Phe Arg Cys His Ser
195 200 205
Ile Met Asn Cys Val Ser Val Cys Pro Lys Gly Leu Asn Pro Thr Arg
210 215 220
Ala Ile Gly His Ile Lys Ser Met Leu Leu Gln Arg Ser Ala
225 230 235
<210>5
<211>3944
<212>DNA
<213>Pantoea ananatis
<220>
<221>CDS
<222>(527)..(913)
<400>5
tataagcagt gaagggtaca gagtgagagg cgcgttccac aggaaatgta aagccgtgac 60
atgtgcatat cgcggttgtt aaagatccgg tcaggacata acttctttgt aaccaataac 120
ctgatggttt taaaagctgt tagtggcaat gttacataac ttgagcttag gagaaagtgt 180
atccccataa cttttgtgca tcataggatt ttatcgggat agtttgtaac tgaattgttg 240
aactattgtc aaatcagata attaaatttg tatgaattgt gaaaaacgtg agaatgatca 300
ctgttccacc caaaattctc caaagagttt gtaggggaat tgtaatcaga atgtgatcgt 360
cctatacttc cggccaggtc tccggaatac cctgacacca ggagccaccc agcgtttgta 420
acacgtcatt taacactgga tgttgctatt ttggtgacgg ataaagtctg aacacgttta 480
gggttcaggt cttatgtccg gaggaagcaa aataaaaaag agctgt gtg ggc aaa 535
Val Gly Lys
1
acc gtg aaa aaa caa aga cct gtc aac ttg gat ctc tcg acg atc cgg 583
Thr Val Lys Lys Gln Arg Pro Val Asn Leu Asp Leu Ser Thr Ile Arg
5 10 15
ttt ccc gtt act gca ata tcg tcc att ctt cac cgc gtc tcc ggc gtc 631
Phe Pro Val Thr Ala Ile Ser Ser Ile Leu His Arg Val Ser Gly Val
20 25 30 35
atc acc ctg gtt gct ctc ggc atc ctg tta tgg ctt ctc ggt ctt tcc 679
Ile Thr Leu Val Ala Leu Gly Ile Leu Leu Trp Leu Leu Gly Leu Ser
40 45 50
ctc tct tct cct gaa ggt ttc cag cac gct gca tct gtc atg aat ggc 727
Leu Ser Ser Pro Glu Gly Phe Gln His Ala Ala Ser Val Met Asn Gly
55 60 65
ttc ttt gcg aag ttc atc atg tgg ggc att ttg act gcg ctg gcc tat 775
Phe Phe Ala Lys Phe Ile Met Trp Gly Ile Leu Thr Ala Leu Ala Tyr
70 75 80
cat gca gtg ggc ggg ata cgt cat atg tta atg gat ttt ggt tat ctg 823
His Ala Val Gly Gly Ile Arg His Met Leu Met Asp Phe Gly Tyr Leu
85 90 95
gcc gaa acg ctc gaa gca ggt aag cgt tct gcg cag gtg acg ttt ggt 871
Ala Glu Thr Leu Glu Ala Gly Lys Arg Ser Ala Gln Val Thr Phe Gly
100 105 110 115
att act gtc gtg ctt tca att tta gca gga gtc ctc gta tgg 913
Ile Thr Val Val Leu Ser Ile Leu Ala Gly Val Leu Val Trp
120 125
taagcaatgc atccgcgttg ggtcgcaatg gcattcagga ctggctgctg ctgcgcgcga 973
cggcaatcct catcacgctt tacatcatct atctcctcgg tttcgtggtg atgacagaca 1033
cgctgacgta tgacatctgg cgtggcttct tcgcttctgc atttacaaaa gtgttcacgc 1093
tcctgacgct gttttccatt ctgatccacg gctggatcgg tatgtggcag gtcttaacgg 1153
actacgttaa accgctggca acgcgtctgc tgttgcagtt ggtcatcgta gtggcgctgt 1213
tgtcatatgc aatttatgga tttgttgtgg tgtggggtgt gtaaatgagt ttaccagtaa 1273
gagaatttga tgccgtggtg atcggcgcag gcggcgcggg tatgcgcgcg gcgttgcaaa 1333
tctcccagtc gggccagacc tgtgccctgt tgtccaaagt cttcccgacc cgttcccata 1393
cggtctccgc gcagggcgga atcaccgttg cgctgggtaa cacccatgac gataactggg 1453
aatggcatat gtatgacacc gtcaaaggtt ccgactacat cggtgaccag gacgcgatcg 1513
aatacatgtg tcacgtcggt ccggaagcga ttctggaact ggagcacatg ggcttgccct 1573
tctcccgtct tgatgacggc cgcgtttatc agcgtccgtt tggtggtcag tcgaaaaact 1633
tcggcggtga gcaggcggcg cgtactgccg cagcggccga ccgtaccggc cacgcactgc 1693
tgcatacgct gtatcagcag aacctgaaaa ataaaaccac tatcttctcc gaatggtatg 1753
cactggatct ggtcaaaaat gacgacggtg ccatcgtagg ctgtacggca atctgcatgg 1813
aaaccggcga aacggtttac ttcaaagcca aggccaccat tctggcgacg ggcggtgcag 1873
gacgtattta tcagtccacg acgaacgccc acatcaatac cggcgacggt gttggcatgg 1933
cgctgcgcgc gggcgtgcct gtgcaggata tggaaatgtg gcagttccac ccaaccggta 1993
tcgccggtgc tggtgtactg gtcaccgaag gctgtcgtgg tgaaggcggt tatctgctga 2053
acaaacacgg tgagcgtttc atggagcgct atgcgcctaa cgccaaagac cttgccggtc 2113
gtgacgttgt ggcccgttcg atgatggtgg agatccgtga aggtcgtggt tgcgacggtc 2173
catggggccc gcacatcaag ctgaaactcg atcacctggg tgcggaagtg ctggaatcgc 2233
gcctgccggg catccttgag ctgtcccgta cctttgctca cgttgacccg attaaagagc 2293
cgattccggt tatcccaacc tgtcactaca tgatgggcgg cgtgccaacc aaagtcaccg 2353
gtcaggcgct gcgtgtgaat gagcagggcg aagatgaagt gattcctggc ctgttcgcgg 2413
tgggtgaaat cgcctgcgta tcggtacacg gggcgaaccg tctgggtggc aactcgctgc 2473
tggacctggt ggtcttcggt cgcgcggctg gcgtgcatct gcttgaatgt ctggaagagc 2533
agggtgaact gcgtgaagcc agccaggaaa acattgatgc cgcgatggcg cgtttcaacc 2593
gctgggaaaa taacaccacg ggtgaagatc cggttgaaat ccgcaaggcg ttgcaacgct 2653
gcatgcagaa caacttctcg gtattccgtg aaggcgatgc gatgcgtgaa gggcttgctg 2713
aactgaaaga gatccgtgag cgtctgaagt ccgcgcgcct ggatgaccgc tcacctgact 2773
tcaatacaca gcgtattgag tgccttgagc tggataacct gatggaaacc gcttatgcca 2833
ccgcagtggc ggccaactac cgcactgaga gccgtggcgc acacagtcgc ttcgactatc 2893
cggaacgtga tgatgccaac tggctgtgcc atagcctcta tgttccgcaa acggaaagca 2953
tgacgcgccg tgaggtgaac atgcaaccga aactgcgtgc ggccttcccg ccgaaagcgc 3013
gtacctacta attgcggaga tcatcatgag acttgaattt tcgatctatc gttacaaccc 3073
ggacgtcgat gacaaaccgc gcatgcagga ctataccctg gaagcggaag acggccgtga 3133
catgatgctg ctggatgcgc tgatccgtct gaaagagaag gatcctaccc tggcgtttcg 3193
tcgctcatgc cgtgaaggcg tgtgcggttc agacggcctg aacatgaacg ggaaaaacgg 3253
cctggcctgt atcacgccgg tctcggcgtt aggcaacggc aagcagaaaa tcgttatccg 3313
tcctctaccg ggcctgccgg tcgtgcgcga cctggtggtg gacatgggcc agttttatgc 3373
ccagtatgag aaaattaagc ctttcttatt gaataacggt gaaaacccgc cagcgcgtga 3433
gcatctgcag tcgccgggcg agcgtgagca tctggacgga ttgtatgagt gtattctctg 3493
cgcctgttgc tcaacctctt gcccgtcgtt ctggtggaac ccggacaagt ttattggtcc 3553
tgcaggcctg ctggccgctt accgcttcct gattgacagc cgtgatacgg aaacggatgc 3613
gcgtctggat aatctgagcg atgcattcag cgttttccgc tgtcacagca tcatgaactg 3673
tgtaagtgtt tgtcctaaag ggctaaaccc gacgcgcgct atcggccaca ttaagtcgat 3733
gctgctgcaa cgcagcgcgt agttatacca ccgggaacct caggttcccg gtattttacg 3793
gaagcctctg taaacgcggt cccaaccacg tttacaaagg ttcccttacg ggccgggcgc 3853
gcgctgcgca cagtgctcgt atcgctgaac tcactacggc aaaccgcgaa agcggcaaca 3913
aatgaaacct caaaaaagca taacattgct t 3944
<210>6
<211>129
<212>PRT
<213>Pantoea ananatis
<400>6
Val Gly Lys Thr Val Lys Lys Gln Arg Pro Val Asn Leu Asp Leu Ser
1 5 10 15
Thr Ile Arg Phe Pro Val Thr Ala Ile Ser Ser Ile Leu His Arg Val
20 25 30
Ser Gly Val Ile Thr Leu Val Ala Leu Gly Ile Leu Leu Trp Leu Leu
35 40 45
Gly Leu Ser Leu Ser Ser Pro Glu Gly Phe Gln His Ala Ala Ser Val
50 55 60
Met Asn Gly Phe Phc Ala Lys Phc Ile Met Trp Gly Ile Leu Thr Ala
65 70 75 80
Leu Ala Tyr His Ala Val Gly Gly Ile Arg His Met Leu Met Asp Phe
85 90 95
Gly Tyr Leu Ala Glu Thr Leu Glu Ala Gly Lys Arg Ser Ala Gln Val
100 105 110
Thr Phe Gly Ile Thr Val Val Leu Ser Ile Leu Ala Gly Val Leu Val
115 120 125
Trp
<210>7
<211>4556
<212>DNA
<213>Pantoea ananatis
<220>
<221>CDS
<222>(2)..(121)
<220>
<221>CDS
<222>(322)..(3129)
<220>
<221>CDS
<222>(3145)..(4368)
<220>
<221>CDS
<222>(4437)..(4556)
<400>7
t gca ttc agc gtt ttc cgc tgt cac agc atc atg aac tgt gta agt gtt 49
Ala Phe Ser Val Phe Arg Cys His Ser Ile Met Asn Cys Val Ser Val
1 5 10 15
tgt cct aaa ggg cta aac ccg acg cgc gct atc ggc cac att aag tcg 97
Cys Pro Lys Gly Leu Asn Pro Thr Arg Ala Ile Gly His Ile Lys Ser
20 25 30
atg ctg ctg caa cgc agc gcg tag ttataccacc gggaacctca ggttcccggt 151
Met Leu Leu Gln Arg Ser Ala
35
attttacgga agcctctgta aacgcggtcc caaccacgtt tacaaaggtt cccttacggg 211
ccgggcgcgc gctgcgcaca gtgctcgtat cgctgaactc actacggcaa accgcgaaag 271
cggcaacaaa tgaaacctca aaaaagcata acattgctta agggatcaca atg cag 327
Met Gln
40
aac agc gcg atg aag ccc tgg ctg gac tcc tcc tgg ctg gcc ggc gcg 375
Asn Ser Ala Met Lys Pro Trp Leu Asp Ser Ser Trp Leu Ala Gly Ala
45 50 55
aat cag tct tac ata gag caa ctc tat gag gat ttc ctg acc gat cct 423
Asn Gln Ser Tyr Ile Glu Gln Leu Tyr Glu Asp Phe Leu Thr Asp Pro
60 65 70
gac tct gtg gat gca gtg tgg cgc tcg atg ttc caa cag tta cca ggc 471
Asp Ser Val Asp Ala Val Trp Arg Ser Met Phe Gln Gln Leu Pro Gly
75 80 85
acg gga gtg aaa cct gag cag ttc cac tcc gca act cgc gaa tat ttc 519
Thr Gly Val Lys Pro Glu Gln Phe His Ser Ala Thr Arg Glu Tyr Phe
90 95 100 105
cgt cgc ctg gcg aaa gac gca tct cgt tac acc tcc tca gtt acc gat 567
Arg Arg Leu Ala Lys Asp Ala Ser Arg Tyr Thr Ser Ser Val Thr Asp
110 115 120
ccg gca acc aac tcc aaa caa gtg aaa gtg ctg cag ctg att aac gcg 615
Pro Ala Thr Asn Ser Lys Gln Val Lys Val Leu Gln Leu Ile Asn Ala
125 130 135
ttt cgt ttc cgc gga cat cag gaa gca aat ctc gat ccg ctt ggc ctg 663
Phe Arg Phe Arg Gly His Gln Glu Ala Asn Leu Asp Pro Leu Gly Leu
140 145 150
tgg aaa cag gac cgc gtt gcc gat ctc gat cct gcc ttt cac gat ctg 711
Trp Lys Gln Asp Arg Val Ala Asp Leu Asp Pro Ala Phe His Asp Leu
155 160 165
acc gac gcc gat ttt cag gaa agc ttt aac gta ggt tct ttt gcc att 759
Thr Asp Ala Asp Phe Gln Glu Ser Phe Asn Val Gly Ser Phe Ala Ile
170 175 180 185
ggc aaa gaa acc atg aag ctg gcc gat ctg ttc gac gcg ctg aag cag 807
Gly Lys Glu Thr Met Lys Leu Ala Asp Leu Phe Asp Ala Leu Lys Gln
190 195 200
acc tac tgt ggc tcg att ggt gca gag tat atg cac arc aat aac acc 855
Thr Tyr Cys Gly Ser Ile Gly Ala Glu Tyr Met His Ile Asn Asn Thr
205 210 215
gaa gag aaa cgc tgg atc cag cag cgt atc gaa tcc ggt gcg agc cag 903
Glu Glu Lys Arg Trp Ile Gln Gln Arg Ile Glu Ser Gly Ala Ser Gln
220 225 230
acg tca ttc agt ggc gaa gag aaa aaa ggt ttc ctg aaa gag ctg acc 951
Thr Ser Phe Ser Gly Glu Glu Lys Lys Gly Phe Leu Lys Glu Leu Thr
235 240 245
gcg gca gaa ggg ctg gaa aaa tat ctg ggc gcg aaa ttc ccg ggt gca 999
Ala Ala Glu Gly Leu Glu Lys Tyr Leu Gly Ala Lys Phc Pro Gly Ala
250 255 260 265
aaa cgt ttc tcg ctg gaa ggc ggt gat gcg ctg gtg ccg atg ctg cgc 1047
Lys Arg Phe Ser Leu Glu Gly Gly Asp Ala Leu Val Pro Met Leu Arg
270 275 280
gag atg att cgt cat gcg ggc aaa agc ggc aca cgt gaa gtg gta ctg 1095
Glu Met Ile Arg His Ala Gly Lys Ser Gly Thr Arg Glu Val Val Leu
285 290 295
ggg atg gcg cac cgt ggc cgt ctt aac gta ctg att aac gta ctg ggt 1143
Gly Met Ala His Arg Gly Arg Leu Asn Val Leu Ile Asn Val Leu Gly
300 305 310
aaa aag cca cag gat ctg ttc gac gaa ttc tcc ggt aaa cac aaa gag 1191
Lys Lys Pro Gln Asp Leu Phe Asp Glu Phe Ser Gly Lys His Lys Glu
315 320 325
cat ctg ggc acc ggt gat gtg aag tat cac atg ggc ttc tct tcg gat 1239
His Leu Gly Thr Gly Asp Val Lys Tyr His Met Gly Phe Ser Ser Asp
330 335 340 345
att gaa acc gaa ggt ggt ctg gtg cat ctg gcg ctg gcg ttt aac ccg 1287
Ile Glu Thr Glu Gly Gly Leu Val His Leu Ala Leu Ala Phe Asn Pro
350 355 360
tct cac ctg gaa att gtc agc ccg gtg gtc atg gga tcggta cgt gca 1335
Ser His Leu Glu Ile Val Ser Pro Val Val Met Gly Ser Val Arg Ala
365 370 375
cgt ctc gat cgt ctg gcc gaa ccg gtc agc aat aaa gtg ttg cct atc 1383
Arg Leu Asp Arg Leu Ala Glu Pro Val Ser Asn Lys Val Leu Pro Ile
380 385 390
acc att cac ggt gat gcg gcg gtg att ggt cag ggc gtg gtt cag gaa 1431
Thr Ile His Gly Asp Ala Ala Val Ile Gly Gln Gly Val Val Gln Glu
395 400 405
acc ctg aac atg tct cag gcg cgc ggc tac gaa gtg ggc ggc acg gta 1479
Thr Leu Asn Met Ser Gln Ala Arg Gly Tyr Glu Val Gly Gly Thr Val
410 415 420 425
cgt atc gtc att aac aac cag gtt ggt ttt acc acc tcc aac ccg aaa 1527
Arg Ile Val Ile Asn Asn Gln Val Gly Phe Thr Thr Ser Asn Pro Lys
430 435 440
gat gcg cgt tca acc ccg tac tgt act gac atc ggc aag atg gtg ctg 1575
Asp Ala Arg Ser Thr Pro Tyr Cys Thr Asp Ile Gly Lys Met Val Leu
445 450 455
gca ccg att ttc cac gtc aat gct gac gat ccg gaa gcg gtg gcc ttt 1623
Ala Pro Ile Phe His Val Asn Ala Asp Asp Pro Glu Ala Val Ala Phe
460 465 470
gtt acc cgc ctg gcg ctg gac tat cgc aac acc ttc aaa cgc gat gtg 1671
Val Thr Arg Leu Ala Leu Asp Tyr Arg Asn Thr Phe Lys Arg Asp Val
475 480 485
ttt atc gat ctg gtg tgc tat cgc cgt cat ggt cac aac gag gcg gat 1719
Phe Ile Asp Leu Val Cys Tyr Arg Arg His Gly His Asn Glu Ala Asp
490 495 500 505
gag cca agt gct acc cag ccg ttg atg tac cag aaa atc aaa aag cat 1767
Glu Pro Ser Ala Thr Gln Pro Leu Met Tyr Gln Lys Ile Lys Lys His
510 515 520
ccg acg ccg cgt aaa att tac gcc gat cgt ctg gaa ggc gaa ggt gtc 1815
Pro Thr Pro Arg Lys Ile Tyr Ala Asp Arg Leu Glu Gly Glu Gly Val
525 530 535
gcg tcg cag gaa gat gcc acc gag atg gtg aac ctg tac cgc gat gcg 1863
Ala Ser Gln Glu Asp Ala Thr Glu Met Val Asn Leu Tyr Arg Asp Ala
540 545 550
ctc gat gcg ggc gaa tgc gtg gtg ccg gaa tgg cgt ccg atg agc ctg 1911
Leu Asp Ala Gly Glu Cys Val Val Pro Glu Trp Arg Pro Met Ser Leu
555 560 565
cac tcc ttc acg tgg tcg cct tat ctg aac cac gaa tgg gat gag cct 1959
His Ser Phe Thr Trp Ser Pro Tyr Leu Asn His Glu Trp Asp Glu Pro
570 575 580 585
tat ccg gca cag gtt gac atg aaa cgc ctg aag gaa ctg gca ttg cgt 2007
Tyr Pro Ala Gln Val Asp Met Lys Arg Leu Lys Glu Leu Ala Leu Arg
590 595 600
atc agc cag gtc cct gag cag att gaa gtg cag tcg cgc gtg gcc aag 2055
Ile Ser Gln Val Pro Glu Gln Ile Glu Val Gln Ser Arg Val Ala Lys
605 610 615
atc tat aac gat cgc aag ctg atg gcc gaa ggc gag aaa gcg ttc gac 2103
Ile Tyr Asn Asp Arg Lys Leu Met Ala Glu Gly Glu Lys Ala Phe Asp
620 625 630
tgg ggc ggt gcc gag aat ctg gcg tac gcc acg ctg gtg gat gaa ggt 2151
Trp Gly Gly Ala Glu Asn Leu Ala Tyr Ala Thr Leu Val Asp Glu Gly
635 640 645
att ccg gtt cgc ctc tcg ggt gaa gac tcc ggt cgt gga acc ttc ttc 2199
Ile Pro Val Arg Leu Scr Gly Glu Asp Ser Gly Arg Gly Thr Phe Phe
650 655 660 665
cat cgc cac gcg gtc gtg cac aac cag gct aac ggt tca acc tat acg 2247
His Arg His Ala Val Val His Asn Gln Ala Asn Gly Ser Thr Tyr Thr
670 675 680
ccg ctg cac cat att cat aac agc cag ggc gag ttc aaa gtc tgg gat 2295
Pro Leu His His Ile His Asn Ser Gln Gly Glu Phe Lys Val Trp Asp
685 690 695
tcg gtg ctg tct gaa gaa gcg gtg ctg gcg ttt gaa tac ggt tac gcc 2343
Ser Val Leu Ser Glu Glu Ala Val Leu Ala Phe Glu Tyr Gly Tyr Ala
700 705 710
acg gct gag ccg cgc gtg ctg acc atc tgg gaa gcg cag ttt ggt gac 2391
Thr Ala Glu Pro Arg Val Leu Thr Ile Trp Glu Ala Gln Phe Gly Asp
715 720 725
ttt gcc aac ggt gct cag gtg gtg att gac cag ttc atc agc tct ggc 2439
Phe Ala Asn Gly Ala Gln Val Val Ile Asp Gln Phe Ile Ser Ser Gly
730 735 740 745
gaa cag aag tgg ggc cgt atg tgt ggc ctg gtg atg ttg ctg ccg cat 2487
Glu Gln Lys Trp Gly Arg Met Cys Gly Leu Val Met Leu Leu Pro His
750 755 760
ggc tac gaa ggt cag gga ccg gaa cac tcc tct gcc cgt ctg gaa cgc 2535
Gly Tyr Glu Gly Gln Gly Pro Glu His Ser Ser Ala Arg Leu Glu Arg
765 770 775
tat ctg caa ctt tgc gcc gag cag aac atg cag gtt tgc gtc ccg tcg 2583
Tyr Leu Gln Leu Cys Ala Glu Gln Asn Met Gln Val Cys Val Pro Ser
780 785 790
acg ccg gct cag gtg tat cac atg ctg cgc cgt cag gcg ctg cgc ggg 2631
Thr Pro Ala Gln Val Tyr His Met Leu Arg Arg Gln Ala Leu Arg Gly
795 800 805
atg cgc cgt ccg ctg gtg gtg atg tcg ccg aag tcg ctg tta cgc cat 2679
Met Arg Arg Pro Leu Val Val Met Ser Pro Lys Ser Leu Leu Arg His
810 815 820 825
cca ctg gcg atc tcg tcg ctg gat gaa ctg gca aac ggc agt ttc cag 2727
Pro Leu Ala Ile Ser Ser Leu Asp Glu Leu Ala Asn Gly Ser Phe Gln
830 835 840
ccg gcc att ggt gag atc gac gat ctg gat ccg cag ggc gtg aaa cgc 2775
Pro Ala Ile Gly Glu Ile Asp Asp Leu Asp Pro Gln Gly Val Lys Arg
845 850 855
gtc gtg ctg tgc tcc ggt aag gtt tac tac gat ctg ctg gaa cag cgt 2823
Val Val Leu Cys Ser Gly Lys Val Tyr Tyr Asp Leu Leu Glu Gln Arg
860 865 870
cgt aaa gac gag aaa acc gat gtt gcc atc gtg cgc atc gaa cag ctt 2871
Arg Lys Asp Glu Lys Thr Asp Val Ala Ile Val Arg Ile Glu Gln Leu
875 880 885
tac ccg ttc ccg cat cag gcg gta cag gaa gca ttg aaa gcc tat tct 2919
Tyr Pro Phe Pro His Gln Ala Val Gln Glu Ala Leu Lys Ala Tyr Ser
890 895 900 905
cac gta cag gac ttt gtc tgg tgc cag gaa gag cct ctg aac cag ggc 2967
His Val Gln Asp Phe Val Trp Cys Gln Glu Glu Pro Leu Asn Gln Gly
910 915 920
gcc tgg tac tgt agc cag cat cat ttc cgt gat gtc gtg ccg ttt ggt 3015
Ala Trp Tyr Cys Ser Gln His His Phe Arg Asp Val Val Pro Phe Gly
925 930 935
gcc acc ctg cgt tat gca ggt cgc ccg gca tcg gct tct ccg gcc gtg 3063
Ala Thr Leu Arg Tyr Ala Gly Arg Pro Ala Ser Ala Ser Pro Ala Val
940 945 950
ggt tat atg tcc gta cac caa caa cag cag caa gac ctg gtt aat gac 3111
Gly Tyr Met Ser Val His Gln Gln Gln Gln Gln Asp Leu Val Asn Asp
955 960 965
gca ctg aac gtc aat taa ttaaaaggaa agata atg agt agc gta gat att 3162
Ala Leu Asn Val Asn Met Ser Ser Val Asp Ile
970 975 980
ctc gtt ccc gac ctg cct gaa tcg gtt gca gat gcc aca gta gca acc 3210
Leu Val Pro Asp Leu Pro Glu Ser Val Ala Asp Ala Thr Val Ala Thr
985 990 995
tgg cac aag aaa cca ggc gat gca gtc agc cgc gat gaa gtc atc 3255
Trp His Lys Lys Pro Gly Asp Ala Val Ser Arg Asp Glu Val Ile
1000 1005 1010
gtc gaa att gaa act gac aaa gtc gtg ctg gaa gtg ccg gca tct 3300
Val Glu Ile Glu Thr Asp Lys Val Val Leu Glu Val Pro Ala Ser
1015 1020 1025
gcc gat ggc gtg ctg gaa gcc gtg ctg gaa gac gaa ggg gca acc 3345
Ala Asp Gly Val Leu Glu Ala Val Leu Glu Asp Glu Gly Ala Thr
1030 1035 1040
gtt acg tcc cgc cag atc ctg ggt cgc ctg aaa gaa ggc aac agt 3390
Val Thr Ser Arg Gln Ile Leu Gly Arg Leu Lys Glu Gly Asn Ser
1045 1050 1055
gcg ggt aaa gaa agc agt gcc aaa gcg gaa agc aat gac acc acg 3435
Ala Gly Lys Glu Ser Ser Ala Lys Ala Glu Ser Asn Asp Thr Thr
1060 1065 1070
cca gcc cag cgt cag aca gcg tcg ctt gaa gaa gag agc agc gat 3480
Pro Ala Gln Arg Gln Thr Ala Ser Leu Glu Glu Glu Ser Ser Asp
1075 1080 1085
gcg ctc agc ccg gcg atc cgt cgc ctg att gcg gag cat aat ctt 3525
Ala Leu Ser Pro Ala Ile Arg Arg Leu Ile Ala Glu His Asn Leu
1090 1095 1100
gac gct gcg cag atc aaa ggc acc ggc gta ggc gga cgt tta acg 3570
Asp Ala Ala Gln Ile Lys Gly Thr Gly Val Gly Gly Arg Leu Thr
1105 1110 1115
cgt gaa gac gtt gaa aaa cat ctg gcg aac aaa ccg cag gct gag 3615
Arg Glu Asp Val Glu Lys His Leu Ala Asn Lys Pro Gln Ala Glu
1120 1125 1130
aaa gcc gcc gcg cca gcg gcg ggt gca gca acg gct cag cag cct 3660
Lys Ala Ala Ala Pro Ala Ala Gly Ala Ala Thr Ala Gln Gln Pro
1135 1140 1145
gtt gcc aac cgc agc gaa aaa cgt gtt ccg atg acg cgt tta cgt 3705
Val Ala Asn Arg Ser Glu Lys Arg Val Pro Met Thr Arg Leu Arg
1150 1155 1160
aag cgc gtc gcg gag cgt ctg ctg gaa gcc aag aac agc acc gcc 3750
Lys Arg Val Ala Glu Arg Leu Leu Glu Ala Lys Asn Ser Thr Ala
1165 1170 1175
atg ttg acg acc ttc aac gaa atc aac atg aag ccg att atg gat 3795
Met Leu Thr Thr Phe Asn Glu Ile Asn Met Lys Pro Ile Met Asp
1180 1185 1190
ctg cgt aag cag tac ggc gat gcg ttc gag aag cgt cac ggt gtg 3840
Leu Arg Lys Gln Tyr Gly Asp Ala Phe Glu Lys Arg His Gly Val
1195 1200 1205
cgt ctg ggc ttt atg tct ttc tac atc aag gcc gtg gtc gaa gcg 3885
Arg Leu Gly Phe Met Ser Phe Tyr Ile Lys Ala Val Val Glu Ala
1210 1215 1220
ctg aag cgt tat cca gaa gtc aac gcc tct atc gat ggc gaa gac 3930
Leu Lys Arg Tyr Pro Glu Val Asn Ala Ser Ile Asp Gly Glu Asp
1225 1230 1235
gtg gtg tac cac aac tat ttc gat gtg agt att gcc gtc tct acg 3975
Val Val Tyr His Asn Tyr Phe Asp Val Ser Ile Ala Val Ser Thr
1240 1245 1250
cca cgc gga ctg gtg acg cct gtc ctg cgt gac gtt gat gcg ctg 4020
Pro Arg Gly Leu Val Thr Pro Val Leu Arg Asp Val Asp Ala Leu
1255 1260 1265
agc atg gct gac atc gag aag aaa att aaa gaa ctg gca gtg aaa 4065
Ser Met Ala Asp Ile Glu Lys Lys Ile Lys Glu Leu Ala Val Lys
1270 1275 1280
ggc cgt gac ggc aag ctg acg gtt gac gat ctg acg ggc ggt aac 4110
Gly Arg Asp Gly Lys Leu Thr Val Asp Asp Leu Thr Gly Gly Asn
1285 1290 1295
ttt acc atc acc aac ggt ggt gtg ttc ggt tcg ctg atg tct acg 4155
Phe Thr Ile Thr Asn Gly Gly Val Phe Gly Ser Leu Met Ser Thr
1300 1305 1310
cca atc atc aac ccg cca cag agc gcg att ctg ggc atg cac gcc 4200
Pro Ile Ile Asn Pro Pro Gln Ser Ala Ile Leu Gly Met His Ala
1315 1320 1325
att aaa gat cgt cct atg gcg gtc aat ggt cag gtt gtg atc ctg 4245
Ile Lys Asp Arg Pro Met Ala Val Asn Gly Gln Val Val Ile Leu
1330 1335 1340
cca atg atg tac ctg gct ctc tcc tac gat cac cgt tta atc gat 4290
Pro Met Met Tyr Leu Ala Leu Ser Tyr Asp His Arg Leu Ile Asp
1345 1350 1355
ggt cgt gaa tct gtc ggc tat ctg gtc gcg gtg aaa gag atg ctg 4335
Gly Arg Glu Ser Val Gly Tyr Leu Val Ala Val Lys Glu Met Leu
1360 1365 1370
gaa gat ccg gcg cgt ctg ctg ctg gat gtc tga ttcatcactg 4378
Glu Asp Pro Ala Arg Leu Leu Leu Asp Val
1375 1380
ggcacgcgtt gcgtgcccaa tctcaatact cttttcagat ctgaatggat agaacatc 4436
atg aac tta cac gaa tac cag gct aaa cag ctg ttt gca cgg tat 4481
Met Asn Leu His Glu Tyr Gln Ala Lys Gln Leu Phe Ala Arg Tyr
1385 1390 1395
ggc atg cca gca ccg acc ggc tac gcc tgt act aca cca cgt gaa 4526
Gly Met Pro Ala Pro Thr Gly Tyr Ala Cys Thr Thr Pro Arg Glu
1400 1405 1410
gca gaa gaa gcg gca tcg aaa atc ggt gca 4556
Ala Glu Glu Ala Ala Ser Lys Ile Gly Ala
1415 1420
<210>8
<211>39
<212>PRT
<213>Pantoea ananatis
<400>8
Ala Phe Ser Val Phe Arg Cys His Ser Ile Met Asn Cys Val Ser Val
1 5 10 15
Cys Pro Lys Gly Leu Asn Pro Thr Arg Ala Ile Gly His Ile Lys Ser
20 25 30
Met Leu Leu Gln Arg Ser Ala
35
<210>9
<211>935
<212>PRT
<213>Pantoea ananatis
<400>9
Met Gln Asn Ser Ala Met Lys Pro Trp Leu Asp Ser Ser Trp Leu Ala
1 5 10 15
Gly Ala Asn Gln Ser Tyr Ile Glu Gln Leu Tyr Glu Asp Phe Leu Thr
20 25 30
Asp Pro Asp Ser Val Asp Ala Val Trp Arg Ser Met Phe Gln Gln Leu
35 40 45
Pro Gly Thr Gly Val Lys Pro Glu Gln Phe His Ser Ala Thr Arg Glu
50 55 60
Tyr Phe Arg Arg Leu Ala Lys Asp Ala Ser Arg Tyr Thr Ser Ser Val
65 70 75 80
Thr Asp Pro Ala Thr Asn Ser Lys Gln Val Lys Val Leu Gln Leu Ile
85 90 95
Asn Ala Phe Arg Phe Arg Gly His Gln Glu Ala Asn Leu Asp Pro Leu
100 105 110
Gly Leu Trp Lys Gln Asp Arg Val Ala Asp Leu Asp Pro Ala Phe His
115 120 125
Asp Leu Thr Asp Ala Asp Phe Gln Glu Ser Phe Asn Val Gly Ser Phe
130 135 140
Ala Ile Gly Lys Glu Thr Met Lys Leu Ala Asp Leu Phe Asp Ala Leu
145 150 155 160
Lys Gln Thr Tyr Cys Gly Ser Ile Gly Ala Glu Tyr Met His Ile Asn
165 170 175
Asn Thr Glu Glu Lys Arg Trp Ile Gln Gln Arg Ile Glu Ser Gly Ala
180 185 190
Ser Gln Thr Ser Phe Ser Gly Glu Glu Lys Lys Gly Phe Leu Lys Glu
195 200 205
Leu Thr Ala Ala Glu Gly Leu Glu Lys Tyr Leu Gly Ala Lys Phe Pro
210 215 220
Gly Ala Lys Arg Phe Ser Leu Glu Gly Gly Asp Ala Leu Val Pro Met
225 230 235 240
Leu Arg Glu Met Ile Arg His Ala Gly Lys Ser Gly Thr Arg Glu Val
245 250 255
Val Leu Gly Met Ala His Arg Gly Arg Leu Asn Val Leu Ile Asn Val
260 265 270
Leu Gly Lys Lys Pro Gln Asp Leu Phe Asp Glu Phe Ser Gly Lys His
275 280 285
Lys Glu His Leu Gly Thr Gly Asp Val Lys Tyr His Met Gly Phe Ser
290 295 300
Ser Asp Ile Glu Thr Glu Gly Gly Leu Val His Leu Ala Leu Ala Phe
305 310 315 320
Asn Pro Ser His Leu Glu Ile Val Ser Pro Val Val Met Gly Ser Val
325 330 335
Arg Ala Arg Leu Asp Arg Leu Ala Glu Pro Val Ser Asn Lys Val Leu
340 345 350
Pro Ile Thr Ile His Gly Asp Ala Ala Val Ile Gly Gln Gly Val Val
355 360 365
Gln Glu Thr Leu Asn Met Ser Gln Ala Arg Gly Tyr Glu Val Gly Gly
370 375 380
Thr Val Arg Ile Val Ile Asn Asn Gln Val Gly Phe Thr Thr Ser Asn
385 390 395 400
Pro Lys Asp Ala Arg Ser Thr Pro Tyr Cys Thr Asp Ile Gly Lys Met
405 410 415
Val Leu Ala Pro Ile Phe His Val Asn Ala Asp Asp Pro Glu Ala Val
420 425 430
Ala Phe Val Thr Arg Leu Ala Leu Asp Tyr Arg Asn Thr Phe Lys Arg
435 440 445
Asp Val Phe Ile Asp Leu Val Cys Tyr Arg Arg His Gly His Asn Glu
450 455 460
Ala Asp Glu Pro Ser Ala Thr Gln Pro Leu Met Tyr Gln Lys Ile Lys
465 470 475 480
Lys His Pro Thr Pro Arg Lys Ile Tyr Ala Asp Arg Leu Glu Gly Glu
485 490 495
Gly Val Ala Ser Gln Glu Asp Ala Thr Glu Met Val Asn Leu Tyr Arg
500 505 510
Asp Ala Leu Asp Ala Gly Glu Cys Val Val Pro Glu Trp Arg Pro Met
515 520 525
Ser Leu His Ser Phe Thr Trp Ser Pro Tyr Leu Asn His Glu Trp Asp
530 535 540
Glu Pro Tyr Pro Ala Gln Val Asp Met Lys Arg Leu Lys Glu Leu Ala
545 550 555 560
Leu Arg Ile Ser Gln Val Pro Glu Gln Ile Glu Val Gln Ser Arg Val
565 570 575
Ala Lys Ile Tyr Asn Asp Arg Lys Leu Met Ala Glu Gly Glu Lys Ala
580 585 590
Phe Asp Trp Gly Gly Ala Glu Asn Leu Ala Tyr Ala Thr Leu Val Asp
595 600 605
Glu Gly Ile Pro Val Arg Leu Ser Gly Glu Asp Ser Gly Arg Gly Thr
610 615 620
Phe Phe His Arg His Ala Val Val His Asn Gln Ala Asn Gly Ser Thr
625 630 635 640
Tyr Thr Pro Leu His His Ile His Asn Ser Gln Gly Glu Phe Lys Val
645 650 655
Trp Asp Ser Val Leu Ser Glu Glu Ala Val Leu Ala Phe Glu Tyr Gly
660 665 670
Tyr Ala Thr Ala Glu Pro Arg Val Leu Thr Ile Trp Glu Ala Gln Phe
675 680 685
Gly Asp Phe Ala Asn Gly Ala Gln Val Val Ile Asp Gln Phe Ile Ser
690 695 700
Ser Gly Glu Gln Lys Trp Gly Arg Met Cys Gly Leu Val Met Leu Leu
705 710 715 720
Pro His Gly Tyr Glu Gly Gln Gly Pro Glu His Ser Ser Ala Arg Leu
725 730 735
Glu Arg Tyr Leu Gln Leu Cys Ala Glu Gln Asn Met Gln Val Cys Val
740 745 750
Pro Ser Thr Pro Ala Gln Val Tyr His Met Leu Arg Arg Gln Ala Leu
755 760 765
Arg Gly Met Arg Arg Pro Leu Val Val Met Ser Pro Lys Ser Leu Leu
770 775 780
Arg His Pro Leu Ala Ile Ser Ser Leu Asp Glu Leu Ala Asn Gly Ser
785 790 795 800
Phe Gln Pro Ala Ile Gly Glu Ile Asp Asp Leu Asp Pro Gln Gly Val
805 810 815
Lys Arg Val Val Leu Cys Ser Gly Lys Val Tyr Tyr Asp Leu Leu Glu
820 825 830
Gln Arg Arg Lys Asp Glu Lys Thr Asp Val Ala Ile Val Arg Ile Glu
835 840 845
Gln Leu Tyr Pro Phe Pro His Gln Ala Val Gln Glu Ala Leu Lys Ala
850 855 860
Tyr Ser His Val Gln Asp Phe Val Trp Cys Gln Glu Glu Pro Leu Asn
865 870 875 880
Gln Gly Ala Trp Tyr Cys Ser Gln His His Phe Arg Asp Val Val Pro
885 890 895
Phe Gly Ala Thr Leu Arg Tyr Ala Gly Arg Pro Ala Ser Ala Ser Pro
900 905 910
Ala Val Gly Tyr Met Ser Val His Gln Gln Gln Gln Gln Asp Leu Val
915 920 925
Asn Asp Ala Leu Asn Val Asn
930 935
<210>10
<211>407
<212>PRT
<213>Pantoea ananatis
<400>10
Met Ser Ser Val Asp Ile Leu Val Pro Asp Leu Pro Glu Ser Val Ala
1 5 10 15
Asp Ala Thr Val Ala Thr Trp His Lys Lys Pro Gly Asp Ala Val Ser
20 25 30
Arg Asp Glu Val Ile Val Glu Ile Glu Thr Asp Lys Val Val Leu Glu
35 40 45
Val Pro Ala Ser Ala Asp Gly Val Leu Glu Ala Val Leu Glu Asp Glu
50 55 60
Gly Ala Thr Val Thr Ser Arg Gln Ile Leu Gly Arg Leu Lys Glu Gly
65 70 75 80
Asn Ser Ala Gly Lys Glu Ser Ser Ala Lys Ala Glu Ser Asn Asp Thr
85 90 95
Thr Pro Ala Gln Arg Gln Thr Ala Ser Leu Glu Glu Glu Ser Ser Asp
100 105 110
Ala Leu Ser Pro Ala Ile Arg Arg Leu Ile Ala Glu His Asn Leu Asp
115 120 125
Ala Ala Gln Ile Lys Gly Thr Gly Val Gly Gly Arg Leu Thr Arg Glu
130 135 140
Asp Val Glu Lys His Leu Ala Asn Lys Pro Gln Ala Glu Lys Ala Ala
145 150 155 160
Ala Pro Ala Ala Gly Ala Ala Thr Ala Gln Gln Pro Val Ala Asn Arg
165 170 175
Ser Glu Lys Arg Val Pro Met Thr Arg Leu Arg Lys Arg Val Ala Glu
180 185 190
Arg Leu Leu Glu Ala Lys Asn Ser Thr Ala Met Leu Thr Thr Phe Asn
195 200 205
Glu Ile Asn Met Lys Pro Ile Met Asp Leu Arg Lys Gln Tyr Gly Asp
210 215 220
Ala Phe Glu Lys Arg His Gly Val Arg Leu Gly Phe Met Ser Phe Tyr
225 230 235 240
Ile Lys Ala Val Val Glu Ala Leu Lys Arg Tyr Pro Glu Val Asn Ala
245 250 255
Ser Ile Asp Gly Glu Asp Val Val Tyr His Asn Tyr Phe Asp Val Ser
260 265 270
Ile Ala Val Ser Thr Pro Arg Gly Leu Val Thr Pro Val Leu Arg Asp
275 280 285
Val Asp Ala Leu Ser Met Ala Asp Ile Glu Lys Lys Ile Lys Glu Leu
290 295 300
Ala Val Lys Gly Arg Asp Gly Lys Leu Thr Val Asp Asp Leu Thr Gly
305 310 315 320
Gly Asn Phe Thr Ile Thr Asn Gly Gly Val Phe Gly Ser Leu Met Ser
325 330 335
Thr Pro Ile Ile Asn Pro Pro Gln Ser Ala Ile Leu Gly Met His Ala
340 345 350
Ile Lys Asp Arg Pro Met Ala Val Asn Gly Gln Val Val Ile Leu Pro
355 360 365
Met Met Tyr Leu Ala Leu Ser Tyr Asp His Arg Leu Ile Asp Gly Arg
370 375 380
Glu Ser Val Gly Tyr Leu Val Ala Val Lys Glu Met Leu Glu Asp Pro
385 390 395 400
Ala Arg Leu Leu Leu Asp Val
405
<210>11
<211>40
<212>PRT
<213>Pantoea ananatis
<400>11
Met Asn Leu His Glu Tyr Gln Ala Lys Gln Leu Phe Ala Arg Tyr Gly
1 5 10 15
Met Pro Ala Pro Thr Gly Tyr Ala Cys Thr Thr Pro Arg Glu Ala Glu
20 25 30
Glu Ala Ala Ser Lys Ile Gly Ala
35 40
<210>12
<211>4394
<212>DNA
<2l3>谷氨酸棒状杆菌(Corynebacterium glutamicum)
<220>
<221>CDS
<222>(443)..(4213)
<400>12
gtcgacaagc aaaatcgaag cggcagcacg ccgcgtcgga gccttaaacg ccatcgccgc 60
catccctgat ggtttcaatc atcaagtcgg tgaacgcggg cgcaacctgt catccggaca 120
gcgccaactg atcgcgctgg cgcgcgccga actcatcgag ccttccatca tgcttctcga 180
cgaagccacc tccaccctcg accccgccac cgaagccgtt atcctcaacg cctccgatcg 240
agtcactaag ggacgcacca gcatcatcgt cgcgcaccgc ttggcaaccg ctaaaagggc 300
cgaccgtatt cttgttgttg aacaaggacg tatcattgag gacggatctc acgacgcgtt 360
gttgtctgct aacggcacct acgcccgcat gtggcattta atggcctgac acgttatttt 420
taggagaact gtcaacaaat ta atg cta caa ctg ggg ctt agg cat aat cag 472
Met Leu Gln Leu Gly Leu Arg His Asn Gln
1 5 10
cca acg acc aac gtt aca gtg gat aaa ata aag ctc aat aaa ccc tca 520
Pro Thr Thr Asn Val Thr Val Asp Lys Ile Lys Leu Asn Lys Pro Ser
15 20 25
aga agc aag gaa aag agg cga gta cct gcc gtg agc agc gct agt act 568
Arg Ser Lys Glu Lys Arg Arg Val Pro Ala Val Ser Ser Ala Ser Thr
30 35 40
ttc ggc cag aat gcg tgg ctg gta gac gag atg ttc cag cag ttc cag 616
Phe Gly Gln Asn Ala Trp Leu Val Asp Glu Met Phe Gln Gln Phe Gln
45 50 55
aag gac ccc aag tcc gtg gac aag gaa tgg aga gaa ctc ttt gag gcg 664
Lys Asp Pro Lys Ser Val Asp Lys Glu Trp Arg Glu Leu Phe Glu Ala
60 65 70
cag ggg gga cca aat gct acc ccc gct aca aca gaa gca cag cct tca 712
Gln Gly Gly Pro Asn Ala Thr Pro Ala Thr Thr Glu Ala Gln Pro Ser
75 80 85 90
gcg ccc aag gag tct gcg aaa cca gca cca aag gct gcc cct gca gcc 760
Ala Pro Lys Glu Ser Ala Lys Pro Ala Pro Lys Ala Ala Pro Ala Ala
95 100 105
aag gca gca ccg cgc gta gaa acc aag ccg gcc gcc aag acc gcc cct 808
Lys Ala Ala Pro Arg Val Glu Thr Lys Pro Ala Ala Lys Thr Ala Pro
110 115 120
aag gcc aag gag tcc tca gtg cca cag caa cct aag ctt ccg gag cca 856
Lys Ala Lys Glu Ser Ser Val Pro Gln Gln Pro Lys Leu Pro Glu Pro
125 130 135
gga caa acc cca atc agg ggt att ttc aag tcc atc gcg aag aac atg 904
Gly Gln Thr Pro Ile Arg Gly Ile Phe Lys Ser Ile Ala Lys Asn Met
140 145 150
gat atc tcc ctg gaa atc cca acc gca acc tcg gtt cgc gat atg cca 952
Asp Ile Ser Leu Glu Ile Pro Thr Ala Thr Ser Val Arg Asp Met Pro
155 160 165 170
gct cgc ctc atg ttc gaa aac cgc gcg atg gtc aac gat cag ctc aag 1000
Ala Arg Leu Met Phe Glu Asn Arg Ala Met Val Asn Asp Gln Leu Lys
175 180 185
cgc acc cgc ggt ggc aag atc tcc ttc acc cac atc att ggc tac gcc 1048
Arg Thr Arg Gly Gly Lys Ile Ser Phe Thr His Ile Ile Gly Tyr Ala
190 195 200
atg gtg aag gca gtc atg gct cac ccg gac atg aac aac tcc tac gac 1096
Met Val Lys Ala Val Met Ala His Pro Asp Met Asn Asn Ser Tyr Asp
205 210 215
gtc atc gac ggc aag cca acc ctg atc gtg cct gag cac atc aac ctg 1144
Val Ile Asp Gly Lys Pro Thr Leu Ile Val Pro Glu His Ile Asn Leu
220 225 230
ggc ctt gcc atc gac ctt cct cag aag gac ggc tcc cgc gca ctt gtc 1192
Gly Leu Ala Ile Asp Leu Pro Gln Lys Asp Gly Ser Arg Ala Leu Val
235 240 245 250
gta gca gcc atc aag gaa acc gag aag atg aac ttc tcc gag ttc ctc 1240
Val Ala Ala Ile Lys Glu Thr Glu Lys Met Asn Phe Ser Glu Phe Leu
255 260 265
gca gca tac gaa gac atc gtg aca cgc tcc cgc aag ggc aag ctc acc 1288
Ala Ala Tyr Glu Asp Ile Val Thr Arg Ser Arg Lys Gly Lys Leu Thr
270 275 280
atg gat gac tac cag ggc gtt acc gtt tcc ttg acc aac cca ggt ggc 1336
Met Asp Asp Tyr Gln Gly Val Thr Val Ser Leu Thr Asn Pro Gly Gly
285 290 295
atc ggt acc cgc cac tct gtc cca cgt ctg acc aag ggc cag ggc acc 1384
Ile Gly Thr Arg His Ser Val Pro Arg Leu Thr Lys Gly Gln Gly Thr
300 305 310
atc atc ggt gtc ggt tcc atg gat tac cca gca gag ttc cag ggc gct 1432
Ile Ile Gly Val Gly Ser Met Asp Tyr Pro Ala Glu Phe Gln Gly Ala
315 320 325 330
tcc gaa gac cgc ctt gca gag ctc ggc gtt gga aag ctt gtc acc atc 1480
Ser Glu Asp Arg Leu Ala Glu Leu Gly Val Gly Lys Leu Val Thr Ile
335 340 345
acc tcc acc tac gat cac cgc gtg atc cag ggt gct gtg tcc ggt gaa 1528
Thr Ser Thr Tyr Asp His Arg Val Ile Gln Gly Ala Val Ser Gly Glu
350 355 360
ttc ctg cgt acc atg tct cgc ctg ctc acc gat gat tcc ttc tgg gat 1576
Phe Leu Arg Thr Met Ser Arg Leu Leu Thr Asp Asp Ser Phe Trp Asp
365 370 375
gag atc ttc gac gca atg aac gtt cct tac acc cca atg cgt tgg gca 1624
Glu Ile Phe Asp Ala Met Asn Val Pro Tyr Thr Pro Met Arg Trp Ala
380 385 390
cag gac gtt cca aac acc ggt gtt gat aag aac acc cgc gtc atg cag 1672
Gln Asp Val Pro Asn Thr Gly Val Asp Lys Asn Thr Arg Val Met Gln
395 400 405 410
ctc att gag gca tac cgc tcc cgt gga cac ctc atc gct gac acc aac 1720
Leu Ile Glu Ala Tyr Arg Ser Arg Gly His Leu Ile Ala Asp Thr Asn
415 420 425
cca ctt tca tgg gtt cag cct ggc atg cca gtt cca gac cac cgc gac 1768
Pro Leu Ser Trp Val Gln Pro Gly Met Pro Val Pro Asp His Arg Asp
430 435 440
ctc gac atc gag acc cac agc ctg acc atc tgg gat ctg gac cgt acc 1816
Leu Asp Ile Glu Thr His Ser Leu Thr Ile Trp Asp Leu Asp Arg Thr
445 450 455
ttc agc gtc ggt ggc ttc ggc ggc aag gag acc atg acc ctg cgc gag 1864
Phe Ser Val Gly Gly Phe Gly Gly Lys Glu Thr Met Thr Leu Arg Glu
460 465 470
gta ctg tcc cgc ctg cgc gct gcc tac acc ttg aag gtc ggc tcc gaa 1912
Val Leu Ser Arg Leu Arg Ala Ala Tyr Thr Leu Lys Val Gly Ser Glu
475 480 485 490
tac acc cac atc ctg gac cgc gac gag cgc acc tgg ctg cag gac cgc 1960
Tyr Thr His Ile Leu Asp Arg Asp Glu Arg Thr Trp Leu Gln Asp Arg
495 500 505
ctc gaa gcc gga atg cca aag cca acc cag gca gag cag aag tac atc 2008
Leu Glu Ala Gly Met Pro Lys Pro Thr Gln Ala Glu Gln Lys Tyr Ile
510 515 520
ctg cag aag ctg aac gcc gca gag gct ttc gag aac ttc ctg cag acc 2056
Leu Gln Lys Leu Asn Ala Ala Glu Ala Phe Glu Asn Phe Leu Gln Thr
525 530 535
aag tac gtc ggc cag aag cgc ttc tcc ctc gaa ggt gca gaa gct ctc 2104
Lys Tyr Val Gly Gln Lys Arg Phe Ser Leu Glu Gly Ala Glu Ala Leu
540 545 550
atc cca ctg atg gac tcc gcc atc gac acc gcc gca ggc cag ggc ctc 2152
Ile Pro Leu Met Asp Ser Ala Ile Asp Thr Ala Ala Gly Gln Gly Leu
555 560 565 570
gac gaa gtt gtc atc ggt atg cca cac cgt ggt cgc ctc aac gtg ctg 2200
Asp Glu Val Val Ile Gly Met Pro His Arg Gly Arg Leu Asn Val Leu
575 580 585
ttc aac atc gtg ggc aag cca ctg gca tcc atc ttc aac gag ttt gaa 2248
Phe Asn Ile Val Gly Lys Pro Leu Ala Ser Ile Phe Asn Glu Phe Glu
590 595 600
ggc caa atg gag cag ggc cag atc ggt ggc tcc ggt gac gtg aag tac 2296
Gly Gln Met Glu Gln Gly Gln Ile Gly Gly Ser Gly Asp Val Lys Tyr
605 610 615
cac ctc ggt tcc gaa ggc cag cac ctg cag atg ttc ggc gac ggc gag 2344
His Leu Gly Ser Glu Gly Gln His Leu Gln Met Phe Gly Asp Gly Glu
620 625 630
atc aag gtc tcc ctg act gct aac ccg tcc cac ctg gaa gct gtt aac 2392
Ile Lys Val Ser Leu Thr Ala Asn Pro Ser His Leu Glu Ala Val Asn
635 640 645 650
cca gtg atg gaa ggt atc gtccgc gca aag cag gac tac ctg gac aag 2440
Pro Val Met Glu Gly Ile Val Arg Ala Lys Gln Asp Tyr Leu Asp Lys
655 660 665
ggc gta gac ggc aag act gtt gtg cca ctg ctg ctc cac ggt gac gct 2488
Gly Val Asp Gly Lys Thr Val Val Pro Leu Leu Leu His Gly Asp Ala
670 675 680
gca ttc gca ggc ctg ggc atc gtg cca gaa acc atc aac ctg gct aag 2536
Ala Phe Ala Gly Leu Gly Ile Val Pro Glu Thr Ile Asn Leu Ala Lys
685 690 695
ctg cgt ggc tac gac gtc gga ggc acc atc cac atc gtg gtg aac aac 2584
Leu Arg Gly Tyr Asp Val Gly Gly Thr Ile His Ile Val Val Asn Asn
700 705 710
cag atc ggc ttc acc acc acc cca gac tcc agc cgc tcc atg cac tac 2632
Gln Ile Gly Phe Thr Thr Thr Pro Asp Ser Ser Arg Ser Met His Tyr
715 720 725 730
gca acc gac tac gcc aag gca ttc ggc tgc cca gtc ttc cac gtc aat 2680
Ala Thr Asp Tyr Ala Lys Ala Phe Gly Cys Pro Val Phe His Val Asn
735 740 745
ggt gat gac cca gag gca gtt gtc tgg gtt ggc cag ctg gca acc gag 2728
Gly Asp Asp Pro Glu Ala Val Val Trp Val Gly Gln Leu Ala Thr Glu
750 755 760
tac cgt cgt cgc ttc ggc aag gac gtc ttc atc gac ctc gtt tgc tac 2776
Tyr Arg Arg Arg Phe Gly Lys Asp Val Phe Ile Asp Leu Val Cys Tyr
765 770 775
cgc ctc cgc ggc cac aac gaa gct gat gat cct tcc atg acc cag cca 2824
Arg Leu Arg Gly His Asn Glu Ala Asp Asp Pro Ser Met Thr Gln Pro
780 785 790
aag atg tat gag ctc atc acc ggc cgc gag acc gtt cgt gct cag tac 2872
Lys Met Tyr Glu Leu Ile Thr Gly Arg Glu Thr Val Arg Ala Gln Tyr
795 800 805 810
acc gaa gac ctg ctc gga cgt gga gac ctc tcc aac gaa gat gca gaa 2920
Thr Glu Asp Leu Leu Gly Arg Gly Asp Leu Ser Asn Glu Asp Ala Glu
815 820 825
gca gtc gtc cgc gac ttc cac gac cag atg gaa tct gtg ttc aac gaa 2968
Ala Val Val Arg Asp Phe His Asp Gln Met Glu Ser Val Phe Asn Glu
830 835 840
gtc aag gaa ggc ggc aag aag cag gct gag gca cag acc ggc atc acc 3016
Val Lys Glu Gly Gly Lys Lys Gln Ala Glu Ala Gln Thr Gly Ile Thr
845 850 855
ggc tcc cag aag ctt cca cac ggc ctt gag acc aac atc tcc cgt gaa 3064
Gly Ser Gln Lys Leu Pro His Gly Leu Glu Thr Asn Ile Ser Arg Glu
860 865 870
gag ctc ctg gaa ctg gga cag gct ttc gcc aac acc cca gaa ggc ttc 3112
Glu Leu Leu Glu Leu Gly Gln Ala Phe Ala Asn Thr Pro Glu Gly Phe
875 880 885 890
aac tac cac cca cgt gtg gct cca gtt gct aag aag cgc gtc tcc tct 3160
Asn Tyr His Pro Arg Val Ala Pro Val Ala Lys Lys Arg Val Ser Ser
895 900 905
gtc acc gaa ggt ggc atc gac tgg gca tgg ggc gag ctc ctc gcc ttc 3208
Val Thr Glu Gly Gly Ile Asp Trp Ala Trp Gly Glu Leu Leu Ala Phe
910 915 920
ggt tcc ctg gct aac tcc ggc cgc ttg gtt cgc ctt gca ggt gaa gat 3256
Gly Ser Leu Ala Asn Ser Gly Arg Leu Val Arg Leu Ala Gly Glu Asp
925 930 935
tcc cgc cgc ggt acc ttc acc cag cgc cac gca gtt gcc atc gac cca 3304
Ser Arg Arg Gly Thr Phe Thr Gln Arg His Ala Val Ala Ile Asp Pro
940 945 950
gcg acc gct gaa gag ttc aac cca ctc cac gag ctt gca cag tcc aag 3352
Ala Thr Ala Glu Glu Phe Asn Pro Leu His Glu Leu Ala Gln Ser Lys
955 960 965 970
ggc aac aac ggt aag ttc ctg gtc tac aac tcc gca ctg acc gag tac 3400
Gly Asn Asn Gly Lys Phe Leu Val Tyr Asn Ser Ala Leu Thr Glu Tyr
975 980 985
gca ggc atg ggc ttc gag tac ggc tac tcc gta gga aac gaa gac tcc 3448
Ala Gly Met Gly Phe Glu Tyr Gly Tyr Ser Val Gly Asn Glu Asp Ser
990 995 1000
gtc gtt gca tgg gaa gca cag ttc ggc gac ttc gcc aac ggc gct 3493
Val Val Ala Trp Glu Ala Gln Phe Gly Asp Phe Ala Asn Gly Ala
1005 1010 1015
cag acc atc atc gat gag tac gtc tcc tca ggc gaa gct aag tgg 3538
Gln Thr Ile Ile Asp Glu Tyr Val Ser Ser Gly Glu Ala Lys Trp
1020 1025 1030
ggc cag acc tcc aag ctg atc ctt ctg ctg cct cac ggc tac gaa 3583
Gly Gln Thr Ser Lys Leu Ile Leu Leu Leu Pro His Gly Tyr Glu
1035 1040 1045
ggc cag ggc cca gac cac tct tcc gca cgt atc gag cgc ttc ctg 3628
Gly Gln Gly Pro Asp His Ser Ser Ala Arg Ile Glu Arg Phe Leu
1050 1055 1060
cag ctg tgc gct gag ggt tcc atg act gtt gct cag cca tcc acc 3673
Gln Leu Cys Ala Glu Gly Ser Met Thr Val Ala Gln Pro Ser Thr
1065 1070 1075
cca gca aac cac ttc cac ctg ctg cgt cgt cac gct ctg tcc gac 3718
Pro Ala Asn His Phe His Leu Leu Arg Arg His Ala Leu Ser Asp
1080 1085 1090
ctg aag cgt cca ctg gtt atc ttc acc ccg aag tcc atg ctg cgt 3763
Leu Lys Arg Pro Leu Val Ile Phe Thr Pro Lys Ser Met Leu Arg
1095 1100 1105
aac aag gct gct gcc tac gca cca gaa gac ttc act gag gtc acc 3808
Asn Lys Ala Ala Ala Ser Ala Pro Glu Asp Phe Thr Glu Val Thr
1110 1115 1120
aag ttc caa tcc gtg atc gac gat cca aac gtt gca gat gca gcc 3853
Lys Phe Gln Ser Val Ile Asp Asp Pro Asn Val Ala Asp Ala Ala
1125 1130 1135
aag gtg aag aag gtc atg ctg gtc tcc ggc aag ctg tac tac gaa 3898
Lys Val Lys Lys Val Met Leu Val Ser Gly Lys Leu Tyr Tyr Glu
1140 1145 1150
ttg gca aag cgc aag gag aag gac gga cgc gac gac atc gcg atc 3943
Leu Ala Lys Arg Lys Glu Lys Asp Gly Arg Asp Asp Ile Ala Ile
1155 1160 1165
gtt cgt atc gaa atg ctc cac cca att ccg ttc aac cgc atc tcc 3988
Val Arg Ile Glu Met Leu His Pro Ile Pro Phe Asn Arg Ile Ser
1170 1175 1180
gag gct ctt gcc ggc tac cct aac gct gag gaa gtc ctc ttc gtt 4033
Glu Ala Leu Ala Gly Tyr Pro Asn Ala Glu Glu Val Leu Phe Val
1185 1190 1195
cag gat gag cca gca aac cag ggc cca tgg ccg ttc tac cag gag 4078
Gln Asp Glu Pro Ala Asn Gln Gly Pro Trp Pro Phe Tyr Gln Glu
1200 1205 1210
cac ctc cca gag ctg atc ccg aac atg cca aag atg cgc cgc gtt 4123
His Leu Pro Glu Leu Ile Pro Asn Met Pro Lys Met Arg Arg Val
1215 1220 1225
tcc cgc cgc gct cag tcc tcc acc gca act ggt gtt gct aag gtg 4168
Ser Arg Arg Ala Gln Ser Ser Thr Ala Thr Gly Val Ala Lys Val
1230 1235 1240
cac cag ctg gag gag aag cag ctt atc gac gag gct ttc gag gct 4213
His Gln Leu Glu Glu Lys Gln Leu Ile Asp Glu Ala Phe Glu Ala
1245 1250 1255
taagtcttta tagtcctgca ctagcctaga gggccttatg cagtgtgaat cacacagcat 4273
aaggcccttt ttgctgccgt ggttgcctaa ggtggaaggc atgaaacgaa tctgtgcggt 4333
cacgatctct tcagtacttt tgctaagtgg ctgctcctcc acttccacca cgcagctcga 4393
g 4394
<210>13
<211>1257
<212>PRT
<213>谷氨酸棒状杆菌
<400>13
Met Leu Gln Leu Gly Leu Arg His Asn Gln Pro Thr Thr Asn Val Thr
1 5 10 15
Val Asp Lys Ile Lys Leu Asn Lys Pro Ser Arg Ser Lys Glu Lys Arg
20 25 30
Arg Val Pro Ala Val Ser Ser Ala Ser Thr Phe Gly Gln Asn Ala Trp
35 40 45
Leu Val Asp Glu Met Phe Gln Gln Phe Gln Lys Asp Pro Lys Ser Val
50 55 60
Asp Lys Glu Trp Arg Glu Leu Phe Glu Ala Gln Gly Gly Pro Asn Ala
65 70 75 80
Thr Pro Ala Thr Thr Glu Ala Gln Pro Ser Ala Pro Lys Glu Ser Ala
85 90 95
Lys Pro Ala Pro Lys Ala Ala Pro Ala Ala Lys Ala Ala Pro Arg Val
100 105 110
Glu Thr Lys Pro Ala Ala Lys Thr Ala Pro Lys Ala Lys Glu Ser Ser
115 120 125
Val Pro Gln Gln Pro Lys Leu Pro Glu Pro Gly Gln Thr Pro Ile Arg
130 135 140
Gly Ile Phe Lys Ser Ile Ala Lys Asn Met Asp Ile Ser Leu Glu Ile
145 150 155 160
Pro Thr Ala Thr Ser Val Arg Asp Met Pro Ala Arg Leu Met Phe Glu
165 170 175
Asn Arg Ala Met Val Asn Asp Gln Leu Lys Arg Thr Arg Gly Gly Lys
180 185 190
Ile Ser Phe Thr His Ile Ile Gly Tyr Ala Met Val Lys Ala Val Met
195 200 205
Ala His Pro Asp Met Asn Asn Ser Tyr Asp Val Ile Asp Gly Lys Pro
210 215 220
Thr Leu Ile Val Pro Glu His Ile Asn Leu Gly Leu Ala Ile Asp Leu
225 230 235 240
Pro Gln Lys Asp Gly Ser Arg Ala Leu Val Val Ala Ala Ile Lys Glu
245 250 255
Thr Glu Lys Met Asn Phe Ser Glu Phe Leu Ala Ala Tyr Glu Asp Ile
260 265 270
Val Thr Arg Ser Arg Lys Gly Lys Leu Thr Met Asp Asp Tyr Gln Gly
275 280 285
Val Thr Val Ser Leu Thr Asn Pro Gly Gly Ile Gly Thr Arg His Ser
290 295 300
Val Pro Arg Leu Thr Lys Gly Gln Gly Thr Ile Ile Gly Val Gly Ser
305 310 315 320
Met Asp Tyr Pro Ala Glu Phe Gln Gly Ala Ser Glu Asp Arg Leu Ala
325 330 335
Glu Leu Gly Val Gly Lys Leu Val Thr Ile Thr Ser Thr Tyr Asp His
340 345 350
Arg Val Ile Gln Gly Ala Val Ser Gly Glu Phe Leu Arg Thr Met Ser
355 360 365
Arg Leu Leu Thr Asp Asp Ser Phe Trp Asp Glu Ile Phe Asp Ala Met
370 375 380
Asn Val Pro Tyr Thr Pro Met Arg Trp Ala Gln Asp Val Pro Asn Thr
385 390 395 400
Gly Val Asp Lys Asn Thr Arg Val Met Gln Leu Ile Glu Ala Tyr Arg
405 410 415
Ser Arg Gly His Leu Ile Ala Asp Thr Asn Pro Leu Ser Trp Val Gln
420 425 430
Pro Gly Met Pro Val Pro Asp His Arg Asp Leu Asp Ile Glu Thr His
435 440 445
Ser Leu Thr Ile Trp Asp Leu Asp Arg Thr Phe Ser Val Gly Gly Phe
450 455 460
Gly Gly Lys Glu Thr Met Thr Leu Arg Glu Val Leu Ser Arg Leu Arg
465 470 475 480
Ala Ala Tyr Thr Leu Lys Val Gly Ser Glu Tyr Thr His Ile Leu Asp
485 490 495
Arg Asp Glu Arg Thr Trp Leu Gln Asp Arg Leu Glu Ala Gly Met Pro
500 505 510
Lys Pro Thr Gln Ala Glu Gln Lys Tyr Ile Leu Gln Lys Leu Asn Ala
515 520 525
Ala Glu Ala Phe Glu Asn Phe Leu Gln Thr Lys Tyr Val Gly Gln Lys
530 535 540
Arg Phe Ser Leu Glu Gly Ala Glu Ala Leu Ile Pro Leu Met Asp Ser
545 550 555 560
Ala Ile Asp Thr Ala Ala Gly Gln Gly Leu Asp Glu Val Val Ile Gly
565 570 575
Met Pro His Arg Gly Arg Leu Asn Val Leu Phe Asn Ile Val Gly Lys
580 585 590
Pro Leu Ala Ser Ile Phe Asn Glu Phe Glu Gly Gln Met Glu Gln Gly
595 600 605
Gln Ile Gly Gly Ser Gly Asp Val Lys Tyr His Leu Gly Ser Glu Gly
610 615 620
Gln His Leu Gln Met Phe Gly Asp Gly Glu Ile Lys Val Ser Leu Thr
625 630 635 640
Ala Asn Pro Ser His Leu Glu Ala Val Asn Pro Val Met Glu Gly Ile
645 650 655
Val Arg Ala Lys Gln Asp Tyr Leu Asp Lys Gly Val Asp Gly Lys Thr
660 665 670
Val Val Pro Leu Leu Leu His Gly Asp Ala Ala Phe Ala Gly Leu Gly
675 680 685
Ile Val Pro Glu Thr Ile Asn Leu Ala Lys Leu Arg Gly Tyr Asp Val
690 695 700
Gly Gly Thr Ile His Ile Val Val Asn Asn Gln Ile Gly Phe Thr Thr
705 710 715 720
Thr Pro Asp Ser Ser Arg Ser Met His Tyr Ala Thr Asp Tyr Ala Lys
725 730 735
Ala Phe Gly Cys Pro Val Phe His Val Asn Gly Asp Asp Pro Glu Ala
740 745 750
Val Val Trp Val Gly Gln Leu Ala Thr Glu Tyr Arg Arg Arg Phe Gly
755 760 765
Lys Asp Val Phe Ile Asp Leu Val Cys Tyr Arg Leu Arg Gly His Asn
770 775 780
Glu Ala Asp Asp Pro Ser Met Thr Gln Pro Lys Met Tyr Glu Leu Ile
785 790 795 800
Thr Gly Arg Glu Thr Val Arg Ala Gln Tyr Thr Glu Asp Leu Leu Gly
805 810 815
Arg Gly Asp Leu Ser Asn Glu Asp Ala Glu Ala Val Val Arg Asp Phe
820 825 830
His Asp Gln Met Glu Ser Val Phe Asn Glu Val Lys Glu Gly Gly Lys
835 840 845
Lys Gln Ala Glu Ala Gln Thr Gly Ile Thr Gly Ser Gln Lys Leu Pro
850 855 860
His Gly Leu Glu Thr Asn Ile Ser Arg Glu Glu Leu Leu Glu Leu Gly
865 870 875 880
Gln Ala Phe Ala Asn Thr Pro Glu Gly Phe Asn Tyr His Pro Arg Val
885 890 895
Ala Pro Val Ala Lys Lys Arg Val Ser Ser Val Thr Glu Gly Gly Ile
900 905 910
Asp Trp Ala Trp Gly Glu Leu Leu Ala Phe Gly Ser Leu Ala Asn Ser
915 920 925
Gly Arg Leu Val Arg Leu Ala Gly Glu Asp Ser Arg Arg Gly Thr Phe
930 935 940
Thr Gln Arg His Ala Val Ala Ile Asp Pro Ala Thr Ala Glu Glu Phe
945 950 955 960
Asn Pro Leu His Glu Leu Ala Gln Ser Lys Gly Asn Asn Gly Lys Phe
965 970 975
Leu Val Tyr Asn Ser Ala Leu Thr Glu Tyr Ala Gly Met Gly Phe Glu
980 985 990
Tyr Gly Tyr Ser Val Gly Asn Glu Asp Ser Val Val Ala Trp Glu Ala
995 1000 1005
Gln Phe Gly Asp Phe Ala Asn Gly Ala Gln Thr Ile Ile Asp Glu
1010 1015 1020
Tyr Val Ser Ser Gly Glu Ala Lys Trp Gly Gln Thr Ser Lys Leu
1025 1030 1035
Ile Leu Leu Leu Pro His Gly Tyr Glu Gly Gln Gly Pro Asp His
1040 1045 1050
Ser Ser Ala Arg Ile Glu Arg Phe Leu Gln Leu Cys Ala Glu Gly
1055 1060 1065
Ser Met Thr Val Ala Gln Pro Ser Thr Pro Ala Asn His Phe His
1070 1075 1080
Leu Leu Arg Arg His Ala Leu Ser Asp Leu Lys Arg Pro Leu Val
1085 1090 1095
Ile Phe Thr Pro Lys Ser Met Leu Arg Asn Lys Ala Ala Ala Ser
1100 1105 1110
Ala Pro Glu Asp Phe Thr Glu Val Thr Lys Phe Gln Ser Val Ile
1115 1120 1125
Asp Asp Pro Asn Val Ala Asp Ala Ala Lys Val Lys Lys Val Met
1130 1135 1140
Leu Val Ser Gly Lys Leu Tyr Tyr Glu Leu Ala Lys Arg Lys Glu
1145 1150 1155
Lys Asp Gly Arg Asp Asp Ile Ala Ile Val Arg Ile Glu Met Leu
1160 1165 1170
His Pro Ile Pro Phe Asn Arg Ile Ser Glu Ala Leu Ala Gly Tyr
1175 1180 1185
Pro Asn Ala Glu Glu Val Leu Phe Val Gln Asp Glu Pro Ala Asn
1190 1195 1200
Gln Gly Pro Trp Pro Phe Tyr Gln Glu His Leu Pro Glu Leu Ile
1205 1210 1215
Pro Asn Met Pro Lys Met Arg Arg Val Ser Arg Arg Ala Gln Ser
1220 1225 1230
Ser Thr Ala Thr Gly Val Ala Lys Val His Gln Leu Glu Glu Lys
1235 1240 1245
Gln Leu Ile Asp Glu Ala Phe Glu Ala
1250 1255
<210>14
<211>2028
<212>DNA
<213>谷氨酸棒状杆菌
<220>
<221>CDS
<222>(1)..(2028)
<400>14
atg gcg ttc tcc gta gag atg ccc gag ctg ggc gaa tca gta acc gaa 48
Met Ala Phe Ser Val Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu
1 5 10 15
ggc acg atc acc cag tgg ttg aag tct gtt ggt gac act gtt gag gta 96
Gly Thr Ile Thr Gln Trp Leu Lys Ser Val Gly Asp Thr Val Glu Val
20 25 30
gat gag ccg ttg ctc gag gtc tca act gac aag gtc gac acc gag att 144
Asp Glu Pro Leu Leu Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile
35 40 45
ccc tct cct gtc gcc ggt gtc atc cta gag att aag gct gaa gag gat 192
Pro Ser Pro Val Ala Gly Val Ile Leu Glu Ile Lys Ala Glu Glu Asp
50 55 60
gac acc gtc gac gtc ggc ggt gtc att gca ata atc ggc gat gct gat 240
Asp Thr Val Asp Val Gly Gly Val Ile Ala Ile Ile Gly Asp Ala Asp
65 70 75 80
gag act cct gcc aac gaa gct cct gcc gac gag gca cca gct cct gcc 288
Glu Thr Pro Ala Asn Glu Ala Pro Ala Asp Glu Ala Pro Ala Pro Ala
85 90 95
gaa gag gaa gaa cca gtt aag gaa gag cca aag aag gag gca gct cct 336
Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys Glu Ala Ala Pro
100 105 110
gaa gct cca gca gca act ggc gcc gca acc gat gtg gaa atg cca gaa 384
Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val Glu Met Pro Glu
115 120 125
ctc ggc gaa tcc gtc acc gaa ggc acc att acc cag tgg ctc aag gct 432
Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln Trp Leu Lys Ala
130 135 140
gtc ggc gac acc gtc gaa gta gac gaa cca ctt ctt gag gtc tcc acc 480
Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu Glu Val Ser Thr
145 150 155 160
gac aag gtc gac acc gaa atc cca tcc cca gta gca ggc acc atc gtg 528
Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala Gly Thr Ile Val
165 170 175
gag atc ctt gca gac gaa gac gac acc gtc gac gtc ggc gca gtc atc 576
Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val Gly Ala Val Ile
180 185 190
gcc cgc atc ggt gac gca aac gca gct gca gca cct gcc gaa gag gaa 624
Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro Ala Glu Glu Glu
195 200 205
gca gct cct gcc gaa gag gaa gaa cca gtt aag gaa gag cca aag aag 672
Ala Ala Pro Ala Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys
210 215 220
gag gca gct cct gaa gct cca gca gca act ggc gcc gca acc gat gtg 720
Glu Ala Ala Pro Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val
225 230 235 240
gaa atg cca gaa ctc ggc gaa tcc gtc acc gaa ggc acc att acc cag 768
Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln
245 250 255
tgg ctc aag gct gtc ggc gac acc gtc gaa gta gac gaa cca ctt ctt 816
Trp Leu Lys Ala Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu
260 265 270
gag gtc tcc acc gac aag gtc gac acc gaa atc cca tcc cca gta gca 864
Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala
275 280 285
ggc acc atc gtg gag atc ctt gca gac gaa gac gac acc gtc gac gtc 912
Gly Thr Ile Val Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val
290 295 300
ggc gca gtc atc gcc cgc atc ggt gac gca aac gca gct gca gca cct 960
Gly Ala Val Ile Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro
305 310 315 320
gcc gaa gag gaa gca gct cct gcc gaa gag gag gaa cca gtt aag gaa 1008
Ala Glu Glu Glu Ala Ala Pro Ala Glu Glu Glu Glu Pro Val Lys Glu
325 330 335
gag cca aag aag gaa gag ccc aag aag gaa gag ccc aag aag gaa gca 1056
Glu Pro Lys Lys Glu Glu Pro Lys Lys Glu Glu Pro Lys Lys Glu Ala
340 345 350
gct act aca cct gct gcg gca tcc gca act gtg tcc gct tct ggc gac 1104
Ala Thr Thr Pro Ala Ala Ala Ser Ala Thr Val Ser Ala Ser Gly Asp
355 360 365
aac gtt cca tac gtc acc cca ctg gtg cgc aag ctt gct gaa aag cac 1152
Asn Val Pro Tyr Val Thr Pro Leu Val Arg Lys Leu Ala Glu Lys His
370 375 380
ggc gtt gac ttg aac acc gtg acc ggt acc ggt atc ggt ggc cgt atc 1200
Gly Val Asp Leu Asn Thr Val Thr Gly Thr Gly Ile Gly Gly Arg Ile
385 390 395 400
cgc aag cag gat gtt ttg gct gct gcg aac ggc gag gct gca cct gct 1248
Arg Lys Gln Asp Val Leu Ala Ala Ala Asn Gly Glu Ala Ala Pro Ala
405 410 415
gag gct gct gct cct gtt tcc gct tgg tcc act aag tct gtt gac cct 1296
Glu Ala Ala Ala Pro Val Ser Ala Trp Ser Thr Lys Ser Val Asp Pro
420 425 430
gag aag gct aag ctc cgt ggt acc act cag aag gtc aac cgc atc cgt 1344
Glu Lys Ala Lys Leu Arg Gly Thr Thr Gln Lys Val Asn Arg Ile Arg
435 440 445
gag atc acc gcg atg aag acc gtc gag gct ctg cag att tct gct cag 1392
Glu Ile Thr Ala Met Lys Thr Val Glu Ala Leu Gln Ile Ser Ala Gln
450 455 460
ctc acc cag ctg cac gag gtc gat atg act cgc gtt gct gag ctg cgt 1440
Leu Thr Gln Leu His Glu Val Asp Met Thr Arg Val Ala Glu Leu Arg
465 470 475 480
aag aag aac aag ccc gcg ttc atc gag aag cac ggt gtg aac ctc act 1488
Lys Lys Asn Lys Pro Ala Phe Ile Glu Lys His Gly Val Asn Leu Thr
485 490 495
tac ctg cca ttc ttc gtg aag gca gtt gtc gag gct ttg gtt tcc cat 1536
Tyr Leu Pro Phe Phe Val Lys Ala Val Val Glu Ala Leu Val Ser His
500 505 510
cca aac gtc aac gcg tct ttc aac gcg aag acc aag gag atg acc tac 1584
Pro Asn Val Asn Ala Ser Phe Asn Ala Lys Thr Lys Glu Met Thr Tyr
515 520 525
cac tcc tcc gtt aac ctc tcc atc gct gtt gat acc cca gct ggt ctg 1632
His Ser Ser Val Asn Leu Ser Ile Ala Val Asp Thr Pro Ala Gly Leu
530 535 540
ttg acc cca gtc att cac gat gct cag gat ctc tcc atc cca gag atc 1680
Leu Thr Pro Val Ile His Asp Ala Gln Asp Leu Ser Ile Pro Glu Ile
545 550 555 560
gca aag gca att gtt gac ctg gct gat cgt tca cgc aac aac aag ctg 1728
Ala Lys Ala Ile Val Asp Leu Ala Asp Arg Ser Arg Asn Asn Lys Leu
565 570 575
aag cca aac gat ctg tcc ggt ggc acc ttc acc atc acc aac att ggt 1776
Lys Pro Asn Asp Leu Ser Gly Gly Thr Phe Thr Ile Thr Asn Ile Gly
580 585 590
tct gaa ggc gca ctg tct gat acc cca atc ctg gtt cca cca cag gct 1824
Ser Glu Gly Ala Leu Ser Asp Thr Pro Ile Leu Val Pro Pro Gln Ala
595 600 605
ggc atc ttg ggc acc ggc gcg atc gtg aag cgt cca gtt gtc atc acc 1872
Gly Ile Leu Gly Thr Gly Ala Ile Val Lys Arg Pro Val Val Ile Thr
610 615 620
gag gat gga att gat tcc atc gcg atc cgt cag atg gtc ttc cta cca 1920
Glu Asp Gly Ile Asp Ser Ile Ala Ile Arg Gln Met Val Phe Leu Pro
625 630 635 640
ctg acc tac gac cac cag gtt gta gat ggc gca gat gct ggt cgc ttc 1968
Leu Thr Tyr Asp His Gln Val Val Asp Gly Ala Asp Ala Gly Arg Phe
645 650 655
ctg acc acc atc aag gac cgc ctt gag acc gct aac ttc gaa ggc gat 2016
Leu Thr Thr Ile Lys Asp Arg Leu Glu Thr Ala Asn Phe Glu Gly Asp
660 665 670
ctg cag ctc taa 2028
Leu Gln Leu
675
<210>15
<211>675
<212>PRT
<213>谷氨酸棒状杆菌
<400>15
Met Ala Phe Ser Val Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu
1 5 10 15
Gly Thr Ile Thr Gln Trp Leu Lys Ser Val Gly Asp Thr Val Glu Val
20 25 30
Asp Glu Pro Leu Leu Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile
35 40 45
Pro Ser Pro Val Ala Gly Val lle Leu Glu Ile Lys Ala Glu Glu Asp
50 55 60
Asp Thr Val Asp Val Gly Gly Val Ile Ala Ile Ile Gly Asp Ala Asp
65 70 75 80
Glu Thr Pro Ala Asn Glu Ala Pro Ala Asp Glu Ala Pro Ala Pro Ala
85 90 95
Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys Glu Ala Ala Pro
100 105 110
Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val Glu Met Pro Glu
115 120 125
Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln Trp Leu Lys Ala
130 135 140
Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu Glu Val Ser Thr
145 150 155 160
Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala Gly Thr Ile Val
165 170 175
Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val Gly Ala Val Ile
180 185 190
Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro Ala Glu Glu Glu
195 200 205
Ala Ala Pro Ala Glu Glu Glu Glu Pro Val Lys Glu Glu Pro Lys Lys
210 215 220
Glu Ala Ala Pro Glu Ala Pro Ala Ala Thr Gly Ala Ala Thr Asp Val
225 230 235 240
Glu Met Pro Glu Leu Gly Glu Ser Val Thr Glu Gly Thr Ile Thr Gln
245 250 255
Trp Leu Lys Ala Val Gly Asp Thr Val Glu Val Asp Glu Pro Leu Leu
260 265 270
Glu Val Ser Thr Asp Lys Val Asp Thr Glu Ile Pro Ser Pro Val Ala
275 280 285
Gly Thr Ile Val Glu Ile Leu Ala Asp Glu Asp Asp Thr Val Asp Val
290 295 300
Gly Ala Val Ile Ala Arg Ile Gly Asp Ala Asn Ala Ala Ala Ala Pro
305 310 315 320
Ala Glu Glu Glu Ala Ala Pro Ala Glu Glu Glu Glu Pro Val Lys Glu
325 330 335
Glu Pro Lys Lys Glu Glu Pro Lys Lys Glu Glu Pro Lys Lys Glu Ala
340 345 350
Ala Thr Thr Pro Ala Ala Ala Ser Ala Thr Val Ser Ala Ser Gly Asp
355 360 365
Asn Val Pro Tyr Val Thr Pro Leu Val Arg Lys Leu Ala Glu Lys His
370 375 380
Gly Val Asp Leu Asn Thr Val Thr Gly Thr Gly Ile Gly Gly Arg Ile
385 390 395 400
Arg Lys Gln Asp Val Leu Ala Ala Ala Asn Gly Glu Ala Ala Pro Ala
405 410 415
Glu Ala Ala Ala Pro Val Ser Ala Trp Ser Thr Lys Ser Val Asp Pro
420 425 430
Glu Lys Ala Lys Leu Arg Gly Thr Thr Gln Lys Val Asn Arg Ile Arg
435 440 445
Glu Ile Thr Ala Met Lys Thr Val Glu Ala Leu Gln Ile Ser Ala Gln
450 455 460
Leu Thr Gln Leu His Glu Val Asp Met Thr Arg Val Ala Glu Leu Arg
465 470 475 480
Lys Lys Asn Lys Pro Ala Phe Ile Glu Lys His Gly Val Asn Leu Thr
485 490 495
Tyr Leu Pro Phe Phe Val Lys Ala Val Val Glu Ala Leu Val Ser His
500 505 510
Pro Asn Val Asn Ala Ser Phe Asn Ala Lys Thr Lys Glu Met Thr Tyr
515 520 525
His Ser Ser Val Asn Leu Ser Ile Ala Val Asp Thr Pro Ala Gly Leu
530 535 540
Leu Thr Pro Val Ile His Asp Ala Gln Asp Leu Ser Ile Pro Glu Ile
545 550 555 560
Ala Lys Ala Ile Val Asp Leu Ala Asp Arg Ser Arg Asn Asn Lys Leu
565 570 575
Lys Pro Asn Asp Leu Ser Gly Gly Thr Phe Thr Ile Thr Asn Ile Gly
580 585 590
Ser Glu Gly Ala Leu Ser Asp Thr Pro Ile Leu Val Pro Pro Gln Ala
595 600 605
Gly Ile Leu Gly Thr Gly Ala Ile Val Lys Arg Pro Val Val Ile Thr
610 615 620
Glu Asp Gly Ile Asp Ser Ile Ala Ile Arg Gln Met Val Phe Leu Pro
625 630 635 640
Leu Thr Tyr Asp His Gln Val Val Asp Gly Ala Asp Ala Gly Arg Phe
645 650 655
Leu Thr Thr Ile Lys Asp Arg Leu Glu Thr Ala Asn Phe Glu Gly Asp
660 665 670
Leu Gln Leu
675
<210>16
<211>1308
<212>DNA
<213>Pantoea ananatis
<400>16
atgagcagaa tcatgacgcc cgtgaactgg gaagcctgca gcagcgaggc gcagcaggcg 60
ctgttggcac gccctgcgct cgcctcgtct gacagcatca gccagatcgt gcgcgatgtg 120
ttggtcagag tgaaagagga aggcgatgcg gctttacgag aattcagcgc gcgctttgac 180
aaggttgaaa cagacgacct gcgcgttacg ccacagcaga tgcaggcggc cagcgatcgc 240
cttggtgacg agctgaaaca ggcgatggcc gtggccattg gcaatattga aacctttcac 300
cgtgcgcaga tcctgccgcc ggtggatgtg gaaacgcagc ccggcgtgcg ctgtcagcaa 360
attacgcgcc cgatgaaatc ggtgggcttg tatattccgg gcggttctgc cccgctgttt 420
tctaccgttc tgatgctggc taccccggcg cggattgcgg gctgtggtcg cgtggtgctg 480
tgctcgcccc cgccgattgc tgatgaaatt ctctacgcgg ccaaactttg cggtgtggaa 540
gaagtgttcc aggtgggtgg atcacaggcg attgccgccc tggcttttgg caccgaaagc 600
atccctaagg tagataaaat ttttggtccg ggcaacgcgt gggttaccga agccaaacgt 660
caggtcagcc agcgccttga tggcgcggcg attgatatgc ccgctggccc gtcggaagtg 720
ctggtgattg ccgatgaagg tgccacaccg gccttcgttg cctctgatct gctgtcgcag 780
gcggaacacg gccctgactc gcaggtgatt ttactgacgc cttcgctggc gctggccgag 840
cgcgtcgccg aggcggtgga ggatcagctg gcccagttgc cacgtgcggc gacagcccgc 900
caggcactgg aaagcagccg cctgatcgtc gcccgggata tgcagcaatg cattgcgatc 960
tccaaccgct atggtccgga gcacctgatt ctgcaaaccc gcacgccacg ggatctggtg 1020
gaacagatta ccagcgccgg ttcggttttc ctgggcgact ggtcaccgga atccgcagga 1080
gattatgctt cgggcaccaa ccacgtgctg ccgacctacg gctataccgc gacatgctcc 1140
agcctgggcc tggccgactt tcagaaacgc atgacggtac aggagctgac gccgcagggc 1200
ttcctgaacc tggcggcgac catcgaaacc ctggcggccg ctgaacagct gcacgcccac 1260
aaaaatgccg tcacgttgcg cgttgccgca ctcaaggagc aagcatga 1308
<210>17
<211>68
<212>DNA
<213>人工的
<220>
<223>引物
<400>17
ccatagcggt tggagatcgc aatgcattgc tgcatatccc tgaagcctgc ttttttatac 60
taagttgg 68
<210>l8
<211>68
<212>DNA
<213>人工的
<220>
<223>引物
<400>18
gcccgccagg cactggaaag cagccgcctg atcgtcgccc cgctcaagtt agtataaaaa 60
agctgaac 68
<210>19
<211>33
<212>DNA
<213>人工的
<220>
<223>引物
<400>19
tagcgagatc tctgatgtcc ggcggtgctt ttg 33
<210>20
<211>32
<212>DNA
<213>人工的
<220>
<223>引物
<400>20
aaaaagagct cttacgcccc gccctgccac tc 32
<210>21
<211>34
<212>DNA
<213>人工的
<220>
<223>引物
<400>21
caggatctag aaggagacat gaacgatgaa catc 34
<210>22
<211>36
<212>DNA
<213>人工的
<220>
<223>引物
<400>22
gataaggatc cgaaataaaa gaaaatgcca atagga 36
<210>23
<211>31
<212>DNA
<213>人工的
<220>
<223>引物
<400>23
cctttgagct cgcgggcagt gagcgcaacg c 31
<210>24
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<400>24
ctagagcggc cgccgatcgg gatcctcctg tgtgaaattg ttatccgc 48
<210>25
<211>42
<212>DNA
<213>人工的
<220>
<223>引物
<400>25
ctctacgatc gaggaggtta taaaaaatgg atattaatac tg 42
<210>26
<211>36
<212>DNA
<213>人工的
<220>
<223>引物
<400>26
tcaaagcggc cgcttcttcg tctgtttcta ctggta 36
<210>27
<211>31
<212>DNA
<213>人工的
<220>
<223>引物
<400>27
cctttggtac cgcgggcagt gagcgcaacg c 31
<210>28
<211>34
<212>DNA
<213>人工的
<220>
<223>引物
<400>28
aacaggaatt ctttgcctgg cggcagtagc gcgg 34
<210>29
<211>40
<212>DNA
<213>人工的
<220>
<223>引物P1
<400>29
ctagtaagat cttgaagcct gcttttttat actaagttgg 40
<210>30
<211>41
<212>DNA
<213>人工的
<220>
<223>引物P2
<400>30
atgatcgaat tcgaaatcaa ataatgattt tattttgact g 41
<210>31
<211>120
<212>DNA
<213>人工的
<220>
<223>包含attL的DNA片段
<400>31
agatcttgaa gcctgctttt ttatactaag ttggcattat aaaaaagcat tgcttatcaa 60
tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttga tttcgaattc 120
<210>32
<211>41
<212>DNA
<213>人工的
<220>
<223>引物P3
<400>32
atgccactgc agtctgttac aggtcactaa taccatctaa g 41
<210>33
<211>46
<212>DNA
<213>人工的
<220>
<223>引物P4
<400>33
accgttaagc tttctagacg ctcaagttag tataaaaaag ctgaac 46
<210>34
<211>184
<212>DNA
<213>人工的
<220>
<223>包含attR的DNA片段
<400>34
ctgcagtctg ttacaggtca ctaataccat ctaagtagtt gattcatagt gactgcatat 60
gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga 120
tatttatatc attttacgtt tctcgttcag cttttttata ctaacttgag cgtctagaaa 180
gctt 184
<210>35
<211>38
<212>DNA
<213>人工的
<220>
<223>引物P5
<400>35
ttcttagacg tcaggtggca cttttcgggg aaatgtgc 38
<210>36
<211>37
<212>DNA
<213>人工的
<220>
<223>引物P6
<400>36
taacagagat ctcgcgcaga aaaaaaggat ctcaaga 37
<210>37
<211>46
<212>DNA
<213>人工的
<220>
<223>引物P7
<400>37
aacagagatc taagcttaga tcctttgcct ggcggcagta gcgcgg 46
<210>38
<211>35
<212>DNA
<213>人工的
<220>
<223>引物P8
<400>38
ataaactgca gcaaaaagag tttgtagaaa cgcaa 35
<210>39
<211>1388
<212>DNA
<213>人工的
<220>
<223>包含Tc基因和ter_thrL的DNA片段
<400>39
gaattctcat gtttgacagc ttatcatcga taagctttaa tgcggtagtt tatcacagtt 60
aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct 120
cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct 180
cttgcgggat atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct 240
atatgcgttg atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg 300
ccgccgccca gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc 360
gaccacaccc gtcctgtgga tcctctacgc cggacgcatc gtggccggca tcaccggcgc 420
cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg 480
ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg 540
gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg 600
cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgacc 660
gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat 720
cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc 780
gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc 840
gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac 900
caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta 960
cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc 1020
ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga 1080
ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcactgg 1140
accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg 1200
gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag 1260
ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca 1320
actagaaagc ttaacacaga aaaaagcccg cacctgacag tgcgggcttt ttttttcgac 1380
cactgcag 1388
<210>40
<211>36
<212>DNA
<213>人工的
<220>
<223>引物P9
<400>40
agtaattcta gaaagcttaa cacagaaaaa agcccg 36
<210>41
<211>43
<212>DNA
<213>人工的
<220>
<223>引物P10
<400>41
ctagtaggat ccctgcagtg gtcgaaaaaa aaagcccgca ctg 43
<210>42
<211>33
<212>DNA
<213>人工的
<220>
<223>引物1
<400>42
ggaagatcta tttgccttcg cacatcaacc tgg 33
<210>43
<211>30
<212>DNA
<213>人工的
<220>
<223>引物2
<400>43
cggggtacct tgtaaatatt ttaacccgcc 30
<210>44
<211>56
<212>DNA
<213>人工的
<220>
<223>引物3
<400>44
ggaagatcta aggagacctt aaatgagcga cacaacgatc ctgcaaaaca gtaccc 56
<210>45
<211>39
<212>DNA
<213>人工的
<220>
<223>引物4
<400>45
cggggtacct cgtagaggtt tactggcgct tatccagcg 39
<210>46
<211>80
<212>DNA
<213>人工的
<220>
<223>引物
<400>46
gcatatgtat gacaccgtca aaggttccga ctacatcggt gaccaggacg tgaagcctgc 60
ttttttatac taagttggca 80
<210>47
<211>80
<212>DNA
<213>人工的
<220>
<223>引物
<400>47
tccagctcaa ggcactcaat acgctgtgta ttgaagtcag gtgagcggtc cgctcaagtt 60
agtataaaaa agctgaacga 80
<210>48
<211>20
<212>DNA
<213>人工的
<220>
<223>引物
<400>48
ccaggcactc gtcctcggtt 20
<210>49
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<400>49
aggctagtgc aggactataa agaccagttc tcctaaaaat aacgtgtc 48
<210>50
<211>20
<212>DNA
<213>人工的
<220>
<223>引物
<400>50
tccatcgtgg ccaccgatcc 20
<210>51
<211>48
<212>DNA
<213>人工的
<220>
<223>引物
<400>51
gacacgttat ttttaggaga actggtcttt atagtcctgc actagcct 48
<210>52
<211>25
<212>DNA
<213>人工的
<220>
<223>引物
<400>52
cgggatcccc accggcgtac tcgtg 25
<210>53
<211>28
<212>DNA
<213>人工的
<220>
<223>引物
<400>53
ccacggatcc ttccaatgct attggttg 28
<210>54
<211>28
<212>DNA
<213>人工的
<220>
<223>引物
<400>54
gggagctcga ctttctggct cctttact 28
<210>55
<211>28
<212>DNA
<213>人工的
<220>
<223>引物
<400>55
gggagctcgc cgatgactaa taatgaga 28
<210>56
<211>2432
<212>DNA
<213>谷氨酸棒状杆菌
<220>
<221>CDS
<222>(507)..(2096)
<400>56
aggggcggcg gatcgaccac ggcttgcaac cgtggcggga gtgggctgtt gagaagctgc 60
cacattcacg actttctggc tcctttacta aataaggatt ttcacaggac ccgtccaagc 120
caagccgatt tcaactcagc ctaaagacaa agccctcatt taaaattgtt ccgacgcgga 180
tgcgtgtgca cgcagtgcga cagatgtctg ttgcaaagtt ggctacttgg gtcataacca 240
acaagaaagc cctcgttcca acactgtggt gagtgttgtc gagggcgctt gacgagacga 300
cttggaaggc cgttacggca ggcgccgcgc ggttactact acaagtcgaa taatggtcat 360
ggtgtgtcat gctacacaca tcgagtttcc aattccacaa cgcacgaaaa ttcccacccc 420
caaaactccc ccacttcggt taaggaatca ggattctcac aaagttcagg caggctcccg 480
ctacttttca gcgctaatct tggctc atg att tta ggc gta ccc att caa tat 533
Met Ile Leu Gly Val Pro Ile Gln Tyr
1 5
ttg ctc tat tca ttg tgg aat tgg att gtc gat acc ggt ttt gat gta 581
Leu Leu Tyr Ser Leu Trp Asn Trp Ile Val Asp Thr Gly Phe Asp Val
10 15 20 25
gca att atc ctg gtc ttg gcg ttt ttg att cca cgt atc ggc cga ctg 629
Ala Ile Ile Leu Val Leu Ala Phe Leu Ile Pro Arg Ile Gly Arg Leu
30 35 40
gcc atg cgt att atc aag cgg cga gtg gag tct gca gcc gat gcg gac 677
Ala Met Arg Ile Ile Lys Arg Arg Val Glu Ser Ala Ala Asp Ala Asp
45 50 55
acc act aag aac cag ctc gcg ttc gct ggc gtt ggc gtt tat atc gcg 725
Thr Thr Lys Asn Gln Leu Ala Phe Ala Gly Val Gly Val Tyr Ile Ala
60 65 70
caa att gtg gcg ttt ttc atg ctt gcc gtc tcc gcg atg cag gct ttt 773
Gln Ile Val Ala Phe Phe Met Leu Ala Val Ser Ala Met Gln Ala Phe
75 80 85
ggt ttc tct ctc gcg ggc gct gcg att ccg gca acc att gcg tca gct 821
Gly Phe Ser Leu Ala Gly Ala Ala Ile Pro Ala Thr Ile Ala Ser Ala
90 95 100 105
gcc att ggc ctt ggt gcg cag tcg att gtt gcg gac ttc ttg gcc gga 869
Ala Ile Gly Leu Gly Ala Gln Ser Ile Val Ala Asp Phe Leu Ala Gly
110 115 120
ttt ttc atc ctg acg gaa aag caa ttc ggc gtg ggt gac tgg gtg cgt 917
Phe Phe Ile Leu Thr Glu Lys Gln Phe Gly Val Gly Asp Trp Val Arg
125 130 135
ttt gag ggc aac ggc atc gtt gtc gaa ggc acc gtc att gag atc acc 965
Phe Glu Gly Asn Gly Ile Val Val Glu Gly Thr Val Ile Glu Ile Thr
140 145 150
atg cgc gcg acc aaa att cgc acg att gca caa gag acc gtg atc atc 1013
Met Arg Ala Thr Lys Ile Arg Thr Ile Ala Gln Glu Thr Val Ile Ile
155 160 165
ccc aac tcc acg gcg aaa gtg tgc atc aac aat tct aat aac tgg tcg 1061
Pro Asn Ser Thr Ala Lys Val Cys Ile Asn Asn Ser Asn Asn Trp Ser
170 175 180 185
cgt gcg gtt gtc gtt att ccg atc ccc atg ttg ggt tct gaa aac atc 1109
Arg Ala Val Val Val Ile Pro Ile Pro Met Leu Gly Ser Glu Asn Ile
190 195 200
aca gat gtc atc gcg cgc tct gaa gct gcg act cgt cgc gca ctt ggc 1157
Thr Asp Val Ile Ala Arg Ser Glu Ala Ala Thr Arg Arg Ala Leu Gly
205 210 215
cag gag aaa atc gca ccg gaa atc ctc ggt gaa ctc gat gtg cac cca 1205
Gln Glu Lys Ile Ala Pro Glu Ile Leu Gly Glu Leu Asp Val His Pro
220 225 230
gcc acg gaa gtc aca ccg cca acg gtg gtc ggc atg ccg tgg atg gtc 1253
Ala Thr Glu Val Thr Pro Pro Thr Val Val Gly Met Pro Trp Met Val
235 240 245
acc atg cgt ttc ctc gtg caa gtc acc gcc ggc aat caa tgg ctg gtc 1301
Thr Met Arg Phe Leu Val Gln Val Thr Ala Gly Asn Gln Trp Leu Val
250 255 260 265
gaa cgc gcc atc cgc aca gaa atc atc aac gaa ttc tgg gaa gaa tac 1349
Glu Arg Ala Ile Arg Thr Glu Ile Ile Asn Glu Phe Trp Glu Glu Tyr
270 275 280
ggc agc gca acc act aca tcg gga acc ctc att gat tcc tta cac gtt 1397
Gly Ser Ala Thr Thr Thr Ser Gly Thr Leu Ile Asp Ser Leu His Val
285 290 295
gag cat gaa gag cca aag acc tcg ctt atc gac gcc tcc ccc cag gct 1445
Glu His Glu Glu Pro Lys Thr Ser Leu Ile Asp Ala Ser Pro Gln Ala
300 305 310
ctt aag gaa ccg aag ccg gag gct gcg gcg acg gtt gca tcg cta gct 1493
Leu Lys Glu Pro Lys Pro Glu Ala Ala Ala Thr Val Ala Ser Leu Ala
315 320 325
gca tcg tct aac gac gat gca gac aat gca gac gcc tcg gcg atc aat 1541
Ala Ser Ser Asn Asp Asp Ala Asp Asn Ala Asp Ala Ser Ala Ile Asn
330 335 340 345
gca ggc aat cca gag aag gaa ctt gat tcc gat gtg ctg gaa caa gaa 1589
Ala Gly Asn Pro Glu Lys Glu Leu Asp Ser Asp Val Leu Glu Gln Glu
350 355 360
ctc tcc agc gaa gaa ccg gaa gaa aca gca aaa cca gat cac tct ctc 1637
Leu Ser Ser Glu Glu Pro Glu Glu Thr Ala Lys Pro Asp His Ser Leu
365 370 375
cga ggc ttc ttc cgc act gat tac tac cca aat cgg tgg cag aag atc 1685
Arg Gly Phe Phe Arg Thr Asp Tyr Tyr Pro Asn Arg Trp Gln Lys Ile
380 385 390
ctg tcg ttt ggc gga cgt gtc cgc atg agc act tcc ctg ttg ttg ggt 1733
Leu Ser Phe Gly Gly Arg Val Arg Met Ser Thr Ser Leu Leu Leu Gly
395 400 405
gcg ctg ctc ttg ctg tca cta ttt aag gtc atg act gtg gaa cca agt 1781
Ala Leu Leu Leu Leu Ser Leu Phe Lys Val Met Thr Val Glu Pro Ser
410 415 420 425
gag aat tgg caa aac tcc agt gga tgg ctg tca cca agc act gcc acc 1829
Glu Asn Trp Gln Asn Ser Ser Gly Trp Leu Ser Pro Ser Thr Ala Thr
430 435 440
tca act gcg gtg acc acc tcc gaa act tcc gcg cca gta agc act tct 1877
Ser Thr Ala Val Thr Thr Ser Glu Thr Ser Ala Pro Val Ser Thr Ser
445 450 455
tcg atg aca gtg ccc act acg gtg gag gag acc cca acg atg gaa tct 1925
Ser Met Thr Val Pro Thr Thr Val Glu Glu Thr Pro Thr Met Glu Ser
460 465 470
agc gtc gaa acg cag cag gaa acc tca acc cct gca acc gca acg ccc 1973
Ser Val Glu Thr Gln Gln Glu Thr Ser Thr Pro Ala Thr Ala Thr Pro
475 480 485
cag cga gcc gac acc atc gaa ccg acc gag gaa gcc acg tcg cag gag 2021
Gln Arg Ala Asp Thr Ile Glu Pro Thr Glu Glu Ala Thr Ser Gln Glu
490 495 500 505
gaa acg act gcg tcg cag acg cag tct cca gca acc gcg gtt caa gag 2069
Glu Thr Thr Ala Ser Gln Thr Gln Ser Pro Ala Thr Ala Val Gln Glu
510 515 520
aca gtt gcg ccg acg tcc acc cct tag gacgctgatt acagacgtgt 2116
Thr Val Ala Pro Thr Ser Thr Pro
525
cccatttctt tactactatt ggaaattatg agttcagacg cagaaaaggc atccgtggag 2176
ctttccgaaa aatttcaccc agaacgcacc catattttgg gcgccgttgt ttttggcctg 2236
atctcattat tagtcatcgg cgcagcccct cagtacctgt tttggctgct cgcgctccct 2296
gtcatcttcg gttactgggt tctaaaatca tccacgatcg ttgatgaaca gggcatcacc 2356
gcaaactacg ccttcaaggg caaaaaggtt gtggcctggg aagacctcgc aggaatcgga 2416
ttcaagggtg cccgca 2432
<210>57
<211>529
<212>PRT
<213>谷氨酸棒状杆菌
<400>57
Met Ile Leu Gly Val Pro Ile Gln Tyr Leu Leu Tyr Ser Leu Trp Asn
1 5 10 15
Trp Ile Val Asp Thr Gly Phe Asp Val Ala Ile Ile Leu Val Leu Ala
20 25 30
Phe Leu Ile Pro Arg Ile Gly Arg Leu Ala Met Arg Ile Ile Lys Arg
35 40 45
Arg Val Glu Ser Ala Ala Asp Ala Asp Thr Thr Lys Asn Gln Leu Ala
50 55 60
Phe Ala Gly Val Gly Val Tyr Ile Ala Gln Ile Val Ala Phe Phe Met
65 70 75 80
Leu Ala Val Ser Ala Met Gln Ala Phe Gly Phe Ser Leu Ala Gly Ala
85 90 95
Ala Ile Pro Ala Thr Ile Ala Ser Ala Ala Ile Gly Leu Gly Ala Gln
100 105 110
Ser Ile Val Ala Asp Phe Leu Ala Gly Phe Phe Ile Leu Thr Glu Lys
115 120 125
Gln Phe Gly Val Gly Asp Trp Val Arg Phe Glu Gly Asn Gly Ile Val
130 135 140
Val Glu Gly Thr Val Ile Glu Ile Thr Met Arg Ala Thr Lys Ile Arg
145 150 155 160
Thr Ile Ala Gln Glu Thr Val Ile Ile Pro Asn Ser Thr Ala Lys Val
165 170 175
Cys Ile Asn Asn Ser Asn Asn Trp Ser Arg Ala Val Val Val Ile Pro
180 185 190
Ile Pro Met Leu Gly Ser Glu Asn Ile Thr Asp Val Ile Ala Arg Ser
195 200 205
Glu Ala Ala Thr Arg Arg Ala Leu Gly Gln Glu Lys Ile Ala Pro Glu
210 215 220
Ile Leu Gly Glu Leu Asp Val His Pro Ala Thr Glu Val Thr Pro Pro
225 230 235 240
Thr Val Val Gly Met Pro Trp Met Val Thr Met Arg Phe Leu Val Gln
245 250 255
Val Thr Ala Gly Asn Gln Trp Leu Val Glu Arg Ala Ile Arg Thr Glu
260 265 270
Ile Ile Asn Glu Phe Trp Glu Glu Tyr Gly Ser Ala Thr Thr Thr Ser
275 280 285
Gly Thr Leu Ile Asp Ser Leu His Val Glu His Glu Glu Pro Lys Thr
290 295 300
Ser Leu Ile Asp Ala Ser Pro Gln Ala Leu Lys Glu Pro Lys Pro Glu
305 310 315 320
Ala Ala Ala Thr Val Ala Ser Leu Ala Ala Ser Ser Asn Asp Asp Ala
325 330 335
Asp Asn Ala Asp Ala Ser Ala Ile Asn Ala Gly Asn Pro Glu Lys Glu
340 345 350
Leu Asp Ser Asp Val Leu Glu Gln Glu Leu Ser Ser Glu Glu Pro Glu
355 360 365
Glu Thr Ala Lys Pro Asp His Ser Leu Arg Gly Phe Phe Arg Thr Asp
370 375 380
Tyr Tyr Pro Asn Arg Trp Gln Lys Ile Leu Ser Phe Gly Gly Arg Val
385 390 395 400
Arg Met Ser Thr Ser Leu Leu Leu Gly Ala Leu Leu Leu Leu Ser Leu
405 410 415
Phe Lys Val Met Thr Val Glu Pro Ser Glu Asn Trp Gln Asn Ser Ser
420 425 430
Gly Trp Leu Ser Pro Ser Thr Ala Thr Ser Thr Ala Val Thr Thr Ser
435 440 445
Glu Thr Ser Ala Pro Val Ser Thr Ser Ser Met Thr Val Pro Thr Thr
450 455 460
Val Glu Glu Thr Pro Thr Met Glu Ser Ser Val Glu Thr Gln Gln Glu
465 470 475 480
Thr Ser Thr Pro Ala Thr Ala Thr Pro Gln Arg Ala Asp Thr Ile Glu
485 490 495
Pro Thr Glu Glu Ala Thr Ser Gln Glu Glu Thr Thr Ala Ser Gln Thr
500 505 510
Gln Ser Pro Ala Thr Ala Val Gln Glu Thr Val Ala Pro Thr Ser Thr
515 520 525
Pro
<210>58
<211>2432
<212>DNA
<213>谷氨酸棒状杆菌
<220>
<221>CDS
<222>(507)..(2096)
<400>58
aggggcggcg gatcgaccac ggcttgcaac cgtggcggga gtgggctgtt gagaagctgc 60
cacattcacg actttctggc tcctttacta aataaggatt ttcacaggac ccgtccaagc 120
caagccgatt tcaactcagc ctaaagacaa agccctcatt taaaattgtt ccgacgcgga 180
tgcgtgtgca cgcagtgcga cagatgtctg ttgcaaagtt ggctacttgg gtcataacca 240
acaagaaagc cctcgttcca acactgtggt gagtgttgtc gagggcgctt gacgagacga 300
cttggaaggc cgttacggca ggcgccgcgc ggttactact acaagtcgaa taatggtcat 360
ggtgtgtcat gctacacaca tcgagtttcc aattccacaa cgcacgaaaa ttcccacccc 420
caaaactccc ccacttcggt taaggaatca ggattctcac aaagttcagg caggctcccg 480
ctacttttca gcgctaatct tggctc atg att tta ggc gta ccc att caa tat 533
Met Ile Leu Gly Val Pro Ile Gln Tyr
1 5
ttg ctc tat tca ttg tgg aat tgg att gtc gat acc ggt ttt gat gta 581
Leu Leu Tyr Ser Leu Trp Asn Trp Ile Val Asp Thr Gly Phe Asp Val
10 15 20 25
gca att atc ctg gtc ttg gcg ttt ttg att cca cgt atc ggc cga ctg 629
Ala Ile Ile Leu Val Leu Ala Phe Leu Ile Pro Arg Ile Gly Arg Leu
30 35 40
gcc atg cgt att atc aag cgg cga gtg gag tct gca gcc gat gcg gac 677
Ala Met Arg Ile Ile Lys Arg Arg Val Glu Ser Ala Ala Asp Ala Asp
45 50 55
acc act aag aac cag ctc gcg ttc gct ggc gtt ggc gtt tat atc gcg 725
Thr Thr Lys Asn Gln Leu Ala Phe Ala Gly Val Gly Val Tyr Ile Ala
60 65 70
caa att gtg gcg ttt ttc atg ctt gcc gtc tcc gcg atg cag gct ttt 773
Gln Ile Val Ala Phe Phe Met Leu Ala Val Ser Ala Met Gln Ala Phe
75 80 85
ggt ttc tct ctc gcg ggc gct gcg att ccg gca acc att gcg tca gct 821
Gly Phe Ser Leu Ala Gly Ala Ala Ile Pro Ala Thr Ile Ala Ser Ala
90 95 100 105
gcc att ggc ctt ggt gcg cag tcg att gtt gcg gac ttc ttg gcc gga 869
Ala Ile Gly Leu Gly Ala Gln Ser Ile Val Ala Asp Phe Leu Ala Gly
110 115 120
ttt ttc atc ctg acg gaa aag caa ttc ggc gtg ggt gac tgg gtg cgt 917
Phe Phe Ile Leu Thr Glu Lys Gln Phe Gly Val Gly Asp Trp Val Arg
125 130 135
ttt gag ggc aac ggc atc gtt gtc gaa ggc acc gtc att gag atc acc 965
Phe Glu Gly Asn Gly Ile Val Val Glu Gly Thr Val Ile Glu Ile Thr
140 145 150
atg cgc gcg acc aaa att cgc acg att gca caa gag acc gtg atc atc 1013
Met Arg Ala Thr Lys Ile Arg Thr Ile Ala Gln Glu Thr Val Ile Ile
155 160 165
ccc aac tcc acg gcg aaa gtg tgc atc aac aat tct aat aac tgg tcg 1061
Pro Asn Ser Thr Ala Lys Val Cys Ile Asn Asn Ser Asn Asn Trp Ser
170 175 180 185
cgt gcg gtt gtc gtt att ccg atc ccc atg ttg ggt tct gaa aac atc 1109
Arg Ala Val Val Val Ile Pro Ile Pro Met Leu Gly Ser Glu Asn Ile
190 195 200
aca gat gtc atc gcg cgc tct gaa gct gcg act cgt cgc gca ctt ggc 1157
Thr Asp Val Ile Ala Arg Ser Glu Ala Ala Thr Arg Arg Ala Leu Gly
205 210 215
cag gag aaa atc gca ccg gaa atc ctc ggt gaa ctc gat gtg cac cca 1253
Ala Thr Glu Val Thr Pro Pro Thr Val Val Gly Met Pro Trp Met Val
235 240 245
acc atg cgt ttc ctc gtg caa gtc acc gcc ggc aat caa tgg ctg gtc 1301
Thr Met Arg Phe Leu Val Gln Val Thr Ala Gly Asn Gln Trp Leu Val
250 255 260 265
gaa cgc gcc atc cgc aca gaa atc atc aac gaa ttc tgg gaa gaa tac 1349
Glu Arg Ala Ile Arg Thr Glu Ile Ile Asn Glu Phe Trp Glu Glu Tyr
270 275 280
ggc agc gca acc act aca tcg gga acc ctc att gat tcc tta cac gtt 1397
Gly Ser Ala Thr Thr Thr Ser Gly Thr Leu Ile Asp Ser Leu His Val
285 290 295
gag cat gaa gag cca aag acc tcg ctt atc gac gcc tcc ccc cag gct 1445
Glu His Glu Glu Pro Lys Thr Ser Leu Ile Asp Ala Ser Pro Gln Ala
300 305 310
ctt aag gaa ccg aag ccg gag gct gcg gcg acg gtt gca tcg cta gct 1493
Leu Lys Glu Pro Lys Pro Glu Ala Ala Ala Thr Val Ala Ser Leu Ala
315 320 325
gca tcg tct aac gac gat gca gac aat gca gac gcc tcg gcg atc aat 1541
Ala Ser Ser Asn Asp Asp Ala Asp Asn Ala Asp Ala Ser Ala Ile Asn
330 335 340 345
gca ggc aat cca gag aag gaa ctt gat tcc gat gtg ctg gaa caa gaa 1589
Ala Gly Asn Pro Glu Lys Glu Leu Asp Ser Asp Val Leu Glu Gln Glu
350 355 360
ctc tcc agc gaa gaa ccg gaa gaa aca gca aaa cca gat cac tct ctc 1637
Leu Ser Ser Glu Glu Pro Glu Glu Thr Ala Lys Pro Asp His Ser Leu
365 370 375
cga ggc ttc ttc cgc act gat tac tac cca aat cgg tgg cag aag atc 1685
Arg Gly Phe Phe Arg Thr Asp Tyr Tyr Pro Asn Arg Trp Gln Lys Ile
380 385 390
ctg tcg ttt ggc gga cgt gtc cgc atg agc act tcc ctg ttg ttg ggt 1733
Leu Ser Phe Gly Gly Arg Val Arg Met Ser Thr Ser Leu Leu Leu Gly
395 400 405
gcg ctg ctc ttg ctg tca cta ttt aag gtc atg act gtg gaa cca agt 1781
Ala Leu Leu Leu Leu Ser Leu Phe Lys Val Met Thr Val Glu Pro Ser
410 415 420 425
gag aat tgg caa aac tcc agt gga tgg ctg tca cca agc act gcc acc 1829
Glu Asn Trp Gln Asn Ser Ser Gly Trp Leu Ser Pro Ser Thr Ala Thr
430 435 440
tca act gcg gtg acc acc tcc gaa act tcc gcg cca gta agc act tct 1877
Ser Thr Ala Val Thr Thr Ser Glu Thr Ser Ala Pro Val Ser Thr Ser
445 450 455
tcg atg aca gtg ccc act acg gtg gag gag acc cca acg atg gaa tct 1925
Ser Met Thr Val Pro Thr Thr Val Glu Glu Thr Pro Thr Met Glu Ser
460 465 470
agc gtc gaa acg cag cag gaa acc tca acc cct gca acc gca acg ccc 1973
Ser Val Glu Thr Gln Gln Glu Thr Ser Thr Pro Ala Thr Ala Thr Pro
475 480 485
cag cga gcc gac acc atc gaa ccg acc gag gaa gcc acg tcg cag gag 2021
Gln Arg Ala Asp Thr Ile Glu Pro Thr Glu Glu Ala Thr Ser Gln Glu
490 495 500 505
gaa acg act gcg tcg cag acg cag tct cca gca acc gcg gtt caa gag 2069
Glu Thr Thr Ala Ser Gln Thr Gln Ser Pro Ala Thr Ala Val Gln Glu
510 515 520
aca gtt gcg ccg acg tcc acc cct tag gacgctgatt acagacgtgt 2116
Thr Val Ala Pro Thr Ser Thr Pro
525
cccatttctt tactactatt ggaaattatg agttcagacg cagaaaaggc atccgtggag 2176
ctttccgaaa aatttcaccc agaacgcacc catattttgg gcgccgttgt ttttggcctg 2236
atctcattat tagtcatcgg cgcagcccct cagtacctgt tttggctgct cgcgctccct 2296
gtcatcttcg gttactgggt tctaaaatca tccacgatcg ttgatgaaca gggcatcacc 2356
gcaaactacg ccttcaaggg caaaaaggtt gtggcctggg aagacctcgc aggaatcgga 2416
ttcaagggtg cccgca 2432
<210>59
<211>529
<212>PRT
<213>谷氨酸棒状杆菌
<400>59
Met Ile Leu Gly Val Pro Ile Gln Tyr Leu Leu Tyr Ser Leu Trp Asn
1 5 10 15
Trp Ile Val Asp Thr Gly Phe Asp Val Ala Ile Ile Leu Val Leu Ala
20 25 30
Phe Leu Ile Pro Arg Ile Gly Arg Leu Ala Met Arg Ile Ile Lys Arg
35 40 45
Arg Val Glu Ser Ala Ala Asp Ala Asp Thr Thr Lys Asn Gln Leu Ala
50 55 60
Phe Ala Gly Val Gly Val Tyr Ile Ala Gln Ile Val Ala Phe Phe Met
65 70 75 80
Leu Ala Val Ser Ala Met Gln Ala Phe Gly Phe Ser Leu Ala Gly Ala
85 90 95
Ala Ile Pro Ala Thr Ile Ala Ser Ala Ala Ile Gly Leu Gly Ala Gln
100 105 110
Ser Ile Val Ala Asp Phe Leu Ala Gly Phe Phe Ile Leu Thr Glu Lys
115 120 125
Gln Phe Gly Val Gly Asp Trp Val Arg Phe Glu Gly Asn Gly Ile Val
130 135 140
Val Glu Gly Thr Val Ile Glu Ile Thr Met Arg Ala Thr Lys Ile Arg
145 150 155 160
Thr Ile Ala Gln Glu Thr Val Ile Ile Pro Asn Ser Thr Ala Lys Val
165 170 175
Cys Ile Asn Asn Ser Asn Asn Trp Ser Arg Ala Val Val Val Ile Pro
180 185 190
Ile Pro Met Leu Gly Ser Glu Asn Ile Thr Asp Val Ile Ala Arg Ser
195 200 205
Glu Ala Ala Thr Arg Arg Ala Leu Gly Gln Glu Lys Ile Ala Pro Glu
210 215 220
Ile Leu Gly Glu Leu Asp Val His Pro Ala Thr Glu Val Thr Pro Pro
225 230 235 240
Thr Val Val Gly Met Pro Trp Met Val Thr Met Arg Phe Leu Val Gln
245 250 255
Val Thr Ala Gly Asn Gln Trp Leu Val Glu Arg Ala Ile Arg Thr Glu
260 265 270
Ile Ile Asn Glu Phe Trp Glu Glu Tyr Gly Ser Ala Thr Thr Thr Ser
275 280 285
Gly Thr Leu Ile Asp Ser Leu His Val Glu His Glu Glu Pro Lys Thr
290 295 300
Ser Leu Ile Asp Ala Ser Pro Gln Ala Leu Lys Glu Pro Lys Pro Glu
305 310 315 320
Ala Ala Ala Thr Val Ala Ser Leu Ala Ala Ser Ser Asn Asp Asp Ala
325 330 335
Asp Asn Ala Asp Ala Ser Ala Ile Asn Ala Gly Asn Pro Glu Lys Glu
340 345 350
Leu Asp Ser Asp Val Leu Glu Gln Glu Leu Ser Ser Glu Glu Pro Glu
355 360 365
Glu Thr Ala Lys Pro Asp His Ser Leu Arg Gly Phe Phe Arg Thr Asp
370 375 380
Tyr Tyr Pro Asn Arg Trp Gln Lys Ile Leu Ser Phe Gly Gly Arg Val
385 390 395 400
Arg Met Ser Thr Ser Leu Leu Leu Gly Ala Leu Leu Leu Leu Ser Leu
405 410 415
Phe Lys Val Met Thr Val Glu Pro Ser Glu Asn Trp Gln Asn Ser Ser
420 425 430
Gly Trp Leu Ser Pro Ser Thr Ala Thr Ser Thr Ala Val Thr Thr Ser
435 440 445
Glu Thr Ser Ala Pro Val Ser Thr Ser Ser Met Thr Val Pro Thr Thr
450 455 460
Val Glu Glu Thr Pro Thr Met Glu Ser Ser Val Glu Thr Gln Gln Glu
465 470 475 480
Thr Ser Thr Pro Ala Thr Ala Thr Pro Gln Arg Ala Asp Thr Ile Glu
485 490 495
Pro Thr Glu Glu Ala Thr Ser Gln Glu Glu Thr Thr Ala Ser Gln Thr
500 505 510
Gln Ser Pro Ala Thr Ala Val Gln Glu Thr Val Ala Pro Thr Ser Thr
515 520 525
Pro
<210>60
<211>22
<212>DNA
<213>人工的
<220>
<223>引物
<400>60
cagttgtggc tgatcgccaa gg 22
<210>61
<211>52
<212>DNA
<213>人工的
<220>
<223>引物
<400>61
tcaattatag cagtgtcgca cagatatggc cacaaagaat taaaattgtt tg 52
<210>62
<211>23
<212>DNA
<213>人工的
<220>
<223>引物
<400>62
ccatgcgacg gtagtggcca aac 23
<210>63
<211>52
<212>DNA
<213>人工的
<220>
<223>引物
<400>63
ttgtggccat atctgtgcga cactgctata attgaacgtg agcatttacc ag 52
<210>64
<211>31
<212>DNA
<213>人工的
<220>
<223>引物
<400>64
cgccccgggt gaccgcgtct gcgatcaaaa 31
<210>65
<211>28
<212>DNA
<213>人工的
<220>
<223>引物
<400>65
cgatcccggg gccaccaact ccgatgtc 28
<210>66
<211>440
<212>DNA
<213>人工的
<220>
<223>gdh的突变启动子
<400>66
cgaaaaggtc ggaaagtgcc cgaatgtgcg ttgttctagc tagcctcggg agctccagga 60
gattgtgaaa aacggctcaa atttctccga tgtagcgcct ataaaagtcg caccaattcc 120
atttgagggc gctcaagtgt ggccaggtta tataaccagt cagtcaactg gtctcattcg 180
ctggtcggat gaatttaatt aaagaagaga cttcatgcag ttaccgcgcg ttttggcgat 240
acaaaattga taaacctaaa gaaattttca aacaatttta attctttgtg gccatatctg 300
tgcgacactg ctataattga acgtgagcat ttaccagcct aaatgtccgc agtgagttaa 360
gtctcaaagc aagaagttgc tctttagggc atccgtagtt taaaactatt aaccgttagg 420
tatgacaagc cggttgatgt 440
<210>67
<211>20
<212>DNA
<213>人工的
<220>
<223>引物
<400>67
tcctcccaca cggctcagtc 20
<210>68
<211>42
<212>DNA
<213>人工的
<220>
<223>引物
<400>68
tacttgtagt tccttgtctg aaattcttcc taacctttac gc 42
<210>69
<211>20
<212>DNA
<213>人工的
<220>
<223>引物
<400>69
ggcagcgaca catgcaccac 20
<2l0>70
<211>42
<212>DNA
<2l3>人工的
<220>
<223>引物
<400>70
gcgtaaaggt taggaagaat ttcagacaag gaactacaag ta 42
<210>71
<211>25
<212>DNA
<2l3>人工的
<220>
<223>引物
<400>71
ctggatccta cgtattatcc tgctg 25
<210>72
<211>24
<212>DNA
<213>人工的
<220>
<223>引物
<400>72
gcggatccag gctgcgtggt caag 24
<210>73
<211>4200
<212>DNA
<213>谷氨酸棒状杆菌
<220>
<221>CDS
<222>(449)..(1219)
<220>
<221>CDS
<222>(1239)..(3257)
<220>
<221>CDS
<222>(3260)..(4006)
<400>73
aggtgttgca aacttgttga ttttcgcttt tcgacgcagc ccgccgccac caggtgcccg 60
gcgtggtcgg gccacatccg ccccgggaac tttttaggca cctacggtgc aactgttggg 120
ataattgtgt cacctgcgca aagttgctcc ctggatcgga aggttgggct gtctaaactt 180
tttggttgat accaaacggg gttagaaact gttcggatcg gtatcctgtg aggaagctca 240
ccttggtttt agaatgttga aaaggcctca cgtttccgca ggtagagcac actcaattaa 300
atgagcgtca aacgacaata aagtaaggct atcctaataa gtggggtttt atgtctctaa 360
acagccagtt gggggtcatg ggggagcgcc ccgtgactgg ttaatgcccc gatctgggac 420
gtacagtaac aacgacactg gaggtgcc atg act gtt aga aat ccc gac cgt 472
Met Thr Val Arg Asn Pro Asp Arg
1 5
gag gca atc cgt cac gga aaa att acg acg gag gcg ctg cgt gag cgt 520
Glu Ala Ile Arg His Gly Lys Ile Thr Thr Glu Ala Leu Arg Glu Arg
10 15 20
ccc gca tac ccg acc tgg gca atg aag ctg acc atg gcc atc act ggc 568
Pro Ala Tyr Pro Thr Trp Ala Met Lys Leu Thr Met Ala Ile Thr Gly
25 30 35 40
cta atg ttt ggt ggc ttc gtt ctt gtt cac atg atc gga aac ctg aaa 616
Leu Met Phe Gly Gly Phe Val Leu Val His Met Ile Gly Asn Leu Lys
45 50 55
atc ttc atg ccg gac tac gca gcc gat tct gcg cat ccg ggt gaa gca 664
Ile Phe Met Pro Asp Tyr Ala Ala Asp Ser Ala His Pro Gly Glu Ala
60 65 70
caa gta gat gtc tac ggc gag ttc ctg cgt gag atc gga tcc ccg atc 712
Gln Val Asp Val Tyr Gly Glu Phe Leu Arg Glu Ile Gly Ser Pro Ile
75 80 85
ctc cca cac ggc tca gtc ctc tgg atc cta cgt att atc ctg ctg gtc 760
Lcu Pro His Gly Ser Val Leu Trp Ile Leu Arg Ile Ile Leu Leu Val
90 95 100
gca ttg gtt ctg cac atc tac tgt gca ttc gca ttg acc ggc cgt tct 808
Ala Leu Val Leu His Ile Tyr Cys Ala Phe Ala Leu Thr Gly Arg Ser
105 110 115 120
cac cag tcc cgc gga aag ttc cgc cgt acc aac ctc gtt ggc ggc ttc 856
His Gln Ser Arg Gly Lys Phe Arg Arg Thr Asn Leu Val Gly Gly Phe
125 130 135
aac tcc ttc gcg acc cgc tcc atg ctg gtg acc gga atc gtt ctc ctt 904
Asn Ser Phe Ala Thr Arg Ser Met Leu Val Thr Gly Ile Val Leu Leu
140 145 150
gcg ttc att atc ttc cac atc ctc gac ctg acc atg ggt gtt gct cca 952
Ala Phe Ile Ile Phe His Ile Leu Asp Leu Thr Met Gly Val Ala Pro
155 160 165
gca gcc cca acc tca ttc gag cac ggc gaa gta tac gca aac atg gtg 1000
Ala Ala Pro Thr Ser Phe Glu His Gly Glu Val Tyr Ala Asn Met Val
170 175 180
gct tcc ttt agc cgc tgg cct gta gca att tgg tac atc att gcc aac 1048
Ala Ser Phe Ser Arg Trp Pro Val Ala Ile Trp Tyr Ile Ile Ala Asn
185 190 195 200
ctg gtc ctg ttc gtc cac ctg tca cac ggc atc tgg ctt gca gtc tct 1096
Leu Val Leu Phe Val His Leu Ser His Gly Ile Trp Leu Ala Val Ser
205 210 215
gac ctg gga atc acc gga cgc cgc tgg agg gca atc ctc ctc gca gtt 1144
Asp Leu Gly Ile Thr Gly Arg Arg Trp Arg Ala Ile Leu Leu Ala Val
220 225 230
gcg tac atc gtt cct gca ctg gtc ctg atc ggc aac atc acc att ccg 1192
Ala Tyr Ile Val Pro Ala Leu Val Leu Ile Gly Asn Ile Thr Ile Pro
235 240 245
ttc gcc atc gct gtt ggc tgg att gcg taaaggttag gaagaattt atg agc 1244
Phe Ala Ile Ala Val Gly Trp Ile Ala Met Ser
250 255
act cac tct gaa acc acc cgc cca gag ttc atc cac cca gtc tca gtc 1292
Thr His Ser Glu Thr Thr Arg Pro Glu Phe Ile His Pro Val Ser Val
260 265 270 275
ctc cca gag gtc tca gct ggt acg gtc ctt gac gct gca gag cca gca 1340
Leu Pro Glu Val Ser Ala Gly Thr Val Leu Asp Ala Ala Glu Pro Ala
280 285 290
ggc gtt ccc acc aaa gat atg tgg gaa tac caa aaa gac cac atg aac 1388
Gly Val Pro Thr Lys Asp Met Trp Glu Tyr Gln Lys Asp His Met Asn
295 300 305
ctg gtc tcc cca ctg aac cga cgc aag ttc cgt gtc ctc gtc gtt ggc 1436
Leu Val Ser Pro Leu Ash Arg Arg Lys Phe Arg Val Leu Val Val Gly
310 315 320
acc ggc ctg tcc ggt ggt gct gca gca gca gcc ctc ggc gaa ctc gga 1484
Thr Gly Leu Ser Gly Gly Ala Ala Ala Ala Ala Leu Gly Glu Leu Gly
325 330 335
tac gac gtc aag gcg ttc acc tac cac gac gca cct cgc cgt gcg cac 1532
Tyr Asp Val Lys Ala Phe Thr Tyr His Asp Ala Pro Arg Arg Ala His
340 345 350 355
tcc att gct gca cag ggt ggc gtt aac tcc gcc cgc ggc aag aag gta 1580
Ser Ile Ala Ala Gln Gly Gly Val Asn Ser Ala Arg Gly Lys Lys Val
360 365 370
gac aac gac ggc gca tac cgc cac gtc aag gac acc gtc aag ggc ggc 1628
Asp Asn Asp Gly Ala Tyr Arg His Val Lys Asp Thr Val Lys Gly Gly
375 380 385
gac tac cgt ggt cgc gag tcc gac tgc tgg cgt ctc gcc gtc gag tcc 1676
Asp Tyr Arg Gly Arg Glu Ser Asp Cys Trp Arg Leu Ala Val Glu Ser
390 395 400
gtc cgc gtc atc gac cac atg aac gcc atc ggt gca cca ttc gcc cgc 1724
Val Arg Val Ile Asp His Met Asn Ala Ile Gly Ala Pro Phe Ala Arg
405 410 415
gaa tac ggt ggc gcc ttg gca acc cgt tcc ttc ggt ggt gtg cag gtc 1772
Glu Tyr Gly Gly Ala Leu Ala Thr Arg Ser Phe Gly Gly Val Gln Val
420 425 430 435
tcc cgt acc tac tac acc cgt gga caa acc gga cag cag ctg cag ctc 1820
Ser Arg Thr Tyr Tyr Thr Arg Gly Gln Thr Gly Gln Gln Leu Gln Leu
440 445 450
tcc acc gca tcc gca cta cag cgc cag atc cac ctc ggc tcc gta gaa 1868
Set Thr Ala Ser Ala Leu Gln Arg Gln Ile His Leu Gly Ser Val Glu
455 460 465
atc ttc acc cat aac gaa atg gtt gac gtc att gtc acc gaa cgt aac 1916
Ile Phe Thr His Asn Glu Met Val Asp Val Ile Val Thr Glu Arg Asn
470 475 480
ggt gaa aag cgc tgc gaa ggc ctg atc atg cgc aac ctg atc acc ggc 1964
Gly Glu Lys Arg Cys Glu Gly Leu Ile Met Arg Asn Leu Ile Thr Gly
485 490 495
gag ctc acc gca cac acc ggc cat gcc gtt atc ctg gca acc ggt ggc 2012
Glu Leu Thr Ala His Thr Gly His Ala Val Ile Leu Ala Thr Gly Gly
500 505 510 515
tac ggc aac gtg tac cac atg tcc acc ctg gcc aag aac tcc aac gcc 2060
Tyr Gly Asn Val Tyr His Met Ser Thr Leu Ala Lys Asn Ser Asn Ala
520 525 530
tcg gcc atc atg cgt gca tac gaa gcc ggc gca tac ttc gcg tcc cca 2108
Ser Ala Ile Met Arg Ala Tyr Glu Ala Gly Ala Tyr Phe Ala Ser Pro
535 540 545
tcg ttc atc cag ttc cac cca acc ggc ctg cct gtg aac tcc acc tgg 2156
Ser Phe Ile Gln Phe His Pro Thr Gly Leu Pro Val Asn Ser Thr Trp
550 555 560
cag tcc aag acc att ctg atg tcc gag tcg ctg cgt aac gac ggc cgc 2204
Gln Ser Lys Thr Ile Leu Met Ser Glu Ser Leu Arg Asn Asp Gly Arg
565 570 575
atc tgg tcc cct aag gaa ccg aac gat aac cgc gat cca aac acc atc 2252
Ile Trp Set Pro Lys Glu Pro Asn Asp Asn Arg Asp Pro Asn Thr Ile
580 585 590 595
cct gag gat gag cgc gac tac ttc ctg gag cgc cgc tac cca gca ttc 2300
Pro Glu Asp Glu Arg Asp Tyr Phe Leu Glu Arg Arg Tyr Pro Ala Phe
600 605 610
ggt aac ctc gtc cca cgt gac gtt gct tcc cgt gcg atc tcc cag cag 2348
Gly Asn Leu Val Pro Arg Asp Val Ala Ser Arg Ala Ile Ser Gln Gln
615 620 625
atc aat gct ggt ctc ggt gtt gga cct ctg aac aac gct gca tac ctg 2396
Ile Asn Ala Gly Leu Gly Val Gly Pro Leu Asn Asn Ala Ala Tyr Leu
630 635 640
gac ttc cgc gac gcc acc gag cgc ctc gga cag gac acc atc cgc gag 2444
Asp Phe Arg Asp Ala Thr Glu Arg Leu Gly Gln Asp Thr Ile Arg Glu
645 650 655
cgt tac tcc aac ctc ttc acc atg tac gaa gag gca att ggc gag gac 2492
Arg Tyr Ser Asn Leu Phe Thr Met Tyr Glu Glu Ala Ile Gly Glu Asp
660 665 670 675
cca tac tcc agc cca atg cgt att gca ccg acc tgc cac ttc acc atg 2540
Pro Tyr Ser Ser Pro Met Arg Ile Ala Pro Thr Cys His Phe Thr Met
680 685 690
ggt ggc ctc tgg act gac ttc aac gaa atg acg tca ctc cca ggt ctg 2588
Gly Gly Leu Trp Thr Asp Phe Asn Glu Met Thr Ser Leu Pro Gly Leu
695 700 705
ttc tgc gca ggc gaa gca tcc tgg acc tac cac ggt gca aac cgt ctg 2636
Phe Cys Ala Gly Glu Ala Ser Trp Thr Tyr His Gly Ala Asn Arg Leu
710 715 720
ggc gca aac tcc ctg ctc tcc gct tcc gtc gat ggc tgg ttc acc ctg 2684
Gly Ala Asn Ser Leu Leu Ser Ala Ser Val Asp Gly Trp Phe Thr Leu
725 730 735
cca ttc acc atc cct aac tac ctc ggc cca ttg ctt ggc tcc gag cgt 2732
Pro Phe Thr Ile Pro Asn Tyr Leu Gly Pro Leu Leu Gly Ser Glu Arg
740 745 750 755
ctg tca gag gat gca cca gaa gca cag gca gcg att gcg cgt gca cag 2780
Leu Ser Glu Asp Ala Pro Glu Ala Gln Ala Ala Ile Ala Arg Ala Gln
760 765 770
gct cgc att gac cgc ctc atg ggc aac cgc cca gag tgg gtc ggt gac 2828
Ala Arg Ile Asp Arg Leu Met Gly Asn Arg Pro Glu Trp Val Gly Asp
775 780 785
aac gtt cac gga cct gag tac tac cac cgc cag ctt ggc gat atc ctg 2876
Asn Val His Gly Pro Glu Tyr Tyr His Arg Gln Leu Gly Asp Ile Leu
790 795 800
tac ttc tcc tgt ggc gtt tcc cga aac gta gaa gac ctc cag gat ggc 2924
Tyr Phe Ser Cys Gly Val Ser Arg Asn Val Glu Asp Leu Gln Asp Gly
805 810 815
atc aac aag atc cgt gcc ctc cgc gat gac ttc tgg aag aac atg cgc 2972
Ile Asn Lys Ile Arg Ala Leu Arg Asp Asp Phe Trp Lys Asn Met Arg
820 825 830 835
atc acc ggc agc acc gat gag atg aac cag gtt ctc gaa tac gca gca 3020
Ile Thr Gly Ser Thr Asp Glu Met Asn Gln Val Leu Glu Tyr Ala Ala
840 845 850
cgc gta gcc gac tac atc gac ctc ggc gaa ctc atg tgt gtc gac gcc 3068
Arg Val Ala Asp Tyr Ile Asp Leu Gly Glu Leu Met Cys Val Asp Ala
855 860 865
ctc gac cgc gac gag tcc tgt ggc gct cac ttc cgc gac gac cac ctc 3116
Leu Asp Arg Asp Glu Ser Cys Gly Ala His Phe Arg Asp Asp His Leu
870 875 880
tcc gaa gat ggc gaa gca gaa cgt gac gac gaa aac tgg tgc ttc gtc 3164
Ser Glu Asp Gly Glu Ala Glu Arg Asp Asp Glu Asn Trp Cys Phe Val
885 890 895
tcc gca tgg gaa cca ggc gag aac gga acc ttc gtc cgc cac gca gaa 3212
Ser Ala Trp Glu Pro Gly Glu Asn Gly Thr Phe Val Arg His Ala Glu
900 905 910 915
cca ctg ttc ttc gaa tcc gtc cca ctg cag aca agg aac tac aag ta 3259
Pro Leu Phe Phe Glu Ser Val Pro Leu Gln Thr Arg Asn Tyr Lys
920 925 930
atg aaa ctt aca ctt gag atc tgg cgt caa gca ggc cca act gcg gaa 3307
Met Lys Leu Thr Leu Glu Ile Trp Arg Gln Ala Gly Pro Thr Ala Glu
935 940 945
ggc aag ttc gaa acc gtc cag gtt gac gac gcc gtc gcg cag atg tcc 3355
Gly Lys Phe Glu Thr Val Gln Val Asp Asp Ala Val Ala Gln Met Ser
950 955 960
atc ctg gag ctg ctt gac cac gta aac aac aag ttc atc gaa gaa ggc 3403
Ile Leu Glu Leu Leu Asp His Val Asn Asn Lys Phe Ile Glu Glu Gly
965 970 975
aaa gaa cca ttc gcg ttc gcc tct gac tgc cgc gaa ggc att tgt ggt 3451
Lys Glu Pro Phe Ala Phe Ala Ser Asp Cys Arg Glu Gly Ile Cys Gly
980 985 990
acc tgt ggt ctc ctc gtg aac ggt cgc cct cac ggc gcc gac cag 3496
Thr Cys Gly Leu Leu Val Asn Gly Arg Pro His Gly Ala Asp Gln
995 1000 1005
aac aag cct gcc tgt gcg cag cgc ctg gtc agc tac aag gaa ggc 3541
Asn Lys Pro Ala Cys Ala Gln Arg Leu Val Ser Tyr Lys Glu Gly
1010 1015 1020
gac acc ctc aag atc gaa cca ctg cgt tcc gcc gca tac cca gtg 3586
Asp Thr Leu Lys Ile Glu Pro Leu Arg Ser Ala Ala Tyr Pro Val
1025 1030 1035
atc aag gac atg gtc gtc gac cgc tcc gca ctg gac cgt gtc atg 3631
Ile Lys Asp Met Val Val Asp Arg Ser Ala Leu Asp Arg Val Met
1040 1045 1050
gaa cag ggt ggc tac gtg acc atc aac gca ggt acc gca cct gac 3676
Glu Gln Gly Gly Tyr Val Thr Ile Asn Ala Gly Thr Ala Pro Asp
1055 1060 1065
gct gat acc ctc cac gtc aac cac gaa acc gca gaa ctc gca ctt 3721
Ala Asp Thr Leu His Val Asn His Glu Thr Ala Glu Leu Ala Leu
1070 1075 1080
gac cac gca gcc tgc atc ggc tgt ggc gca tgt gtt gct gcc tgc 3766
Asp His Ala Ala Cys Ile Gly Cys Gly Ala Cys Val Ala Ala Cys
1085 1090 1095
cct aac ggc gca gca cac ctg ttc acc ggc gca aag ctt gtt cac 3811
Pro Asn Gly Ala Ala His Leu Phe Thr Gly Ala Lys Leu Val His
1100 1105 1110
ctc tcc ctc ctc cca ctg ggt aag gaa gag cgc gga ctg cgt gca 3856
Leu Ser Leu Leu Pro Leu Gly Lys Glu Glu Arg Gly Leu Arg Ala
1115 1120 1125
cgt aag atg gtt gat gaa atg gaa acc aac ttc gga cac tgc tcc 3901
Arg Lys Met Val Asp Glu Met Glu Thr Asn Phe Gly His Cys Ser
1130 1135 1140
ctc tac ggc gag tgc gca gat gtc tgc ccc gca ggc atc cca ctg 3946
Leu Tyr Gly Glu Cys Ala Asp Val Cys Pro Ala Gly Ile Pro Leu
1145 1150 1155
acc gct gtg gca gct gtc acc aag gaa cgt gcg cgt gca gct ttc 3991
Thr Ala Val Ala Ala Val Thr Lys Glu Arg Ala Arg Ala Ala Phe
1160 1165 1170
cga ggc aaa gac gac tagtctttaa tccaagtaag taccggttca gacagttaaa 4046
Arg Gly Lys Asp Asp
1175
ccagaaagac gagtgaacac catgtcctcc gcgaaaaaga aacccgcacc ggagcgtatg 4106
cactacatca agggctatgt acctgtggcg tatagctctc cacactcatc cctcgagcgc 4166
agcgcaacct ggttgggcat gggattcctc ctca 4200
<210>74
<211>257
<212>PRT
<213>谷氨酸棒状杆菌
<400>74
Met Thr Val Arg Asn Pro Asp Arg Glu Ala Ile Arg His Gly Lys Ile
1 5 10 15
Thr Thr Glu Ala Leu Arg Glu Arg Pro Ala Tyr Pro Thr Trp Ala Met
20 25 30
Lys Leu Thr Met Ala Ile Thr Gly Leu Met Phe Gly Gly Phe Val Leu
35 40 45
Val His Met Ile Gly Asn Leu Lys Ile Phe Met Pro Asp Tyr Ala Ala
50 55 60
Asp Ser Ala His Pro Gly Glu Ala Gln Val Asp Val Tyr Gly Glu Phe
65 70 75 80
Leu Arg Glu Ile Gly Ser Pro Ile Leu Pro His Gly Ser Val Leu Trp
85 90 95
Ile Leu Arg Ile Ile Leu Leu Val Ala Leu Val Leu His Ile Tyr Cys
100 105 110
Ala Phe Ala Leu Thr Gly Arg Ser His Gln Ser Arg Gly Lys Phe Arg
115 120 125
Arg Thr Asn Leu Val Gly Gly Phe Asn Ser Phe Ala Thr Arg Ser Met
130 135 140
Leu Val Thr Gly Ile Val Leu Leu Ala Phe Ile Ile Phe His Ile Leu
145 150 155 160
Asp Leu Thr Met Gly Val Ala Pro Ala Ala Pro Thr Ser Phe Glu His
165 170 175
Gly Glu Val Tyr Ala Asn Met Val Ala Ser Phe Ser Arg Trp Pro Val
180 185 190
Ala Ile Trp Tyr Ile Ile Ala Asn Leu Val Leu Phe Val His Leu Ser
195 200 205
His Gly Ile Trp Leu Ala Val Ser Asp Leu Gly Ile Thr Gly Arg Arg
210 215 220
Trp Arg Ala Ile Leu Leu Ala Val Ala Tyr Ile Val Pro Ala Leu Val
225 230 235 240
Leu Ile Gly Asn Ile Thr Ile Pro Phe Ala Ile Ala Val Gly Trp Ile
245 250 255
Ala
<210>75
<211>673
<212>PRT
<213>谷氨酸棒状杆菌
<400>75
Met Ser Thr His Ser Glu Thr Thr Arg Pro Glu Phe Ile His Pro Val
1 5 10 15
Ser Val Leu Pro Glu Val Ser Ala Gly Thr Val Leu Asp Ala Ala Glu
20 25 30
Pro Ala Gly Val Pro Thr Lys Asp Met Trp Glu Tyr Gln Lys Asp His
35 40 45
Met Asn Leu Val Ser Pro Leu Asn Arg Arg Lys Phe Arg Val Leu Val
50 55 60
Val Gly Thr Gly Leu Ser Gly Gly Ala Ala Ala Ala Ala Leu Gly Glu
65 70 75 80
Leu Gly Tyr Asp Val Lys Ala Phe Thr Tyr His Asp Ala Pro Arg Arg
85 90 95
Ala His Ser Ile Ala Ala Gln Gly Gly Val Asn Ser Ala Arg Gly Lys
100 105 110
Lys Val Asp Asn Asp Gly Ala Tyr Arg His Val Lys Asp Thr Val Lys
115 120 125
Gly Gly Asp Tyr Arg Gly Arg Glu Ser Asp Cys Trp Arg Leu Ala Val
130 135 140
Glu Ser Val Arg Val Ile Asp His Met Asn Ala Ile Gly Ala Pro Phe
145 150 155 160
Ala Arg Glu Tyr Gly Gly Ala Leu Ala Thr Arg Ser Phe Gly Gly Val
165 170 175
Gln Val Ser Arg Thr Tyr Tyr Thr Arg Gly Gln Thr Gly Gln Gln Leu
180 185 190
Gln Leu Ser Thr Ala Ser Ala Leu Gln Arg Gln Ile His Leu Gly Ser
195 200 205
Val Glu Ile Phe Thr His Asn Glu Met Val Asp Val Ile Val Thr Glu
210 215 220
Arg Asn Gly Glu Lys Arg Cys Glu Gly Leu Ile Met Arg Asn Leu Ile
225 230 235 240
Thr Gly Glu Leu Thr Ala His Thr Gly His Ala Val Ile Leu Ala Thr
245 250 255
Gly Gly Tyr Gly Asn Val Tyr His Met Ser Thr Leu Ala Lys Asn Ser
260 265 270
Asn Ala Ser Ala Ile Met Arg Ala Tyr Glu Ala Gly Ala Tyr Phe Ala
275 280 285
Ser Pro Ser Phe Ile Gln Phe His Pro Thr Gly Leu Pro Val Asn Ser
290 295 300
Thr Trp Gln Ser Lys Thr Ile Leu Met Ser Glu Ser Leu Arg Asn Asp
305 310 315 320
Gly Arg Ile Trp Ser Pro Lys Glu Pro Asn Asp Asn Arg Asp Pro Asn
325 330 335
Thr Ile Pro Glu Asp Glu Arg Asp Tyr Phe Leu Glu Arg Arg Tyr Pro
340 345 350
Ala Phe Gly Asn Leu Val Pro Arg Asp Val Ala Ser Arg Ala Ile Ser
355 360 365
Gln Gln Ile Asn Ala Gly Leu Gly Val Gly Pro Leu Asn Asn Ala Ala
370 375 380
Tyr Leu Asp Phe Arg Asp Ala Thr Glu Arg Leu Gly Gln Asp Thr Ile
385 390 395 400
Arg Glu Arg Tyr Ser Asn Leu Phe Thr Met Tyr Glu Glu Ala Ile Gly
405 410 415
Glu Asp Pro Tyr Ser Ser Pro Met Arg Ile Ala Pro Thr Cys His Phe
420 425 430
Thr Met Gly Gly Leu Trp Thr Asp Phe Asn Glu Met Thr Ser Leu Pro
435 440 445
Gly Leu Phe Cys Ala Gly Glu Ala Ser Trp Thr Tyr His Gly Ala Asn
450 455 460
Arg Leu Gly Ala Asn Ser Leu Leu Ser Ala Ser Val Asp Gly Trp Phe
465 470 475 480
Thr Leu Pro Phe Thr Ile Pro Asn Tyr Leu Gly Pro Leu Leu Gly Ser
485 490 495
Glu Arg Leu Ser Glu Asp Ala Pro Glu Ala Gln Ala Ala Ile Ala Arg
500 505 510
Ala Gln Ala Arg Ile Asp Arg Leu Met Gly Asn Arg Pro Glu Trp Val
515 520 525
Gly Asp Asn Val His Gly Pro Glu Tyr Tyr His Arg Gln Leu Gly Asp
530 535 540
Ile Leu Tyr Phe Ser Cys Gly Val Ser Arg Asn Val Glu Asp Leu Gln
545 550 555 560
Asp Gly Ile Asn Lys Ile Arg Ala Leu Arg Asp Asp Phe Trp Lys Asn
565 570 575
Met Arg Ile Thr Gly Ser Thr Asp Glu Met Asn Gln Val Leu Glu Tyr
580 585 590
Ala Ala Arg Val Ala Asp Tyr Ile Asp Leu Gly Glu Leu Met Cys Val
595 600 605
Asp Ala Leu Asp Arg Asp Glu Ser Cys Gly Ala His Phe Arg Asp Asp
610 615 620
His Leu Ser Glu Asp Gly Glu Ala Glu Arg Asp Asp Glu Asn Trp Cys
625 630 635 640
Phe Val Ser Ala Trp Glu Pro Gly Glu Asn Gly Thr Phe Val Arg His
645 650 655
Ala Glu Pro Leu Phe Phe Glu Ser Val Pro Leu Gln Thr Arg Asn Tyr
660 665 670
Lys
<210>76
<211>249
<212>PRT
<213>谷氨酸棒状杆菌
<400>76
Met Lys Leu Thr Leu Glu Ile Trp Arg Gln Ala Gly Pro Thr Ala Glu
1 5 10 15
Gly Lys Phe Glu Thr Val Gln Val Asp Asp Ala Val Ala Gln Met Ser
20 25 30
Ile Leu Glu Leu Leu Asp His Val Asn Asn Lys Phe Ile Glu Glu Gly
35 40 45
Lys Glu Pro Phe Ala Phe Ala Ser Asp Cys Arg Glu Gly Ile Cys Gly
50 55 60
Thr Cys Gly Leu Leu Val Asn Gly Arg Pro His Gly Ala Asp Gln Asn
65 70 75 80
Lys Pro Ala Cys Ala Gln Arg Leu Val Ser Tyr Lys Glu Gly Asp Thr
85 90 95
Leu Lys Ile Glu Pro Leu Arg Ser Ala Ala Tyr Pro Val Ile Lys Asp
100 105 110
Met Val Val Asp Arg Ser Ala Leu Asp Arg Val Met Glu Gln Gly Gly
115 120 125
Tyr Val Thr Ile Asn Ala Gly Thr Ala Pro Asp Ala Asp Thr Leu His
130 135 140
Val Asn His Glu Thr Ala Glu Leu Ala Leu Asp His Ala Ala Cys Ile
145 150 155 160
Gly Cys Gly Ala Cys Val Ala Ala Cys Pro Asn Gly Ala Ala His Leu
165 170 175
Phe Thr Gly Ala Lys Leu Val His Leu Ser Leu Leu Pro Leu Gly Lys
180 185 190
Glu Glu Arg Gly Leu Arg Ala Arg Lys Met Val Asp Glu Met Glu Thr
195 200 205
Asn Phe Gly His Cys Ser Leu Tyr Gly Glu Cys Ala Asp Val Cys Pro
210 215 220
Ala Gly Ile Pro Leu Thr Ala Val Ala Ala Val Thr Lys Glu Arg Ala
225 230 235 240
Arg Ala Ala Phe Arg Gly Lys Asp Asp
245
<210>77
<211>4200
<212>DNA
<213>乳发酵短杆菌(Rrevibacterium lactofermentum)
<220>
<221>CDS
<222>(449)..(1219)
<220>
<221>CDS
<222>(1239)..(3257)
<220>
<221>CDS
<222>(3260)..(4006)
<400>77
aggtgttgca aagttgttga ttttcgcttt tcgacgcagc ccgccgccat cgggtgcccg 60
gcgtggtcag gccacatgcg ccccgggaac tttttgggca cctacggtgc aacagttgcg 120
aaaattgtgt cacctgcgca aagccttgct tctattcggg aaattcgggt gtctaaactt 180
tttggttgat accaaacggg gttagaaact gttcagatcg gtatcctgtg aggaagctca 240
ccttggtttt agaatgttga aaaagcctca cgtttccgca ggtagagcac actcaattaa 300
atgagcgtca aacgacaata aagtaaggct accctaataa ctggggtttt atgcctctaa 360
acagtcagtt gggggcggta ggggagcgtc ccatgactgg ttaatgcctc gatctgggac 420
gtacagtaac agcgacactg gaggtgcc atg act gtt aga aat ccc gac cgt 472
Met Thr Val Arg Asn Pro Asp Arg
1 5
gag gca atc cgt cac gga aaa att acg acg gag gcg ctg cgt gag cgt 520
Glu Ala Ile Arg His Gly Lys Ile Thr Thr Glu Ala Leu Arg Glu Arg
10 15 20
ccc gca tac ccg acc tgg gca atg aag ctg acc atg gcc atc act ggc 568
Pro Ala Tyr Pro Thr Trp Ala Met Lys Leu Thr Met Ala Ile Thr Gly
25 30 35 40
cta atc ttc ggt ggc ttc gtt ctt gtt cac atg atc gga aac ctg aaa 616
Leu Ile Phe Gly Gly Phe Val Leu Val His Met Ile Gly Asn Leu Lys
45 50 55
atc ttc atg ccg gac tac gca gcc gat tct gcg cat ccg ggt gaa gca 664
Ile Phe Met Pro Asp Tyr Ala Ala Asp Ser Ala His Pro Gly Glu Ala
60 65 70
caa gta gat gtc tac ggc gag ttc ctg cgt gag atc gga tcc ccg atc 712
Gln Val Asp Val Tyr Gly Glu Phe Leu Arg Glu Ile Gly Ser Pro Ile
75 80 85
ctc cca cac ggc tca gtc ctc tgg atc cta cgt att atc ctg ctg gtc 760
Leu Pro His Gly Ser Val Leu Trp Ile Leu Arg Ile Ile Leu Leu Val
90 95 100
gca ttg gtt ctg cac atc tac tgt gca ttc gca ttg acc ggc cgt tct 808
Ala Leu Val Leu His Ile Tyr Cys Ala Phe Ala Leu Thr Gly Arg Ser
105 110 115 120
cac cag tcc cgc gga aag ttc cgc cgt acc aac ctc gtt ggc ggc ttc 856
His Gln Ser Arg Gly Lys Phe Arg Arg Thr Asn Leu Val Gly Gly Phe
125 130 135
aac tcc ttc gcg acc cgc tcc atg ctg gtg acc gga atc gtt ctc ctt 904
Asn Ser Phe Ala Thr Arg Ser Met Leu Val Thr Gly Ile Val Leu Leu
140 145 150
gcg ttc att atc ttc cac atc ctc gac ctg acc atg ggt gtt gct cca 952
Ala Phe Ile Ile Phe His Ile Leu Asp Leu Thr Met Gly Val Ala Pro
155 160 165
gca gcc cca act tca ttc gag cac ggc gaa gta tac gca aat atg gtg 1000
Ala Ala Pro Thr Ser Phe Glu His Gly Glu Val Tyr Ala Asn Met Val
170 175 180
gct tcc ttt agc cgc tgg cct gta gca att tgg tac atc att gcc aac 1048
Ala Ser Phe Ser Arg Trp Pro Val Ala Ile Trp Tyr Ile Ile Ala Asn
185 190 195 200
ctg gtc ctg ttc gtc cac ctg tct cac ggc atc tgg ctt gca gtc tct 1096
Leu Val Leu Phe Val His Leu Ser His Gly Ile Trp Leu Ala Val Ser
205 210 215
gac ctg gga atc acc gga cgt cgc tgg agg gca atc ctc ctc gca gtt 1144
Asp Leu Gly Ile Thr Gly Arg Arg Trp Arg Ala Ile Leu Leu Ala Val
220 225 230
gcg tac atc gtt cct gca ctg gtc ctg atc ggc aac atc acc att ccg 1192
Ala Tyr Ile Val Pro Ala Leu Val Leu Ile Gly Asn Ile Thr Ile Pro
235 240 245
ttc gcc atc gct gtt ggc tgg att gcg taaaggttag gaagaattt atg agc 1244
Phe Ala Ile Ala Val Gly Trp Ile Ala Met Ser
250 255
act cac tct gaa acc acc cgc cca gag ttc atc cac cca gtc tca gtc 1292
Thr His Ser Glu Thr Thr Arg Pro Glu Phe Ile His Pro Val Ser Val
260 265 270 275
ctc cca gag gtc tca gct ggt acg gtc ctt gac gct gct gag cca gca 1340
Leu Pro Glu Val Ser Ala Gly Thr Val Leu Asp Ala Ala Glu Pro Ala
280 285 290
ggc gtt ccc acc aaa gat atg tgg gaa tac caa aaa gac cac atg aac 1388
Gly Val Pro Thr Lys Asp Met Trp Glu Tyr Gln Lys Asp His Met Asn
295 300 305
ctg gtc tcc cca ctg aac cga cgc aag ttc cgc gtc ctc gtc gtt ggc 1436
Leu Val Ser Pro Leu Asn Arg Arg Lys Phe Arg Val Leu Val Val Gly
310 315 320
acc ggc ctg tcc ggt ggc gct gca gca gca gcc ctc ggc gaa ctc gga 1484
Thr Gly Leu Ser Gly Gly Ala Ala Ala Ala Ala Leu Gly Glu Leu Gly
325 330 335
tac gac gtc aag gcg ttc acc tac cac gac gca cct cgc cgt gcg cac 1532
Tyr Asp Val Lys Ala Phe Thr Tyr His Asp Ala Pro Arg Arg Ala His
340 345 350 355
tcc att gct gca cag ggt ggc gtt aac tcc gcc cgc ggc aag aag gta 1580
Ser Ile Ala Ala Gln Gly Gly Val Asn Ser Ala Arg Gly Lys Lys Val
360 365 370
gac aac gac ggc gca tac cgc cac gtc aag gac acc gtc aag ggc ggc 1628
Asp Asn Asp Gly Ala Tyr Arg His Val Lys Asp Thr Val Lys Gly Gly
375 380 385
gac tac cgt ggc cgc gag tcc gac tgc tgg cgt ctc gcc gtc gag tcc 1676
Asp Tyr Arg Gly Arg Glu Ser Asp Cys Trp Arg Leu Ala Val Glu Ser
390 395 400
gtc cgc gtc atc gac cac atg aac gcc atc ggt gca cca ttc gcc cgc 1724
Val Arg Val Ile Asp His Met Asn Ala Ile Gly Ala Pro Phe Ala Arg
405 410 415
gaa tac ggt ggc gcc ttg gca acc cgt tcc ttc ggt ggt gtg cag gtc 1772
Glu Tyr Gly Gly Ala Leu Ala Thr Arg Ser Phe Gly Gly Val Gln Val
420 425 430 435
tcc cgt acc tac tac acc cgt gga caa acc gga cag cag ctg cag ctc 1820
Ser Arg Thr Tyr Tyr Thr Arg Gly Gln Thr Gly Gln Gln Leu Gln Leu
440 445 450
tcc acc gca tcc gca cta cag cgc cag atc cac ctc ggc tcc gta gag 1868
Ser Thr Ala Ser Ala Leu Gln Arg Gln Ile His Leu Gly Ser Val Glu
455 460 465
atc ttc acc cat aac gaa atg gtt gac gta att gtc acc gaa cgt aat 1916
Ile Phe Thr His Asn Glu Met Val Asp Val Ile Val Thr Glu Arg Asn
470 475 480
ggt gaa aag cgc tgc gaa ggc ctg atc atg cgc aac ctg atc acc ggc 1964
Gly Glu Lys Arg Cys Glu Gly Leu Ile Met Arg Asn Leu Ile Thr Gly
485 490 495
gag ctc acc gca cac acc ggc cat gcc gtt atc ctg gca acc ggt ggc 2012
Glu Leu Thr Ala His Thr Gly His Ala Val Ile Leu Ala Thr Gly Gly
500 505 510 515
tac ggc aac gtg tac cac atg tcc acc ctg gcg aag aac tcc aac gcc 2060
Tyr Gly Asn Val Tyr His Met Ser Thr Leu Ala Lys Asn Ser Asn Ala
520 525 530
tcg gcc atc atg cgt gca tac gaa gcc ggc gca tac ttc gcg tcc cca 2108
Ser Ala Ile Met Arg Ala Tyr Glu Ala Gly Ala Tyr Phe Ala Ser Pro
535 540 545
tcg ttc atc cag ttc cac cca acc ggc ctg cct gtg aac tcc acc tgg 2156
Ser Phe Ile Gln Phe His Pro Thr Gly Leu Pro Val Asn Ser Thr Trp
550 555 560
cag tcc aag acc att ctg atg tcc gag tcg ctg cgt aac gac ggc cgc 2204
Gln Ser Lys Thr Ile Leu Met Ser Glu Ser Leu Arg Asn Asp Gly Arg
565 570 575
atc tgg tcc cct aag gtt aag ggg gat gat cgc gat cca aac acc atc 2252
Ile Trp Ser Pro Lys Val Lys Gly Asp Asp Arg Asp Pro Asn Thr Ile
580 585 590 595
cct gag gat gag cgc gac tac ttc ctg gag cgc cgc tac cca gca ttc 2300
Pro Glu Asp Glu Arg Asp Tyr Phe Leu Glu Arg Arg Tyr Pro Ala Phe
600 605 610
ggt aac ctc gtc cca cgt gac gtt gct tcc cgt gcg atc tcc cag cag 2348
Gly Asn Leu Val Pro Arg Asp Val Ala Ser Arg Ala Ile Ser Gln Gln
615 620 625
atc aac gct ggt ctc ggt gtt gga cct ctg aac aac gct gca tac ctg 2396
Ile Asn Ala Gly Leu Gly Val Gly Pro Leu Asn Asn Ala Ala Tyr Leu
630 635 640
gac ttc cgc gac gct acc gag cgt ctc gga cag gac acc atc cgc gag 2444
Asp Phe Arg Asp Ala Thr Glu Arg Leu Gly Gln Asp Thr Ile Arg Glu
645 650 655
cgt tac tcc aac ctc ttc acc atg tac gaa gag gca att ggt gag gac 2492
Arg Tyr Ser Asn Leu Phe Thr Met Tyr Glu Glu Ala Ile Gly Glu Asp
660 665 670 675
cca tac tcc agc cca atg cgt att gca ccg acc tgc cac ttc acc atg 2540
Pro Tyr Ser Ser Pro Met Arg Ile Ala Pro Thr Cys His Phe Thr Met
680 685 690
ggc ggc ctc tgg act gac ttc aac gaa atg acg tca ctc cca ggt ctg 2588
Gly Gly Leu Trp Thr Asp Phe Asn Glu Met Thr Ser Leu Pro Gly Leu
695 700 705
ttc tgc gca ggc gaa gca tcc tgg acc tac cac ggt gca aac cgt ctg 2636
Phe Cys Ala Gly Glu Ala Ser Trp Thr Tyr His Gly Ala Asn Arg Leu
710 715 720
ggc gca aac tcc ctg ctt tcc gct tcc gtc gat ggc tgg ttc acc ctg 2684
Gly Ala Asn Ser Leu Leu Ser Ala Ser Val Asp Gly Trp Phe Thr Leu
725 730 735
cca ttc acc atc cct aac tac ctc ggc cca ttg ctt ggc gcc gag cgt 2732
Pro Phe Thr Ile Pro Asn Tyr Leu Gly Pro Leu Leu Gly Ala Glu Arg
740 745 750 755
ctg gca gag gat gca cca gaa gca gtc cag gca atc gaa cgc gct caa 2780
Leu Ala Glu Asp Ala Pro Glu Ala Val Gln Ala Ile Glu Arg Ala Gln
760 765 770
gca cgc att gac cgc ctc atg ggc aac cgc cca gag tgg gtc ggt gac 2828
Ala Arg Ile Asp Arg Leu Met Gly Asn Arg Pro Glu Trp Val Gly Asp
775 780 785
aac gtt cac gga cct gag tac tac cac cgc cag ctt ggc gat atc ctg 2876
Asn Val His Gly Pro Glu Tyr Tyr His Arg Gln Leu Gly Asp Ile Leu
790 795 800
tac ttc tcc tgt ggc gtt tct cga aac gta aag gat ctc cag gac ggt 2924
Tyr Phe Ser Cys Gly Val Ser Arg Asn Val Lys Asp Leu Gln Asp Gly
805 810 815
atc gac aag atc cgt gcg ctc cgc gag gac ttc tgg aag aac atg cgc 2972
Ile Asp Lys Ile Arg Ala Leu Arg Glu Asp Phe Trp Lys Asn Met Arg
820 825 830 835
atc acc ggc agc acc gat gag atg aac cag gtt ctc gaa tac gca gca 3020
Ile Thr Gly Ser Thr Asp Glu Met Asn Gln Val Leu Glu Tyr Ala Ala
840 845 850
cgc gta gct gat tac atc gac ctc ggc gaa ctc atg tgt gtc gac gca 3068
Arg Val Ala Asp Tyr Ile Asp Leu Gly Glu Leu Met Cys Val Asp Ala
855 860 865
ctc gac cgc gac gag tcc tgt ggc gct cac ttc cgc gac gac cac ctc 3116
Leu Asp Arg Asp Glu Ser Cys Gly Ala His Phe Arg Asp Asp His Leu
870 875 880
tcc gaa gat ggc gaa gca gaa cgt gac gac gaa aac tgg tgc ttc gtc 3164
Ser Glu Asp Gly Glu Ala Glu Arg Asp Asp Glu Asn Trp Cys Phe Val
885 890 895
tcc gca tgg gaa cca ggc gag aac gga acc ttc gtc cgc cac gca gaa 3212
Ser Ala Trp Glu Pro Gly Glu Asn Gly Thr Phe Val Arg His Ala Glu
900 905 910 915
cca ctg ttc ttc gaa tcc gtc cca etg cag aca agg aac tac aag ta 3259
Pro Leu Phe Phe Glu Ser Val Pro Leu Gln Thr Arg Asn Tyr Lys
920 925 930
atg aaa ctt aca ctt gag atc tgg cgt caa gca ggc cca act gcg gaa 3307
Met Lys Leu Thr Leu Glu Ile Trp Arg Gln Ala Gly Pro Thr Ala Glu
935 940 945
ggc aag ttc gaa acc gtc cag gtt gac gac gcc gtc gcg cag atg tcc 3355
Gly Lys Phe Glu Thr Val Gln Val Asp Asp Ala Val Ala Gln Met Ser
950 955 960
atc ctg gag ctg ctt gac cac gta aac aac aag ttc atc gaa gaa ggc 3403
Ile Leu Glu Leu Leu Asp His Val Asn Asn Lys Phe Ile Glu Glu Gly
965 970 975
aag gaa cca ttc gca ttc gcg tct gac tgc cgc gaa ggc atc tgt ggt 3451
Lys Glu Pro Phe Ala Phe Ala Ser Asp Cys Arg Glu Gly Ile Cys Gly
980 985 990
acc tgt ggt ctc ctc gtg aac ggc cgc cct cac ggc gcc gac cag 3496
Thr Cys Gly Leu Leu Val Asn Gly Arg Pro His Gly Ala Asp Gln
995 1000 1005
aac aag cct gcc tgt gcg cag cgc ttg gtc agc tac aac gaa ggc 3541
Asn Lys Pro Ala Cys Ala Gln Arg Leu Val Ser Tyr Asn Glu Gly
1010 1015 1020
gac acc ctc aag atc gaa cca ctg cgt tcc gcc gca tac cca gtg 3586
Asp Thr Leu Lys Ile Glu Pro Leu Arg Ser Ala Ala Tyr Pro Val
1025 1030 1035
atc aag gac atg gtc gtc gac cgc tcc gca ctg gac cgc gtc atg 3631
Ile Lys Asp Met Val Val Asp Arg Ser Ala Leu Asp Arg Val Met
1040 1045 1050
gaa cag ggt ggc tac gtg acc atc aac gca ggt acc gca cct gac 3676
Glu Gln Gly Gly Tyr Val Thr Ile Asn Ala Gly Thr Ala Pro Asp
1055 1060 1065
gct gat acc ctc cac gtc aac cac gaa acc gca gaa ctc gca ctt 3721
Ala Asp Thr Leu His Val Asn His Glu Thr Ala Glu Leu Ala Leu
1070 1075 1080
gac cac gca gcc tgc atc ggc tgt ggt gca tgt gtc gct gcc tgc 3766
Asp His Ala Ala Cys Ile Gly Cys Gly Ala Cys Val Ala Ala Cys
1085 1090 1095
cct aac ggc gca gca cac ctg ttc acc ggc gca aag ctt gtt cac 3811
Pro Asn Gly Ala Ala His Leu Phe Thr Gly Ala Lys Leu Val His
1100 1105 1110
ctc tcc ctc ctc cca ctg ggt aag gaa gag cgc gga ctg cgt gca 3856
Leu Ser Leu Leu Pro Leu Gly Lys Glu Glu Arg Gly Leu Arg Ala
1115 1120 1125
cgt aag atg gtt gat gaa atg gaa acc aac ttc gga cac tgc tcc 3901
Arg Lys Met Val Asp Glu Met Glu Thr Asn Phe Gly His Cys Ser
1130 1135 1140
ctc tac ggc gag tgc gca gac gtc tgc ccc gca ggc att cca ctg 3946
Leu Tyr Gly Glu Cys Ala Asp Val Cys Pro Ala Gly Ile Pro Leu
1145 1150 1155
acc gct gtg gca gct gtc acc aag gaa cgt gcg cgt gca gct ttc 3991
Thr Ala Val Ala Ala Val Thr Lys Glu Arg Ala Arg Ala Ala Phe
1160 1165 1170
cga ggc aaa gac gac tagtctttaa tccaagtaag taacggttca gacagttaaa 4046
Arg Gly Lys Asp Asp
1175
ccagaaagac gagtgaacac catgtcctcc gagaataaga attccgcacc ggagcgtatg 4106
cactacatca agggctatgt acctgtggcg tacaactctc cacactcatc cctcgagcgc 4166
agcgcaacct ggttgggcat gggattcctc ctca 4200
<210>78
<211>257
<212>PRT
<213>乳发酵短杆菌
<400>78
Met Thr Val Arg Asn Pro Asp Arg Glu Ala Ile Arg His Gly Lys Ile
1 5 10 15
Thr Thr Glu Ala Leu Arg Glu Arg Pro Ala Tyr Pro Thr Trp Ala Met
20 25 30
Lys Leu Thr Met Ala Ile Thr Gly Leu Ile Phe Gly Gly Phe Val Leu
35 40 45
Val His Met Ile Gly Asn Leu Lys Ile Phe Met Pro Asp Tyr Ala Ala
50 55 60
Asp Ser Ala His Pro Gly Glu Ala Gln Val Asp Val Tyr Gly Glu Phe
65 70 75 80
Leu Arg Glu Ile Gly Ser Pro Ile Leu Pro His Gly Ser Val Leu Trp
85 90 95
Ile Leu Arg Ile Ile Leu Leu Val Ala Leu Val Leu His Ile Tyr Cys
100 105 110
Ala Phe Ala Leu Thr Gly Arg Ser His Gln Ser Arg Gly Lys Phe Arg
115 120 125
Arg Thr Asn Leu Val Gly Gly Phe Asn Ser Phe Ala Thr Arg Ser Met
130 135 140
Leu Val Thr Gly Ile Val Leu Leu Ala Phe Ile Ile Phe His Ile Leu
145 150 155 160
Asp Leu Thr Met Gly Val Ala Pro Ala Ala Pro Thr Ser Phe Glu His
165 170 175
Gly Glu Val Tyr Ala Asn Met Val Ala Ser Phe Ser Arg Trp Pro Val
180 185 190
Ala Ile Trp Tyr Ile Ile Ala Asn Leu Val Leu Phe Val His Leu Ser
195 200 205
His Gly Ile Trp Leu Ala Val Ser Asp Leu Gly Ile Thr Gly Arg Arg
210 215 220
Trp Arg Ala Ile Leu Leu Ala Val Ala Tyr Ile Val Pro Ala Leu Val
225 230 235 240
Leu Ile Gly Asn Ile Thr Ile Pro Phe Ala Ile Ala Val Gly Trp Ile
245 250 255
Ala
<210>79
<211>673
<212>PRT
<213>乳发酵短杆菌
<400>79
Met Ser Thr His Ser Glu Thr Thr Arg Pro Glu Phe Ile His Pro Val
1 5 10 15
Ser Val Leu Pro Glu Val Ser Ala Gly Thr Val Leu Asp Ala Ala Glu
20 25 30
Pro Ala Gly Val Pro Thr Lys Asp Met Trp Glu Tyr Gln Lys Asp His
35 40 45
Met Asn Leu Val Ser Pro Leu Asn Arg Arg Lys Phe Arg Val Leu Val
50 55 60
Val Gly Thr Gly Leu Ser Gly Gly Ala Ala Ala Ala Ala Leu Gly Glu
65 70 75 80
Leu Gly Tyr Asp Val Lys Ala Phe Thr Tyr His Asp Ala Pro Arg Arg
85 90 95
Ala His Ser Ile Ala Ala Gln Gly Gly Val Asn Ser Ala Arg Gly Lys
100 105 110
Lys Val Asp Asn Asp Gly Ala Tyr Arg His Val Lys Asp Thr Mal Lys
115 120 125
Gly Gly Asp Tyr Arg Gly Arg Glu Ser Asp Cys Trp Arg Leu Ala Val
130 135 140
Glu Ser Val Arg Val Ile Asp His Met Asn Ala Ile Gly Ala Pro Phe
145 150 155 160
Ala Arg Glu Tyr Gly Gly Ala Leu Ala Thr Arg Ser Phe Gly Gly Val
165 170 175
Gln Val Ser Arg Thr Tyr Tyr Thr Arg Gly Gln Thr Gly Gln Gln Leu
180 185 190
Gln Leu Ser Thr Ala Ser Ala Leu Gln Arg Gln Ile His Leu Gly Ser
195 200 205
Val Glu Ile Phe Thr His Asn Glu Met Val Asp Val Ile Val Thr Glu
210 215 220
Arg Asn Gly Glu Lys Arg Cys Glu Gly Leu Ile Met Arg Asn Leu Ile
225 230 235 240
Thr Gly Glu Leu Thr Ala His Thr Gly His Ala Val Ile Leu Ala Thr
245 250 255
Gly Gly Tyr Gly Asn Val Tyr His Met Ser Thr Leu Ala Lys Asn Ser
260 265 270
Asn Ala Ser Ala Ile Met Arg Ala Tyr Glu Ala Gly Ala Tyr Phe Ala
275 280 285
Ser Pro Ser Phe Ile Gln Phe His Pro Thr Gly Leu Pro Val Asn Ser
290 295 300
Thr Trp Gln Ser Lys Thr Ile Leu Met Ser Glu Ser Leu Arg Asn Asp
305 310 315 320
Gly Arg Ile Trp Ser Pro Lys Val Lys Gly Asp Asp Arg Asp Pro Asn
325 330 335
Thr Ile Pro Glu Asp Glu Arg Asp Tyr Phe Leu Glu Arg Arg Tyr Pro
340 345 350
Ala Phe Gly Asn Leu Val Pro Arg Asp Val Ala Ser Arg Ala Ile Ser
355 360 365
Gln Gln Ile Asn Ala Gly Leu Gly Val Gly Pro Leu Asn Asn Ala Ala
370 375 380
Tyr Leu Asp Phe Arg Asp Ala Thr Glu Arg Leu Gly Gln Asp Thr Ile
385 390 395 400
Arg Glu Arg Tyr Ser Asn Leu Phe Thr Met Tyr Glu Glu Ala Ile Gly
405 410 415
Glu Asp Pro Tyr Ser Ser Pro Met Arg Ile Ala Pro Thr Cys His Phe
420 425 430
Thr Met Gly Gly Leu Trp Thr Asp Phe Asn Glu Met Thr Ser Leu Pro
435 440 445
Gly Leu Phe Cys Ala Gly Glu Ala Ser Trp Thr Tyr His Gly Ala Ash
450 455 460
Arg Leu Gly Ala Asn Ser Leu Leu Ser Ala Ser Val Asp Gly Trp Phe
465 470 475 480
Thr Leu Pro Phe Thr Ile Pro Asn Tyr Leu Gly Pro Leu Leu Gly Ala
485 490 495
Glu Arg Leu Ala Glu Asp Ala Pro Glu Ala Val Gln Ala Ile Glu Arg
500 505 510
Ala Gln Ala Arg Ile Asp Arg Leu Met Gly Asn Arg Pro Glu Trp Val
515 520 525
Gly Asp Asn Val His Gly Pro Glu Tyr Tyr His Arg Gln Leu Gly Asp
530 535 540
Ile Leu Tyr Phe Ser Cys Gly Val Ser Arg Asn Val Lys Asp Leu Gln
545 550 555 560
Asp Gly Ile Asp Lys Ile Arg Ala Leu Arg Glu Asp Phe Trp Lys Asn
565 570 575
Met Arg Ile Thr Gly Ser Thr Asp Glu Met Asn Gln Val Leu Glu Tyr
580 585 590
Ala Ala Arg Val Ala Asp Tyr Ile Asp Leu Gly Glu Leu Met Cys Val
595 600 605
Asp Ala Leu Asp Arg Asp Glu Ser Cys Gly Ala His Phe Arg Asp Asp
610 615 620
His Leu Ser Glu Asp Gly Glu Ala Glu Arg Asp Asp Glu Asn Trp Cys
625 630 635 640
Phe Val Ser Ala Trp Glu Pro Gly Glu Asn Gly Thr Phe Val Arg His
645 650 655
Ala Glu Pro Leu Phe Phe Glu Ser Val Pro Leu Gln Thr Arg Asn Tyr
660 665 670
Lys
<210>80
<211>249
<212>PRT
<213>乳发酵短杆菌
<400>80
Met Lys Leu Thr Leu Glu Ile Trp Arg Gln Ala Gly Pro Thr Ala Glu
1 5 10 15
Gly Lys Phe Glu Thr Val Gln Val Asp Asp Ala Val Ala Gln Met Ser
20 25 30
Ile Leu Glu Leu Leu Asp His Val Asn Asn Lys Phe Ile Glu Glu Gly
35 40 45
Lys Glu Pro Phe Ala Phe Ala Ser Asp Cys Arg Glu Gly Ile Cys Gly
50 55 60
Thr Cys Gly Leu Leu Val Asn Gly Arg Pro His Gly Ala Asp Gln Asn
65 70 75 80
Lys Pro Ala Cys Ala Gln Arg Leu Val Ser Tyr Asn Glu Gly Asp Thr
85 90 95
Leu Lys Ile Glu Pro Leu Arg Ser Ala Ala Tyr Pro Val Ile Lys Asp
100 105 110
Met Val Val Asp Arg Ser Ala Leu Asp Arg Val Met Glu Gln Gly Gly
115 120 125
Tyr Val Thr Ile Asn Ala Gly Thr Ala Pro Asp Ala Asp Thr Leu His
130 135 140
Val Asn His Glu Thr Ala Glu Leu Ala Leu Asp His Ala Ala Cys Ile
145 150 155 160
Gly Cys Gly Ala Cys Val Ala Ala Cys Pro Asn Gly Ala Ala His Leu
165 170 175
Phe Thr Gly Ala Lys Leu Val His Leu Ser Leu Leu Pro Leu Gly Lys
180 185 190
Glu Glu Arg Gly Leu Arg Ala Arg Lys Met Val Asp Glu Met Glu Thr
195 200 205
Asn Phe Gly His Cys Ser Leu Tyr Gly Glu Cys Ala Asp Val Cys Pro
210 215 220
Ala Gly Ile Pro Leu Thr Ala Val Ala Ala Val Thr Lys Glu Arg Ala
225 230 235 240
Arg Ala Ala Phe Arg Gly Lys Asp Asp
245
Claims (1)
1.一种生产L-氨基酸的方法,包括在培养基中培养具有L-氨基酸生产能力、且通过破坏编码琥珀酸脱氢酶的基因和编码α-酮戊二酸脱氢酶的基因而进行了修饰而使得琥珀酸脱氢酶活性和α-酮戊二酸脱氢酶活性降低的微生物,从而使L-氨基酸在该培养基中或微生物体内生成并蓄积,并且从该培养基或微生物收集L-氨基酸,其中所述L-氨基酸是L-谷氨酸,其中所述微生物为Pantoea ananatis,
其中所述编码琥珀酸脱氢酶基因是选自sdhA基因、sdhB基因、sdhC基因和sdhD基因中的1种或2种以上的基因,和
其中编码α-酮戊二酸脱氢酶的基因是sucA基因。
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JP2006341019A JP2010041920A (ja) | 2006-12-19 | 2006-12-19 | L−アミノ酸の製造法 |
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PCT/JP2007/067387 WO2008075483A1 (ja) | 2006-12-19 | 2007-09-06 | L-アミノ酸の製造法 |
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EP (1) | EP2100957B1 (zh) |
JP (1) | JP2010041920A (zh) |
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JP2010041920A (ja) | 2010-02-25 |
EP2100957A1 (en) | 2009-09-16 |
BRPI0721063B1 (pt) | 2017-11-14 |
EP2100957B1 (en) | 2016-10-26 |
US20090286290A1 (en) | 2009-11-19 |
CN101563453A (zh) | 2009-10-21 |
US8058035B2 (en) | 2011-11-15 |
WO2008075483A1 (ja) | 2008-06-26 |
BRPI0721063A2 (pt) | 2014-02-25 |
EP2100957A4 (en) | 2012-01-25 |
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