磷酸转酮酶用于生产有用的代谢物的用途的制作方法

专利名称：磷酸转酮酶用于生产有用的代谢物的用途的制作方法
技术领域：
本发明涉及生产有用的代谢物，特别是源自乙酰辅酶A(乙酰CoA)的那些代谢物的方法。本发明还涉及用于所述生产方法的新的细菌。
相关技术简述通常，通过一些方法来生产细菌代谢的有用代谢物例如L-氨基酸、它们的中间体和其他化学物质，在所述方法中将分离自天然来源的细菌菌株或其突变体修饰以增强它们的生产力。
在适合于发酵的微生物中糖类是主要的碳源。Embden-Meyerhof和戊糖磷酸(戊糖-P)途径是微生物中中间糖代谢的两个初始途径。第三种途径，Entner-Doudoroff途径也是已知的，作为与羧酸途径的一些联系。在糖酵解期间，葡萄糖被代谢为主要的中间体化合物，例如磷酸烯醇丙酮酸、丙酮酸和乙酰辅酶A，它们在许多细胞化合物例如L-氨基酸、嘌呤和嘧啶、维生素等等的形成中被用作组分。并且，在糖酵解期间出现能量的产生(ATP和NADH)。糖酵解后形成的丙酮酸常常通过由pps基因编码的磷酸烯醇丙酮酸合酶转变回磷酸烯醇丙酮酸(PEP)，或通过由pdh基因等编码的丙酮酸脱氢酶转变回乙酰CoA。上述化合物之一，乙酰辅酶A，通过丙酮酸脱氢酶从丙酮酸形成，并伴有CO2的释放。一个碳原子的损失导致来源于乙酰CoA的有用化合物的生产得率(production yield)降低。
已经报道了双歧途径的两个酶，D-木酮糖-5-磷酸磷酸转酮酶(也称为“磷酸转酮酶”和果糖-6-磷酸磷酸转酮酶。D-木酮糖-5-磷酸磷酸转酮酶(EC 4.1.2.9)催化木酮糖-5-磷酸的消耗磷酸性转化变成甘油醛-3-磷酸和乙酰磷酸，带有一个水分子的相伴性释放。果糖-6-磷酸磷酸转酮酶(EC 4.1.2.22)催化果糖-6-磷酸的耗磷酸性转化变成赤藓糖-4-磷酸和乙酰磷酸，带有一个水分子的相伴性释放。两种酶都形成乙酰CoA的前体-乙酰磷酸，而不经由CO2损失碳。已经在属于醋杆菌属(Acetobacter)(Schramm，M.等，J.Biol.Chem.，233(6)，1283-8(1958))，双歧杆菌属(Bifidobacterium)(Sgorbati，B.等，Antonie VanLeeuwenhoek.42(1-2)，49-57(1976)；Grill，J.P.等Curr Microbiol.，31(1)，49-54(1995))，乳杆菌属(Lactobacillus)(Posthuma，C.C.等，Appl.Environ.Microbiol.，68(2)，831-7(2002))，硫杆菌属(Thiobacillus)(Greenley，D.E.andSmith，D.W.，Arch.Microbiol.，122，257-261(1979))的细菌中，在属于假丝酵母属(Candida)、红酵母属(Rhodotorula)、红冬孢酵母属(Rhodosporidium)、毕赤酵母属(Pichia)、西洋蓍霉(Yarrowia)、汉逊酵母属(Hansenula)、汉逊酵母属、克鲁维酵母属(Kluyveromyces)、酵母属(Saccharomyces)、丝孢酵母属(Trichosporon)、温酵母属(Wingea)的酵母中(Evans，C.T.and Ratledge，C.，Arch.Microbiol.，139，48-52(1984)；Ratledge，C.and Holdsworth，J.E.，Appl.Microbiol.Biotechnol.，22，217-221(1985))报道了D-木酮糖-5-磷酸磷酸转酮酶(EC 4.1.2.9)。已经在细菌，例如木醋杆菌(Acetobacter xylinum)(Schramm，M.等，J.Biol.Chem.，233(6)，1283-8(1958))，球形双歧杆菌(Bifidobacteriumglobosum)和齿双歧杆菌(Bifidobacterium dentium)(Sgorbath，B.等，AntonieLeeuwenhoek，42，49-57(1976))，双歧双歧杆菌(Bifidobacterium bifidum)、阴道加德纳氏菌(Gardnerella vaginalis)(Gavini，F.等，Anaerobe，2，191-193(1996))中，和酵母，例如牧草红酵母(Rhodotorula graminis)、红酵母(Rhodotorulaglutinis)、假丝酵母属菌种(Candida sp.)、热带假丝酵母(Candida tropicalis)、Saccharomyces pastorianus(Whitworth，D.A.and Ratledge，C.，J.Gen.Microbiol.，102，397-401(1977))中报道了果糖-6-磷酸磷酸转酮酶(EC4.1.2.22)。已经报道了在某些生物体中由一种酶表现了两种活性(参见，例如，Schramm，M.等(J.Biol.Chem.，233(6)，1283-8(1958))；Sgorbati，B.等(AntonieVan Leeuwenhoek.42(1-2)，49-57(1976))；Meile，L.等(J.Bacteriol.，183(9)，2929-36(2001))的文章)。
已经克隆了来自两个物种的磷酸转酮酶基因并测定了它们的序列。它们是编码来自乳酸双歧杆菌(Bifidobacterium lactis)的D-木酮糖-5-磷酸磷酸转酮酶/果糖-6-磷酸磷酸转酮酶的xfp基因(Meile，L.等，J.Bacteriol.，183(9)，2929-36(2001))，和编码来自戊糖乳杆菌(Lactobacillus pentosus)的D-木酮糖-5-磷酸磷酸转酮酶的xpkA基因(Posthuma，C.C.等，Appl.Environ.Microbiol.，68(2)，831-7(2002))。对国立生物技术信息中心(National Center forBiotechnology information)提供的微生物基因组数据库(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi？CMD＝&DB＝genome)的搜索揭示了编码推定的磷酸转酮酶的几个基因。
通过导入木糖还原酶、木糖醇脱氢酶和另外的磷酸转酮酶的基因，来改善酵母从木糖生产乙醇的能力的方法是已知的(WO2003078643)。然而，从来没有报道过使用磷酸转酮酶基因用于消除二氧化碳的效果。

发明内容
本发明的目的是通过具有生产代谢物的能力的细菌菌株来增强有用代谢物的产生，以及提供使用这些菌株生产代谢物的方法。
本发明的目的是提供具有生产有用代谢物的能力的细菌，其中所述细菌被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶的活性。
本发明的进一步的目的是提供如上所述的细菌，其中所述有用代谢物源自乙酰辅酶A。
本发明的进一步的目的是提供如上所述的细菌，其中所述有用代谢物选自由L-谷氨酸、L-谷氨酰胺、L-脯氨酸、L-精氨酸、L-亮氨酸、L-半胱氨酸、琥珀酸酯(succinate)和聚羟基丁酸酯(polyhydroxybutyrate)构成的组。
本发明的目的是提供具有生产有用代谢物的能力的细菌，其中所述细菌天然不具有D-木酮糖-5-磷酸磷酸转酮酶或果糖-6-磷酸磷酸转酮酶的活性，其中所述细菌已经用编码D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶的DNA片段转化。
本发明的进一步的目的是提供如上所述的细菌，其中所述有用代谢物源自乙酰辅酶A。
本发明的进一步的目的是提供如上所述的细菌，其中所述有用代谢物选自由L-谷氨酸、L-谷氨酰胺、L-脯氨酸、L-精氨酸、L-亮氨酸、L-半胱氨酸、琥珀酸酯(succinate)和聚羟基丁酸酯(polyhydroxybutyrate)构成的组。
本发明的进一步的目的是提供如上所述的细菌，其中所述细菌选自由肠杆菌科(Enterobacteriaceae)、棒状杆菌型细菌(Coryneform bacterium)和芽孢杆菌属(Bacillus)细菌构成的组。
本发明的进一步的目的是提供如上所述的细菌，其中所述细菌属于埃希氏菌属(Escherichia)或泛菌属(Pantoea)。
本发明的进一步的目的是提供生产有用代谢物的方法，包括在培养基中培养所述细菌，并从所述培养基中收集所述有用代谢物。
本发明的进一步的目的是提供如上所述的方法，其中所述有用代谢物源自乙酰辅酶A。
本发明的进一步的目的是提供如上所述的方法，其中所述有用代谢物选自由L-谷氨酸、L-谷氨酰胺、L-脯氨酸、L-精氨酸、L-亮氨酸、L-半胱氨酸、琥珀酸酯和聚羟基丁酸酯构成的组。
通过在具有生产有用代谢物，特别是源自乙酰CoA的有用代谢物的能力的细菌中增强D-木酮糖-5-磷酸磷酸转酮酶或果糖-6-磷酸磷酸转酮酶的能力，实现了这些目的，这使得更有效地利用碳和增加细菌的有用代谢物产量成为可能。由此，完成了本发明。
以下将详细描述本发明。


图1显示来自戊糖乳杆菌(L.pentosus)和植物乳杆菌(L.plantarum)的D-木酮糖-5-磷酸磷酸转酮酶的比对(同一性98.5％)。
图2显示质粒pBS3的构建方法。
图3显示质粒pBS4S的构建方法。
图4显示质粒pBS5T的构建方法。
图5显示扩增了磷酸转酮酶基因的菌株和对照菌株在含有醋酸盐的基本培养基上的生长曲线。
图6显示扩增了磷酸转酮酶基因的菌株和对照菌株在基本培养基中的生长曲线。
图7(照片)显示含有磷酸转酮酶蛋白的细胞提取物的电泳。
图8显示使用扩增了磷酸转酮酶基因的菌株在生物素充足的条件下生产L-谷氨酸。
图9显示使用扩增了磷酸转酮酶基因的菌株在添加青霉素的情况下生产L-谷氨酸。
图10显示使用其中导入了磷酸转酮酶基因并降低了6-磷酸果糖激酶活性的菌株生产L-谷氨酸。
图11显示使用其中导入了磷酸转酮酶基因并降低了6-磷酸果糖激酶活性的菌株在存在青霉素的情况下生产L-谷氨酸。
图12显示使用扩增了磷酸转酮酶基因的菌株生产L-谷氨酰胺(Gln)。
优选实施方式的说明本发明的细菌包括具有生产有用代谢物的能力的细菌，其中所述细菌已经被修饰以增加D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶的活性。
本发明的细菌包括天然不具有D-木酮糖-5-磷酸磷酸转酮酶和果糖-6-磷酸磷酸转酮酶的活性的细菌和天然具有D-木酮糖-5-磷酸磷酸转酮酶和果糖-6-磷酸磷酸转酮酶的活性的细菌。通过将编码D-木酮糖-5-磷酸磷酸转酮酶或果糖-6-磷酸磷酸转酮酶的基因导入这种细菌，细菌中乙酰CoA的产生力得到增强，引起有用代谢物产量的提高。
在本发明的细菌中，D-木酮糖-5-磷酸磷酸转酮酶或果糖-6-磷酸磷酸转酮酶的活性之一或两者可以被增强。
在本发明中，术语“磷酸转酮酶”包括D-木酮糖-5-磷酸磷酸转酮酶和果糖-6-磷酸磷酸转酮酶。
在本发明中，用语“D-木酮糖-5-磷酸磷酸转酮酶的活性”是指一种活性，其催化木酮糖-5-磷酸变为甘油醛-3-磷酸和乙酰磷酸的磷酸消耗性转化反应并带有一个水分子的相伴性释放。这种活性可以通过Goldberg，M.等(MethodsEnzymol.，9，515-520(1966)或L.Meile(J.Bacteriol.(2001)183；2929-2936)描述的方法来测量。
在本发明中，用语“果糖-6-磷酸磷酸转酮酶的活性”是指一种活性，其催化果糖-6-磷酸变为赤藓糖-4-磷酸和乙酰磷酸的磷酸消耗性转化反应并带有一个水分子的相伴性释放。果糖-6-磷酸磷酸转酮酶活性可以通过Racker，E.(Methods Enzymol.，5，276-280(1962)或L.Meile(J.Bacteriol.(2001)183；2929-2936)描述的方法来测量。
在天然具有磷酸转酮酶活性的细菌中磷酸转酮酶的活性可以被增强超过野生型或未修饰的菌株，优选不少于野生型或未修饰的菌株的1.5倍，更优选不少于2倍，和最优选的不少于3倍。此外，在本发明中，天然不具有D-木酮糖-5-磷酸磷酸转酮酶和果糖-6-磷酸磷酸转酮酶的活性的细菌可以被用作亲本菌株。在天然不具有磷酸转酮酶活性的细菌中磷酸转酮酶的活性可以通过用编码D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶的DNA片段转化所述细菌来增强。无论亲本细菌是否天然具有磷酸转酮酶活性，都可以用如上所述相同的方法来测量。
是否已经导入了磷酸转酮酶活性可以使用丙酮酸脱氢酶(PDH)缺陷菌株来证实。PDH缺陷菌株是醋酸盐(acetate)营养缺陷的。另一方面，如果导入了磷酸转酮酶，PDH缺陷菌株对于醋酸盐将不再是营养缺陷的。因此，根据宿主菌株对于醋酸盐营养缺陷型的补偿可以证实编码磷酸转酮酶的基因的导入。作为PDH缺陷菌株，可以使用实施例中描述的ΔaceE菌株。
“D-木酮糖-5-磷酸磷酸转酮酶”可以是一种酶，其源自具有D-木酮糖-5-磷酸磷酸转酮酶活性的那些细菌，包括乳酸细菌、甲醇同化细菌、甲烷同化细菌、链球菌属(Streptococcus)细菌，特别是属于醋杆菌属、双歧杆菌属、乳杆菌属、硫杆菌属、链球菌属、甲基球菌属(Methylococus)、丁酸杆菌属(Butyrivibrio)、丝状杆菌属(Fibrobacter)的那些细菌和/或属于假丝酵母属、红酵母属、红冬孢酵母属(Rhodosporidium)、毕赤酵母属、西洋蓍霉、汉逊酵母属、克鲁维酵母属、酵母属、丝孢酵母属、温酵母属(Wingea)等的那些酵母。
“果糖-6-磷酸磷酸转酮酶”可以是一种酶，其源自具有果糖-6-磷酸磷酸转酮酶活性、属于醋杆菌属、双歧杆菌属、绿菌属(Chlorobium)、布鲁氏菌属(Brucella)、甲基球菌属(Methylococus)、加德纳氏菌属(Gardnerella)的那些细菌，和/或属于红酵母属、假丝酵母属、酵母属等的酵母。
也可能的是通过一个酶，D-木酮糖-5-磷酸/果糖-6-磷酸磷酸转酮酶来呈现两种活性，。
编码戊糖乳杆菌MD363的D-木酮糖-5-磷酸磷酸转酮酶的xpkA基因的核苷酸序列已经在EMBL/GenBank数据库中以登记号码AJ309011登记(Posthuma，C.C.等，Appl.Environ.Microbiol.，68(2)，831-7(2002))，如SEQ IDNO1所示。编码植物乳杆菌的D-木酮糖-5-磷酸磷酸转酮酶的xpk1基因的核苷酸序列已经在EMBL/GenBank数据库中以登记号码NC_004567 Region互补物(2362936..2365302)登记(Kleerebezem，M.，等，Proc.Natl.Acad.Sci.U.S.A.100(4)，1990-1995(2003))，如SEQ ID NO3所示。xpk和xpk同源基因的核苷酸序列在表1和序列表中示出。SEQ ID NO2和4的氨基酸序列的比对在图1中示出。
表1

编码乳酸双歧杆菌的D-木酮糖-5-磷酸/果糖-6-磷酸磷酸转酮酶的xfp基因的核苷酸序列已经在EMBL/GenBank数据库中以登记号AJ293946登记(Meile，L.等，J.Bacteriol.，183(9)，2929-36(2001))，在SEQ ID NO5中示出，由xfp基因编码的氨基酸序列如SEQ ID NO6所示。xfp和xfp同源基因的核苷酸序列在表2和序列表中示出。
表2


xpkA、xpk1和xfp基因都可以使用根据已知基因的核苷酸序列设计的引物通过PCR(聚合酶链式反应，参考White，T.J.等，Trends Genet.，5，185(1989))获得。可以使用pfam和motif(http://pfam.wustl.edu/，http://www.genome.jp/dbget-bin/www_bget？pfam+XFP)来获得含有磷酸转酮酶的基序的基因。通过使用SEQ ID NO7和No8所示的引物可以获得来自乳杆菌属的xpk1基因。通过使用SEQ ID NO11和No12所示的引物可以获得来自双歧杆菌属的xfp基因。通过使用SEQ ID NO19和No24所示的引物可以获得来自乳杆菌属的xpkA基因。
可通过用编码磷酸转酮酶的DNA转化亲本菌株来增强磷酸转酮酶的活性。通过使用编码如上所述的磷酸转酮酶的DNA的常规方法可以进行用编码磷酸转酮酶的DNA转化细菌。
编码D-木酮糖-5-磷酸磷酸转酮酶的DNA可以是编码D-木酮糖-5-磷酸磷酸转酮酶的变体的DNA，根据天然的多样性，其可存在于不同的细菌菌株。编码这种变体的DNA可以通过分离DNA来获得，所述DNA在严格条件下与xpkA基因(例如，SEQ ID NO1或3)或该基因的部分杂交、并且编码具有D-木酮糖-5-磷酸磷酸转酮酶活性的蛋白。本文使用的术语“严格条件”包括这样的条件，在所述条件下形成所谓的特异性杂合体，而不形成非特异性杂合体。例如，严格条件可以包括这样的条件，在这些条件下具有高同源性的DNA，例如相互具有不低于70％的同源性、优选80％或更高、更优选90％或更高、最优选95％或更高同源性的DNA杂交。做为选择，通过一些条件例示了严格条件，其包括Southern杂交中的普通洗涤条件，例如，60℃，1×SSC，0.1％SDS，优选60℃，0.1×SSC，0.1％SDS。洗涤的持续时间取决于用于印迹的膜类型，通常，是由制造商推荐的。例如，HybondTMN+尼龙膜(Amersham)在严格条件下建议的洗涤持续时间是15分钟。对于筛选变体xpkA基因的探针，可以使用表1中公开的核苷酸序列的部分序列。使用基于表1中公开的核苷酸序列的寡核苷酸作为引物，和含有表1中公开的核苷酸序列的DNA片段作为模板，通过PCR可以制备这样的探针。当具有约300 bp长度的DNA片段被用作探针时，杂交后的洗涤可以在以下条件下进行50℃，2×SSC和0.1％SDS至少两次。
使用表2中公开的核苷酸序列作为探针，通过与以上描述的类似的方法，可以获得编码果糖-6-磷酸磷酸转酮酶的DNA。
已知的是，在mRNA分子群体内发现的61种氨基酸密码子之中存在着同义密码子的某些分布偏爱，同族tRNA的水平似乎与密码子利用的频率成正比(参见，例如Kane，J.F.，Curr.Opin.Biotechnol.，6(5)，494-500(1995))。根据这个观点，很容易预测含有过多稀有tRNA密码子的大量mRNA种类的翻译问题。在例如大肠杆菌(E.coli)宿主中克隆的异源基因的转录开始之后，这种情况可能出现。新近的研究表明，AGG/AGA、CUA、AUA、CGA或CCC密码子的群集(cluster)可能降低合成的蛋白质的数量和质量。此外，可能的是任何这些密码子的过量，即使没有群集，可能产生翻译困难。所以，当编码目标蛋白质的异源DNA被转移到细菌时，需要用更频繁使用的密码子替换稀有密码子以避免这些翻译困难。在超过11,000种生物体中密码子利用的频率在“密码子利用率数据库”中呈现(http://www.kazusa.or.jp/codon；Nakamura，Y.等，Nucl.Acids Res.，28，292(2000))。
此外，编码本发明的磷酸转酮酶的基因不限于野生型基因，而可以是在表1和2所示核苷酸序列中具有修饰的突变体或人工修饰的基因。编码的蛋白质可以在表1和2所示氨基酸序列中的一个或多个位置包括一个或几个氨基酸残基的取代、缺失或插入，只要维持了编码的磷酸转酮酶蛋白的功能。尽管本文所指的“几个”氨基酸残基的数目取决于三维结构中的位置或氨基酸残基的类型而不同，但其可以是2到20个，优选2到10个，更优选2到5个。这种氨基酸取代包括功能上中性的(neutral)有义突变，优选为保守性取代。对于芳香族氨基酸来说，保守性取代包括相互可交换的phe、trp和tyr。对于疏水性氨基酸来说，保守性取代包括相互可交换的leu、ile和val。对于极性氨基酸来说，保守性取代包括相互可交换的gln和asn。对于碱性氨基酸来说，保守性取代包括相互可交换的arg、lys和his。对于酸性氨基酸来说，保守性取代包括相互可交换的asp和glu。对于含羟基氨基酸来说，保守性取代包括相互可交换的ser和thr。保守性取代还包括ser或thr对ala的取代，gln、his或lys对arg的取代，glu、gln、lys、his或asp对asn的取代，asn、glu或gln对asp的取代，ser或ala对cys的取代，asn、glu、lys、his、asp或arg对gln的取代，gly、asn、gln、lys或asp对glu的取代，pro对gly的取代，asn、lys、gln、arg或tyr对his的取代，leu、met、val或phe对ile的取代，ile、met、val或phe对leu的取代，asn、glu、gln、his或arg对lys的取代，ile、leu、val或phe对met的取代，trp、tyr、met、ile或leu对phe的取代，thr或ala对ser的取代，ser或ala对于thr的取代，phe或tyr对trp的取代，his、phe或trp对tyr的取代，met、ile或leu对val的取代。如上所述引起取代、缺失或插入一个或几个氨基酸的突变还包括起因于携带磷酸转酮酶基因(突变体或变体)的微生物的个体差异和物种差异的天然存在的突变。通过用MOTIF和Pfam在PKT中搜索保守区域(基序)可以选择要取代的区域。
可以通过例如，定点突变来修饰表1或2中所示的核苷酸序列来获得这样的基因，从而将一个或多个取代、缺失或插入导入到由该基因编码的蛋白质的特定位点上。
此外，这种基因也可以通过常规的诱变处理，例如以下所述的那些来获得。诱变处理的实例包括在体外用羟胺处理具有表1或2中所示的核苷酸序列或SEQ ID NO1中49至948位核苷酸的核苷酸序列的基因，和用紫外线照射或用于通常的突变处理的诱变试剂例如N-甲基-N′-硝基-N-亚硝基胍(NTG)或EMS(甲磺酸乙酯(ethyl methanesulfonate))处理带有该基因的细菌，例如埃希氏菌属(Escherichia)细菌。
可以通过，例如，使用遗传重组技术增加细胞中pkt基因的拷贝数来增强磷酸转酮酶基因(以下称为pkt基因，包括xpk或xfp基因)的表达。例如，可以通过将含有pkt基因的基因片段连接到能在宿主细菌中复制的载体，优选多拷贝载体，并将得到的载体导入到宿主细菌中来制备重组DNA。
当使用动物双歧杆菌(Bifidobacterium animalis)的xfp基因时，可以，例如使用根据动物双歧杆菌的xfp基因的核苷酸序列(SEQ ID NO9或11)设计的引物，例如分别具有SEQ ID NO13或14的核苷酸序列的引物，并使用动物双歧杆菌的染色体DNA作为模板，通过PCR方法(聚合酶链式反应，参见White，T.J.等.，Trends Genet.，13，1813(1989))来获得。也可以使用来自其他微生物的pkt基因，可以使用根据它们的pkt基因序列或其同源序列设计的寡核苷酸引物通过PCR，或使用根据如表1或2所示的这种序列信息制备的寡核苷酸探针通过杂交，从它们的染色体DNA或染色体DNA文库获得pkt基因。可以通过例如Saito和Miura的方法(参见H.Saito and K.Miura，Biochem.Biophys.Acta，72，619(1963)，Text for Bioengineering Experiments，Edited by the Society forBioscience and Bioengineering，Japan，pp.97-98，Baifukan，1992))从微生物制备作为DNA供体的染色体DNA。
然后，将pkt基因连接到在宿主细菌中可操作的载体DNA来制备重组DNA。优选地，使用在宿主细菌中可自主复制的载体。
能在大肠杆菌(Escherichia coli)中自主复制的载体的实例包括pUC19、pUC18、pHSG299、pHSG399、pHSG398、pACYC184、(pHSG和pACYC可从Takara Bio获得)、RSF1010、pBR322、pMW118和pMW219(pMW可从NipponGene获得)，等等。也可以使用噬菌体载体，例如11059、IBF101、M13mp9。
在棒状杆菌型细菌中可自主复制的载体的实例包括pAM330(EP0077548B)、pHM1519(EP 0078537)、pGA1(EP 10977998)和pYM2(US6,905,819)此外，也可以使用在大肠杆菌和棒状杆菌型细菌中都可自主复制的所谓穿梭载体，例如pVK9、pVK7(US2003-0175912)、pSFK6(JP2000-262288A)和pHK4(JP5-007491A)。
pVK9是大肠杆菌-棒状杆菌穿梭载体，其通过从pHK4(JP-A-5-007491)切除含有可复制起点的BamHI-KpnI片段并将所述片段导入pHSG299(Takara-Bio的产品)的AvaII位点来获得。
为了通过将pkt基因和任何上述载体连接来制备重组DNA，通常通过使用连接酶，例如T4 DNA连接酶来连接所述载体和含有所述pkt基因的片段。
为了将如上所述制备的重组DNA导入细菌中，可以采用迄今为止报道的任何已知的转化方法。可以采用例如，用氯化钙处理受体细胞以提高DNA的透过性的方法，针对大肠杆菌已经报道了(Mandel，M.and Higa，A.，J.Mol.Biol.，53，159(1970))，和使用从生长细胞制备的感受态细胞来导入DNA的方法，对于枯草芽孢杆菌(Bacillus subtilis)已经报道了(Duncan，C.H.，Wilson，G.A.and Young，F.E.，Gene，1，153(1977))。除这些方法之外，可以采用已经被报道适用于枯草芽孢杆菌、放线菌类和酵母的将重组DNA导入原生质体样或球形体样受体细胞的方法(Chang，S.and Choen，S.N.，Molec.Gen.Genet.，168，111(1979)；Bibb，M.J.，Ward，J.M.and Hopwood，O.A.，Nature，274，398(1978)；Hinnen，A.，Hicks，J.B.and Fink，G.R.，Proc.Natl.Sci.，USA，75，1929(1978))。此外，棒状杆菌型细菌的转化也可以通过电脉冲方法(Sugimoto等，JP2-207791A)进行。
可以通过将单拷贝或多拷贝的基因整合到细菌的染色体DNA中来导入pkt基因。此外，也可以通过将多拷贝的基因整合到细菌的染色体DNA中来增加pkt基因的拷贝数。为了将pkt基因的一个或多个拷贝整合到细菌的染色体DNA上，可以通过靶向以多拷贝存在于染色体DNA上的序列来进行同源重组。转座子末端的重复DNA和反向重复序列(inverted repeats)可被用作以多拷贝存在于染色体DNA上的序列。做为选择，如在JP2-109985A中公开的，也有可能将pkt基因掺入到转座子中，并容许其被转移，从而将所述基因整合到染色体DNA中。此外，通过转座例如Mu整合可以实现基因表达的增强。例如，一轮Mu整合容许向细菌染色体中导入多达3个拷贝的基因。可以使用转座子例如Mu、Tn10和Tn5。可以通过使用具有pkt基因的部分序列的探针进行Southern杂交来确认pkt基因整合到染色体中。
也可以通过用更强的表达调节序列取代染色体DNA或质粒上的表达调节序列，包括pkt基因的启动子，如WO00/18935所述，扩增提高pkt基因表达的调节因子，或缺失或削弱降低pkt基因表达的调节因子，来实现pkt基因表达的增强。此外，还有可能的是向pkt基因的启动子区导入几个核苷酸取代使得所述启动子更强。在Goldstein等(Prokaryotic promoters in biotechnology.Biotechnol.Annu.Rev.，1995，1，105-128)中公开了评估启动子效力的方法和强启动子的实例。此外，由于已知的是，在核糖体结合位点(RBS)和翻译起始密码子之间的间隔区序列，特别是紧靠起始密码子上游的几个核苷酸，对翻译效率有很大的影响，因此可以修饰这个序列。pkt基因的表达调节序列可以使用用于启动子鉴定的载体或基因分析软件例如GENETYX来鉴定。
这种强启动子的实例包括lac启动子、trp启动子、trc启动子、tac启动子、lambda噬菌体的PR启动子和PL启动子、tet启动子、amyE启动子、spac启动子，等等。棒状杆菌属的强启动子的实例包括PS2启动子(Appl Environ Microbiol.2003 Jan；69(1)358-66；Mol Microbiol.1993 Jul；9(1)97-109；WO93/03158)、trc启动子、tac启动子、lacUV5启动子、araBAD启动子。启动子强度被定义为RNA合成起始作用的频率。评估启动子强度的方法由例如Deuschle U.，Kammerer W.，Gentz R.，Bujard H.(Promoters in Escherichia colia hierarchy ofin vivo strength indicates alternate structures.EMBO J.，5，2987-2994(1986))描述。
通过这样的启动子取代或修饰来增强pkt基因的表达。表达调节序列的取代也可以通过例如使用温度敏感质粒来实现。用于棒状杆菌型细菌的温度敏感质粒的实例包括p48K和pSFKT2(JP2000-262288A)、pHSC4(参考法国专利Laid-open Publication No.2667875，1992和JP5-7491A)，pBS5T等等。在棒状杆菌型细菌中这些质粒至少可以在25℃的温度下自主复制，但在37℃的温度下不能自主复制。表达调节序列的修饰可以与增加pkt基因的拷贝数相组合。
使用对棒状杆菌属是致命的果聚糖蔗糖酶作为同源重组的标记物的方法也是适合的。编码果聚糖蔗糖酶的sacB基因可用于选择在其中载体被有效地从染色体缺失的菌株。(Schafer，A.等Gene 145(1994)69-73)。可以使用如下所述的sacB基因和sacB的同源基因。
枯草芽孢杆菌sacB GenBank登记号X02730(SEQ ID NO40)解淀粉芽孢杆菌(Bacillus amyloliqufaciens)sacB GenBank登记号X52988运动发酵单孢菌(Zymomonas mobilis)sacB GenBank登记号L33402嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)surB GenBank登记号U34874旧金山乳杆菌(Lactobacillus sanfranciscensis)frfA GenBank登记号AJ508391木醋杆菌(Acetobacter xylinus)lsxA GenBank登记号AB034152Gluconacetobacter diazotrophicuslsdA GenBank登记号L41732制备质粒DNA、消化和连接DNA、转化、选择寡核苷酸作为引物等等，可以通过本领域技术人员公知的普通方法来进行。例如，在Sambrook，J.，Fritsch，E.F.，and Maniatis，T.，″Molecular Cloning A Laboratory Manual，SecondEdition″，Cold Spring Harbor Laboratory Press(1989)中描述了这些方法。
本发明的细菌不仅包括天然不具有D-木酮糖-5-磷酸磷酸转酮酶和果糖-6-磷酸磷酸转酮酶的活性的细菌，还包括天然具有这些蛋白质的活性、并且被修饰从而所述活性被增强的细菌。前者是优选使用的。
在本发明中使用的细菌的具体实例包括属于肠杆菌科，例如埃希氏菌属(Escherichia)、泛菌属(Pantoea)的细菌，棒状杆菌型细菌例如谷氨酸棒杆菌(Corynebacterium glutamicum)，芽孢杆菌属细菌例如枯草芽孢杆菌，等等。然而，本发明的细菌不限于这些实例。
肠杆菌科包括属于肠杆菌属(Enterobacter)、欧文氏菌属(Erwinia)、埃希氏菌属、克雷伯氏菌属(Klebsiella)、泛菌属、普罗威登斯菌属(Providencia)、沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、志贺氏菌属(Shigella)、摩根氏菌属(Morganella)等等的细菌。具体地，可以使用根据NCBI(国家生物技术信息中心)数据库中使用的分类系统(http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg？mode＝Tree&id＝1236&lvl＝3&keep＝1&srchmode＝1&unlock)分类到肠杆菌科中的那些。属于埃希氏菌属或泛菌属的细菌是优选的。
可以使用在Neidhardt等(Neidhardt，F.C.等，Escherichia coli and SalmonellaTyphimurium，American Society for Microbiology，Washington D.C.，1208，表1)中报道的埃希氏菌属细菌，例如大肠杆菌。大肠杆菌野生型菌株的实例包括，但不限于，K12菌株和其衍生物、MG1655菌株(ATCC No.47076)，和W3110菌株(ATCC No.27325)。这些菌株可从美国典型培养物保藏中心(ATCC，地址P.O.Box 1549，Manassas，VA 20108，United States of America)获得。
术语“属于泛菌属的细菌”是指根据微生物学领域的技术人员已知的分类法分类为泛菌属的细菌，还包括近来根据16S rRNA等等的核苷酸序列分析重新分类入成团泛菌(Pantoea agglomerans)、Pantoea ananatis、斯氏泛菌(Pantoeastewartii)等等的成团肠杆菌(Enterobacter agglomerans)的某些菌种。
在本发明中，棒状杆菌型细菌包括根据微生物学领域的技术人员已知的分类法被分类为棒状杆菌型细菌的细菌，迄今被分类为短杆菌属(Brevibacterium)、但现在重新分类入棒杆菌属(Corynebacterium)(Int.J.Syst.Bacteriol.，41，255(1991))的细菌，以及属于短杆菌属的细菌，其是棒杆菌属的近亲。这种棒状杆菌型细菌的实例如下所列。
嗜乙酰乙酸棒杆菌(Corynebacterium acetoacidophylum)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(Corynebacterium glutamicum)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)
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)玫瑰色短杆菌(Brevibacterium roseum)解糖短杆菌(Brevibacterium saccharolyticum)生硫短杆菌(Brevibacterium thiogenitalis)产氨棒杆菌(Corynebacterium ammoniagenes)白色短杆菌(Brevibacterium album)蜡状短杆菌(Brevibacterium cerinum)嗜氨微杆菌(Microbacterium ammoniaphilum)具体地，包括下列菌株嗜乙酰乙酸棒杆菌ATCC 13870醋谷棒杆菌ATCC 15806烷醇棒杆菌ATCC 21511美棒杆菌ATCC 15991谷氨酸棒杆菌ATCC 13020，13032，13060百合花棒杆菌ATCC 15990栖糖蜜棒杆菌ATCC 17965Corynebacterium efficiens AJ12340(FERM BP-1539)力士棒状杆菌ATCC 13868二歧短杆菌ATCC 14020黄色短杆菌ATCC 13826，ATCC 14067，AJ12418(FERM BP-2205)Brevibacterium immariophilum ATCC 14068乳发酵短杆菌(谷氨酸棒杆菌)ATCC 13869玫瑰色短杆菌ATCC 13825解糖短杆菌ATCC 14066生硫短杆菌ATCC 19240产氨棒杆菌ATCC 6871，ATCC 6872白色短杆菌ATCC 15111
蜡状短杆菌ATCC 15112嗜氨微杆菌ATCC 15354。
这些菌株可从例如美国典型培养物保藏中心(ATCC，地址P.O.Box 1549，Manassas，VA 20108，United States of America)获得。每个菌株被分配了登记号，人们可以根据它的登记号请求提供每种菌株。在美国典型培养物保藏中心的目录中指明了每个菌株的登记号。根据布达佩斯条约的规定，于1987年10月27日将AJ12340菌株保藏在通商产业省工业技术院生命工学工业技术研究所(the National Institute of Bioscience and Human-Technology，Agency ofIndustrial Science and Technology，Ministry of International Trade and Industry)(目前为，独立行政法人，产业技术综合研究所，特许微生物保藏中心(theindependent administrative agency，National Institute of Advanced IndustrialScience and Technology，International Patent Organism Depositary)(TsukubaCentral 6，1-1，Higashi 1-Chome，Tsukuba-shi，Ibaraki-ken，Japan，邮编305-5466))，收到的登记号是FERM BP-1539。根据根据布达佩斯条约的规定，于1989年1月5日将AJ12418菌株保藏在通商产业省工业技术院生命工学工业技术研究所，收到的登记号是FERM BP-2205。
术语“芽孢杆菌属细菌”是指根据微生物学领域的技术人员已知的分类法被分类为芽孢杆菌属的细菌。在本发明中，芽孢杆菌属细菌包括，但不限于，枯草芽孢杆菌168 Marburg菌株(ATCC 6051)、枯草芽孢杆菌PY79菌株(Plasmid，1984，12，1-9)等等，解淀粉芽孢杆菌的实例包括，但不限于，解淀粉芽孢杆菌T菌株(ATCC 23842)、解淀粉芽孢杆菌N菌株(ATCC 23845)等等。
通过将上述DNA(pkt基因)导入到已经具有生产有用代谢物的能力的细菌中，可以获得本发明的细菌。做为选择，通过使已经导入了pkt基因的细菌赋予生产有用代谢物的能力，可以获得本发明的细菌。在后者的情况中，导入pkt基因和赋予生产有用代谢物的能力可以按任何顺序进行。
用语“具有生产有用代谢物的能力的细菌”是指一种细菌，当本发明的细菌在培养基中培养时它具有产生和引起有用代谢物在培养基中积累的能力。通过培育，包括诱变处理和遗传修饰，可以赋予或增强生产代谢物的能力。用语“ 细菌具有生产有用代谢物的能力”是指细菌能够产生和引起代谢物以大于野生型菌株或未突变菌株的量在培养基中积累。在此使用的用语“细菌具有生产有用代谢物的能力”还指一种细菌，其能够产生和引起目标代谢物以不低于0.5g/L、更优选不低于1.0g/L的量在培养基中积累。
用语“有用代谢物”是指初级代谢物，例如L-氨基酸、有机酸、维生素、糖类和/或Embden-Meyerhof、戊糖磷酸(pentose-P)途径、Entner-Doudoroff途径、柠檬酸循环和氨基酸生物合成的中间物。在本发明中要生产的L-氨基酸没有特别的限制，包括L-赖氨酸、L-精氨酸、L-鸟氨酸、L-组氨酸、L-瓜氨酸(L-citrulline)、L-异亮氨酸、L-丙氨酸、L-缬氨酸、L-亮氨酸、和L-甘氨酸、L-苏氨酸、L-丝氨酸、L-脯氨酸、L-苯丙氨酸、L-酪氨酸和L-色氨酸、L-半胱氨酸、L-胱氨酸、L-甲硫氨酸、L-鸟氨酸、L-谷氨酸、L-天冬氨酸、L-谷氨酰胺和L-天冬酰胺。更优选地，本发明的细菌是具有生产源自乙酰CoA的代谢物的能力的细菌。这种代谢物选自由L-谷氨酸、L-谷氨酰胺、L-脯氨酸、L-精氨酸、L-亮氨酸、L-半胱氨酸、琥珀酸酯(succinate)和聚羟基丁酸酯构成的组。
以下，将详细描述赋予了生产有用代谢物的能力的菌株的实例和赋予这种能力的方法。
在下文中，将说明给如上所述的亲本菌株赋予生产有用的代谢物能力的方法。
为了赋予生产有用代谢物的能力，可以使用通常用来培育属于埃希氏菌属或棒状杆菌型细菌的生产有用代谢物的细菌的方法。例如，可以使用用于获得具有生产有用代谢物能力的营养缺陷突变菌株、类似物抗性菌株或代谢调节突变菌株的方法，和用于产生具有增强的有用代谢物生物合成酶活性的重组菌株的方法(″Amino Acid Fermentation″，the Japan Scientific SocietiesPress[Gakkai Shuppan Center]，1st Edition，published on May 30，1986，pp.77-100)。当使用这些方法培育生产有用代谢物的细菌时，可以赋予一种或多种性质，包括营养缺陷型、类似物抗性和代谢调节突变。
当产生重组菌株时，可以增强单个或多个有用代谢物生物合成酶的活性。此外，赋予营养缺陷型、类似物抗性和代谢调节突变性质的方法可以与增强有用代谢物生物合成酶活性的方法组合。
具有生产有用代谢物的能力的营养缺陷突变菌株(有用代谢物例如L-氨基酸)、类似物抗性菌株或代谢调节突变菌株，可以通过使亲本或野生型菌株经过通常的诱变处理，例如X射线或紫外线照射、用诱变试剂例如N-甲基-N′-硝基-N-亚硝基胍(NTG)处理来获得。然后，可以从突变的菌株中挑选具有生产有用代谢物能力的营养缺陷菌株、类似物抗性菌株或代谢调节突变菌株。
为如上所述的细菌赋予生产L-谷氨酸的能力的方法包括，例如，修饰所述细菌使得编码涉及L-谷氨酸生物合成的酶的基因表达增强和/或过量表达。涉及L-谷氨酸生物合成的酶的实例包括谷氨酸脱氢酶(以下也称为“GDH”)、谷氨酰胺合成酶、谷氨酸合酶、异柠檬酸脱氢酶、乌头酸水合酶、柠檬酸合酶(以下也称为“CS”)、磷酸烯醇丙酮酸羧化酶(以下也称为“PEPC”)、丙酮酸羧化酶、丙酮酸脱氢酶、丙酮酸激酶、磷酸烯醇丙酮酸合酶、烯醇化酶、磷酸甘油酸变位酶、磷酸甘油酸激酶、甘油醛-3-磷酸脱氢酶、磷酸丙糖异构酶、果糖二磷酸醛缩酶、磷酸果糖激酶、葡萄糖磷酸异构酶，等等。在这些酶中，优选CS、PEPC和GDH中一种或多种的活性被增强，更优选的是所有这三种酶的活性被增强。
通过如上所述方法修饰、从而CS基因、PEPC基因和/或GDH基因的表达被增强的细菌的实例包括在US6,197,559、US6,331,419和欧洲专利公开No.0999282和No.1078989中公开的细菌。
修饰细菌来赋予L-谷氨酸生产能力可以通过降低或失活酶的活性来进行，所述酶催化从L-谷氨酸生物合成途径分支的、并产生L-谷氨酸以外的化合物的反应。催化从L-谷氨酸生物合成途径分支的并产生L-谷氨酸以外的化合物的反应的酶的实例包括，2-酮戊二酸脱氢酶、异柠檬酸裂解酶、谷氨酸脱羧酶、1-吡咯啉脱氢酶，等等。在这些酶中，优选的是降低或消除2-酮戊二酸脱氢酶的活性。
为了降低或失活上述酶的活性，可以通过普通的诱变处理或遗传工程来导入降低或失活酶的细胞内活性的突变。诱变处理的实例包括通过X-射线或紫外线照射、用诱变剂例如N-甲基-N’-硝基-N-亚硝基胍处理，等等。降低或消除酶的细胞内活性的方法的实例包括突变或缺失微生物的细胞内编码所述酶的基因，使得与非突变菌株相比细胞内活性被降低或消除。突变或缺失基因的方法的实例包括修饰表达调节序列，例如启动子和Shine-Dalgarno(SD)序列，向开放阅读框中导入错义突变、无义突变、或移码突变，和缺失基因的部分(J Biol Chem.1997 272(13)8611-7)。可以通过使用同源重组技术或通过使用转座子或IS因子将突变的基因导入到微生物中，在所述同源重组技术中染色体上的野生型基因被取代为所述突变的基因。同源重组技术包括使用线性DNA、温度敏感质粒和非可复制质粒的方法。在Proc Natl Acad Sci USA.2000 Jun 6；97(12)6640-5、美国专利No.6303383、JP05-007491A等中描述了这些方法。
通过使用获自候选菌株的细胞提取物或其纯化的级分测量酶活性并将它与野生型或未修饰的菌株比较，可以确认目标酶的细胞内活性和活性下降的程度。例如，2-酮戊二酸脱氢酶活性可以通过Reed等的方法(Reed L.J.和Mukherjee B.B.，Methods in Enzymology，13，pp.55-61，1969)来测量。在美国专利5,378,616和5,573,945中描述了在α-酮戊二酸脱氢酶活性上有缺陷或具有降低的α-酮戊二酸脱氢酶活性、属于埃希氏菌属的细菌的实例，和获得它们的方法。具体地，这些菌株包括如下大肠杆菌W3110sucA::Kmr大肠杆菌AJ12624(FERM BP-3853)大肠杆菌AJ12628(FERM BP-3854)大肠杆菌AJ12949(FERM BP-4881)通过破坏大肠杆菌W3110中的α-酮戊二酸脱氢酶基因(以下称为“sucA基因”)获得了大肠杆菌W3110sucA::Kmr。这个菌株是在α-酮戊二酸脱氢酶上完全缺陷的。
在WO95/34672和美国专利5,977,331中详细阐述了在其中使用同源重组破坏了sucA基因的棒状杆菌型细菌。
生产L-谷氨酸的细菌的其他实例包括属于埃希氏菌属并对天冬氨酸抗代谢物具有抗性的那些。这些菌株也可以是在α-酮戊二酸脱氢酶活性中有缺陷的，并包括，例如，菌株AF13199(FERM BP-5807)(美国专利5,908,768)，或菌株FFRM P-12379，其被额外修饰以具有降低的分解L-谷氨酸的能力(美国专利5,393,671)；大肠杆菌菌株AJ13138(FERM BP-5565)(美国专利6,110,714)等等。
属于泛菌属的生产L-谷氨酸的细菌的实例包括在α-酮戊二酸脱氢酶活性中有缺陷的或具有降低的α-酮戊二酸脱氢酶活性的突变菌株，而且可以如上所述获得。这种菌株包括Pantoea ananatis AJ13356或AJ13601(美国专利6,331,419)。Pantoea ananatis AJ13356菌株于1998年2月19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(目前为，产业技术综合研究所，特许微生物保藏中心，Central 6，1-1，Higashi 1-Chome，Tsukuba-shi，Ibaraki-ken，305-8566，日本)，收到的登记号是FERM P-16645。然后它于1999年1月11日根据布达佩斯条约的规定转为国际保藏，接受的登记号是FERM BP-6615。Pantoea ananatis AJ13601于1999年8月18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(目前为，产业技术综合研究所，特许微生物保藏中心，Central 6，1-1，Higashi 1-Chome，Tsukuba-shi，Ibaraki-ken，305-8566，日本)，收到的登记号是FERM P-17516。然后它于2000年7月6日根据布达佩斯条约的规定转为国际保藏，接受的登记号是FERM BP-7207。作为破坏编码αKGDH的F1亚基的基因(sucA基因)的结果，Pantoea ananatis AJ13356和AJ13601在α-KGDH活性中是有缺陷的。当分离上述菌株时它们被鉴定为成团肠杆菌，并分别作为成团肠杆菌AJ13356菌株和AJ13601菌株保藏。然而，在16S rRNA的核苷酸测序等的基础上，它们近来被重新分类为Pantoea ananatis。虽然AJ13356、AJ13601和它们的亲本AJ13355(FERM BP-6614)都作为成团肠杆菌在上述保藏单位保藏，为了本说明书的目的它们被描述为Pantoea ananatis。
具有生产L-谷氨酸的能力的棒状杆菌型细菌的其它实例包括有机酸类似物抗性突变体和escletin抗性突变体(JP56-1889A、JP56140895A、JP5702689A、JP88994A)。
类似物抗性的生产L-谷氨酸的棒状杆菌型细菌的实例包括以下菌株黄色短杆菌AJ11355(FERM P-5007，JP56-1889A)谷氨酸短杆菌(Brevibacterium glutamicum)AJ11368(FERM P-P-5020，JP56-1889A)黄色短杆菌AJ11217(FERM P-4318，JP57-2689A)黄色短杆菌AJ11218(FERM-P 4319，JP57-2689A)黄色短杆菌AJ11564(FERM P-5472，JP56-140895A)黄色短杆菌AJ11439(FERM P-5316，JP56-35981A)谷氨酸棒杆菌H7684(FERM BP-3004，JP56-151495A)可以用作亲本菌株来修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性的生产L-谷氨酰胺的细菌的实例包括，生产L-谷氨酰胺的棒状杆菌型细菌，其被修饰以具有增强的谷氨酸脱氢酶活性、谷氨酰胺合成酶活性(EP 1229121A)，或具有降低的谷氨酰胺酶活性(EP1424397A)。并且，可以使用携带突变谷氨酰胺合成酶、属于埃希氏菌属的细菌，在所述突变谷氨酰胺合成酶中相应于野生型谷氨酰胺合成酶中397位的酪氨酸氨基酸残基被任何氨基酸残基取代。
赋予6-重氮-5-氧-正亮氨酸抗性(JP3-232497A)、嘌呤类似物抗性、甲硫氨酸亚砜抗性(JP 61-202694A)、α-Ketomalinic acid抗性(JP56-151495)的方法可以用于通过培育来赋予或增强生产L-谷氨酰胺的能力。具有生产L-谷氨酰胺的能力的棒状杆菌型细菌的具体实例包括，但不限于黄色短杆菌AJ11573(FERM P-5492，JP56-161495A)黄色短杆菌AJ11576(FERMBP-10381，JP56-161495A)黄色短杆菌AJ12212(FERM P-8123，JP61-202694A)用作亲本菌株、被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性的生产L-脯氨酸的细菌的实例包括属于埃希氏菌属的生产L-脯氨酸的细菌，所述细菌携带在L-脯氨酸的反馈抑制中脱敏的(desensitized)γ-谷氨酰激酶，和/或其中L-脯氨酸降解系统被破坏。通过将编码在L-脯氨酸的反馈抑制中脱敏的γ-谷氨酰激酶的DNA导入细胞中(Dandekar，A.M.，Uratsu，S.L.，J.Bacteriol.，170，12，5943-5(1988))，例示了培育具有在L-脯氨酸的反馈抑制中脱敏的γ-谷氨酰激酶的细菌的方法。通过将突变导入脯氨酸脱氢酶基因中从而不表达活性的脯氨酸脱氢酶，例示了破坏L-脯氨酸降解系统的方法。并且，通过获得在L-脯氨酸同化能力中有缺陷的菌株并且通过使用L-脯氨酸营养缺陷型作为指标选择在细胞外过度生产L-脯氨酸的菌株，可以获得L-脯氨酸降解系统被破坏的细菌。属于埃希氏菌属的生产L-脯氨酸的细菌包括大肠杆菌菌株NRRL B-12403和NRRL B-12404(GB专利2075056)、VKPM B-8012(美国专利Laid-open Publication 2002-0058315)，和DE专利3127361中描述的质粒突变体、Bloom F.R.等描述的质粒突变体(The15thMiami winter symposium，1983，p.34)等等可以用于获得这种菌株。
可以用作亲本菌株被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性的生产L-精氨酸的细菌的实例包括属于埃希氏菌属的生产L-精氨酸的细菌，例如大肠杆菌菌株237(VKPM B-7925)和具有突变N-乙酰谷氨酸合成酶的它的衍生菌株(美国专利Laid-open Publication2002-0034793)，其中导入了编码N-乙酰谷氨酸合成酶的argA基因的生产精氨酸的菌株(JP57-5693A)等等。
生产L-精氨酸的细菌的其他实例是具有编码L-精氨酸生物合成酶的扩增的基因的菌株，所述L-精氨酸生物合成酶例如N-乙酰基谷氨酸合酶(argA)、N-乙酰谷氨酰磷酸还原酶(N-acetylglutamyl phosphate reductase)(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)、鸟氨酸氨甲酰转移酶(argF)、精氨琥珀酸合酶(argG)、精氨琥珀酸裂解酶(argH)和氨甲酰基磷酸酯合酶(carAB)。编码这些酶的基因的名称分别在酶的名字后的括号中给出。
可以用作亲本菌株被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性的生产L-亮氨酸的细菌的实例包括属于埃希氏菌属的生产L-亮氨酸的细菌，例如对4-氮亮氨酸(4-azaleucine)或5，5，5-三氟亮氨酸有抗性的大肠杆菌菌株H-9068(ATCC 21530)、H-9070(FERM BP-4704)和H-9072(FERM BP-4706)(美国专利5,744,331)，其中L-亮氨酸的异丙基苹果酸合酶反馈抑制被脱敏的大肠杆菌菌株(EP1067191B)、对β-2-噻吩丙氨酸和β-羟基亮氨酸有抗性的大肠杆菌菌株AJ11478(美国专利5,763,231)，大肠杆菌菌株57(VKPM B-7386，俄国专利No.2140450)，等等。
可以用作亲本菌株被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性的生产L-半胱氨酸的细菌的实例包括属于埃希氏菌属的生产L-半胱氨酸的细菌，例如大肠杆菌菌株JM15，其用编码反馈抗性丝氨酸酰基转移酶的不同cysE等位基因转化(美国专利6,218,168)；大肠杆菌菌株W3110，其过量表达适合于分泌细胞毒性物质的蛋白质的基因(美国专利5,972,663)；具有降低的半胱氨酸脱硫水化酶(cysteine desulfohydrase)活性的大肠杆菌菌株(JP11-155571A2)；对由cysB基因编码的半胱氨酸调节子使用阳性转录调节物活性增加的大肠杆菌菌株W3110(WO01/27307A1)等等。
可以用作亲本菌株被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性的生产琥珀酸酯的细菌的实例包括属于埃希氏菌属和棒状杆菌型细菌的生产琥珀酸的细菌，例如黄色短杆菌MJ233Δ1dh菌株(JP11-206385A)，和黄色短杆菌MJ233/pPCPYC菌株(WO01/27258)。
可以用作亲本菌株被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性的生产L-赖氨酸的细菌的实例包括乳发酵短杆菌AJ11082的S-(2-氨基乙基)半胱氨酸(以下“AEC”)抗性突变菌株(NRRLB-11470)(在JP56-1914B、JP56-1915B、JP57-14157B、JP57-14158B、JP57-30474B、JP58-10075B、JP59-4993B、JP61-35840B、JP62-24074B、JP62-36673B、JP5-11958B、JP7-112437B和JP7-112438B中描述)，对氨基酸例如L-高丝氨酸有营养缺陷的突变菌株(JP48-28078B和JP56-6499B)，对AEC有抗性和对氨基酸例如L-亮氨酸、L-高丝氨酸、L-脯氨酸、L-丝氨酸、L-精氨酸、L-丙氨酸和L-缬氨酸有营养缺陷的突变菌株(美国专利Nos.3,708,395和3,825,472)，对DL-α-氨基-ε-己内酰胺、α-氨基-月桂基内酰胺(α-amino-lauryllactam)、天冬氨酸类似物、磺胺药(sulfa drug)、醌型化合物(quinoid)、和N-月桂酰亮氨酸有抗性的生产L-赖氨酸的突变菌株，对草酰乙酸酯脱羧酶抑制物或呼吸道酶抑制物有抗性的生产L-赖氨酸的突变菌株(JP50-53588A、JP50-31093A、JP52-102498A、JP53-9394A、JP53-86089A、JP55-9783A、JP55-9759A、JP56-32995A、JP56-39778A、JP53-43591B和JP53-1833B)，对环己六醇或乙酸有营养缺陷的生产L-赖氨酸的突变菌株(JP55-9784A和JP56-8692A)，在34℃或更高温度下对氟丙酮酸(fluoropyruvicacid)敏感的生产L-赖氨酸的突变菌株(JP55-9783A和JP53-86090A)，对乙二醇有抗性的短杆菌属或棒杆菌属细菌的生产L-赖氨酸的突变菌株(美国专利No.4,411,997)，等等。
可以用作亲本菌株被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性的生产L-赖氨酸的细菌的其它实例包括大肠杆菌WC196的S-(2-氨基乙基)半胱氨酸(以下称“AEC”)抗性突变菌株(WO96/17930)。WC196菌株被命名为大肠杆菌AJ13069菌株，于1994年12月6日保藏在独立行政法人，产业技术综合研究所，特许微生物保藏中心(TsukubaCentral 6，1-1，Higashi 1-Chome，Tsukuba-shi，Ibaraki-ken，Japan，邮政编码305-8566))，收到的登记号是FERM P-14690。之后，该保藏于1995年9月29日根据布达佩斯条约的规定转为国际保藏，收到的登记号是FERM BP-5252。
具有生产L-苏氨酸的能力的微生物的实例包括6-二甲基氨基嘌呤-抗性突变体(JP5-304969A)，其中具有增强酶活性的突变的苏氨酸生物合成酶基因用质粒来扩增的菌株(JP1-29559B，JP05-227977A)，其中苏氨酸操纵子用质粒来扩增的菌株(JP2-109985A)，其中扩增了编码丙酮酸羧化酶的基因和编码烟酰胺核苷酸转氢酶(nicotinamide nucleotide transhydrogenase)的基因的菌株(JP2002-51787A)，等等。
大肠杆菌VKPM B-3996菌株(美国专利No.5,175,107)也可以被用作生产L-苏氨酸的菌株。VKPM B-3996于1987年11月19日保藏在俄国国立工业微生物保藏中心(Russian National Collection of Industrial Microorganisms)(VKPM)，GNII Genetika，登记号为VKPM B-3996。VKPM B-3996菌株携带质粒pVIC40(国际专利公开WO90/04636)，该质粒是通过向具有链霉素抗性标记基因的质粒pAYC32中插入苏氨酸操纵子(thrABC)获得的(Chistorerdov，A.Y.，Tsygankov，Y.D.，Plasmid，1986，16，161-167)。由包含在pVIC40中的突变thrA基因编码的天冬氨酸激酶I-高丝氨酸脱氢酶I不受(is released from)L-苏氨酸的反馈抑制。
大肠杆菌VKPM B-5318菌株(EP 0593792B)也可被用作生产L-苏氨酸的菌株。VKPM B-5318于1987年11月19日保藏在俄国国立工业微生物保藏中心(VKPM)，GNII Genetika，登记号为VKPM B-5318。VKPM B-5318菌株对于L-异亮氨酸是营养缺陷的，编码苏氨酸生物合成酶的苏氨酸操纵子位于C1温度敏感阻遏物、PR-启动子和来源于λ噬菌体的Cro蛋白质N末端的下游。此外，这个菌株含有质粒DNA，其被构建使得苏氨酸生物合成基因的表达被来自λ噬菌体的启动子和阻遏物调节。
此外，大肠杆菌MG442菌株(US4,278,765)也可以用作生产L-苏氨酸的菌株。MG442菌株作为CMIMB-1628保藏在俄国国立工业微生物保藏中心(VKPM)。
具有生产L-苯丙氨酸的能力的埃希氏菌属细菌的实例包括其中破坏了tyrA和tyrR基因的大肠杆菌AJ12739(tyrA::Tn10，TyrR；VKPM B-8197)，其中导入突变的pheA的大肠杆菌HW1089(美国专利No.5,354,672)，和其中扩增了yddG和yedA基因的大肠杆菌菌株(WO 03/044192)。具有生产L-苯丙氨酸的能力的棒状杆菌型细菌的实例包括对于酪氨酸是营养缺陷的和对L-苯丙氨酰-L-酪氨酸有抗性的菌株(JP5-49489A)。
具有生产L-色氨酸的能力的细菌可以通过增强L-色氨酸生物合成酶，包括磷酸甘油酸脱氢酶和邻氨基苯甲酸合酶的活性来获得。这些酶可以是对L-色氨酸或L-丝氨酸的反馈抑制有抗性的。例如，具有这些反馈抗性酶的细菌可以通过将质粒pGH5导入大肠杆菌SV164菌株来获得，所述质粒pGH5含有编码L-色氨酸抗性磷酸甘油酸脱氢酶的突变serA基因，所述大肠杆菌SV164菌株带有编码L-丝氨酸抗性邻氨基苯甲酸合酶的基因(WO94/08031)。
具有生产L-色氨酸的能力的细菌也可以通过增强由色氨酸操纵子编码的L-色氨酸生物合成酶的活性来获得。L-色氨酸操纵子中的这些酶包括色氨酸合酶和邻氨基苯甲酸合酶。这些细菌的实例包括其中导入了色氨酸操纵子的大肠杆菌菌株，所述色氨酸操纵子含有编码L-丝氨酸抗性邻氨基苯甲酸合酶的基因(JP57-71397A、JP62-244382A和美国专利No.4,371,614)。
此外，具有生产L-色氨酸的能力的细菌的实例包括对L-苯丙氨酸和L-酪氨酸是营养缺陷的大肠杆菌AGX17(pGX44)[NRRL B-12263]，和携带含有色氨酸操纵子的质粒pGX50的AGX6(pGX50)aroP[NRRL B-12264)(美国专利No.4,371,614)。
具有生产L-异亮氨酸的能力的埃希氏菌属细菌的实例包括对6-二甲基氨基嘌呤有抗性的突变菌株(JP5-304969A)，对L-异亮氨酸异羟肟酸(L-isoleucinehydroxamate)、硫代异亮氨酸(thiaisoleucine)、DL-乙硫氨酸(ethionine)或精氨酸异羟肟酸(argininehydroxamate)有抗性的突变菌株(JP5-130882A)，和其中编码苏氨酸脱氨酶和乙酰羟酸合酶(acetohydroxylicacid synthase)的基因用质粒扩增了的重组菌株(JP2-458A、JP2-42988A和JP8-47397A)。
具有生产L-缬氨酸的能力的细菌可以通过增强L-缬氨酸生物合成酶的活性来获得，所述L-缬氨酸生物合成酶包括由ilvGMEDA操纵子编码的那些，特别是由ilvG基因编码的乙酰羟酸合酶(acetohydroxylate synthase)(JP02-748418B)。这些酶可以是对L-缬氨酸的反馈抑制有抗性的。
具有生产L-缬氨酸的能力的细菌包括具有减少的乙酰乳酸合酶III基因(ilvIH基因)的表达的那些细菌。
具有生产L-缬氨酸的能力的细菌可以对氨基酸类似物具有抗性。这种细菌的实例包括对L-异亮氨酸和L-甲硫氨酸是营养缺陷的并且对D-核糖、嘌呤核苷或嘧啶核糖核苷有抗性的突变菌株(FERM P-1841，P-5556；JP53-025034A)，和对类聚酮(polyketonoid)有抗性的突变菌株(FERM P-9325；JP04-045314B)。
具有生产L-丙氨酸的能力的细菌的实例包括在H+-ATPase活性方面有缺陷的棒状杆菌型细菌(Appl Microbiol Biotechnol.2001 Nov；57(4)534-40)，或其中扩增了天冬氨酸β-脱羧酶基因的棒状杆菌型细菌菌株(JP07-163383A)。
此外，优选本发明的细菌被进一步修饰以具有降低的6-磷酸果糖激酶活性。可以通过例如Denise Kotlars和Henri Buc描述的方法(Methods inEnzymology(1982)9060-70)来测量6-磷酸果糖激酶活性。在本发明的细菌中将6-磷酸果糖激酶的活性降低至低于野生型或未修饰菌株，优选低于野生型或未修饰菌株的90％、更优选低于70％、最优选不低于50％。通过突变或破坏编码这个酶的基因，可以降低6-磷酸果糖激酶的活性，其中所述方法可以类似于用于降低α-酮戊二酸脱氢酶活性的方法。
在本发明中，通过在培养基中培养上述细菌、容许有用代谢物积累到培养基和/或细菌细胞中、并从培养基和/或细菌细胞收集所述有用代谢物，可以生产有用代谢物。
培养、收集和从培养基纯化有用代谢物，可以通过使用细菌生产有用代谢物的常规发酵方法进行。培养基可以是合成的或天然的，只要培养基含有碳源和氮源和矿物质，而且如有必要，含有细菌生长所需的适量营养物。碳源包括各种碳水化物，例如葡萄糖和蔗糖，和各种有机酸。取决于所选细菌的同化方式，可以使用醇，包括乙醇和甘油。对于氮源，可以使用氨，硫酸铵，其他氮化合物例如胺、天然的氮源例如蛋白胨、大豆水解产物和消化的发酵微生物。对于矿物质，可以使用单磷酸钾(potassium monophosphate)、硫酸镁、氯化钠、硫酸亚铁、硫酸锰、氯化钙等等。在本发明中，更可取的是向培养基中添加盐酸硫胺素(thiamine hydrochloride)(维生素B1)。盐酸硫胺素的浓度是大于10μg/L，优选大于10mg/L，更优选低于100mg/L。
培养优选在有氧条件，例如通气的摇动培养和搅拌培养，在20到42℃、优选37到40℃的温度下进行。培养基的pH值通常在5和9之间，优选在6.5和7.2之间。可以用氨、碳酸钙、各种酸、各种碱和缓冲液来调节培养基的pH值。通常，1到5天的培养足以在液体培养基中积累目标的有用代谢物。
培养之后，可以通过离心或膜过滤从液体培养基除去固体例如细胞，然后可以通过离子交换、浓缩和/或结晶方法等收集和纯化目标的有用代谢物。
实施例以下将参考以下非限制性实施例更具体地说明本发明。缩写Pyr-丙酮酸(pyruvate)，PEP-磷酸烯醇丙酮酸，GA-3P-甘油醛-3-磷酸，AceCoA-乙酰CoA。理论重量得率(theoretical weight yield)(Y)计算为Y＝(产物的分子量×摩尔数)/(底物的分子量×摩尔数)。反应式被简化并仅显示碳化合物(carbon compounds)和能量分子。
实施例1.计算具有或不具有磷酸转酮酶活性的细菌的乙酰CoA形成1.不包含磷酸转酮酶活性的乙酰CoA生物合成途径的反应其后是糖酵解的PTS系统给出以下反应式葡萄糖＝PEP+Pyr+ATP+2 NADH由ppc基因编码的磷酸烯醇丙酮酸羧化酶催化以下反应PEP+CO2＝草酰乙酸由pdh基因编码的丙酮酸脱氢酶催化以下反应Pyr＝乙酰CoA+CO2+NADH最终反应式是葡萄糖＝乙酰CoA+草酰乙酸+ATP+3NADH(A)2.包含磷酸转酮酶活性的乙酰CoA生物合成途径的反应其后是糖酵解的PTS系统给出以下反应式葡萄糖+PEP＝果糖-6-磷酸+Pyr磷酸烯醇丙酮酸合酶催化以下反应Pyr+ATP＝PEP由ppc基因编码的磷酸烯醇丙酮酸羧化酶催化以下反应PEP+CO2＝草酰乙酸果糖-6-磷酸磷酸转酮酶磷酸+果糖-6-磷酸＝H2O+赤藓糖-4-磷酸+乙酰磷酸转醛醇酶(transaldolase)和转酮醇酶(transketolase)果糖-6-磷酸+赤藓糖-4-磷酸＝2木酮糖-5-磷酸D-木酮糖-5-磷酸磷酸转酮酶磷酸+木酮糖-5-磷酸＝甘油醛-3-磷酸+乙酰磷酸糖酵解甘油醛-3-磷酸＝PEP+2ATP+2NADH由pta基因编码的磷酸转乙酰酶乙酰磷酸＝乙酰CoA最终反应式是2葡萄糖+2CO2＝3乙酰CoA+2草酰乙酸+NADH(B)比较反应式(A)和(B)显示，使用磷酸转酮酶的活性容许从1分子葡萄糖产生1.5分子的乙酰CoA并保存1个CO2分子，相比之下，在没有磷酸转酮酶活性的葡萄糖代谢中从1分子葡萄糖获得1分子的乙酰CoA。
实施例2.计算使用具有或不具有磷酸转酮酶活性的细菌时L-谷氨酸生产的理论得率。
1.包括PTS、糖酵解和TCA循环的L-谷氨酸生物合成途径的反应。
PTS+糖酵解葡萄糖＝乙酰CoA+草酰乙酸+ATP+3NADH(A)TCA循环乙酰CoA+草酰乙酸＝2-酮戊二酸+NADPH+CO2谷氨酸脱氢酶2-酮戊二酸+NH3+NADPH＝谷氨酸最终反应式葡萄糖＝谷氨酸+ATP+3NADH+CO2(C)L-谷氨酸的理论重量得率是81.7％2.包括糖酵解、非氧化性PPC和仅D-木酮糖-5-磷酸磷酸转酮酶的L-谷氨酸生物合成途径的反应糖酵解和非氧化性PPC5葡萄糖+5PEP＝5果糖-6-磷酸+5Pyr，其中果糖-6-磷酸+ATP＝2GA-3P2果糖-6-磷酸+2GA-3P＝2木酮糖-5-磷酸+2赤藓糖-4-磷酸2果糖-6-磷酸+2赤藓糖-4-磷酸＝4木酮糖-5-磷酸总反应式5葡萄糖+5PEP+ATP＝6木酮糖-5-磷酸+5Pyr然后，D-木酮糖-5-磷酸磷酸转酮酶木酮糖-5-磷酸+磷酸＝GA-3P+乙酰磷酸糖酵解和磷酸转乙酰酶GA-3P＝PEP+ATP+NADH乙酰磷酸＝乙酰CoA总反应式
5葡萄糖＝6乙酰CoA+PEP+5ATP+6NADH+5PYR (1)磷酸烯醇丙酮酸合酶PYR+2ATP＝PEP+磷酸假定NADH＝2ATP总反应式5葡萄糖＝6乙酰CoA+6PEP+7ATP磷酸烯醇丙酮酸羧化酶(ppc)PEP+CO2＝草酰乙酸总反应式5葡萄糖+6CO2＝6乙酰CoA+6草酰乙酸+7ATP(2)TCA循环草酰乙酸+乙酰CoA＝酮戊二酸+CO2+NADPH总反应式5葡萄糖＝6酮戊二酸+6NADPH+7ATP谷氨酸脱氢酶酮戊二酸+NADPH＝谷氨酸最终反应式5葡萄糖＝6谷氨酸+7ATP(D)L-谷氨酸的理论重量得率是98％3.包括糖酵解、非氧化性PPC、两种磷酸转酮酶和乙醛酸支路的L-谷氨酸生物合成途径的反应PTS+糖酵解2葡萄糖+2PEP＝2果糖-6-磷酸+2PYR果糖-6-磷酸磷酸转酮酶2果糖-6-磷酸+磷酸＝赤藓糖-4-磷酸+乙酰磷酸转醛醇酶和转酮醇酶果糖-6-磷酸+赤藓糖-4-磷酸＝2木酮糖-5-磷酸木酮糖-5-磷酸磷酸转酮酶2木酮糖-5-磷酸+2磷酸＝2GA-3P+2乙酰磷酸总反应式
2葡萄糖+2PEP＝2GA-3P+3乙酰磷酸+2PYR糖酵解GA-3P＝PEP+ATP+NADH总反应式2葡萄糖＝3乙酰磷酸+2PYR+2ATP+2NADH(3)磷酸烯醇丙酮酸合酶PYR+2ATP＝PEP磷酸转乙酰酶乙酰磷酸＝乙酰CoA总反应式2葡萄糖＝3乙酰CoA+2PEP+NADH磷酸烯醇丙酮酸羧化酶PEP+CO2＝草酰乙酸总反应式2葡萄糖+2CO2＝3乙酰CoA+2草酰乙酸+NADH或(4)6葡萄糖+6CO2＝9乙酰CoA+6草酰乙酸+3NADH乙醛酸支路2乙酰CoA＝琥珀酸+NADHTCA循环琥珀酸＝草酰乙酸+ATP+NADH总反应式6葡萄糖+6CO2＝7乙酰CoA+7草酰乙酸+ATP+5NADHTCA循环乙酰CoA+草酰乙酸＝2-酮戊二酸+NADPH+CO2谷氨酸脱氢酶2-酮戊二酸+NH3+NADPH＝谷氨酸最终反应式6葡萄糖＝7谷氨酸+ATP+5 NADH+CO2或6葡萄糖＝7谷氨酸+11ATP+CO2(E)L-谷氨酸的理论重量得率是95.3％
比较反应式(C)、(D)和(E)显示，使用磷酸转酮酶活性显著地增加了L-谷氨酸生物合成的理论得率，与在没有磷酸转酮酶活性的葡萄糖代谢中从1分子葡萄糖获得1分子L-谷氨酸相比，容许产生比利用的葡萄糖分子多1个分子的L-谷氨酸，并防止了CO2释放。
实施例3.计算使用具有或不具有磷酸转酮酶活性的细菌时琥珀酸生产的理论得率。
1.包括PTS、糖酵解和TCA循环的琥珀酸生物合成途径的反应。
PTS+糖酵解葡萄糖＝PEP+PYR+ATP+2NADH磷酸烯醇丙酮酸羧化酶PEP+CO2＝草酰乙酸丙酮酸脱氢酶PYR＝乙酰CoA+CO2+NADH总反应式葡萄糖＝草酰乙酸+乙酰CoA+ATP+3NADHTCA循环草酰乙酸+乙酰CoA＝琥珀酸+NADPH+NADH+ATP+2CO2最终反应式葡萄糖＝琥珀酸+NADPH+4NADH+2ATP+2CO2或假定NADH＝NADPH＝2ATP葡萄糖＝琥珀酸+12ATP+2CO2(F)琥珀酸的理论重量得率是65％。
2.包括糖酵解、非氧化性PPC和仅D-木酮糖-5-磷酸磷酸转酮酶的琥珀酸生物合成途径的反应糖酵解、非氧化性PPC、D-木酮糖-5-磷酸磷酸转酮酶、磷酸转乙酰酶和磷酸烯醇丙酮酸羧化酶给出总反应式(参见实施例2，反应式2)5葡萄糖+6CO2＝6乙酰CoA+6草酰乙酸+7ATPTCA循环
草酰乙酸+乙酰CoA＝琥珀酸+NADPH+NADH+ATP+2CO2最终反应式5葡萄糖＝6琥珀酸+6CO2+6NADPH+25ATP或假定NADH＝NADPH＝2ATP5葡萄糖＝6琥珀酸+6CO2+37ATP(G)琥珀酸的理论重量得率是79％3.包括糖酵解、非氧化性PPC、两种磷酸转酮酶和乙醛酸支路的琥珀酸生物合成途径的反应糖酵解、非氧化性PPC、果糖-6-磷酸磷酸转酮酶、D-木酮糖-5-磷酸磷酸转酮酶、磷酸转乙酰酶和磷酸烯醇丙酮酸羧化酶给出总反应式(参见实施例2，反应式4)2葡萄糖+2CO2＝3乙酰CoA+2草酰乙酸+NADH或4葡萄糖+4CO2＝6乙酰CoA+4草酰乙酸+2NADHTCA循环草酰乙酸+乙酰CoA＝琥珀酸+NADPH+NADH+ATP+2CO2总反应式4葡萄糖＝4琥珀酸+2乙酰CoA+4CO2+4NADPH+6NADH乙醛酸支路2乙酰CoA＝琥珀酸+NADH最终反应式4葡萄糖＝5琥珀酸+4CO2+4NADPH+7NADH或假定NADH＝NADPH＝2ATP4葡萄糖＝5琥珀酸+4CO2+22ATP(H)琥珀酸的理论重量得率是Y＝82％比较反应式(F)、(G)和(H)显示，使用磷酸转酮酶活性显著地提高了琥珀酸生物合成的理论得率，与在没有磷酸转酮酶活性的葡萄糖代谢中从1分子葡萄糖获得1分子琥珀酸相比，容许产生比利用的葡萄糖分子多1个分子的琥珀酸，并防止了CO2释放。
实施例4.计算使用具有或不具有磷酸转酮酶活性的细菌时L-亮氨酸生产的理论得率。
1.包括PTS和糖酵解的L-亮氨酸生物合成途径的反应。
糖酵解葡萄糖＝2PYR+2ATP+2NADHL-亮氨酸生物合成2PYR+NADPH＝2-酮-异戊酸+CO22-酮-异戊酸+乙酰CoA+L-谷氨酸＝亮氨酸+NADH+2-酮戊二酸+CO2或2-酮-异戊酸+乙酰CoA＝亮氨酸+NADH-NADPH+CO2其中NADPH用于L-谷氨酸的再生丙酮酸脱氢酶PYR＝乙酰CoA+NADH+CO2总反应式3PYR＝亮氨酸+2NADH-2NADPH+3CO2最终反应式3葡萄糖＝2亮氨酸+4NADH-4NADPH+6CO2+6ATP+6NADH或假定NADH＝NADPH＝2ATP3葡萄糖＝2亮氨酸+18ATP+6CO2(I)L-亮氨酸的理论重量得率是Y＝48％2.包括糖酵解、非氧化性PPC和仅D-木酮糖-5-磷酸磷酸转酮酶的L-亮氨酸生物合成途径的反应糖酵解、非氧化性PPC、D-木酮糖-5-磷酸磷酸转酮酶和磷酸转乙酰酶给出总反应式(参见实施例2，反应式1)5葡萄糖＝6乙酰CoA+PEP+5ATP+6NADH+5PYR丙酮酸激酶PEP＝PYR+ATP总反应式5葡萄糖＝6乙酰CoA+6PYR+6ATP+6NADH
L-亮氨酸生物合成2PYR+乙酰CoA＝亮氨酸+NADH-2NADPH+2CO2总反应式5葡萄糖＝3亮氨酸++3乙酰CoA+6ATP+9NADH-6NADPH+6CO2(5)糖酵解的另一个作用给出3葡萄糖＝6PYR+18ATP(6)将反应式5和6相加3葡萄糖＝6PYR+18ATP+5葡萄糖＝3亮氨酸++3乙酰CoA+6ATP+9NADH-6NADPH+6CO28葡萄糖＝3亮氨酸+(6PYR+3Ace-CoA)+9NADH-6NADPH+24ATP+6CO2最终反应式8葡萄糖＝6亮氨酸+24ATP+12NADH-12NADPH+12CO2或假定NADH＝NADPH8葡萄糖＝6亮氨酸+12CO2+24ATP(J)L-亮氨酸的理论重量得率是Y＝55％3.包括糖酵解、非氧化性PPC、两种磷酸转酮酶的L-亮氨酸生物合成途径的反应。
糖酵解、非氧化性PPC、果糖-6-磷酸磷酸转酮酶、D-木酮糖-5-磷酸磷酸转酮酶给出总反应式(参见实施例2，反应式3)2葡萄糖＝3乙酰磷酸+2PYR+2ATP+2NADH磷酸转乙酰酶乙酰磷酸＝乙酰CoA总反应式2葡萄糖＝3乙酰CoA+2PYR+2ATP+2NADHL-亮氨酸生物合成
2PYR+乙酰CoA＝亮氨酸+NADH-2NADPH+2CO2总反应式2葡萄糖＝亮氨酸+2乙酰CoA+2ATP+3NADH+-2NADPH+2CO2(7)另一个糖酵解作用给出2葡萄糖＝3PYR+12ATP(8)将反应式7和8相加2葡萄糖＝亮氨酸+2乙酰CoA+2ATP+3NADH+-2NADPH+2CO2+2葡萄糖＝4PYR+12ATP＝4葡萄糖＝亮氨酸+(4PYR+2乙酰CoA)+14ATP+3NADH+-2NADPH+2CO2L-亮氨酸生物合成4PYR+2乙酰CoA＝2亮氨酸+2NADH-4NADPH+4CO2最终反应式4葡萄糖＝3亮氨酸+14ATP+5NADH+-6NADPH+6CO2或假定NADH＝NADPH＝2ATP4葡萄糖＝3亮氨酸+6CO2+12ATP(K)L-亮氨酸的理论重量得率是Y＝55％比较反应式(I)、(J)和(K)显示，使用磷酸转酮酶活性显著地增加了L-亮氨酸生物合成的理论产量。
实施例4.从植物乳杆菌克隆磷酸转酮酶基因(xpk1)并评估xpk1基因扩增对大肠杆菌生产有用代谢物的影响xpk1基因可以从植物乳杆菌菌株8PA3(VKPM B-7495)的染色体DNA克隆。根据报道的核苷酸序列，可以合成用于扩增xpk1基因的、SEQ ID NO7(引物1)和No.8(引物2)中描述的引物。引物1含有导入其5’-末端的HindIII识别位点。引物2含有导入其5’-末端的EcoRI识别位点。
植物乳杆菌菌株8PA3的染色体DNA可以用作PCR的模板，可以通过普通方法制备。可以使用“Applied Biosystems GeneAmp PCR系统2400”使用Fermentas Taq聚合酶(Fermentas，Lithuania)在以下条件下进行PCR在95℃初始DNA变性5min；然后30个循环的95℃变性30sec、55℃退火60sec和72℃延伸120sec；最后在72℃多聚化7min。可以用HindIII和EcoRI处理获得的PCR片段并将其插入到预先用相同酶处理的载体pMW119中，所述PCR片段含有带其自身SD序列且没有启动子序列的xpk1基因。因而，可以获得质粒pMW-xpk1。
用pMW-xpk1质粒转化生产有用代谢物的细菌菌株可以通过普通方法进行，以获得含有扩增的xpk1基因的菌株。
通过大肠杆菌菌株VL334thrC+-pMW-xpk1生产L-谷氨酸。
用pMW-xpk1质粒转化大肠杆菌L-谷氨酸生产菌株VL334thrC+(欧洲专利公开No.1172433)可以通过普通方法进行，以获得菌株VL334thrC+-pMW-xpk1。
两种菌株VL334thrC+和VL334thrC+-pMW-xpk1可以在L-琼脂平板上在37℃生长18-24小时。然后，可以将一环细胞转移到含有2ml发酵培养基的试管中。发酵培养基应含有60g/l葡萄糖、25g/l硫酸铵、2g/l KH2PO4、1g/lMgSO4、0.1mg/ml硫胺素、70μg/ml L-异亮氨酸和25g/l白垩(chalk)(pH7.2)。葡萄糖和白垩应单独地灭菌。培养可以在30℃摇动进行3天。在培养之后，积累的L-谷氨酸的积累量可以通过纸层析(液相组合物丁醇-乙酸-水＝4∶1∶1)及随后用茚三酮(ninhydrin)(丙酮中1％溶液)染色和进一步在含有0.5％CdCl2的50％乙醇中洗脱化合物来测定。
通过大肠杆菌菌株702ilvA-pMW-xpk1生产L-脯氨酸用pMW-xpk1质粒转化大肠杆菌L-脯氨酸生产菌株702ilvA(VKPMB-8012，俄国专利申请2000124295，欧洲专利公开NO.1172433)可以通过普通方法进行，以获得菌株702ilvA-pMW-xpk1。
两种菌株702ilvA和702ilvA-pMW-xpk1可以在L-琼脂平板上在37℃生长18-24小时。然后，这些菌株可以在如上所述的相同条件中培养。
通过大肠杆菌菌株57-pMW-xpk1生产L-亮氨酸用pMW-xpk1质粒转化大肠杆菌L-亮氨酸生产菌株57(VKPM B-7386，美国专利6,124,121)可以通过普通方法进行，以获得菌株57-pMW-xpk1。
两种菌株57和57-pMW-xpk1可以在L-琼脂平板上在37℃生长18-24小时。然后，这些菌株可以在如上所述的相同条件下培养，培养基中不添加异亮氨酸。
通过大肠杆菌菌株JM15(ydeD)-pMW-xpk1生产L-半胱氨酸用pMW-xpk1质粒转化大肠杆菌L-半胱氨酸生产菌株JM15(ydeD)可以通过普通方法进行，以获得菌株JM15(ydeD)-pMW-xpk1。
大肠杆菌菌株JM15(ydeD)是大肠杆菌菌株JM15(美国专利6,218,168)的衍生物，其可以用具有编码膜蛋白的ydeD基因的DNA转化，所述膜蛋白不涉及任何L-氨基酸的生物合成途径(美国专利5,972,663)。
用于评估L-半胱氨酸生产的发酵条件在美国专利6,218,168的实施例6中详细描述。
通过Pantoea ananatis菌株AJ13356-xpk1生产L-谷氨酸。
用pMW-xpk1质粒转化生产L-谷氨酸的成团肠杆菌AJ13356菌株(美国专利6,331,419)(近来重分类为Pantoea ananatis)可以通过普通方法进行，以获得菌株AJ133556-pMW-xpk1。
可以将每种AJ13356和AJ13356-xpk1菌株接种到500ml体积烧瓶中，其含有20ml培养基，所述培养基包含40g/L葡萄糖、20g/L硫酸铵、0.5g/L七水合硫酸镁、2g/L磷酸二氢钾、0.5g/L氯化钠、0.25g/L七水合氯化钙、0.02g/L七水合硫酸亚铁、0.02g/L四水合硫酸锰、0.72mg/L二水合硫酸锌、0.64mg/L五水合硫酸铜、0.72mg/L六水合氯化钴、0.4mg/L硼酸、1.2mg/L二水合钼酸钠(sodium molybdate dihydrate)、2g/L酵母提取物、30g/L碳酸钙、200mg/L L-赖氨酸一氢氯化物(L-lysine monohydrochloride)、200mg/L L-甲硫氨酸和200mg/L DL-α-ε-二氨基庚二酸(DAP)，可以在37℃摇动培养直到培养基中包含的葡萄糖被完全消耗。在培养完成之后，可如上述测量培养基中积累的L-谷氨酸。
实施例5使用棒状杆菌型细菌确认磷酸转酮酶基因的生理活性(5-1)磷酸转酮酶基因的克隆和表达质粒的构建(1)构建pVK9-xfp从菌株动物双歧杆菌JCM1190的染色体DNA克隆xfp基因。根据动物双歧杆菌ATCC 27674的xfp基因的报道的核苷酸序列(GenBank登记No.AY518213，SEQ ID NO9)，可以合成SEQ ID NO13和No14中描述的引物并用于xfp基因的扩增。动物双歧杆菌JCM1190可以从日本微生物保藏中心(Japan Collection of Microorganisms，JCM)获得。
来自动物双歧杆菌JCM1190的xfp基因的DNA序列在SEQ ID NO11中示出。
为了从动物双歧杆菌JCM1190扩增xfp基因(SEQ ID NO11)并将它克隆到pVK9大肠杆菌-棒状杆菌穿梭载体中，使用Wizard Genomic纯化试剂盒(Promega)提取动物双歧杆菌JCM1190的染色体DNA。pVK9是大肠杆菌-棒状杆菌穿梭载体，是通过用BamHI和KpnI消化pHK4(JP 5-007491A)以获得包括它的可复制起点的区域并将该区域导入pHSG299(Takara Bio Inc.的产品)的AvaII位点而获得的。使用动物双歧杆菌的染色体DNA作为模板并使用SEQ IDNO13和NO14所示引物作为引物，通过PCR扩增包含启动子区域的xfp基因片段。通过常规方法进行PCR。
按常规方式纯化得到的PCR产物，并用Xba I消化。通过使用连接试剂盒(Takara Bio Inc的产品)将消化的PCR产物连接到已经用Xba I消化的pVK9。连接混合物用于转化大肠杆菌DH5α(Takara Bio Inc.的产品)的感受态细胞。将细胞铺在含有100μM IPTG、40mg/ml X-Gal和25mg/ml Km的LB培养基(1L中10g细菌用胰蛋白胨、5g细菌用酵母提取物和10g NaCl)上，培养过夜。然后，选择出现的白色菌落，分成单个菌落来获得转化体。从转化体分离含有xfp基因的目标质粒pVK9-xfp。
(2)构建pVK9-PS2_xfp根据重叠PCR法(R.M.Horton，H.D.Hunts，S.N.Ho，J.K.Pullen，和L.R.Pease，Gene，77，61-68(1989))获得DNA片段，其中动物双歧杆菌xfp基因的天然启动子区域被PS2启动子取代(Peyret JL，Mol Microbiol.1993Jul；9(1)97-109，WO93/03158)。以下具体描述该方法。
首先，使用含有PS2启动子的pPSTGl(Y.Kikuchi，M.Date，K.Yokoyama，Y.Umezawa and H.Matsui，Appl.Environ.Microbiol.Appl.Environ.Microbiol69，358-366(2003))作为模板以及SEQ ID NO15和NO16的合成DNA作为引物进行PCR，以获得PS2启动子的扩增产物。然后，为了获得xfp基因(编码区)的序列的扩增产物，使用pVK9-xfp作为模板以及SEQ ID NO17和NO18的合成DNA作为引物进行PCR。SEQ ID NO16和NO18是相互互补的。
然后，为了获得含有PS2启动子和动物双歧杆菌xfp基因的片段，以基本上相等的量混合PS2启动子和动物双歧杆菌xfp基因的上述基因片段，使用这种混合物作为模板以及SEQ ID NO14和NO19的合成DNA作为引物进行PCR。
按常规方式纯化得到的PCR产物，并用Xba I消化。通过使用连接试剂盒(Takara Bio Inc的产品)将消化的PCR产物连接到已经用Xba I消化的pVK9。连接混合物用于转化大肠杆菌DH5α(Takara Bio Inc.的产品)的感受态细胞。将细胞铺在含有100μM IPTG、40mg/ml X-Gal和25mg/ml Km的LB培养基(1L中10g细菌用胰蛋白胨、5g细菌用酵母提取物和10g NaCl)上，培养过夜。然后，选择出现的白色菌落，分成单个菌落来获得转化体。从转化体分离含有PS2启动子和xfp基因的目的质粒pVK9-PS2_xfp。
(3)构建pVK9-tac_xfp根据重叠PCR法(R.M.Horton，H.D.Hunts，S.N.Ho，J.K.Pullen，和L.R.Pease，Gene，77，61-68(1989))获得DNA片段，其中动物双歧杆菌xfp基因的天然启动子区域被tac启动子取代。以下具体描述该方法。
首先，使用pKK223-3(Pharmacia)作为模板以及SEQ ID NO20和NO21的合成DNA作为引物进行PCR来获得tac启动子的扩增产物。然后，为了获得xfp基因(编码区)的序列的扩增产物，使用动物双歧杆菌JCM1190的染色体DNA作为模板以及SEQ ID NO14和NO22的合成DNA作为引物进行PCR。SEQ ID NO20和NO22的核苷酸序列是相互互补的。
然后，为了获得含有tac启动子和动物双歧杆菌xfp基因的片段，以基本上相等的量混合tac启动子和动物双歧杆菌xfp基因的上述基因片段，使用这种混合物作为模板以及SEQ ID NO14和NO21的合成DNA作为引物进行PCR。
按常规方式纯化得到的PCR产物，并用Xba I消化。通过使用连接试剂盒(Takara Bio Inc的产品)将消化的PCR产物连接到已经用Xba I消化的pVK9。连接混合物用于转化大肠杆菌DH5α的感受态细胞(Takara Bio Inc.的产品)。将细胞铺在含有100μM IPTG、40mg/ml X-Gal和25mg/ml Km的LB培养基(1L中10g细菌用胰蛋白胨、5g细菌用酵母提取物和10g NaCl)上，培养过夜。然后，选择出现的白色菌落，分成单个菌落来获得转化体。从转化体分离含有xfp基因和tac启动子的目的质粒pVK9-tac_xfp。
(4)构建pVK9-PS2_xpkA从菌株戊糖乳杆菌JCM1558的染色体DNA克隆xpkA基因。戊糖乳杆菌JCM1558可以从日本微生物保藏中心(Japan Collection of Microorganisms，JCM)获得。为了从戊糖乳杆菌JCM1558扩增编码磷酸转酮酶的xpkA基因(SEQ ID NO1；AJ309011gi16605513)并将它克隆到pVK9穿梭载体中，使用Wizard基因组纯化试剂盒(Promega)提取戊糖乳杆菌JCM1558的染色体DNA。
根据重叠PCR方法通过用PS2启动子取代编码磷酸转酮酶的戊糖乳杆菌xpkA基因的启动子区域获得DNA片段。在此具体描述该方法。
首先，使用含有PS2启动子的pPSTG1(Y.Kikuchi，M.Date，K.Yokoyama，Y.Umezawa and H.Matsui，Appl.Environ.Microbiol.Appl.Environ.Microbiol69，358-366(2003))作为模板以及SEQ ID NO15和NO23的合成DNA作为引物进行PCR，来获得PS2启动子的扩增产物。然后，为了获得xpkA基因(编码区)的序列的扩增产物，使用戊糖乳杆菌JCM1558的染色体DNA作为模板以及SEQ ID NO24和NO25的合成DNA作为引物进行PCR。SEQ ID NO23和NO25的核苷酸序列是相互互补的。
然后，为了获得含有PS2启动子和戊糖乳杆菌xpkA基因的片段，以基本上相等的量混合PS2启动子和戊糖乳杆菌xpkA基因的上述基因片段，使用这种混合物作为模板以及SEQ ID NO19和NO24的合成DNA作为引物进行PCR。
按常规方式纯化得到的PCR产物，并用Xba I消化。通过使用连接试剂盒(Takara Bio Inc的产品)将消化的PCR产物连接到已经用Xba I消化的pVK9。连接混合物用于转化大肠杆菌DH5α的感受态细胞(Takara Bio Inc.的产品)。将细胞铺在含有100μM IPTG、40mg/ml X-Gal和25mg/ml Km的LB培养基(1L中10g细菌用胰蛋白胨、5g细菌用酵母提取物和10g NaCl)上，培养过夜。然后，选择出现的白色菌落，分成单个菌落来获得转化体。从转化体分离含有PS2启动子和xpkA的目的质粒pVK9-PS2_xpkA。
(5-2)磷酸转酮酶的体内生理活性的确认(1)构建ATCC13869ΔaceE菌株PDH缺陷菌株是醋酸盐(acetate)营养缺陷的。另一方面，其中导入了磷酸转酮酶基因的PDH(丙酮酸脱氢酶)缺陷菌株对于醋酸盐不再是营养缺陷的。因此，证实了导入xfp或xpkA基因的影响。
构建基因破坏的载体(A)构建pBS3用枯草芽孢杆菌的染色体DNA作为PCR的模板和SEQ ID NO42和NO43的合成DNA作为引物，通过PCR获得了sacB基因(SEQ ID NO40)。通过在维持94℃5分钟1个循环之后，重复25次94℃ 30秒、49℃30秒和72℃2分钟的循环，使用LA taq(TaKaRa Bio)，进行PCR反应。通过普通方法纯化之后，用BglII和BamHI消化PCR产物并平端化。将得到的PCR产物与已经用AvaII消化并平端化的pHSG299连接。所述质粒用于转化大肠杆菌JM109(TakaraBio Inc.)的感受态细胞，然后将转化体悬浮在含有25mg/ml卡那霉素的LB培养基中并培养过夜。挑出菌落并进行单菌落分离，然后获得转化体。从转化体分离含有sacB基因的目标质粒pBS3。
pBS3的构建步骤在图2中说明。
(B)构建pBS4S通过重叠PCR，通过破坏质粒pBS3中卡那霉素抗性基因的编码区中SmaI位点来获得质粒。首先，使用pBS3作为模板以及SEQ ID NO44和NO45的合成DNA作为引物，进行PCR，获得含有卡那霉素抗性基因的N-末端区域的PCR产物。另一方面，为了获得含有卡那霉素抗性基因的C-末端区域的PCR产物，使用pBS3作为模板和SEQ ID NO46和NO47的合成DNA作为引物进行PCR。通过在98℃热处理5分钟之后，重复25次98℃10秒、57℃30秒和72℃1分钟的循环，使用Pyrobest DNA聚合酶(Takara Bio Inc.)，可以获得PCR产物。SEQ ID NO45和NO46的核苷酸序列是相互部分地互补的。
通过导入不引起氨基酸取代的突变，破坏这个序列的SmaI位点。为了获得其中SmaI位点被破坏的突变卡那霉素抗性基因片段，将卡那霉素抗性基因的基因产物的N-末端区域和基因产物的C-末端区域混合，形成几乎等摩尔的混合物。使用得到的混合物作为PCR的模板以及SEQ ID NO44和NO47的合成DNA作为引物，进行PCR。获得在卡那霉素抗性基因中具有突变的PCR产物。通过在98℃热处理5分钟的一个循环之后，重复25次包括98℃10秒、57℃30秒和72℃1.5分钟的循环，使用Pyrobest DNA聚合酶(Takara Bio Inc.)，可以获得目标PCR产物。
在纯化之后用BanII消化PCR产物，并插入到上述的pBS3的BanII位点中。获得的DNA用于转化大肠杆菌JM109(Takara Bio Inc.)的感受态细胞，悬浮在含有25mg/ml卡那霉素的LB培养基中并培养过夜。挑出菌落并进行单菌落分离，获得转化体。从转化体分离目的质粒pBS4S。pBS4S的构建步骤在图3中说明。
(C)构建pBS5T通过以下步骤构建具有来自棒状杆菌型细菌的温度敏感复制起点的质粒pBS5T。
通过用BamHI和SmaI消化pHSC4(US5616480A)并将消化的片段平端化来获得温度敏感复制的区域，然后将这个区域连接到pBS4S的平末端化的NdeI位点。获得的DNA用于转化大肠杆菌JM109(Takara Bio Inc.的产品)的感受态细胞，将转化体悬浮在含有25mg/ml Km的LB培养基中并培养过夜。挑出菌落并进行单菌落分离，然后获得转化体。从转化体分离含有sacB片段和温度敏感复制起点的目的质粒pBS5T。图4说明了pBS5T的构建方案。
(2)aceE基因破坏的片段的克隆通过重叠PCR方法，使用根据谷氨酸棒杆菌ATCC13032的aceE基因的报道的核苷酸序列(GenBank数据库登记No.NC 003450；SEQ ID NO38)的合成DNA作为引物，从ATCC13869获得编码丙酮酸脱氢酶(丙酮酸脱氢酶E1元件)的aceE基因破坏的DNA片段。
首先，使用谷氨酸棒杆菌ATCC13869的染色体DNA作为模板以及SEQ IDNO26和NO27的合成DNA作为引物进行PCR，来获得aceE基因的N-末端侧的扩增产物。然后，为了获得aceE基因的C-末端侧的扩增产物，使用谷氨酸棒杆菌ATCC13869的染色体DNA作为模板以及SEQ ID NO28和NO29的合成DNA作为引物进行PCR。SEQ ID NO26和NO28的序列是相互互补的。
然后，为了获得其中内部序列被缺失的aceE片段，以基本上等摩尔的量混合N-和C-末端的上述基因片段，使用这种混合物作为模板以及SEQ ID NO30和NO31的合成DNA作为引物进行PCR。
按常规方式纯化得到的PCR产物，并用Sma I消化。通过使用连接试剂盒(Takara Bio Inc的产品)将消化的PCR产物连接到已经用Sma I消化的上述的pBS5T。连接混合物用于转化大肠杆菌DH5α的感受态细胞(Takara Bio Inc.的产品)。将细胞铺在含有100μM IPTG、40mg/ml X-Gal和25mg/ml Km的LB培养基(1L中10g细菌用胰蛋白胨、5g细菌用酵母提取物和10g NaCl)上，培养过夜。然后，选择出现的白色菌落，分成单个菌落来获得转化体。从转化体分离目的质粒pBS5T-ΔaceE。
(3)构建aceE-破坏的菌株aceE基因编码丙酮酸脱氢酶E1元件。
首先，通过电脉冲方法用高浓度质粒pBS5T-ΔaceE转化ATCC13869菌株，铺在含有25mg/ml卡那霉素的CM-Dex培养基(5g/L葡萄糖、10g/L多蛋白胨(polypeptone)、10g/L酵母提取物、1g/L KH2PO4、0.4g/LMgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/L MnSO4·7H2O、3g/L尿素、1.2g/L大豆水解产物和10μg/L生物素，pH值7.5(NaOH))上，在25℃培养约60小时，分离出现的菌落作为转化体。然后，转化体在34℃在CM-Dex液体培养基上培养过夜。在合适的稀释后，将培养的转化体悬浮在含有25mg/ml卡那霉素的CM-Dex培养基中，在34℃培养约30小时。作为质粒上破坏类型的aceE基因(ΔaceE)与染色体上天然aceE基因之间的单交叉同源重组的结果，获得了可以在这个培养基中生长、并且在染色体上兼有卡那霉素抗性基因和源自质粒的sacB基因的ΔaceE菌株。
接下来，将通过单交叉同源重组获得的ΔaceE菌株在31.5℃在CM-Dex液体培养基上培养过夜。在合适的稀释之后，将单交叉同源重组的ΔaceE菌株悬浮在无卡那霉素并含有10％蔗糖的Dex-S10培养基(10g/L蔗糖、10g/L多蛋白胨、10g/L酵母提取物、1g/L KH2PO4、0.4g/L MgSO4·7H2O、0.01g/LFeSO4·7H2O、0.01g/L MnSO4·4H2O、3g/L尿素、1.2g/L大豆水解产物、10μg/L生物素和2g/L醋酸钠、用KOH调节到pH7.5)中。在34℃进行培养约30小时。作为第二次交叉同源重组的结果，获得了不具有染色体SacB基因且对蔗糖不敏感的菌株。
以此方式获得的菌株包括具有破坏型的aceE基因的那些菌株和具有野生型aceE基因的那些菌株。通过使用在Dex-S10琼脂培养基上培养获得的细胞直接进行PCR反应，来确认aceE基因是破坏型还是野生型。选出含有破坏型的aceE基因的菌株，命名为“ATCC13869ΔaceE”。
(4)评估ΔaceE菌株中磷酸转酮酶的生理活性将pVK9(用于对照的质粒)、pVK9-xfp(用于扩增动物双歧杆菌的xfp基因的质粒)、pVK9-PS2_xfp(用于扩增动物双歧杆菌的xfp基因的质粒，其中它的天然启动子被PS2启动子取代)和pVK9-PS2_xpkA(扩增戊糖乳杆菌的xpkA基因的质粒，其中它的天然启动子被PS2启动子取代)各自导入ATCC13869ΔaceE菌株来获得表达磷酸转酮酶的菌株，并将pVK9导入ATCC13869菌株作为对照。具体地，通过电脉冲方法各自转化ATCC13869ΔaceE菌株和ATCC13869菌株，铺在含有25mg/ml卡那霉素的CM-Dex培养基(5g/L葡萄糖、10g/L多蛋白胨、10g/L酵母提取物、1g/LKH2PO4、0.4g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/L MnSO4·7H2O、3g/L尿素、1.2g/L大豆水解产物和10μg/L生物素，通过NaOH调节pH值7.5)上，在31.5℃培养约30小时。将出现的菌落分离为转化体，分别称为ATCC13869ΔaceE(pVK9)、ATCC13869ΔaceE(pVK9-xfp)、ATCC13869ΔaceE(pVK9-PS2_xfp)、ATCC13869ΔaceE(pVK9-PS2_xpkA)和ATCC1869(pVK9)。
将这些菌株在微摇动器(“Biophotorecorder TN-1506”，ADVANTEC的产品)中培养，以时间的函数测量OD。在此采用两种培养基，也就是，含有25mg/ml卡那霉素的基本液体培养基(由10g/L葡萄糖、2.5g/L(NH4)2SO4、0.5g/L KH2PO4、0.25g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/LMnSO4·7H2O、2g/L尿素、50μg/L生物素、100mL VB1-HCl、15mg/L原儿茶酸、0.02mg/L CuSO4、10mg/L CaCl2、和40g/L MOPS组成，通过KOH调节pH7.0)，和含有醋酸盐(2g/L醋酸钠)的基本液体培养基。分别在图6和5中说明在基本培养基上培养的结果和在含有醋酸盐的基本培养基上培养的结果。ATCC13869ΔaceE(pVK9)菌株可以在含有醋酸盐的基本培养基上生长，而不能在基本培养基上生长，因而展现了醋酸盐营养缺陷。携带pvK9-xfp、pVK9-PS2_xfp和pVK9-PS2_xpkA的ATCC13869ΔaceE菌株分别可以在基本培养基上生长，说明导入xfp或xpkA产生了对PDH缺陷菌株的醋酸盐营养缺陷的补偿。因此，证实了被导入谷氨酸棒杆菌中的磷酸转酮酶的生理活性。
实施例6.确认磷酸转酮酶的酶活性棒状杆菌型细菌可以在生物素有限、或培养基中添加青霉素或表面活性剂的情况下生产L-谷氨酸(参见WO95/34672)。另一方面，已知的是，被修饰以降低α-酮戊二酸脱氢酶活性(E1.2.4.2 sucA；2-酮戊二酸脱氢酶)的菌株甚至可以不在这种条件下生产L-谷氨酸(Kimura E.，Adv.Biochem.Eng.Biotechno1.，79，37-57(2003)，“Metabolic engineering of glutamic acid production”)。因此，我们研究了通过使用sucA缺陷菌株谷氨酸棒杆菌ATCC13869导入磷酸转酮酶基因，来改善L-谷氨酸的发酵产量。
(6-1)构建ATCC13869ΔsucA菌株(1)克隆破坏sucA基因的片段使用根据谷氨酸棒杆菌ATCC13032的sucA基因核苷酸序列(GenBank数据库登记No.NC_003450；SEQ ID NO48)设计的合成DNA作为引物，通过重叠PCR法，获得了源自菌株谷氨酸棒杆菌ATCC13869的缺失sucA的片段。
首先，使用谷氨酸棒杆菌ATCC13869的染色体DNA作为模板以及SEQ IDNO32和NO33的合成DNA作为引物进行PCR，来获得sucA基因的N-末端的扩增产物。然后，为了获得sucA基因的C-末端的扩增产物，使用谷氨酸棒杆菌ATCC13869的染色体DNA作为模板以及SEQ ID NO34和NO35的合成DNA作为引物进行PCR。SEQ ID NO33和NO34的序列是相互互补的。
然后，为了获得其中它的内部序列被缺失的sucA片段，以基本上等摩尔的量混合N-和C-末端的上述基因片段，使用这种混合物作为模板以及SEQ IDNO36和NO37的合成DNA作为引物进行PCR。
按常规方式纯化得到的PCR产物，并用BamHI消化。通过使用连接试剂盒(Takara Bbamhio Inc的产品)将消化的PCR产物连接到已经用BamHI消化的以上描述的pBS3。连接混合物用于转化大肠杆菌DH5α的感受态细胞(TakaraBio Inc.的产品)。将细胞铺在含有100μM IPTG、40mg/ml X-Gal和25mg/mlKm的LB培养基(1L中10g细菌用胰蛋白胨、5g细菌用酵母提取物和10gNaCl)上，培养过夜。然后，选择出现的白色菌落，分成单个菌落来获得转化体。从转化体分离目标质粒pBS3ΔsucA。
(2)制备破坏了sucA的菌株在(A)中制备的pBS3ΔsucA不含有在棒状杆菌型细菌的细胞中可自主复制的区域。因此，如果用这个质粒转化棒状杆菌，通过同源重组将这个质粒整合到其染色体中的菌株将作为转化体出现，虽然它以极低的频率发生。通过电脉冲法用高浓度的质粒pBS3ΔsucA转化谷氨酸棒杆菌ATCC13869，铺在含有25mg/ml卡那霉素的CM-Dex培养基(5g/L葡萄糖、10g/L多蛋白胨、10g/L酵母提取物、1g/L KH2PO4、0.4g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/L MnSO4·7H2O、3g/L尿素、1.2g/L大豆水解产物和10μg/L生物素，通过NaOH调节pH值7.5)上，在31.5℃培养约30小时，然后将出现的菌落分离作为转化体。作为在质粒的sucA基因片段和染色体上天然基因之间同源重组的结果，这些转化体具有源自质粒的卡那霉素抗性基因和SacB基因。
接下来，将获得的第一次重组菌株在31.5℃在无卡那霉素的CM-Dex液体培养基上培养过夜。在合适的稀释之后，将培养的转化体铺在含有10％蔗糖的无卡那霉素Dex-S10培养基(10g/L蔗糖、10g/L多蛋白胨、10g/L酵母提取物、1g/L KH2PO4、0.4g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/LMnSO4·4H2O、3g/L尿素、1.2g/L大豆水解产物和10μg/L生物素，通过KOH调节pH值7.5)上，在31.5℃培养约30小时，将出现的菌落分离作为转化体。作为第二次交叉同源重组的结果，获得了染色体丢失了SacB基因并且对蔗糖不敏感的菌株。
照这样获得的菌株包括具有破坏型sucA基因的那些和具有野生型sucA基因的那些。通过使用在Dex-S10琼脂培养基上培养获得的细胞直接进行PCR反应，来确认sucA基因是破坏类还是野生型。选择具有破坏型sucA基因的菌株。
按以下方法评估破坏了sucA的菌株的L-谷氨酸生产。将通过在CM2B平板培养基上培养获得的破坏了sucA的菌株的细胞接种到烧瓶中，所述烧瓶含有1L纯水中80g葡萄糖、1g KH2PO4、0.4g MgSO4、30g(NH4)2SO4、0.01gFeSO4·7H2O、0.01g MnSO4·7H2O、15ml大豆水解产物溶液、200mg盐酸硫胺素、60μg生物素和50g CaCO3(用KOH调节到pH8.0)，在31.5℃摇动培养直到糖类被完全消耗。完成培养之后，测量培养液中积累的L-谷氨酸的量。选择可以比ATCC13869产生更高L-谷氨酸发酵产量的破坏了sucA的菌株，作为ATCC13869的破坏了sucA的菌株，并命名为ATCC13869ΔsucA。
(6-2)在破坏了sucA的ATCC13869菌株中确认磷酸转酮酶基因表达和酶活性通过使用已经被修饰以增强磷酸转酮酶表达的菌株，研究了磷酸转酮酶基因的表达和它的酶活性。
将pVK9(用于对照的质粒)、pVK9-xfp(用于扩增动物双歧杆菌的xfp基因的质粒)、pVK9-tac_xfp(用于扩增动物双歧杆菌的xfp基因的质粒，其中它的天然启动子被tac启动子取代)、pVK9-PS2_xfp(用于扩增动物双歧杆菌的xfp基因的质粒，其中它的天然启动子被PS2启动子取代)和pVK9-PS2_xpkA(用于扩增戊糖乳杆菌的xpkA基因的质粒，其中它的天然启动子被PS2启动子取代)各自导入以上描述的谷氨酸棒杆菌ATCC13869ΔsucA来获得表达磷酸转酮酶的菌株。具体地，通过电脉冲方法用每种质粒转化ATCC13869ΔsucA菌株，铺在含有25mg/ml卡那霉素的CM-Dex培养基(5g/L葡萄糖、10g/L多蛋白胨、10g/L酵母提取物、1g/L KH2PO4、0.4g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/L MnSO4·7H2O、3g/L尿素、1.2g/L大豆水解产物和10μg/L生物素，通过NaOH调节pH7.5)上，在31.5℃培养约30小时。分离出现的菌落作为转化体，分别称为ATCC13869ΔsucA(pVK9)、ATCC13869ΔsucA(pVK9-xfp)、ATCC13869ΔsucA(pVK9-tac_xfp)、ATCC13869ΔsucA(pVK9-PS2_xfp)和ATCC13869ΔsucA(pVK9-PS2_xpkA)。
为了获得粗酶溶液，将通过在CM-Dex平板培养基上培养每个菌株获得的上述菌株的细胞接种到烧瓶中，所述烧瓶含有1L纯水中的30g葡萄糖、1gKH2PO4、0.4g MgSO4、15g(NH4)2SO4、0.01g FeSO4·7H2O、0.01g MnSO4·7H2O、13.7ml大豆水解产物溶液、200mg盐酸硫胺素、300μg生物素和50g CaCO3(用KOH调节pH8.0)，在31.5℃摇动培养。如通过“Hitachi分光光度计U-2000A”测量的，当细胞浓度的OD620变成15时终止培养。通过在3000rpm中度离心30秒除去碳酸钙之后，收集细胞。以下步骤在0到4℃进行。将收集的细胞用0.85NNaCl溶液洗涤两次，重悬浮在4ml/g(湿重)缓冲液A(100mM KPO4(pH6.5)、30mM KCl、0.1mM EDTA、1mM MgCl2、0.2mM PMSF、2mM DTT)中。通过超声波细胞破碎机(“Bioruptor”)破坏细胞。通过离心(15,000g，60min))除去未破坏的细胞来获得粗酶溶液。通过使用CBB溶液(蛋白质分析CBB溶液，Nacalai Tesque的产品)用牛血清白蛋白作为标准样品，定量粗酶溶液中的蛋白质浓度。
通过SDS-PAGE，分离粗酶溶液中的蛋白质。接下来将描述分离的具体过程。在浓度调整之后，将粗酶溶液与样品缓冲液(“Laemmli样品缓冲液”，BIORAD的产品)以1∶1混合。在95℃加热2分钟后，将得到的混合物上样到“Ready GelJ 10％”(BIORAD的产品)上，使得上样的蛋白质的量为10μg，随后在200V的恒定电压下在MiniProtean III Cell电泳槽(BIORAD的产品)中电泳40分钟。对于电泳缓冲液，使用tris/甘氨酸/SDS(BIORAD)。对于染色，使用考马斯亮蓝(“Bio Safe Coomassei”，BIORAD)。结果在以下的图7中示出(标记物BIORAD的产品，精确度加蛋白质标准(precision plus protein standards)(#161-0363))。
图7的泳道1到5分别显示了ATCC13869ΔsucA(pVK9)、ATCC13869ΔsucA(pVK9-xfp)、ATCC13869ΔsucA(pVK9-tac_xfp)、ATCC13869ΔsucA(pVK9-PS2_xfp)和ATCC13869ΔsucA(pVK9-PS2_xpkA))。与对照条带(pVK9)相比，相应于磷酸转酮酶基因产物的大约90kD条带，在被修饰以增强磷酸转酮酶活性的菌株中变得浓稠(denser)。这清楚地表明了，在棒状杆菌型细菌中出现了磷酸转酮酶基因的足量的表达。
根据Meile的方法(L.Meile，L.M.Rohr，T.A.Geissmann，M.Herenspergerand M.Teuber，J.Bacteriol.183，2929-2936(2001))，测量磷酸转酮酶基因的酶活性。在与0.075ml粗酶溶液(33.3mM KPO4(pH 6.5)、1.9mM盐酸L-半胱氨酸、23mM氟化钠、8mM碘乙酸钠和27mM D-果糖6-磷酸)在37℃反应30分钟后，添加0.075ml盐酸羟胺(2M，pH6.5)。容许混合物在室温下反应10分钟，然后用0.05ml 15％(wt/vol)三氯乙酸、0.05ml 4M HCl和0.05ml FeCl3·6H2O(0.1m HCl中，5％wt/vol)染色。通过离心分离除去结晶之后，使用酶活性读数器(“Spectra max 190”，Molecular Devices的产品)测量OD505。
结果以下在表3中示出。在对照(pVK9)中没有检测到酶活性，而在磷酸转酮酶活性已经被增强的菌株中检测到酶活性。
表3

实施例7评估具有增强的磷酸转酮酶活性的细菌生产L-谷氨酸的能力(7-1)在含有足够量生物素的条件下评估通过使用菌株谷氨酸棒杆菌ATCC13869ΔsucA，评估了导入磷酸转酮酶基因对L-谷氨酸的发酵产量的影响。将通过在CM-Dex平板培养基上培养获得的ATCC13869ΔsucA(pVK9)、ATCC13869ΔsucA(pVK9-xfp)、ATCC13869ΔsucA(pVK9-PS2_xfp)和ATCC13869ΔsucA(pVK9-PS2_xpkA)菌株的细胞接种到烧瓶中，所述烧瓶含有1L纯水中30g葡萄糖、1g KH2PO4、0.4gMgSO4、15g(NH4)2SO4、0.01g FeSO4·7H2O、0.01g MnSO4·7H2O、13.7ml大豆水解产物溶液、200mg盐酸硫胺素、300μg生物素和50g CaCO3(用KOH调节到pH8.0)，在31.5℃摇动培养直到糖类被完全消耗。完成培养之后，测量培养液中积累的L-谷氨酸的量和OD。结果在图8中示出。结果显示，与对照相比，其中扩增了xfp基因的所有菌株显示了高L-谷氨酸产量。
(7-2)添加青霉素时的评估为了在补偿的(equalized)最终OD下评估，通过添加青霉素G停止细胞的增殖。当培养开始后OD达到10到14的范围内时在多个生长点处，通过向如上文(1)中描述的培养基中添加青霉素G，使得它的终浓度是4U/ml，来比较L-谷氨酸的产量。结果在图9中示出。这些结果显示，即使在它们的最终细胞计数相等的点上在它们之中比较L-谷氨酸的产量，与对照菌株相比，在xfp基因或xpk基因扩增的菌株中L-谷氨酸产量更高。上述结果显示，导入磷酸转酮酶对于改善L-谷氨酸发酵产量是有效的。
实施例8.动物双歧杆菌的xfp基因的表达对Pantoea ananatis菌株的L-谷氨酸积累的影响使用Bio-Rad MicroPulser通过电穿孔将表达动物双歧杆菌的xfp基因的pVK9-PS2-xfp质粒和对照pVK9穿梭载体导入NP106/RSFCPG生产L-谷氨酸的菌株。通过将AJ13601菌株铺在不含有氯霉素作为选择标记物的培养基上，来从AJ13601菌株(FERM BP-7207；欧洲专利公开1078989)消除(curing)pSTVCB质粒，从而获得NP106/RSFCPG菌株。
为了用pVK9-PS2-xfp质粒或pVK9载体转化NP106/RSFCPG菌株，在添加四环素(10μg/ml)的LBG-M9培养基(胰蛋白胨(trypton)(10g/L)、酵母提取物(5g/L)、NaCl(5g/L)、葡萄糖(5g/L)、0.5×M9盐溶液)上过夜培养获得的细胞，用新鲜的LBG-M9培养基1∶100稀释，在34℃通气培育直至OD595＝0.6。通过离心收获来自10ml培养物的细胞，用冰冷的去离子水和用冰冷的10％甘油洗涤三次，重悬浮在10％甘油中。将10ng质粒DNA添加到细胞悬液，施加电脉冲(E＝20kV/em，t＝5msec)。在电穿孔后立即添加1ml LBG-M9培养基。将细胞在34℃通气培育2小时，铺在含40μg/ml卡那霉素的固体LBG-M9培养基上。将平板在34℃下培育48小时。将生长的克隆接种到含有40μg/ml卡那霉素和10μg/ml四环素的2ml LBG-MES培养基(胰蛋白胨(10g/L)、酵母提取物(5g/L)、NaCl(5g/L)、葡萄糖(5g/L)、MES 0.1M、pH7.0)中，在34℃下通气培育过夜。将含有细菌细胞的80μl过夜培养的培养基接种到测试试管中的2ml发酵培养基(葡萄糖(40g/L)、MgSO4·7H2O(0.5g/L)、(NH4)2SO4(16g/L)、KH2PO4(0.3g/L)、KCl(1.0g/L)、MES(10g/L)、甜菜碱(1.0g/L)、FeSO4·7H2O(10mg/L)、MnSO4·5H2O(10mg/L)、赖氨酸、甲硫氨酸和DAP各100mg/L、胰蛋白胨(1g/L)、酵母提取物(1g/L)、泛酸钙(10mg/L)、CaCO3(30g/L))中，并通气培育26小时。通过TLC方法测定积累的L-谷氨酸的量。4个独立实验的平均数据在表4中示出。
表4.

可以从表4中看出，携带pVK9-PS2-xfp质粒的菌株显示L-谷氨酸积累比用载体pVK9转化的对照菌株高大约2.6g/L，其对应于至少6％的收率增加。
实施例8.通过将磷酸转酮酶活性的增强和6-磷酸果糖激酶(PFK)活性的降低进行组合来改善L-谷氨酸的发酵产量8-1.制备sucA和PFK双破坏的菌株(1)构建用于破坏pfk基因的质粒使用根据谷氨酸棒杆菌ATCC 13032的相应基因的核苷酸序列(GenBank数据库登记No.NC_003450；SEQ ID NO61)设计的合成DNA作为引物，通过重叠PCR法获得了缺乏源自菌株ATCC13869的pfk的ORF的基因片段。具体地，使用谷氨酸棒杆菌菌株ATCC13869的染色体DNA作为模板以及SEQ IDNO50和NO51的合成DNA作为引物，通过常规PCR方法获得了pfk基因的N-末端区域的扩增产物。另一方面，为了获得pfk基因的C-末端区域的扩增产物，使用菌株ATCC13869的染色体DNA作为模板以及SEQ ID NO52和NO53的合成DNA作为引物进行常规PCR。SEQ ID NO51和NO53的序列是相互互补的。
然后，为了获得其中它的内部序列被缺失的pfk基因片段，以基本上等摩尔的量混合pfk的N-末端和C-末端区域的上述扩增产物，使用这种混合物作为模板以及SEQ ID NO54和NO55的合成DNA作为引物通过常规PCR获得基因扩增产物。按普通方法纯化PCR产物，然后用BamH I消化，并插入到上述pBS5T的BamH I位点。这个DNA用于转化大肠杆菌DH5α的感受态细胞(Takara Bio Inc.的产品)，将细胞铺在补充有100μM IPTG、40μg/ml X-Gal和25μg/ml卡那霉素的LB培养基上并培养过夜。然后，挑选出现的白色菌落，分成单个菌落来获得转化体。从获得的转化体分离质粒，选择具有插入目标PCR产物的质粒，并命名为pBS5T-Δpfk。
(2)制备破坏的菌株首先通过电脉冲法用高浓度的质粒pBS5T-Δpfk转化上述菌株ATCC13869ΔsucA，铺在补充有25μg/ml卡那霉素的CM-Dex培养基(5g/L葡萄糖、10g/L多蛋白胨、10g/L酵母提取物、1g/L KH2PO4、0.4g/LMgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/L MnSO4·7H2O、3g/L尿素、1.2g/L大豆水解产物和10μg/L生物素、用NaOH调节pH7.5)上，在25℃培养约60小时。得到的转化体在34℃摇动培养过夜，然后适当地稀释，平铺在补充有25μg/ml卡那霉素的CM-Dex培养基上并培养30小时。作为所述质粒上的pfk基因片段和位于菌株ATCC13869的染色体上的相同基因之间同源重组的结果，可以在这种培养基上生长的菌株是在其中卡那霉素抗性基因和来自所述质粒的SacB基因被整合到其染色体中的菌株。
然后，将第一重组体在31.5℃在无卡那霉素的液体CM-Dex培养基中培养过夜，在合适的稀释之后，将它们铺在无卡那霉素、含10％蔗糖的Dex-S10培养基(10g/L葡萄糖、10g/L多蛋白胨、10g/L酵母提取物、1g/LKH2PO4、0.4g/L MgSO4·7H2O、0.01g/L FeSO4·7H2O、0.01g/LMnSO4·4H2O、3g/L尿素、1.2g/L大豆水解产物、10μg/L生物素和2g/L醋酸钠、用KOH调节pH7.5)上，在34℃培养约30小时。结果，获得了菌株，其由于第二次同源重组看起来丧失了SacB基因并对蔗糖不敏感。
获得的菌株包括具有源自pBS5T-Δpfk的破坏型的pfk基因的那些，和具有野生型pfk基因的那些。为了证实pfk基因是破坏型还是野生型，使用在Dex-10琼脂培养基上培养的细菌细胞进行直接PCR分析。选择仅具有破坏型pfk的菌株，命名为ATCC13869ΔsucAΔpfk。
8-2获取编码具有降低活性的PFK的突变pfk基因(1)具有降低活性的突变PFK对于具有降低的活性的PFK，获得了PFK*1(SEQ ID NO57)和PFK*2(SEQ ID NO59)。PFK*1是通过在野生型PFK(SEQ ID NO61)的位置171处用赖氨酸取代谷氨酸产生的。PFK*2是通过在野生型PFK(SEQ ID NO61)的位置171处用精氨酸取代谷氨酸产生的。使用野生型pfk基因作为模板以及用于导入所述突变的合成DNA作为引物通过交叉PCR可以获得这些序列。
(2)构建突变pfk基因和磷酸转酮酶基因的表达载体为了构建PFK表达载体，使用谷氨酸棒杆菌菌株ATCC13869的染色体DNA作为模板以及SEQ ID NO54和NO55的合成DNA作为引物进行常规PCR。按普通方法纯化PCR产物，然后用Xba I和Kpn I消化，将含有pfk基因的基因片段插入到上述pVK9的Xba I-Kpn I位点。这个DNA用于转化大肠杆菌DH5α的感受态细胞(Takara Bio Inc.的产品)，将细胞铺在补充有100μM IPTG、40μg/ml X-Gal和25μg/ml Km的LB培养基上并培养过夜。然后，挑选出现的白色菌落，分成单个菌落来获得转化体。从获得的转化体分离质粒，选择具有目标基因的插入的质粒，命名为pVK9-PFK。
然后，为了构建同时表达突变PFK和磷酸转酮酶的质粒，用Xba I消化上述pVK9-PS2_xfp，然后纯化含有PS2_xfp的基因片段并插入到pVK9-PFK的Xba I位点中。这个DNA用于转化大肠杆菌DH5α的感受态细胞(Takara Bio Inc.的产品)，将细胞铺在补充有100μM IPTG、40μg/ml X-Gal和25μg/ml Km的LB培养基上并培养过夜。然后，挑选出现的白色菌落，分成单个菌落来获得转化体。从获得的转化体分离质粒，选择具有PS2_xfp和pfk的插入的质粒，命名为pVK9-PS2_xfp_PFK。
为了构建同时表达突变pfk基因和磷酸转酮酶基因的质粒，根据与pVK9-PS2_xfp_PFK的构建中采用的相同方法，产生pVK9-PS2_xfp_PFK*1和pVK9-PS2_xfp_PFK*。
(3)分析活性PFK和磷酸转酮酶的上述表达质粒(pVK9-PS2_xfp_PFK，pVK9-PS2_xfp_PFK*1和pVK9-PS2_xfp_PFK*2)用于转化上述菌株ATCC13869ΔsucAΔpfk来检查PFK的酶活性。通过电脉冲法进行转化，通过将细菌细胞铺在补充有25μg/ml卡那霉素的CM-Dex培养基(5g/L葡萄糖、10g/L多蛋白胨、10g/L酵母提取物、1g/L KH2PO4、0.4g/L MgSO4·7H2O、0.01g/LFeSO4·7H2O、0.01g/L MnSO4·7H2O、3g/L尿素、1.2g/L大豆水解产物和10μg/L生物素，用NaOH调节pH7.5)上，并在31.5℃培养约30小时，来获得转化体。
按以下方式获得粗酶溶液。收集对数生长期中的细菌细胞，用100mM Kpi缓冲液(pH8.2)洗涤，然后溶于相同的缓冲液中，随后超声破碎。通过超速离心(60000rpm，30min)分离可溶组分来获得粗酶溶液。以下显示在粗酶溶液中定量蛋白质浓度的方法。将粗酶溶液和已知浓度用于产生标准曲线的BSA溶液各自与CBB溶液(蛋白质分析CBB溶液，Nacalai Tesque)反应来显色，随后使用spectra max 190(Molecular Divices)测量OD595nm。
然后，根据常规方法(Denise Kotlars和Henri Buc，Methods inEnzymology(1982)9060-70)测量PFK活性。具体方法显示如下。将粗酶溶液添加到100mM Tris-HCl(pH8.2)、10mM MgCl2、1mM ATP、1U/ml甘油-3-磷酸脱氢酶、1U/ml磷酸丙糖异构酶、1U/ml醛缩酶、0.2mM NADH和10mM果糖-6-磷酸，随后使用spectra max 190(Molecular Divices)测量OD340nm中的时间-过程变化，从而测定酶活性。当野生型PFK的活性被设为1时，降低的PFK的相对活性在以下的表5中示出。
表5.

8-3通过降低PFK活性增加L-谷氨酸的产量(1)在含有足量生物素的条件下评估通过使用菌株谷氨酸棒杆菌ATCC13869ΔsucAΔpfk，评估了PFK活性降低对L-谷氨酸发酵产量的影响。将通过在CM-Dex平板上培养菌株获得的每个菌株的细胞接种到培养基中，所述培养基在1L纯水中含有30g葡萄糖、1gKH2PO4、0.4g MgSO4、15g(NH4)2SO4、0.01g FeSO4·7H2O、0.01gMnSO4·7H2O、13.7ml大豆水解产物溶液、200μg盐酸硫胺素、300μg生物素和50g CaCO3(用KOH调节pH8.0)，并在31.5℃培养。完成培养之后，测量培养液中积累的L-谷氨酸的量和细菌细胞浓度(OD)。结果在图10中示出。结果显示，与表达野生型pfk基因的菌株相比，表达突变pfk基因的菌株显示了更高的L-谷氨酸产量。
(2)添加青霉素时的评估当培养开始后OD达到10到14的范围内时在多个生长点处，通过向上述培养基中添加青霉素G到4U/ml的终浓度，以停止细胞生长来获得均等的最终细菌量，来比较L-谷氨酸的产量。结果在图11中示出。上述结果显示，在导入磷酸转酮酶的菌株中PFK活性的降低对于改善L-谷氨酸的发酵产量是有效的。
实施例9使用其中磷酸转酮酶活性被增强的细菌评估L-谷氨酰胺生产使用菌株黄色短杆菌AJ11576(JP56-16479A)，评估了导入磷酸转酮酶基因对于改善L-谷氨酰胺发酵产量的影响。
AJ11576菌株对具有维生素P活性的化合物有抗性。这个菌株于1980年5月6日保藏在产业技术综合研究所，特许微生物保藏中心(Central 6，1-1，Higashi 1-Chome，Tsukuba-shi，Ibaraki-ken，305-8566，Japan)，收到的登记号是FERM P-05502。然后它根据布达佩斯条约的规定转为国际保藏，接受的登记号是FERM BP-10381。
将pVK9(用于对照的质粒)和pVK9-PS2_xfp(用于扩增动物双歧杆菌的xfp基因的质粒，其中天然启动子被PS2启动子取代)各自导入上述菌株黄色短杆菌AJ11576来获得表达磷酸转酮酶的菌株。具体地，通过电脉冲法用每种质粒转化菌株，铺在含有25mg/ml卡那霉素的CM2G培养基(5g/L葡萄糖、10g/L多蛋白胨、10g/L酵母提取物、5g/L NaCl、用KOH调节pH7.0)上，在31.5℃培养约30小时。分离出现的菌株作为转化体，分别称为AJ11576(pVK9)和AJ11576(pVK9-PS2_xfp)。
将通过在CM2G平板培养基上培养获得的AJ11576(pVK9)和AJ11576(pVK9-PS2_xfp)菌株的细胞接种到烧瓶培养基中，所述培养基在1L纯水中含有100g葡萄糖、2.5g KH2PO4、0.4g MgSO4、60g(NH4)2SO4、0.01gFeSO4·7H2O、5.7ml大豆水解产物溶液、2mg盐酸硫胺素、4μg生物素、0.02ml GD-113和50g CaCO3(用NaOH调节到pH6.8)，在31.5℃摇动培养直到糖类被完全消耗。完成培养之后，测量培养液中积累的L-谷氨酰胺的量和OD。结果在图12中示出。显示了与对照菌株相比，所有扩增了xfp基因的菌株都显示了高的L-谷氨酰胺产量。
虽然已经参考本发明的优选的实施方案详细地描述了本发明，但是对于本领域技术人员显而易见的是可以进行各种变化和采用同等物，而不背离本发明的范围。所有在此引用的参考文件，包括优先权文件、RU 2004124226和US 60/644,562，都并入为本申请的部分以作参考。
序列表<110>味之素株式会社(Ajinomoto Co.，Inc.)<120>磷酸转酮酶用于生产有用代谢产物的用途<130>C272-C5130<150>RU2004124226<151>2004-08-10<150>US 60/644562<151>2005-01-19<160>81<170>PatentIn version 3.1<210>1<211>2367<212>DNA<213>戊糖乳杆菌<220>
<221>CDS<222>(1)..(2367)<223>
<400>1atg tct aca gat tac tca tca cca gca tat ttg caa aaa gtt gat aag 48Met Ser Thr Asp Tyr Ser Ser Pro Ala Tyr Leu Gln Lys Val Asp Lys1 5 10 15tac tgg cgt gct gcc aac tat tta tca gtt ggt caa ctt tat tta aaa 96Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Tyr Leu Lys20 25 30gat aat cct tta tta caa cgg cca tta aag gct ag tgac gtt aag gtt144Asp Asn Pro Leu Leu Gln Arg Pro Leu Lys Ala Ser Asp Val Lys Val35 40 45cac cca atc ggt cac tgg ggc acg att gcc ggc caa aac ttc atc tat192His Pro Ile Gly His Trp Gly Thr Ile Ala Gly Gln Asn Phe Ile Tyr50 55 60gcg cat ctt aac cgg gtc atc aac aag tac ggt ttg aag atg ttc tac240Ala His Leu Asn Arg Val Ile Asn Lys Tyr Gly Leu Lys Met Phe Tyr65 70 75 80gtt gaa ggt cca ggt cat ggt ggc caa gtg atg gtc tcc aac tca tac288Val Glu Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr85 90 95ctt gat ggg act tac acg gat att tat cct gaa att acg cag gat gtt336Leu Asp Gly Thr Tyr Thr Asp Ile Tyr Pro Glu Ile Thr Gln Asp Val100 105 110gaa ggg atg caa aaa ctc ttc aag caa ttc tca ttc cca ggt ggc gtg384Glu Gly Met Gln Lys Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val115 120 125gct tcc cat gct gct cct gaa aca cca ggc tca atc cac gaa ggt ggc432Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly130 135 140gaa ctt ggt tac tca att tca cac ggt gtt ggg gca atc ctt gac aac480Glu Leu Gly Tyr Ser Ile Ser His Gly Val Gly Ala Ile Leu Asp Asn145 150 155 160
cct gat gaa atc gcc gca gtc gtt gtt ggt gat ggg gaa tcc gaa acc528Pro Asp Glu Ile Ala Ala Val Val Val Gly Asp Gly Glu Ser Glu Thr165 170 175ggc cca tta gca act tca tgg caa tca acg aag ttc atc aac cca atc576Gly Pro Leu Ala Thr Ser Trp Gln Ser Thr Lys Phe Ile Asn Pro Ile180 185 190aac gat ggg gca gtg tta cca atc ttg aac ctt aac ggc ttt aag att624Asn Asp Gly Ala Val Leu Pro Ile Leu Asn Leu Asn Gly Phe Lys Ile195 200 205tct aac cca acg att ttt ggt cgg act tct gat gaa aag atc aag caa672Ser Asn Pro Thr Ile Phe Gly Arg Thr Ser Asp Glu Lys Ile Lys Gln210 215 220tac ttc gaa agc atg aac tgg gaa cca atc ttt gtt gaa ggt gac gat720Tyr Phe Glu Ser Met Asn Trp Glu Pro Ile Phe Val Glu Gly Asp Asp225 230 235 240cct gaa aag gtt cac cca gct tta gct aag gcc atg gat gaa gcc gtc768Pro Glu Lys Val His Pro Ala Leu Ala Lys Ala Met Asp Glu Ala Val245 250 255gaa aag atc aaa gcc att caa aag aac gct cgt gaa aac gat gac gct816Glu Lys Ile Lys Ala Ile Gln Lys Asn Ala Arg Glu Asn Asp Asp Ala260 265 270act tta cca gta tgg ccg atg atc gtc ttc cgc gca cct aag ggc tgg864Thr Leu Pro Val Trp Pro Met Ile Val Phe Arg Ala Pro Lys Gly Trp275 280 285act ggt cct aag tca tgg gat ggc gac aag atc gaa ggt tca ttc cga912Thr Gly Pro Lys Ser Trp Asp Gly Asp Lys Ile Glu Gly Ser Phe Arg290 295 300gct cac caa att cca att cct gtt gac caa acc gac atg gaa cat gcc960Ala His Gln Ile Pro Ile Pro Val Asp Gln Thr Asp Met Glu His Ala305 310 315 320gat gcg tta gtt gac tgg ttg gaa tca tat caa cca aag gaa ctc ttc 1008Asp Ala Leu Val Asp Trp Leu Glu Ser Tyr Gln Pro Lys Glu Leu Phe325 330 335aat gaa gat ggt tct ttg aag gat gat atc aaa gaa att atc cca act 1056Asn Glu Asp Gly SerLeu Lys Asp Asp Ile Lys Glu Ile Ile ProThr340345 350ggc gat gca cgg atg gcc gct aac cca atc act aat ggt ggg gtt gat 1104Gly Asp Ala Arg Met Ala Ala Asn Pro Ile Thr Asn Gly Gly Val Asp355 360 365cca aag gcc ttg aac tta cct aac ttc cgt gat tac gcc gtt gat acg 1152Pro Lys Ala Leu Asn Leu Pro Asn Phe Arg Asp Tyr Ala Val Asp Thr370 375 380tct aag cat ggt gcc aac gtt aag caa gat atg atc gtt tgg tca gac 1200Ser Lys His Gly Ala Asn Val Lys Gln Asp Met Ile Val Trp Ser Asp385 390 395 400tac ttg cgt gat gtt atc aag aag aac cca gat aac ttc cgg tta ttt 1248Tyr Leu Arg Asp Val Ile Lys Lys Asn Pro Asp Asn Phe Arg Leu Phe405 410 415ggc cct gat gaa acc atg tca aac cgg tta tat ggt gtc ttt gaa acc 1296Gly Pro Asp Glu Thr Met Ser Asn Arg Leu Tyr Gly Val Phe Glu Thr420 425 430act aac cgt caa tgg atg gaa gat att cac cca gat agt gac caa tac 1344Thr Asn Arg Gln Trp Met Glu Asp Ile His Pro Asp Ser Asp Gln Tyr435 440 445gaa gca cct gct ggc cgg gtc ttg gat gct caa tta tct gaa cac caa 1392Glu Ala Pro Ala Gly Arg Val Leu Asp Ala Gln Leu Ser Glu His Gln450 455 460gct gaa ggt tgg tta gaa ggt tac gtc tta act ggt cgt cat ggc ttg 1440
Ala Glu Gly Trp Leu Glu Gly Tyr Val Leu Thr Gly Arg His Gly Leu465 470 475 480ttt gca agt tac gaa gcc ttc tta cgg gtt gtc gac tca atg ttg acg1488Phe Ala Ser Tyr Glu Ala Phe Leu Arg Val Val Asp Ser Met Leu Thr485 490 495caa cac ttc aag tgg tta cgt aag gcc aac gaa ctt gac tgg cgg aag1536Gln His Phe Lys Trp Leu Arg Lys Ala Asn Glu Leu Asp Trp Arg Lys500 505 510aag tac ccg tca ctc aac att atc gcg gct tca act gtg ttc caa caa1584Lys Tyr Pro Ser Leu Asn Ile Ile Ala Ala Ser Thr Val Phe Gln Gln515 520 525gac cat aat ggg tac acc cac caa gat cca ggt gcc ttg act cat ttg1632Asp His Asn Gly Tyr Thr His Gln Asp Pro Gly Ala Leu Thr His Leu530 535 540gct gaa aag aag cct gaa tat atc cgc gaa tat tta cca gcc gac gcc1680Ala Glu Lys Lys Pro Glu Tyr Ile Arg Glu Tyr Leu Pro Ala Asp Ala545 550 555 560aac tcc ttg tta gct gtt ggg gac gtc atc ttc cgt agc caa gaa aag1728Asn Ser Leu Leu Ala Val Gly Asp Val Ile Phe Arg Ser Gln Glu Lys565 570 575atc aac tac gtg gtt acg tcg aag cac cca cgt caa caa tgg ttc agc1776Ile Asn Tyr Val Val Thr Ser Lys His Pro Arg Gln Gln Trp Phe Ser580 585 590att gaa gaa gct aag caa tta gtt gac aac ggt ctt ggt atc att gac1824Ile Glu Glu Ala Lys Gln Leu Val Asp Asn Gly Leu Gly Ile Ile Asp595 600 605tgg gca agc acg gac caa ggt agc gaa cca gat atc gtg ttt gct gct1872Trp Ala Ser Thr Asp Gln Gly Ser Glu Pro Asp lle Val Phe Ala Ala610 615 620gcc gga acg gaa cca acg ctt gaa acg ttg gct gca atc caa ttg ctc1920Ala Gly Thr Glu Pro Thr Leu Glu Thr Leu Ala Ala Ile Gln Leu Leu625 630 635 640cat gat agc ttc cca gac atg aag att cgt ttc gtg aac gtg gtc gac1968His Asp Ser Phe Pro Asp Met Lys Ile Arg Phe Val Asn Val Val Asp645 650 655atc ttg aag tta cgt agc cct gaa aag gac cct cgt ggc ttg tca gat2016Ile Leu Lys Leu Arg Ser Pro Glu Lys Asp Pro Arg Gly Leu Ser Asp660 665 670gct gaa ttt gac cat tac ttc act aag gac aaa cca gtt gtc ttc gcc2064Ala Glu Phe Asp His Tyr Phe Thr Lys Asp Lys Pro Val Val Phe Ala675 680 685ttc cat ggt tac gaa gac ctg gtt cgt gac atc ttc ttt gat cgt cac2112Phe His Gly Tyr Glu Asp Leu Val Arg Asp Ile Phe Phe Asp Arg His690 695 700aac cac aac tta cac gtg cat ggc tac cgt gaa aat ggt gac att acg2160Asn His Asn Leu His Val His Gly Tyr Arg Glu Asn Gly Asp Ile Thr705 710 715 720aca cca ttc gat gtc cgg gtc atg aac caa atg gac cgt ttc gac tta2208Thr Pro Phe Asp Val Arg Val Met Asn Gln Met Asp Arg Phe Asp Leu725 730 735gca aaa tct gca att gcg gcg caa cca gca atg gaa aac acc ggt gca2256Ala Lys Ser Ala Ile Ala Ala Gln Pro Ala Met Glu Asn Thr Gly Ala740 745 750gcc ttt gtt caa gac atg gat aac atg ctt gca aaa cac aac gca tac2304Ala Phe Val Gln Asp Met Asp Asn Met Leu Ala Lys His Asn Ala Tyr755 760 765atc cgt gac gcc gga acc gac ttg cca gaa gtt aac gac tgg caa tgg2352Ile Arg Asp Ala Gly Thr Asp Leu Pro Glu Val Asn Asp Trp Gln Trp
770 775 780aaa ggt ttg aaa taa 2367Lys Gly Leu Lys785<210>2<211>788<212>PRT<213>戊糖乳杆菌<400>2Met Ser Thr Asp Tyr Ser Ser Pro Ala Tyr Leu Gln Lys Val Asp Lys1 5 10 15Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Tyr Leu Lys20 25 30Asp Asn Pro Leu Leu Gln Arg Pro Leu Lys Ala Ser Asp Val Lys Val35 40 45His Pro Ile Gly His Trp Gly Thr Ile Ala Gly Gln Asn Phe Ile Tyr50 55 60Ala His Leu Asn Arg Val Ile Asn Lys Tyr Gly Leu Lys Met Phe Tyr65 70 75 80Val Glu Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr85 90 95Leu Asp Gly Thr Tyr Thr Asp Ile Tyr Pro Glu Ile Thr Gln Asp Val100 105 110Glu Gly Met Gln Lys Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val115 120 125Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly130 135 140Glu Leu Gly Tyr Ser Ile Ser His Gly Val Gly Ala Ile Leu Asp Asn145 150 155 160Pro Asp Glu Ile Ala Ala Val Val Val Gly Asp Gly Glu Ser Glu Thr165 170 75Gly Pro Leu Ala Thr Ser Trp Gln Ser Thr Lys Phe Ile Asn Pro Ile180 185 190Asn Asp Gly Ala Val Leu Pro Ile Leu Asn Leu Asn Gly Phe Lys Ile195 200 205Ser Asn Pro Thr Ile Phe Gly Arg Thr Ser Asp Glu Lys Ile Lys Gln210 215 220Tyr Phe Glu Ser Met Asn Trp Glu Pro Ile Phe Val Glu Gly Asp Asp225 230 235 240Pro Glu Lys Val His Pro Ala Leu Ala Lys Ala Met Asp Glu Ala Val245 250 255Glu Lys Ile Lys Ala Ile Gln Lys Asn Ala Arg Glu Asn Asp Asp Ala260 265 270Thr Leu Pro Val Trp Pro Met Ile Val Phe Arg Ala Pro Lys Gly Trp275 280 285Thr Gly Pro Lys Ser Trp Asp Gly Asp Lys Ile Glu Gly Ser Phe Arg290 295 300Ala His Gln Ile Pro Ile Pro Val Asp Gln Thr Asp Met Glu His Ala305 310 315 320Asp Ala Leu Val Asp Trp Leu Glu Ser Tyr Gln Pro Lys Glu Leu Phe325 330 335Asn Glu Asp Gly Ser Leu Lys Asp Asp Ile Lys Glu Ile Ile Pro Thr340 345 350
Gly Asp Ala Arg Met Ala Ala Asn Pro Ile Thr Asn Gly Gly Val Asp355 360 365Pro Lys Ala Leu Asn Leu Pro Asn Phe Arg Asp Tyr Ala Val Asp Thr370 375 380Ser Lys His Gly Ala Asn Val Lys Gln Asp Met Ile Val Trp Ser Asp385 390 395 400Tyr Leu Arg Asp Val Ile Lys Lys Asn Pro Asp Asn Phe Arg Leu Phe405 4l0 415Gly Pro Asp Glu Thr Met Ser Asn Arg Leu Tyr Gly Val Phe Glu Thr420 425 430Thr Asn Arg Gln Trp Met Glu Asp Ile His Pro Asp Ser Asp Gln Tyr435 440 445Glu Ala Pro Ala Gly Arg Val Leu Asp Ala Gln Leu Ser Glu His Gln450 455 460Ala Glu Gly Trp Leu Glu Gly Tyr Val Leu Thr Gly Arg His Gly Leu465 470 475 480Phe Ala Ser Tyr Glu Ala Phe Leu Arg Val Val Asp Ser Met Leu Thr485 490 495Gln His Phe Lys Trp Leu Arg Lys Ala Asn Glu Leu Asp Trp Arg Lys500 505 5l0Lys Tyr Pro Ser Leu Asn Ile Ile Ala Ala Ser Thr Val Phe Gln Gln515 520 525Asp His Asn Gly Tyr Thr His Gln Asp Pro Gly Ala Leu Thr His Leu530 535 540Ala Glu Lys Lys Pro Glu Tyr Ile Arg Glu Tyr Leu Pro Ala Asp Ala545 550 555 560Asn Ser Leu Leu Ala Val Gly Asp Val Ile Phe Arg Ser Gln Glu Lys565 570 575Ile Asn Tyr Val Val Thr Ser Lys His Pro Arg Gln Gln Trp Phe Ser580 585 590Ile Glu Glu Ala Lys Gln Leu Val Asp Asn Gly Leu Gly Ile Ile Asp595 600 605Trp Ala Ser Thr Asp Gln Gly Ser Glu Pro Asp Ile Val Phe Ala Ala610 615 620Ala Gly Thr Glu Pro Thr Leu Glu Thr Leu Ala Ala Ile Gln Leu Leu625 630 635 640His Asp Ser Phe Pro Asp Met Lys Ile Arg Phe Val Asn Val Val Asp645 650 655Ile Leu Lys Leu Arg Ser Pro Glu Lys Asp Pro Arg Gly Leu Ser Asp660 665 670Ala Glu Phe Asp His Tyr Phe Thr Lys Asp Lys Pro Val Val Phe Ala675 680 685Phe His Gly Tyr Glu Asp Leu Val Arg Asp Ile Phe Phe Asp Arg His690 695 700Asn His Asn Leu His Val His Gly Tyr Arg Glu Asn Gly Asp Ile Thr705 710 715 720Thr Pro Phe Asp Val Arg Val Met Asn Gln Met Asp Arg Phe Asp Leu725 730 735Ala Lys Ser Ala Ile Ala Ala Gln Pro Ala Met Glu Asn Thr Gly Ala740 745 750Ala Phe Val Gln Asp Met Asp Asn Met Leu Ala Lys His Asn Ala Tyr755 760 765Ile Arg Asp Ala Gly Thr Asp Leu Pro Glu Val Asn Asp Trp Gln Trp770 775 780Lys Gly Leu Lys785
<210>3<211>2367<212>DNA<213>植物乳杆菌<220>
<221>CDS<222>(1)..(2367)<223>
<400>1atg aca aca gat tac tca tca cca gca tat ttg caa aaa gtt gat aag 48Met Thr Thr Asp Tyr Ser Ser Pro Ala Tyr Leu Gln Lys Val Asp Lys1 5 10 15tac tgg cgt gct gcc aac tac tta tca gtt ggt caa ctt tat tta aaa 96Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Tyr Leu Lys20 25 30gat aat cca cta tta caa cgg cca ttg aag gcc agt gac gtt aag gtt144Asp Asn Pro Leu Leu Gln Arg Pro Leu Lys Ala Ser Asp Val Lys Val35 40 45cat cca att ggt cac tgg ggg acg att gcc ggt caa aac ttt atc tat192His Pro Ile Gly His Trp Gly Thr Ile Ala Gly Gln Asn Phe Ile Tyr50 55 60gct cat ctt aac cgg gtc atc aac aag tac ggt ttg aag atg ttc tac240Ala His Leu Asn Arg Val Ile Asn Lys Tyr Gly Leu Lys Met Phe Tyr65 70 75 80gtt gaa ggt cca ggt cat ggt ggt caa gtg atg gtt tca aac tct tac288Val Glu Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr85 90 95ctt gac ggt act tac acc gat att tat cca gaa att acg cag gat gtt336Leu Asp Gly Thr Tyr Thr Asp Ile Tyr Pro Glu Ile Thr Gln Asp Val100 105 110gaa ggg atg caa aag ctc ttc aag caa ttc tca ttc cca ggt ggg gtt384Glu Gly Met Gln Lys Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val115 120 125gct tcc cat gcg gca cct gaa aca ccc ggt tca atc cac gaa ggt ggc432Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly130 135 140gaa ctt ggt tac tca att tea cac ggg gtt ggg gca att ctt gac aat480Glu Leu Gly Tyr Ser Ile Ser His Gly Val Gly Ala Ile Leu Asp Asn145 150 155 160cct gac gaa atc gcc gcg gtt gtt gtt ggt gat ggg gaa tcc gaa acg528Pro Asp Glu Ile Ala Ala Val Val Val Gly Asp Gly Glu Ser Glu Thr165 170 175ggt cca tta gca act tca tgg caa tca acg aag ttc att aac cca atc576Gly Pro Leu Ala Thr Ser Trp Gln Ser Thr Lys Phe Ile Asn Pro Ile180 185 190aac gac ggg gct gtt tta cca atc ttg aac tta aat ggt ttt aag att624Asn Asp Gly Ala Val Leu Pro Ile Leu Asn Leu Asn Gly Phe Lys Ile195 200 205tct aat cca acg att ttt ggt cgg act tct gat gct aag att aag gaa672Ser Asn Pro Thr Ile Phe Gly Arg Thr Ser Asp Ala Lys Ile Lys Glu210 215 220tac ttc gaa agc atg aat tgg gaa cca atc ttc gtt gaa ggt gac gat720Tyr Phe Glu Ser Met Asn Trp Glu Pro Ile Phe Val Glu Gly Asp Asp225 230 235 240cct gaa aag gtt cac cca gcc tta gct aag gcc atg gat gaa gcc gtt768Pro Glu Lys Val His Pro Ala Leu Ala Lys Ala Met Asp Glu Ala Val
245 250 255gaa aag atc aag gca atc cag aag cat gct cgc gaa aat aac gat gca 816Glu Lys Ile Lys Ala Ile Gln Lys His Ala Arg Glu Asn Asn Asp Ala260 265 270aca ttg cca gta tgg cca atg atc gtc ttc cgc gca cct aag ggc tgg 864Thr Leu Pro Val Trp Pro Met Ile Val Phe Arg Ala Pro Lys Gly Trp275 280 285act ggt ccg aag tca tgg gac ggt gat aag atc gaa ggt tca ttc cgt 912Thr Gly Pro Lys Ser Trp Asp Gly Asp Lys Ile Glu Gly Ser Phe Arg290 295 300gct cat caa att ccg att cct gtt gat caa aat gac atg gaa cat gcg 960Ala His Gln Ile Pro Ile Pro Val Asp Gln Asn Asp Met Glu His Ala305 310 315 320gat gct tta gtt gat tgg ctc gaa tca tat caa cca aaa gaa ctc ttc 1008Asp Ala Leu Val Asp Trp Leu Glu Ser Tyr Gln Pro Lys Glu Leu Phe325 330 335aat gaa gat ggc tct ttg aag gat gat att aaa gaa att att cct act1056Asn Glu Asp Gly Ser Leu Lys Asp Asp Ile Lys Glu Ile Ile Pro Thr340 345 350ggg gac agt cgg atg gct gct aac cca atc acc aat ggt ggg gtc gat1104Gly Asp Ser Arg Met Ala Ala Asn Pro Ile Thr Asn Gly Gly Val Asp355 360 365ccg aaa gcc ttg aac tta cca aac ttc cgt gat tat gcg gtc gat acg1152Pro Lys Ala Leu Asn Leu Pro Asn Phe Arg Asp Tyr Ala Val Asp Thr370 375 380tcc aaa gaa ggc gcg aat gtt aag caa gat atg atc gtt tgg tca gac1200Ser Lys Glu Gly Ala Asn Val Lys Gln Asp Met Ile Val Trp Ser Asp385 390 395 400tat ttg cgg gat gtc atc aag aaa aat cct gat aac ttc cgg ttg ttc1248Tyr Leu Arg Asp Val Ile Lys Lys Asn Pro Asp Asn Phe Arg Leu Phe405 410 415gga cct gat gaa acc atg tct aac cgt tta tat ggt gtc ttc gaa acc1296Gly Pro Asp Glu Thr Met Ser Asn Arg Leu Tyr Gly Val Phe Glu Thr420 425 430act aat cgt caa tgg atg gaa gac att cat cca gat agt gac caa tat1344Thr Asn Arg Gln Trp Met Glu Asp Ile His Pro Asp Ser Asp Gln Tyr435 440 445gaa gca cca gct ggc cgg gtc tta gat gct cag tta tct gaa cac caa1392Glu Ala Pro Ala Gly Arg Val Leu Asp Ala Gln Leu Ser Glu His Gln450 455 460gct gaa ggt tgg tta gaa ggt tac gtc tta act gga cgt cat ggg tta1440Ala Glu Gly Trp Leu Glu Gly Tyr Val Leu Thr Gly Arg His Gly Leu465 470 475 480ttt gcc agt tat gaa gcc ttc cta cgc gtt gtg gac tca atg ttg acg1488Phe Ala Ser Tyr Glu Ala Phe Leu Arg Val Val Asp Ser Met Leu Thr485 490 495caa cac ttc aag tgg tta cgt aaa gcc aat gaa ctt gat tgg cgt aaa1536Gln His Phe Lys Trp Leu Arg Lys Ala Asn Glu Leu Asp Trp Arg Lys500 505 510aag tac cca tca ctt aac att atc gcg gct tca act gta ttc caa caa1584Lys Tyr Pro Ser Leu Asn Ile Ile Ala Ala Ser Thr Val Phe Gln Gln515 520 525gac cat aat ggt tat acc cac caa gat cca ggt gca tta act cat ttg1632Asp His Asn Gly Tyr Thr His Gln Asp Pro Gly Ala Leu Thr His Leu530 535 540gcc gaa aag aaa cca gaa tac att cgt gaa tat tta cca gcc gat gcc1680Ala Glu Lys Lys Pro Glu Tyr Ile Arg Glu Tyr Leu Pro Ala Asp Ala545 550 555 560
aac acg tta tta gct gtc ggt gac gtc att ttc cgg agc caa gaa aag1728Asn Thr Leu Leu Ala Val Gly Asp Val Ile Phe Arg Ser Gln Glu Lys565 570 575atc aac tac gtg gtt acg tca aaa cac cca cgt caa caa tgg ttc agc1776Ile Asn Tyr Val Val Thr Ser Lys His Pro Arg Gln Gln Trp Phe Ser580 585 590att gaa gaa gct aag caa tta gtt gac aat ggt ctt ggt atc att gat1824Ile Glu Glu Ala Lys Gln Leu Val Asp Asn Gly Leu Gly Ile Ile Asp595 600 605tgg gca agt acg gac caa ggt agc gaa cca gac att gtc ttt gca gct1872Trp Ala Ser Thr Asp Gln Gly Ser Glu Pro Asp Ile Val Phe Ala Ala610 615 620gct ggg acg gaa cca acg ctt gaa acg ttg gct gcc atc caa tta cta1920Ala Gly Thr Glu Pro Thr Leu Glu Thr Leu Ala Ala Ile Gln Leu Leu625 630 635 640cac gac agt ttc cca gag atg aag att cgt ttc gtg aac gtg gtc gac1968His Asp Ser Phe Pro Glu Met Lys Ile Arg Phe Val Asn Val Val Asp645 650 655atc ttg aag tta cgt agt cct gaa aag gat ccg cgg ggc ttg tca gat2016Ile Leu Lys Leu Arg Ser Pro Glu Lys Asp Pro Arg Gly Leu Ser Asp660 665 670gct gag ttt gac cat tac ttt act aag gac aaa cca gtg gtc ttt gct2064Ala Glu Phe Asp His Tyr Phe Thr Lys Asp Lys Pro Val Val Phe Ala675 680 685ttc cac ggt tac gaa gac tta gtt cgt gac atc ttc ttt gat cgt cac2112Phe His Gly Tyr Glu Asp Leu Val Arg Asp Ile Phe Phe Asp Arg His690 695 700aac cat aac tta tac gtc cac ggt tac cgt gaa aat ggt gat att acc2160Asn His Asn Leu Tyr Val His Gly Tyr Arg Glu Asn Gly Asp Ile Thr705 710 715 720aca cca ttc gac gta cgg gtc atg aac cag atg gac cgc ttc gac tta2208Thr Pro Phe Asp Val Arg Val Met Asn Gln Met Asp Arg Phe Asp Leu725 730 735gct aag tcg gca att gcg gcg caa cca gca atg gaa aac act ggt gcg2256Ala Lys Ser Ala Ile Ala Ala Gln Pro Ala Met Glu Asn Thr Gly Ala740 745 750gcc ttc gtt caa tcc atg gat aat atg ctt gct aaa cac aat gcc tat2304Ala Phe Val Gln Ser Met Asp Asn Met Leu Ala Lys His Asn Ala Tyr755 760 765atc cgg gat gcc gga act gac ttg cca gaa gtt aat gat tgg caa tgg2352Ile Arg Asp Ala Gly Thr Asp Leu Pro Glu Val Asn Asp Trp Gln Trp770 775 780aag ggt tta aaa taa2367Lys Gly Leu Lys785<210>4<211>788<212>PRT<213>植物乳杆菌<400>4Met Thr Thr Asp Tyr Ser Ser Pro Ala Tyr Leu Gln Lys Val Asp Lys1 5 10 15Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Tyr Leu Lys20 25 30
Asp Asn Pro Leu Leu Gln Arg Pro Leu Lys Ala Ser Asp Val Lys Val35 40 45His Pro Ile Gly His Trp Gly Thr Ile Ala Gly Gln Asn Phe Ile Tyr50 55 60Ala His Leu Asn Arg Val Ile Asn Lys Tyr Gly Leu Lys Met Phe Tyr65 70 75 80Val Glu Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr85 90 95Leu Asp Gly Thr Tyr Thr Asp Ile Tyr Pro Glu Ile Thr Gln Asp Val100 105 110Glu Gly Met Gln Lys Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val115 120 125Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly130 135 140Glu Leu Gly Tyr Ser Ile Ser His Gly Val Gly Ala Ile Leu Asp Asn145 150 155 160Pro Asp Glu Ile Ala Ala Val Val Val Gly Asp Gly Glu Ser Glu Thr165 170 175Gly Pro Leu Ala Thr Ser Trp Gln Ser Thr Lys Phe Ile Asn Pro Ile180 185 190Asn Asp Gly Ala Val Leu Pro Ile Leu Asn Leu Asn Gly Phe Lys Ile195 200 205Ser Asn Pro Thr Ile Phe Gly Arg Thr Ser Asp Ala Lys Ile Lys Glu210 215 220Tyr Phe Glu Ser Met Asn Trp Glu Pro Ile Phe Val Glu Gly Asp Asp225 230 235 240Pro Glu Lys Val His Pro Ala Leu Ala Lys Ala Met Asp Glu Ala Val245 250 255Glu Lys Ile Lys Ala Ile Gln Lys His Ala Arg Glu Asn Asn Asp Ala260 265 270Thr Leu Pro Val Trp Pro Met Ile Val Phe Arg Ala Pro Lys Gly Trp275 280 285Thr Gly Pro Lys Ser Trp Asp Gly Asp Lys Ile Glu Gly Ser Phe Arg290 295 300Ala His Gln Ile Pro Ile Pro Val Asp Gln Asn Asp Met Glu His Ala305 310 315 320Asp Ala Leu Val Asp Trp Leu Glu Ser Tyr Gln Pro Lys Glu Leu Phe325 330 335Asn Glu Asp Gly Ser Leu Lys Asp Asp Ile Lys Glu Ile Ile Pro Thr340 345 350Gly Asp Ser Arg Met Ala Ala Asn Pro Ile Thr Asn Gly Gly Val Asp355 360 365Pro Lys Ala Leu Asn Leu Pro Asn Phe Arg Asp Tyr Ala Val Asp Thr370 375 380Ser Lys Glu Gly Ala Asn Val Lys Gln Asp Met Ile Val Trp Ser Asp385 390 395 400Tyr Leu Arg Asp Val Ile Lys Lys Asn Pro Asp Asn Phe Arg Leu Phe405 410 415Gly Pro Asp Glu Thr Met Ser Asn Arg Leu Tyr Gly Val Phe Glu Thr420 425 430Thr Asn Arg Gln Trp Met Glu Asp Ile His Pro Asp Ser Asp Gln Tyr435 440 445Glu Ala Pro Ala Gly Arg Val Leu Asp Ala Gln Leu Ser Glu His Gln450 455 460Ala Glu Gly Trp Leu Glu Gly Tyr Val Leu Thr Gly Arg His Gly Leu465 470 475 480Phe Ala Ser Tyr Glu Ala Phe Leu Arg Val Val Asp Ser Met Leu Thr485 490 495
Gln His Phe Lys Trp Leu Arg Lys Ala Asn Glu Leu Asp Trp Arg Lys500 505 510Lys Tyr Pro Ser Leu Asn Ile Ile Ala Ala Ser Thr Val Phe Gln Gln515 520 525Asp His Asn Gly Tyr Thr His Gln Asp Pro Gly Ala Leu Thr His Leu530 535 540Ala Glu Lys Lys Pro Glu Tyr Ile Arg Glu Tyr Leu Pro Ala Asp Ala545 550 555 560Asn Thr Leu Leu Ala Val Gly Asp Val Ile Phe Arg Ser Gln Glu Lys565 570 575Ile Asn Tyr Val Val Thr Ser Lys His Pro Arg Gln Gln Trp Phe Ser580 585 590Ile Glu Glu Ala Lys Gln Leu Val Asp Asn Gly Leu Gly Ile Ile Asp595 600 605Trp Ala Ser Thr Asp Gln Gly Ser Glu Pro Asp Ile Val Phe Ala Ala610 615 620Ala Gly Thr Glu Pro Thr Leu Glu Thr Leu Ala Ala Ile Gln Leu Leu625 630 635 640His Asp Ser Phe Pro Glu Met Lys Ile Arg Phe Val Asn Val Val Asp645 650 655Ile Leu Lys Leu Arg Ser Pro Glu Lys Asp Pro Arg Gly Leu Ser Asp660 665 670Ala Glu Phe Asp His Tyr Phe Thr Lys Asp Lys Pro Val Val Phe Ala675 680 685Phe His Gly Tyr Glu Asp Leu Val Arg Asp Ile Phe Phe Asp Arg His690 695 700Asn His Asn Leu Tyr Val His Gly Tyr Arg Glu Asn Gly Asp Ile Thr705 710 715 720Thr Pro Phe Asp Val Arg Val Met Asn Gln Met Asp Arg Phe Asp Leu725 730 735Ala Lys Ser Ala Ile Ala Ala Gln Pro Ala Met Glu Asn Thr Gly Ala740 745 750Ala Phe Val Gln Ser Met Asp Asn Met Leu Ala Lys His Asn Ala Tyr755 760 765Ile Arg Asp Ala Gly Thr Asp Leu Pro Glu Val Asn Asp Trp Gln Trp770 775 780Lys Gly Leu Lys785<210>5<211>2660<212>DNA<213>乳酸双歧杆菌<220>
<221>CDS<222>(128)..(2605)<223>
<400>5aggtcagcgt attcgcgtaa cataatcagc gatcgggcac ggagaccggc ctgcaggaca 60gcgccgaagc ccgtgcccaa cggaataaac aaatcgcaca tttatgtgca ggagtacagg120agcacacatg act aat cct gtt att ggt acc cca tgg cag aag ctg gat 169Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln Lys Leu Asp1 5 10cgt ecg gtt tcc gaa gag gcc atc gaa ggc atg gac aag tac tgg cgc 217Arg Pro Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys Tyr Trp Arg
15 20 25 30gtc gcc aac tac atg tct atc ggc cag atc tac ctg cgt agc aac ccg265Val Ala Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro35 40 45ctg atg aag gag ccc ttc acc cgc gat gac gtg aag cac cgt ctg gtc313Leu Met Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His Arg Leu Val50 55 60ggc cac tgg ggc acc acc ccg ggc ctg aac ttc ctt ctc gcc cac atc361Gly His Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu Ala His Ile65 70 75aac cgc ctg atc gcc gat cac cag cag aac acc gtg ttc atc atg ggt409Asn Arg Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe Ile Met Gly80 85 90cct ggc cac ggc ggc cct gca ggt acc gct cag tcc tac atc gac ggc457Pro Gly His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr Ile Asp Gly95 100 105 110acc tac acc gag tac tac ccg aac atc acc aag gac gaa gct ggc ctg505Thr Tyr Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu115 120 125cag aag ttc ttc cgc cag ttc tcc tac ccg ggt ggc att cct tcc cac553Gln Lys Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His130 135 140ttc gct ccg gag acg ccg ggc tcc atc cac gaa ggc ggc gag ctg ggc601Phe Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly145 150 155tac gcc ctg tcg cac gcc tac ggc gcg atc atg gac aac ccg agc ctc649Tyr Ala Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu160 165 170ttc gtc ccg tgc atc atc ggt gac ggc gaa gcc gag acc ggc cct ctg697Phe Val Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu175 180 185 190gcc acc ggc tgg cag tcc aac aag ctc gtc aac ccg cgc acc gac ggc745Ala Thr Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly195 200 205atc gtc ctg ccg atc ctg cac ctc aac ggc tac aag atc gcc aac ccg793Ile Val Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro210 215 220acg atc ctc gcc cgc atc tcc gac gag gag ctg cac gac ttc ttc cgc841Thr Ile Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp Phe Phe Arg225 230 235ggc atg ggt tac cac ccg tac gag ttc gtc gcc ggc ttc gac aac gag889Gly Met Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu240 245 250gat cac ctg tcg atc cac cgt cgc ttc gcc gag ctc ttc gag acc atc937Asp His Leu Ser Ile His Arg Arg Phe Ala Glu Leu Phe Glu Thr Ile255 260 265 270ttc gac gag atc tgc gat atc aag gct gcg gct cag acc gac gac atg985Phe Asp Glu Ile Cys Asp Ile Lys Ala Ala Ala Gln Thr Asp Asp Met275 280 285acc cgt ccg ttc tac ccg atg ctc atc ttc cgc acc ccg aag ggc tgg 1033Thr Arg Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp290 295 300acc tgc ccg aag ttc atc gac ggc aag aag acc gaa ggc tcc tgg cgt 1081Thr Cys Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg305 310 315gca cac cag gtc ccg ctg gct tcc gcc cgc gac acc gag gcc cac ttc 1129Ala His Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe320 325 330
gaa gtc ctc aag ggc tgg atg gaa tcc tac aag ccg gag gag ctc ttc1177Glu Val Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe335 340 345 350aac gcc gac ggc tcc atc aag gag gac gtc acc gca ttc atg cct aag1225Asn Ala Asp Gly Ser Ile Lys Glu Asp Val Thr Ala Phe Met Pro Lys355 360 365ggc gaa ctg cgc atc ggc gcc aac ccg aat gcc aac ggc ggc cgc atc1273Gly Glu Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Arg Ile370 375 380cgc gag gat ctg aag ctc cct gag ctc gat cag tac gag atc acc ggc1321Arg Glu Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu Ile Thr Gly385 390 395gtc aag gaa tac ggc cac ggt tgg ggc cag gtc gag gct ccg cgt tcc1369Val Lys Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala Pro Arg Ser400 405 410ctc ggc gcg tac tgc cgc gac atc atc aag aac aac ccg gat tcg ttc1417Leu Gly Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro Asp Ser Phe415 420 425 430cgc gtc ttc gga cct gac gag acc gcg tcc aac cgt ctg aac gcg acc1465Arg Val Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Asn Ala Thr435 440 445tac gag gtc acc aag aag cag tgg gac aac gga tac ctc tcg gct ctc1513Tyr Glu Val Thr Lys Lys Gln Trp Asp Asn Gly Tyr Leu Ser Ala Leu450 455 460gtc gac gag aac atg gcc gtc acc ggc cag gtt gtc gag cag ctc tcc1561Val Asp Glu Asn Met Ala Val Thr Gly Gln Val Val Glu Gln Leu Ser465 470 475gag cat cag tgc gaa ggc ttc ctc gag gcc tac ctg ctc acc ggc cgt1609Glu His Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg480 485 490cac ggc atc tgg agc tcc tac gag tcc ttc gtg cac gtg atc gac tcc1657His Gly Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser495 500 505 510atg ctg aac cag cat gcg aag tgg ctc gag gcc acc gtc cgc gag atc1705Met Leu Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile515 520 525ccg tgg cgt aag ccg atc tcc tcg gtg aac ctc ctg gtc tcc tcg cac1753Pro Trp Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val Ser Ser His530 535 540gtg tgg cgt cag gat cac aac ggc ttc tcg cac cag gat ccg ggt gtg1801Val Trp Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val545 550 555acc tcc gtc ctg ctg aac aag acg ttc aac aac gac cac gtg acg aac1849Thr Ser Val Leu Leu Asn Lys Thr Phe Asn Asn Asp His Val Thr Asn560 565 570atc tac ttc gcg acc gat gcc aac atg ctg ctg gcc atc gcc gag aag1897Ile Tyr Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys575 580 585 590tgc ttc aag tcc acc aac aag atc aac gca atc ttc gcc ggc aag cag1945Cys Phe Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ala Gly Lys Gln595 600 605ccg gcc gcg acg tgg atc acc ctc gac gag gta cgc gcc gag ctc gag1993Pro Ala Ala Thr Trp Ile Thr Leu Asp Glu Val Arg Ala Glu Leu Glu610 615 620gct ggt gcc gcc gag tgg aag tgg gct tcc aac gcc aag agc aac gac2041Ala Gly Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Lys Ser Asn Asp625 630 635gag gtc cag gtt gtc ctc gcc gcc gcc ggc gac gtc ccg acc cag gag2089
Glu Val Gln Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu640 645 650atc atg gcc gct tcc gat gcc crc aac aag atg ggc atc aag ttc aag2137Ile Met Ala Ala Ser Asp Ala Leu Asn Lys MetGly Ile Lys Phe Lys655 660 665 670gtc gtc aac gtc gtg gac ctc atc aag ctg cag tcc tcg aag gag aac2185Val Val Asn Val Val Asp Leu Ile Lys Leu Gln Ser Ser Lys Glu Asn675 680 685gac gag gcc atg tct gac gag gac ttc gcc gac ctg ttc acc gcg gac2233Asp Glu Ala Met Ser Asp Glu Asp Phe Ala Asp Leu Phe Thr Ala Asp690 695 700aag ccg gtc ctc ttc gcc tac cac tcc tat gcc cag gac gtt cgt ggc2281Lys Pro Val Leu Phe Ala Tyr His Set Tyr Ala Gln Asp Val Arg Gly705 710 715ctc atc tac gac cgc ccg aac cac gac aac ttc acc gtt gtc gga tac2329Leu Ile Tyr Asp Arg Pro Asn His Asp Asn Phe Thr Val Val Gly Tyr720 725 730aag gag cag ggc tcc acg acg acg ccg ttc gac atg gtg cgt gtc aac2377Lys Glu Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val Arg Val Asn735 740 745 750gac atg gat cgc tac gcc ctt cag gcc aag gcc ctc gag ctc atc gac2425Asp Met Asp Arg Tyr Ala Leu Gln Ala Lys Ala Leu Glu Leu Ile Asp755 760 765gcc gac aag tat gcc gac aag atc aac gag ctc aac gag ttc cgc aag2473Ala Asp Lys Tyr Ala Asp Lys Ile Asn Glu Leu Asn Glu Phe Arg Lys770 775 780acc gcg ttc cag ttc gcc gtc gac aat ggc tat gac att cct gag ttc2521Thr Ala Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile Pro Glu Phe785 790 795acc gat tgg gtg tac ccg gat gtc aag gtc gac gag acc tcc atg ctc2569Thr Asp Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr Ser Met Leu800 805 810tcc gcc acc gcc gcg acc gcc ggc gac aac gag tga gcatagtctc 2615Ser Ala Thr Ala Ala Thr Ala Gly Asp Asn Glu815 820 825atcgcttagc cgatgaaagg cccgggtgtc cgcacccggg ccttt 2660<210>6<211>825<212>PRT<213>乳酸双歧杆菌<400>6Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln Lys Leu Asp Arg Pro1 5 10 15Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys Tyr Trp Arg Val Ala20 25 30Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met35 40 45Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His Arg Leu Val Gly His50 55 60Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu Ala His Ile Asn Arg65 70 75 80Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe Ile Met Gly Pro Gly85 90 95His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr Ile Asp Gly Thr Tyr
100 105 110Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys115 120 125Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala130 135 140Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala145 150 155 160Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu Phe Val165 170 175Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr180 185 190Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val195 200 205Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile210 215 220Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp Phe Phe Arg Gly Met225 230 235 240Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu Asp His245 250 255Leu Ser Ile His Arg Arg Phe Ala Glu Leu Phe Glu Thr Ile Phe Asp260 265 270Glu Ile Cys Asp Ile Lys Ala Ala Ala Gln Thr Asp Asp Met Thr Arg275 280 285Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys290 295 300Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ala His305 310 315 320Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val325 330 335Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asn Ala340 345 350Asp Gly Ser Ile Lys Glu Asp Val Thr Ala Phe Met Pro Lys Gly Glu355 360 365Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Arg Ile Arg Glu370 375 380Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu Ile Thr Gly Val Lys385 390 395 400Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala Pro Arg Ser Leu Gly405 410 415Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro Asp Ser Phe Arg Val420 425 430Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Asn Ala Thr Tyr Glu435 440 445Val Thr Lys Lys Gln Trp Asp Asn Gly Tyr Leu Ser Ala Leu Val Asp450 455 460Glu Asn Met Ala Val Thr Gly Gln Val Val Glu Gln Leu Ser Glu His465 470 475 480Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly485 490 495Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu500 505 510Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp515 520 525Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val Ser Ser His Val Trp530 535 540Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser545 550 555 560Val Leu Leu Asn Lys Thr Phe Asn Asn Asp His Val Thr Asn Ile Tyr
565 570 575Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Phe580 585 590Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ala Gly Lys Gln Pro Ala595 600 605Ala Thr Trp Ile Thr Leu Asp Glu Val Arg Ala Glu Leu Glu Ala Gly610 615 620Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Lys Ser Asn Asp Glu Val625 630 635 640Gln Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met645 650 655Ala Ala Ser Asp Ala Leu Asn Lys Met Gly Ile Lys Phe Lys Val Val660 665 670Asn Val Val Asp Leu Ile Lys Leu Gln Ser Ser Lys Glu Asn Asp Glu675 680 685Ala Met Ser Asp Glu Asp Phe Ala Asp Leu Phe Thr Ala Asp Lys Pro690 695 700Val Leu Phe Ala Tyr His Ser Tyr Ala Gln Asp Val Arg Gly Leu Ile705 710 715 720Tyr Asp Arg Pro Asn His Asp Asn Phe Thr Val Val Gly Tyr Lys Glu725 730 735Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val Arg Val Asn Asp Met740 745 750Asp Arg Tyr Ala Leu Gln Ala Lys Ala Leu Glu Leu Ile Asp Ala Asp755 760 765Lys Tyr Ala Asp Lys Ile Asn Glu Leu Asn Glu Phe Arg Lys Thr Ala770 775 780Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile Pro Glu Phe Thr Asp785 790 795 800Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr Ser Met Leu Ser Ala805 810 815Thr Ala Ala Thr Ala Gly Asp Asn Glu820 825<210>7<211>31<212>DNA<213>人工序列引物<400>7ctgaagcttg tgaaaagcaa attaaggagt g31<210>8<211>31<212>DNA<213>人工序列引物<400>8tcagaattct tattttaaac ccttccattg c31<210>9<211>2841<212>DNA<213>动物双歧杆菌<220>
<221>CDS<222>(320)..(2797)<223>
<400>9ttttcaacac gccgcgcaat atcctcacaa accgcacgcg acaacgacgg cgaaaacgct 60tgcattcgtt ggtatttcaa cgtttctcgc ctttattcac tgattttcca ttttcacaaa120tcgcccgagc aatctcccaa attcgcaaat tatgcgcaca gattcgctca cactgtttca180aaaactgcaa aaaggtcagc gtattcgcgt aacataatca gcgatcgggc acggagaccg240gcctgcagga cagcgccgaa gcccgtgccc aacggaataa acaaatcgca catttatgtg300caggagtaca ggagcacac atg act aat cct gtt att ggt acc cca tgg cag 352Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln1 5 10aag ctg gat cgt ccg gtt tcc gaa gag gcc atc gaa ggc atg gac aag 400Lys Leu Asp Arg Pro Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys15 20 25tac tgg cgc gtc gcc aac tac atg tct atc ggc cag atc tac ctg cgt 448Tyr Trp Arg Val Ala Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg30 35 40agc aac ccg ctg atg aag gag ccc ttc acc cgc gat gac gtg aag cac 496Ser Asn Pro Leu Met Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His45 50 55cgt ctg gtc ggc cac tgg ggc acc acc ccg ggc ctg aac ttc ctt ctc 544Arg Leu Val Gly His Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu60 65 70 75gcc cac atc aac cgc ctg atc gcc gat cac cag cag aac acc gtg ttc 592Ala His Ile Asn Arg Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe80 85 90atc atg ggt cct ggc cac ggc ggc cct gca ggt acc gct cag tcc tac 640Ile Met Gly Pro Gly His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr95 100 105atc gac ggc acc tac acc gag tac tac ccg aac atc acc aag gac gaa 688Ile Asp Gly Thr Tyr Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu110 115 120gct ggc ctg cag aag ttc ttc cgc cag ttc tcc tac ccg ggt ggc att 736Ala Gly Leu Gln Lys Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile125 130 135cct tcc cac ttc gct ccg gag acg ccg ggc tcc atc cac gaa ggc ggc 784Pro Ser His Phe Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly140 145 150 155gag ctg ggc tac gcc ctg tcg cac gcc tac ggc gcg atc atg gac aac 832Glu Leu Gly Tyr Ala Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn160 165 170ccg agc ctc ttc gtc ccg tgc atc atc ggt gac ggc gaa gcc gag acc 880Pro Ser Leu Phe Val Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr175 180 185ggc cct ctg gcc acc ggc tgg cag tcc aac aag ctc gtc aac ccg cgc 928Gly Pro Leu Ala Thr Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg190 195 200acc gac ggc atc gtc ctg ccg atc ctg cac ctc aac ggc tac aag atc 976Thr Asp Gly Ile Val Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile205 210 215gcc aac ccg acg atc ctc gcc cgc atc tcc gac gag gag ctg cac gac 1024Ala Asn Pro Thr Ile Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp220 225 230 235ttc ttc cgc ggc atg ggt tac cac ccg tac gag ttc gtc gcc ggc ttc 1072Phe Phe Arg Gly Met Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe
240 245 250gac aac gag gat cac ctg tcg atc cac cgt cgc ttc gcc gag ctc ttc1120Asp Asn Glu Asp His Leu Ser Ile His Arg Arg Phe Ala Glu Leu Phe255 260 265gag acc atc ttc gac gag atc tgc gat atc aag gct gcg gct cag acc1168Glu Thr Ile Phe Asp Glu Ile Cys Asp Ile Lys Ala Ala Ala Gln Thr270 275 280gac gac atg acc cgt ccg ttc tac ccg atg ctc atc ttc cgc acc ccg1216Asp Asp Met Thr Arg Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro285 290 295aag ggc tgg acc tgc ccg aag ttc atc gac ggc aag aag acc gaa ggc1264Lys Gly Trp Thr Cys Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly300 305 310 315tcc tgg cgt gca cac cag gtc ccg ctg gct tcc gcc cgc gac acc gag1312Ser Trp Arg Ala His Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu320 325 330gcc cac ttc gaa gtc ctc aag ggc tgg atg gaa tcc tac aag ccg gag1360Ala His Phe Glu Val Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu335 340 345gag ctc ttc aac gcc gac ggc tcc atc aag gag gac gtc acc gca ttc1408Glu Leu Phe Asn Ala Asp Gly Ser Ile Lys Glu Asp Val Thr Ala Phe350 355 360atg cct aag ggc gaa ctg cgc atc ggc gcc aac ccg aat gcc aac ggc1456Met Pro Lys Gly Glu Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly365 370 375ggc cgc atc cgc gag gat ctg aag ctc cct gag ctc gat cag tac gag1504Gly Arg Ile Arg Glu Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu380 385 390 395atc acc ggc gtc aag gaa tac ggc cac ggt tgg ggc cag gtc gag gct1552Ile Thr Gly Val Lys Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala400 405 410ccg cgt tcc ctc ggc gcg tac tgc cgc gac atc atc aag aac aac ccg1600Pro Arg Ser Leu Gly Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro415 420 425gat tcg ttc cgc gtc ttc gga cct gac gag acc gcg tcc aac cgt ctg1648Asp Ser Phe Arg Val Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu430 435 440aac gcg acc tac gag gtc acc aag aag cag tgg gac aac gga tac ctc1696Asn Ala Thr Tyr Glu Val Thr Lys Lys Gln Trp Asp Asn Gly Tyr Leu445 450 455tcg gct ctc gtc gac gag aac atg gcc gtc acc ggc cag gtt gtc gag1744Ser Ala Leu Val Asp Glu Asn Met Ala Val Thr Gly Gln Val Val Glu460 465 470 475cag ctc tcc gag cat cag tgc gaa ggc ttc ctc gag gcc tac ctg ctc1792Gln Leu Ser Glu His Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu480 485 490acc ggc cgt cac ggc atc tgg agc tcc tac gag tcc ttc gtg cac gtg1840Thr Gly Arg His Gly Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val495 500 505atc gac tcc atg ctg aac cag cat gcg aag tgg ctc gag gcc acc gtc1888Ile Asp Ser Met Leu Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val510 515 520cgc gag atc ccg tgg cgt aag ccg atc tcc tcg gtg aac ctc ctg gtc1936Arg Glu Ile Pro Trp Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val525 530 535tcc tcg cac gtg tgg cgt cag gat cac aac ggc ttc tcg cac cag gat1984Ser Ser His Val Trp Arg Gln Asp His Asn Gly Phe Ser His Gln Asp540 545 550 555
ccg ggt gtg acc tcc gtc ctg ctg aac aag acg ttc aac aac gac cac2032Pro Gly Val Thr Ser Val Leu Leu Asn Lys Thr Phe Asn Asn Asp His560 565 570gtg acg aac atc tac ttc gcg acc gat gcc aac atg ctg ctg gcc atc2080Val Thr Asn Ile Tyr Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile575 580 585gcc gag aag tgc ttc aag tcc acc aac aag atc aac gca atc ttc gcc2128Ala Glu Lys Cys Phe Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ala590 595 600ggc aag cag ccg gcc gcg acg tgg atc acc ctc gac gag gca cgc gcc2176Gly Lys Gln Pro Ala Ala Thr Trp Ile Thr Leu Asp Glu Ala Arg Ala605 610 615gag ctc gag gct ggt gcc gcc gag tgg aag tgg gct tcc aac gcc aag2224Glu Leu Glu Ala Gly Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Lys620 625 630 635agc aac gac gag gtc cag gtt gtc ctc gcc gcc gcc ggc gac gtc ccg2272Ser Asn Asp Glu Val Gln Val Val Leu Ala Ala Ala Gly Asp Val Pro640 645 650acc cag gag atc atg gcc gct tcc gat gcc ctc aac aag atg ggc atc2320Thr Gln Glu Ile Met Ala Ala Ser Asp Ala Leu Asn Lys Met Gly Ile655 660 665aag ttc aag gtc gtc aac gtc gtg gac ctc atc aag ctg cag tcc tcg2368Lys Phe Lys Val Val Asn Val Val Asp Leu Ile Lys Leu Gln Ser Ser670 675 680aag gag aac gac gag gcc atg tct gac gag gac ttc gcc gac ctg ttc2416Lys Glu Asn Asp Glu Ala Met Ser Asp Glu Asp Phc Ala Asp Leu Phe685 690 695acc gcg gac aag ccg gtc ctc ttc gcc tac cac tcc tat gcc cag gac2464Thr Ala Asp Lys Pro Val Leu Phe Ala Tyr His Ser Tyr Ala Gln Asp700 705 710 715gtt cgt ggc ctc atc tac gac cgc ccg aac cac gac aac ttc acc gtt2512Val Arg Gly Leu Ile Tyr Asp Arg Pro Asn His Asp Asn Phe Thr Val720 725 730gtc gga tac aag gag cag ggc tcc acg acg acg ccg ttc gac atg gtg2560Val Gly Tyr Lys Glu Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val735 740 745cgt gtc aac gac atg gat cgc tac gcc ctt cag gcc aag gcc ctc gag2608Arg Val Asn Asp Met Asp Arg Tyr Ala Leu Gln Ala Lys Ala Leu Glu750 755 760ctc atc gac gcc gac aag tat gcc gac aag atc aac gag ctc aac gag2656Leu Ile Asp Ala Asp Lys Tyr Ala Asp Lys Ile Asn Glu Leu Asn Glu765 770 775ttc cgc aag acc gcg ttc cag ttc gcc gtc gac aat ggc tat gac att2704Phe Arg Lys Thr Ala Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile780 785 790 795cct gag ttc acc gat tgg gtg tac ccg gat gtc aag gtc gac gag acc2752Pro Glu Phe Thr Asp Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr800 805 810tcc atg ctc tcc gcc acc gcc gcg acc gcc ggc gac aac gag tga2797Ser Met Leu Ser Ala Thr Ala Ala Thr Ala Gly Asp Asn Glu815 820 825gcatagtctc atcgcttagc cgatgaaagg ccaagggcga attc 2841<210>10<211>825<212>PRT<213>动物双歧杆菌
<400>10Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln Lys Leu Asp Arg Pro1 5 10 15Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys Tyr Trp Arg Val Ala20 25 30Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met35 40 45Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His Arg Leu Val Gly His50 55 60Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu Ala His Ile Asn Arg65 70 75 80Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe Ile Met Gly Pro Gly85 90 95His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr Ile Asp Gly Thr Tyr100 105 110Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys115 120 125Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala130 135 140Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala145 150 155 160Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu Phe Val165 170 175Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr180 185 190Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val195 200 205Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile210 215 220Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp Phe Phe Arg Gly Met225 230 235 240Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu Asp His245 250 255Leu Ser Ile His Arg Arg Phe Ala Glu Leu Phe Glu Thr Ile Phe Asp260 265 270Glu Ile Cys Asp lle Lys Ala Ala Ala Gln Thr Asp Asp Met Thr Arg275 280 285Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys290 295 300Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ala His305 310 315 320Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val325 330 335Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asn Ala340 345 350Asp Gly Ser Ile Lys Glu Asp Val Thr Ala Phe Met Pro Lys Gly Glu355 360 365Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Arg Ile Arg Glu370 375 380Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu Ile Thr Gly Val Lys385 390 395 400Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala Pro Arg Ser Leu Gly405 410 415Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro Asp Ser Phe Arg Val420 425 430Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Asn Ala Thr Tyr Glu435 440 445
Val Thr Lys Lys Gln Trp Asp Asn Gly Tyr Leu Ser Ala Leu Val Asp450 455 460Glu Asn Met Ala Val Thr Gly Gln Val Val Glu Gln Leu Ser Glu His465 470 475 480Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly485 490 495Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu500 505 510Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp515 520 525Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val Ser Ser His Val Trp530 535 540Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser545 550 555 560Val Leu Leu Asn Lys Thr Phe Asn Asn Asp His Val Thr Asn Ile Tyr565 570 575Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Phe580 585 590Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ala Gly Lys Gln Pro Ala595 600 605Ala Thr Trp Ile Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Ala Gly610 615 620Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Lys Ser Asn Asp Glu Val625 630 635 640Gln Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met645 650 655Ala Ala Ser Asp Ala Leu Asn Lys Met Gly Ile Lys Phe Lys Val Val660 665 670Asn Val Val Asp Leu Ile Lys Leu Gln Ser Ser Lys Glu Asn Asp Glu675 680 685Ala Met Ser Asp Glu Asp Phe Ala Asp Leu Phe Thr Ala Asp Lys Pro690 695 700Val Leu Phe Ala Tyr His Ser Tyr Ala Gln Asp Val Arg Gly Leu Ile705 710 715 720Tyr Asp Arg Pro Asn His Asp Asn Phe Thr Val Val Gly Tyr Lys Glu725 730 735Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val Arg Val Asn Asp Met740 745 750Asp Arg Tyr Ala Leu Gln Ala Lys Ala Leu Glu Leu Ile Asp Ala Asp755 760 765Lys Tyr Ala Asp Lys Ile Asn Glu Leu Asn Glu Phe Arg Lys Thr Ala770 775 780Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile Pro Glu Phe Thr Asp785 790 795 800Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr Ser Met Leu Ser Ala805 810 815Thr Ala Ala Thr Ala Gly Asp Asn Glu820 825<210>11<211>2841<212>DNA<213>动物双歧杆菌<220>
<221>CDS<222>(320)..(2797)
<223>
<400>11ttttcaacac gccgcgcaat atcctcacaa accgcacgcg acaacgacgg cgaaaatgct 60tgcattcgtt ggaatctcaa cgtttctcgc ctttattcac tgattttcca ttttcacaaa120tcgctcgagc aatcgcccaa attcgcaaat tatgcgcaca gattcgccca cactgtttca180aaaactgcaa aaaggtcagc gtattcgcgt aacataatca gcgatcggac acggaaaccg240gcctgcagga cagcaccgaa gcccgtgtcc aatggaataa acaaatcgca catttatgtg300caggagtaca ggagcacac atg act aat cct gtt att ggt acc cca tgg cag 352Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln1 5 10aag ctg gac cgt ccg gtt tcc gaa gag gcc atc gaa ggc atg gac aag 400Lys Leu Asp Arg Pro Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys15 20 25tac tgg cgc gtc gcc aac tac atg tcc atc ggc cag atc tac ctg cgt 448Tyr Trp Arg Val Ala Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg30 35 40agc aac ccg ctg atg aag gag ccc ttc acc cgc gat gac gtg aag cac 496Ser Asn Pro Leu Met Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His45 50 55cgt ctg gtc ggc cac tgg ggc acc acc ccg ggc ctg aac ttc ctt ctc 544Arg Leu Val Gly His Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu60 65 70 75gcc cat atc aac cgc ctg atc gcc gat cac cag cag aac acc gtg ttc 592Ala His Ile Asn Arg Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe80 85 90atc atg ggt cct ggc cac ggc ggc cct gca ggt acc gct cag tcc tac 640Ile Met Gly Pro Gly His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr95 100 105atc gac ggc acc tac acc gag tac tac ccg aac atc acc aag gac gag 688Ile Asp Gly Thr Tyr Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu110 115 120gct ggc ctg cag aag ttc ttc cgc cag ttc tcc tac ccg ggt ggc att 736Ala Gly Leu Gln Lys Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile125 130 135cct tcc cac ttc gct ccg gag acg cca ggc tcc atc cac gaa ggc ggc 784Pro Ser His Phe Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly140 145 150 155gag ctg ggc tac gcc ctg tcg cac gcc tac ggc gcg atc atg aac aac 832Glu Leu Gly Tyr Ala Leu Ser His Ala Tyr Gly Ala Ile Met Asn Asn160 165 170ccg agc ctc ttc gtc ccg tgc atc atc ggt gac ggc gaa gcc gag acc 880Pro Ser Leu Phe Val Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr175 180 185ggc cct ctg gcc acc ggc tgg cag tcc aac aag ctc gtc aac ccg cgc 928Gly Pro Leu Ala Thr Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg190 195 200acc gac ggc atc gtg ctg ccg atc ctg cac ctc aac ggc tac aag atc 976Thr Asp Gly Ile Val Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile205 210 215gcc aac ccg acg ctc ctc gcc cgc atc tcc gac gag gag ctg cac gac 1024Ala Asn Pro Thr Leu Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp220 225 230 235ttc ttc cgc ggt atg ggt tac cac ccg tac gag ttc gtc gcc ggc ttc 1072Phe Phe Arg Gly Met Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe240 245 250gac aac gag gat cac ctg tcg atc cac cgt cgc ttc gcc gag ctc ttc 1120Asp Asn Glu Asp His Leu Ser Ile His Arg Arg Phe Ala Glu Leu Phe
255 260 265gag acc atc ttc gac gag atc tgc gat atc aag gct gcg gct cag acc1168Glu Thr Ile Phe Asp Glu Ile Cys Asp Ile Lys Ala Ala Ala Gln Thr270 275 280gac gac atg acc cgt ccg ttc tac ccg atg ctc atc ttc cgc acc ccg1216Asp Asp Met Thr Arg Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro285 290 295aag ggc tgg acc tgc ccg aag ttc atc gac ggc aag aag acc gaa ggc1264Lys Gly Trp Thr Cys Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly300 305 310 315tcc tgg cgt gca cac cag gtc ccg ctg gct tcc gcc cgc gac acc gag1312Ser Trp Arg Ala His Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu320 325 330gcc cac ttc gaa gtc ctc aag ggc tgg atg gaa tcc tac aag ccg gag1360Ala His Phe Glu Val Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu335 340 345gag ctc ttc aac gcc gac ggc tcc atc aag gac gac gtc acc gca ttc1408Glu Leu Phe Asn Ala Asp Gly Ser Ile Lys Asp Asp Val Thr Ala Phe350 355 360atg cct aag ggc gaa ctg cgc atc ggc gcc aac ccg aac gcc aac ggt1456Met Pro Lys Gly Glu Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly365 370 375ggc cgc atc cgc gag gat ctg aag ctc cct gag ctc gat cag tac gag1504Gly Arg Ile Arg Glu Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu380 385 390 395atc acc ggc gtc aag gaa tac ggc cat ggc tgg ggc cag gtc gag gct1552Ile Thr Gly Val Lys Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala400 405 410ccg cgc tcc ctc ggc gcg tac tgc cgc gac atc atc aag aac aac ccg1600Pro Arg Ser Leu Gly Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro415 420 425gat tcg ttc cgc atc ttc gga cct gat gag acc gca tcc aac cgt ctg1648Asp Ser Phe Arg Ile Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu430 435 440aac gcg acc tac gag gtc acc aag aag cag tgg gac aac ggc tat ctc1696Asn Ala Thr Tyr Glu Val Thr Lys Lys Gln Trp Asp Asn Gly Tyr Leu445 450 455tcg gct ctc gtc gac gag aac atg gct gtc acc ggc cag gtt gtc gag1744Ser Ala Leu Val Asp Glu Asn Met Ala Val Thr Gly Gln Val Val Glu460 465 470 475cag ctc tcc gag cat cag tgc gaa ggc ttc ctc gag gcc tac ctg ctc1792Gln Leu Ser Glu His Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu480 485 490acg ggc cgc cac ggc atg tgg agc acc tat gag tcc ttc gcc cac gtg1840Thr Gly Arg His Gly Met Trp Ser Thr Tyr Glu Ser Phe Ala His Val495 500 505atc gac tcg atg ctc aac cag cat gcg aag tgg ctc gag gcg acc gtc1888Ile Asp Ser Met Leu Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val510 515 520cgc gag atc ccg tgg cgc aag ccg atc tcc tcg gtc aac ctc ctc gtc1936Arg Glu Ile Pro Trp Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val525 530 535tcc tcg cac gtg tgg cgt cag gac cac aac ggc ttc tcg cat cag gac1984Ser Ser His Val Trp Arg Gln Asp His Asn Gly Phe Ser His Gln Asp540 545 550 555ccg ggt gtc acc tcc gtc ctg atc aac aag acg ttc aac aac gac cac2032Pro Gly Val Thr Ser Val Leu Ile Asn Lys Thr Phe Asn Asn Asp His560 565 570
gtg acg aac atc tac ttc gcg acc gac gcc aac atg ctg ctc gcg atc2080Val Thr Asn Ile Tyr Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile575 580 585gcc gag aag tgc ttc aag tcc acc aac aag atc aac gcg atc ttc tcc2128Ala Glu Lys Cys Phe Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ser590 595 600ggc aag cag ccg gct ccg acc tgg att acc ctc gac gag gct cgt gcc2176Gly Lys Gln Pro Ala Pro Thr Trp Ile Thr Leu Asp Glu Ala Arg Ala605 610 615gag ctc gag gcc ggc gcc gcc gag tgg aag tgg gct tcc aac gcc aag2224Glu Leu Glu Ala Gly Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Lys620 625 630 635agc aac gac gag gtc cag att gtc ctc gcc gcc gca ggc gat gtc ccg2272Ser Asn Asp Glu Val Gln Ile Val Leu Ala Ala Ala Gly Asp Val Pro640 645 650acc cag gag atc atg gcc gct tcc gat gcc ctg aac aag gat ggc atc2320Thr Gln Glu Ile Met Ala Ala Ser Asp Ala Leu Asn Lys Asp Gly Ile655 660 665aag ttc aag gtc gtc aac gtt gtt gac ctc ctg aag ctg cag tcc ccg2368Lys Phe Lys Val Val Asn Val Val Asp Leu Leu Lys Leu Gln Ser Pro670 675 680gag aac aac gac gag gcc atg tcg aac gaa gac ttc acc gag ctc ttc2416Glu Asn Asn Asp Glu Ala Met Ser Asn Glu Asp Phe Thr Glu Leu Phe685 690 695acc gcc gac aaa ccg gtt ctg ttc gcc tac cac tcc tat gcc cag gac2464Thr Ala Asp Lys Pro Val Leu Phe Ala Tyr His Ser Tyr Ala Gln Asp700 705 710 715gtt cgt ggt ctt atc tac gac cgc ccg aac cac gac aac ttc aac gtt2512Val Arg Gly Leu Ile Tyr Asp Arg Pro Asn His Asp Asn Phe Asn Val720 725 730gtc ggc tac aag gag cag ggc tcc acg acc acg ccg ttc gac atg gtc2560Val Gly Tyr Lys Glu Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val735 740 745cgc gtc aac gac atg gat cgc tac gcg ctc gaa gct cag gct ctc gag2608Arg Val Asn Asp Met Asp Arg Tyr Ala Leu Glu Ala Gln Ala Leu Glu750 755 760ctg atc gac gcc gac aag tat gcc gac aag atc gac gag ctc aac gcg2656Leu Ile Asp Ala Asp Lys Tyr Ala Asp Lys Ile Asp Glu Leu Asn Ala765 770 775ttc cgc aag acc gcg ttc cag ttc gcc gtc gac aac ggc tac gac atc2704Phe Arg Lys Thr Ala Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile780 785 790 795ccg gag ttc acc gac tgg gtg tac ccg gac gtc aag gtc gac gag acg2752Pro Glu Phe Thr Asp Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr800 805 810cag atg ctc tcc gcg acc gcg gcg acc gct ggc gac aac gag tga2797Gln Met Leu Ser Ala Thr Ala Ala Thr Ala Gly Asp Asn Glu815 820 825gcatagtctc atcgcttagc cgatgaaagg ccaagggcga attc 2841210>12<211>825<212>PRT<213>动物双歧杆菌<400>12Met Thr Asn Pro Val Ile Gly Thr Pro Trp Gln Lys Leu Asp Arg Pro
1 5 10 15Val Ser Glu Glu Ala Ile Glu Gly Met Asp Lys Tyr Trp Arg Val Ala20 25 30Asn Tyr Met Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met35 40 45Lys Glu Pro Phe Thr Arg Asp Asp Val Lys His Arg Leu Val Gly His50 55 60Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Leu Ala His Ile Asn Arg65 70 75 80Leu Ile Ala Asp His Gln Gln Asn Thr Val Phe Ile Met Gly Pro Gly85 90 95His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr Ile Asp Gly Thr Tyr100 105 110Thr Glu Tyr Tyr Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys115 120 125Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala130 135 140Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala145 150 155 160Leu Ser His Ala Tyr Glv Ala Ile Met Asn Asn Pro Ser Leu Phe Val165 170 175Pro Cys Ile Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr180 185 190Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val195 200 205Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Leu210 215 220Leu Ala Arg Ile Ser Asp Glu Glu Leu His Asp Phe Phe Arg Gly Met225 230 235 240Gly Tyr His Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu Asp His245 250 255Leu Ser Ile His Arg Arg phc Ala Glu Leu Phe Glu Thr Ile Phe Asp260 265 270Glu lle Cys Asp Ile Lys Ala Ala Ala Gln Thr Asp Asp Met Thr Arg275 280 285Pro Phc Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys290 295 300Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ala His305 310 315 320Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val325 330 335Leu Lys Gly Trp Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asn Ala340 345 350Asp Gly Ser Ile Lys Asp Asp Val Thr Ala Phc Met Pro Lys Gly Glu355 360 365Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Arg Ile Arg Glu370 375 380Asp Leu Lys Leu Pro Glu Leu Asp Gln Tyr Glu Ile Thr Gly Val Lys385 390 395 400Glu Tyr Gly His Gly Trp Gly Gln Val Glu Ala Pro Arg Ser Leu Gly405 410 415Ala Tyr Cys Arg Asp Ile Ile Lys Asn Asn Pro Asp Ser Phe Arg Ile420 425 430Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Asn Ala Thr Tyr Glu435 440 445Val Thr Lys Lys Gln Trp Asp Asn Gly Tyr Leu Ser Ala Leu Val Asp450 455 460Glu Asn Met Ala Val Thr Gly Gln Val Val Glu Gln Leu Ser Glu His
465 470 475 480Gln Cys Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly485 490 495Met Trp Ser Thr Tyr Glu Ser Phe Ala His Val Ile Asp Ser Met Leu500 505 510Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp515 520 525Arg Lys Pro Ile Ser Ser Val Asn Leu Leu Val Ser Ser His Val Trp530 535 540Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser545 550 555 560Val Leu Ile Asn Lys Thr Phe Asn Asn Asp His Val Thr Asn Ile Tyr565 570 575Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Phe580 585 590Lys Ser Thr Asn Lys Ile Asn Ala Ile Phe Ser Gly Lys Gln Pro Ala595 600 605Pro Thr Trp Ile Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Ala Gly610 615 620Ala Ala Glu Trp Lys Trp Ala Ser Asn Ala Lys Ser Asn Asp Glu Val625 630 635 640Gln Ile Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met645 650 655Ala Ala Ser Asp Ala Leu Asn Lys Asp Gly Ile Lys Phe Lys Val Val660 665 670Asn Val Val Asp Leu Leu Lys Leu Gln Ser Pro Glu Asn Asn Asp Glu675 680 685Ala Met Ser Asn Glu Asp Phe Thr Glu Leu Phe Thr Ala Asp Lys Pro690 695 700Val Leu Phe Ala Tyr His Ser Tyr Ala Gln Asp Val Arg Gly Leu Ile705 710 715 720Tyr Asp Arg Pro Asn His Asp Asn Phe Asn Val Val Gly Tyr Lys Glu725 730 735Gln Gly Ser Thr Thr Thr Pro Phe Asp Met Val Arg Val Asn Asp Met740 745 750Asp Arg Tyr Ala Leu Glu Ala Gln Ala Leu Glu Leu Ile Asp Ala Asp755 760 765Lys Tyr Ala Asp Lys Ile Asp Glu Leu Asn Ala Phe Arg Lys Thr Ala770 775 780Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp Ile Pro Glu Phe Thr Asp785 790 795 800Trp Val Tyr Pro Asp Val Lys Val Asp Glu Thr Gln Met Leu Ser Ala805 810 815Thr Ala Ala Thr Ala Gly Asp Asn Glu820 825<210>13<211>34<212>DNA<213>人工序列<220>
<223>引物<400>13ctagtctaga ttttcaacac gccgcgcaat atcc34
<210>14<211>41<212>DNA<213>人工序列<220>
<223>引物<400>14ctagtctaga gaattcgccc ttggcctttc atcggctaag c 41<210>15<211>24<212>DNA<213>人工序列<220>
<223>引物<400>15aaattcctgt gaattagctg attt24<210>16<211>48<212>DNA<213>人工序列<220>
<223>引物<400>16ggtaccaata acaggattag tcatagaggc gaaggctcct tgaatagg 48<210>17<211>34<212>DNA<213>人工序列<220>
<223>引物<400>17ctagtctaga gaattcgccc ttggcctttc atcg 34<210>18<211>48<212>DNA<213>人工DNA<220>
<223>引物
<400>18cctattcaag gagccttcgc ctctatgact aatcctgtta ttggtacc 48<210>19<211>44<212>DNA<213>人工DNA<220>
<223>引物<400>19ctattctaga aaattcctgt gaattagctg atttagtact tttc 44<210>20<211>48<212>DNA<213>人工DNA<220>
<223>引物<400>20ggtaccaata acaggattag tcataattct gtttcctgtg tgaaattg 48<210>21<211>34<212>DNA<213>人工DNA<220>
<223>引物<400>21cggttctaga ggatccggag cttatcgact gcac 34<210>22<211>48<212>DNA<213>人工DNA<220>
<223>引物<400>22caatttcaca caggaaacag aattatgact aatcctgtta ttggtacc 48<210>23<211>49<212>DNA<213>人工DNA
<220>
<223>引物<400>23ctggtgatga gtaatctgta gacatagagg cgaaggctcc ttgaatagg 49<210>24<211>34<212>DNA<213>人工DNA<220>
<223>引物<400>24ctagtctaga ttatttcaaa cctttccatt gcca 34<210>25<211>49<212>DNA<213>人工DNA<220>
<223>引物<400>25cctattcaag gagccttcgc ctctatgtct acagattact catcaccag 49<210>26<211>48<212>DNA<213>人工DNA<220>
<223>引物<400>26ttatctgtcc cttgaggtga tttattccac acctcctgtt ggaatgtt 48<210>27<211>23<212>DNA<213>人工DNA<220>
<223>引物<400>27caaggtacaa cgcaacgatg cag23
<210>28<211>48<212>DNA<213>人工DNA<220>
<223>引物<400>28aacattccaa caggaggtgt ggaataaatc acctcaaggg acagataa 48<210>29<211>24<212>DNA<213>人工DNA<220>
<223>引物<400>29gatggtttgc tcgcaggtat tttg 24<210>30<211>34<212>DNA<213>人工DNA<220>
<223>引物<400>30cgcacccggg cagagaagcc ttggaggtga tctg 34<210>31<211>32<212>DNA<213>人工DNA<220>
<223>引物<400>31aggtcccggg accatgattg cgttgtggtc gg32<210>32<211>20<212>DNA<213>人工DNA<220>
<223>引物<400>32ccaggcactc gtcctcggtt20
<210>33<211>48<212>DNA<213>人工DNA<220>
<223>引物<400>33aggctagtgc aggactataa agaccagttc tcctaaaaat aacgtgtc 48<210>34<211>48<212>DNA<213>人工DNA<220>
<223>引物<400>34gacacgttat ttttaggaga actggtcttt atagtcctgc actagcct 48<210>35<211>20<212>DNA<213>人工DNA<220>
<223>引物<400>23tccatcgtgg ccaccgatcc 20<210>36<211>25<212>DNA<213>人工DNA<220>
<223>引物<400>36cgggatcccc accggcgtac tcgtg 25<210>37<211>28<212>DNA<213>人工DNA<220>
<223>引物
<400>37ccacggatcc ttccaatgct attggttg 28<210>38<211>2769<212>DNA<213>谷氨酸棒杆菌<220>
<221>CDS<222>(1)..(2769)<223>aceE<400>38atg gcc gat caa gca aaa ctt ggt ggc aag ccc tcg gat gac tct aac 48Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15ttc gcg atg atc cgc gat ggc gtg gca tct tat ttg aac gac tca gat 96Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp20 25 30ccg gag gag acc aac gag tgg atg gat tca ctc gac gga tta ctc cag144Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln35 40 45gag tct tct cca gaa cgt gct cgt tac ctc atg ctt cgt ttg ctt gag192Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu50 55 60cgt gca tct gca aag cgc gta tct ctt ccc cca atg acg tca acc gac240Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80tac gtc aac acc att cca acc tct atg gaa cct gaa ttc cca ggc gat288Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp85 90 95gag gaa atg gag aag cgt tac cgt cgt tgg att cgc tgg aac gca gcc336Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala100 105 110atc atg gtt cac cgc gct cag cga cca ggc atc ggc gtc ggc gga cac384Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His115 120 125att tcc act tac gca ggc gca gcc cct ctg tac gaa gtt ggc ttc aac432Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn130 135 140cac ttc ttc cgc ggc aag gat cac cca ggc ggc ggc gac cag atc ttc480His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe145 150 155 160ttc cag ggc cac gca tca cca ggt atg tac gca cgt gca ttc atg gag528Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu165 170 175ggt cgc ctt tct gaa gac gat ctc gat ggc ttc cgt cag gaa gt ttcc576Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser180 185 190cgt gag cag ggt ggc att ccg tcc tac cct cac cca cac ggt atg aag624Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys195 200 205gac ttc tgg gag ttc cca act gtg tcc atg ggt ctt ggc cca atg gat672Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp210 215 220gcc att tac cag gca cgt ttc aac cgc tac ctc gaa aac cgt ggc atc720
Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240aag gac acc tct gac cag cac gtc tgg gcc ttc ctt ggc gac ggc gaa768Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu245 250 255atg gac gag cca gaa tca cgt ggt ctc atc cag cag gct gca ctg aac816Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn260 265 270aac ctg gac aac ctg acc ttc gtg gtt aac tgc aac ctg cag cgt ctc864Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu275 280 285gac gga cct gtc cgc ggt aac acc aag atc atc cag gaa ctc gag tcc912Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser290 295 300ttc ttc cgt ggc gca ggc tgg tct gtg atc aag gtt gtt tgg ggt cgc960Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320gag tgg gat gaa ctt ctg gag aag gac cag gat ggt gca ctt gtt gag 1008Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu325 330 335atc atg aac aac acc tcc gat ggt gac tac cag acc ttc aag gct aac 1056Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn340 345 350gac ggc gca tat gtt cgt gag cac ttc ttc gga cgt gac cca cgc acc 1104Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr355 360 365gca aag ctc gtt gag aac atg acc gac gaa gaa atc tgg aag ctt cca 1152Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro370 375 380cgt ggc ggc cac gat tac cgc aag gtt tac gca gcc tac aag cga gct 1200Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400ctt gag acc aag gat cgc cca acc gtc atc ctt gct cac acc att aag 1248Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys405 410 415ggc tac gga ctc ggc cac aac ttc gaa ggc cgt aac gca acc cac cag 1296Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln420 425 430atg aag aag ctg acg ctt gat gat ctg aag ttg ttc cgc gac aag cag 1344Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln435 440 445ggc atc cca atc acc gat gag cag ctg gag aag gat cct tac ctt cct 1392Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro450 455 460cct tac tac cac cca ggt gaa gac gct cct gaa atc aag tac atg aag 1440Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480gaa cgt cgc gca gcg ctc ggt ggc tac ctg cca gag cgt cgt gag aac 1488Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn485 490 495tac gat cca att cag gtt cca cca ctg gat aag ctt cgc tct gtc cgt 1536Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg500 505 510aag ggc tcc ggc aag cag cag atc gct acc acc atg gcg act gtt cgt 1584Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg515 520 525acc ttc aag gaa ctg atg cgc gat aag ggc ttg gct gat cgc ctt gtc 1632Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val
530 535 540cca atc att cct gat gag gca cgt acc ttc ggt ctt gac tct tgg ttc1680Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560cca acc ttg aag atc tac aac ccg cac ggt cag aac tac gtg cct gtt1728Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val565 570 575gac cac gac ctg atg ctc tcc tac cgt gag gca cct gaa gga cag atc1776Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile580 585 590ctg cac gaa ggc atc aac gag gct ggt tcc gtg gca tcg ttc atc gct1824Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala595 600 605gcg ggt acc tcc tac gcc acc cac ggc aag gcc atg att ccg ctg tac1872Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr610 615 620atc ttc tac tcg atg ttc gga ttc cag cgc acc ggt gac tcc atc tgg1920Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640gca gca gcc gat cag atg gca cgt ggc ttc ctc ttg ggc gct acc gca1968Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala645 650 655ggt cgc acc acc ctg acc ggt gaa ggc ctc cag cac atg gat gga cac2016Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His660 665 670tcc cct gtc ttg gct tcc acc aac gag ggt gtc gag acc tac gac cca2064Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro675 680 685tcc ttt gcg tac gag atc gca cac ctg gtt cac cgt ggc atc gac cgc2112Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg690 695 700atg tac ggc cca ggc aag ggt gaa gat gtt atc tac tac atc acc atc2160Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720tac aac gag cca acc cca cag cca gct gag cca gaa gga ctg gac gta2208Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val725 730 735gaa ggc ctg cac aag ggc atc tac ctc tac tcc cgc ggt gaa ggc acc2256Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr740 745 750ggc cat gag gca aac atc ttg gct tcc ggt gtt ggt atg cag tgg gct2304Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala755 760 765ctc aag gct gca tcc atc ctt gag gct gac tac gga gtt cgt gcc aac2352Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn770 775 780att tac tcc gct act tct tgg gtt aac ttg gct cgc gat ggc gct gct2400Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800cgt aac aag gca cag ctg cgc aac cca ggt gca gat gct ggc gag gca2448Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala805 810 815ttc gta acc acc cag ctg aag cag acc tcc ggc cca tac gtt gca gtg2496Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val820 825 830tct gac ttc tcc act gat ctg cca aac cag atc cgt gaa tgg gtc cca2544Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln lle Arg Glu Trp Val Pro835 840 845
ggc gac tac acc gtt ctc ggt gca gat ggc ttc ggt ttc tct gat acc2592Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr850 855 860cgc cca gct gct cgt cgc ttc ttc aac atc gac gct gag tcc att gtt2640Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880gtt gca gtg ctg aac tcc ctg gca cgc gaa ggc aag atc gac gtc tcc2688Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser885 890 895gtt gct gct cag gct gct gag aag ttc aag ttg gat gat cct acg agt2736Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser900 905 910gtt tcc gta gat cca aac gct cct gag gaa taa2769Val Ser Val Asp Pro Asn Ala Pro Glu Glu915 920<210>39<211>922<212>PRT<213>谷氨酸棒杆菌<400>39Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn1 5 10 15Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp20 25 30Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln35 40 45Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu50 55 60Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp65 70 75 80Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp85 90 95Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala100 105 110Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His115 120 125Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn130 135 140His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln lle Phe145 150 155 160Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu165 170 175Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser180 185 190Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys195 200 205Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp210 215 220Ala Ile Tyr Gln Ala Arg Phe Asn Arg Tyr Leu Glu Asn Arg Gly Ile225 230 235 240Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu245 250 255Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn260 265 270
Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu275 280 285Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser290 295 300Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg305 310 315 320Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu325 330 335Ile Met Asn Asn Thr Ser Asp Gly Asp Tyr Gln Thr Phe Lys Ala Asn340 345 350Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr355 360 365Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro370 375 380Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala385 390 395 400Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys405 410 415Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln420 425 430Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln435 440 445Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro450 455 460Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys465 470 475 480Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn485 490 495Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg500 505 510Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg515 520 525Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val530 535 540Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe545 550 555 560Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val565 570 575Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile580 585 590Leu His Glu Gly Ile Asn Glu Ala Gly Ser Val Ala Ser Phe Ile Ala595 600 605Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr610 615 620Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp625 630 635 640Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala645 650 655Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His660 665 670Ser Pro Val Leu Ala Ser Thr Asn Glu Gly Val Glu Thr Tyr Asp Pro675 680 685Ser Phe Ala Tyr Glu Ile Ala His Leu Val His Arg Gly Ile Asp Arg690 695 700Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile705 710 715 720Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val725 730 735
Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr740 745 750Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala755 760 765Leu Lys Ala Ala Ser Ile Leu Glu Ala Asp Tyr Gly Val Arg Ala Asn770 775 780Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala785 790 795 800Arg Asn Lys Ala Gln Leu Arg Asn Pro Gly Ala Asp Ala Gly Glu Ala805 810 815Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val820 825 830Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro835 840 845Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr850 855 860Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val865 870 875 880Val Ala Val Leu Asn Ser Leu Ala Arg Glu Gly Lys Ile Asp Val Ser885 890 895Val Ala Ala Gln Ala Ala Glu Lys Phe Lys Leu Asp Asp Pro Thr Ser900 905 910Val Ser Val Asp Pro Asn Ala Pro Glu Glu915 920<210>40<211>2014<212>DNA<213>枯草芽孢杆菌<220>
<221>CDS<222>(464)..(1885)<223>sacB<400>40gatccttttt aacccatcac atatacctgc cgttcactat tatttagtga aatgagatat 60tatgatattt tctgaattgt gattaaaaag gcaactttat gcccatgcaa cagaaactat120aaaaaataca gagaatgaaa agaaacagat agatttttta gttctttagg cccgtagtct180gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa240acgagagtct aatagaatga ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc300aggcaagacc taaaatgtgt aaagggcaaa gtgtatactt tggcgtcacc ccttacatat360tttaggtctt tttttattgt gcgtaactaa cttgccatct tcaaacagga gggctggaag420aagcagaccg ctaacacagt acataaaaaa ggagacat ga acg atg aac atc aaa 475Met Asn Ile Lys1aag ttt gca aaa caa gca aca gta tta acc ttt act acc gca ct g ctg 523Lys Phe Ala Lys Gln Ala Thr Val Leu Thr Phe Thr Thr Ala Leu Leu5 10 15 20gca gga ggc gca act caa gcg ttt gcg aaa gaa acg aac caa aag cca 571Ala Gly Gly Ala Thr Gln Ala Phe Ala Lys Glu Thr Asn Gln Lys Pro25 30 35tat aag gaa aca tac ggc att tcc cat att aca cgc cat gat atg ctg 619Tyr Lys Glu Thr Tyr Gly Ile Ser His Ile Thr Arg His Asp Met Leu40 45 50caa atc cct gaa cag caa aaa aat gaa aaa tat caa gtt cct gaa ttc 667Gln Ile Pro Glu Gln Gln Lys Asn Glu Lys Tyr Gln Val Pro Glu Phe
55 60 65gat tcg tcc aca att aaa aat atc tct tct gca aaa ggc ctg gac gtt715Asp Ser Ser Thr Ile Lys Asn Ile Ser Ser Ala Lys Gly Leu Asp Val70 75 80tgg gac agc tgg cca tta caa aac gct gac ggc act gtc gca aac tat763Trp Asp Ser Trp Pro Leu Gln Asn Ala Asp Gly Thr Val Ala Asn Tyr85 90 95 100cac ggc tac cac atc gtc ttt gca tta gcc gga gat cct aaa aat gcg811His Gly Tyr His Ile Val Phe Ala Leu Ala Gly Asp Pro Lys Asn Ala105 110 115gat gac aca tcg att tac atg ttc tat caa aaa gtc ggc gaa act tct859Asp Asp Thr Ser Ile Tyr Met Phe Tyr Gln Lys Val Gly Glu Thr Ser120 125 130att gac agc tgg aaa aac gct ggc cgc gtc ttt aaa gac agc gac aaa907Ile Asp Ser Trp Lys Asn Ala Gly Arg Val Phe Lys Asp Ser Asp Lys135 140 145ttc gat gca aat gat tct atc cta aaa gac caa aca caa gaa tgg tca955Phe Asp Ala Asn Asp Ser Ile Leu Lys Asp Gln Thr Gln Glu Trp Ser150 155 160ggt tca gcc aca ttt aca tct gac gga aaa atc cgt tta ttc tac act 1003Gly Ser Ala Thr Phe Thr Ser Asp Gly Lys Ile Arg Leu Phe Tyr Thr165 170 175 180gat ttc tcc ggt aaa cat tac ggc aaa caa aca ctg aca act gca caa 1051Asp Phe Ser Gly Lys His Tyr Gly Lys Gln Thr Leu Thr Thr Ala Gln185 190 195gtt aac gta tca gca tca gac agc tct ttg aac atc aac ggt gta gag 1099Val Asn Val Ser Ala Ser Asp Ser Ser Leu Asn Ile Asn Gly Val Glu200 205 210gat tat aaa tca atc ttt gac ggt gac gga aaa acg tat caa aat gta 1147Asp Tyr Lys Ser Ile Phe Asp Gly Asp Gly Lys Thr Tyr Gln Asn Val215 220 225cag cag ttc atc gat gaa ggc aac tac agc tca ggc gac aac cat acg 1195Gln Gln Phe Ile Asp Glu Gly Asn Tyr Ser Ser Gly Asp Asn His Thr230 235 240ctg aga gat cct cac tac gta gaa gat aaa ggc cac aaa tac tta gta 1243Leu Arg Asp Pro His Tyr Val Glu Asp Lys Gly His Lys Tyr Leu Val245 250 255 260ttt gaa gca aac act gga act gaa gat ggc tac caa ggc gaa gaa tct 1291Phe Glu Ala Asn Thr Gly Thr Glu Asp Gly Tyr Gln Gly Glu Glu Ser265 270 275tta ttt aac aaa gca tac tat ggc aaa agc aca tca ttc ttc cgt caa 1339Leu Phe Asn Lys Ala Tyr Tyr Gly Lys Ser Thr Ser Phe Phe Arg Gln280 285 290gaa agt caa aaa ctt ctg caa agc gat aaa aaa cgc acg gct gag tta 1387Glu Ser Gln Lys Leu Leu Gln Ser Asp Lys Lys Arg Thr Ala Glu Leu295 300 305gca aac ggc gct ctc ggt atg att gag cta aac gat gat tac aca ctg 1435Ala Asn Gly Ala Leu Gly Met Ile Glu Leu Asn Asp Asp Tyr Thr Leu310 315 320aaa aaa gtg atg aaa ccg ctg att gca tct aac aca gta aca gat gaa 1483Lys Lys Val Met Lys Pro Leu Ile Ala Ser Asn Thr Val Thr Asp Glu325 330 335 340att gaa cgc gcg aac gtc ttt aaa atg aac ggc aaa tgg tac ctg ttc 1531Ile Glu Arg Ala Asn Val Phe Lys Met Asn Gly Lys Trp Tyr Leu Phe345 350 355act gac tcc cgc gga tca aaa atg acg att gac ggc att acg tct aac 1579Thr Asp Ser Arg Gly Ser Lys Met Thr Ile Asp Gly Ile Thr Ser Asn360 365 370
gat att tac atg ctt ggt tat gtt tct aat tct tta act ggc cca tac1627Asp Ile Tyr Met Leu Gly Tyr Val Ser Asn Ser Leu Thr Gly Pro Tyr375 380 385aag ccg ctg aac aaa act ggc ctt gtg tta aaa atg gat ctt gat cct1675Lys Pro Leu Asn Lys Thr Gly Leu Val Leu Lys Met Asp Leu Asp Pro390 395 400aac gat gta acc ttt act tac tca cac ttc gct gta cct caa gcg aaa1723Ash Asp Val Thr Phe Thr Tyr Ser His Phe Ala Val Pro Gln Ala Lys405 410 415 420gga aac aat gtc gtg att aca agc tat atg aca aac aga gga ttc tac1771Gly Asn Asn Val Val Ile Thr Ser Tyr Met Thr Asn Arg Gly Phe Tyr425 430 435gca gac aaa caa tca acg ttt gcg cca agc ttc ctg ctg aac atc aaa1819Ala Asp Lys Gln Ser Thr Phe Ala Pro Ser Phe Leu Leu Asn Ile Lys440 445 450ggc aag aaa aca tct gtt gtc aaa gac agc atc ctt gaa caa gga caa1867Gly Lys Lys Thr Ser Val Val Lys Asp Ser Ile Leu Glu Gln Gly Gln455 460 465tta aca gtt aac aaa taa aaacgcaaaa gaaaatgccg atatcctatt 1915Leu Thr Val Asn Lys470ggcattttct tttatttctt atcaacataa aggtgaatcc catatgaact atataaaagc 1975aggcaaatgg ctaaccgtat tcctaacctt ttgaagatc 2014<210>41<211>473<212>PRT<213>枯草芽孢杆菌<400>41Met Asn Ile Lys Lys Phe Ala Lys Gln Ala Thr Val Leu Thr Phe Thr1 5 10 15Thr Ala Leu Leu Ala Gly Gly Ala Thr Gln Ala Phe Ala Lys Glu Thr20 25 30Asn Gln Lys Pro Tyr Lys Glu Thr Tyr Gly Ile Ser His Ile Thr Arg35 40 45His Asp Met Leu Gln Ile Pro Glu Gln Gln Lys Asn Glu Lys Tyr Gln50 55 60Val Pro Glu Phe Asp Ser Ser Thr Ile Lys Asn Ile Ser Ser Ala Lys65 70 75 80Gly Leu Asp Val Trp Asp Ser Trp Pro Leu Gln Asn Ala Asp Gly Thr85 90 95Val Ala Asn Tyr His Gly Tyr His Ile Val Phe Ala Leu Ala Gly Asp100 105 110Pro Lys Asn Ala Asp Asp Thr Ser Ile Tyr Met Phe Tyr Gln Lys Val115 120 125Gly Glu Thr Ser Ile Asp Ser Trp Lys Asn Ala Gly Arg Val Phe Lys130 135 140Asp Ser Asp Lys Phe Asp Ala Asn Asp Ser Ile Leu Lys Asp Gln Thr145 150 155 160Gln Glu Trp Ser Gly Ser Ala Thr Phe Thr Ser Asp Gly Lys Ile Arg165 170 175Leu Phe Tyr Thr Asp Phe Ser Gly Lys His Tyr Gly Lys Gln Thr Leu180 185 190Thr Thr Ala Gln Val Asn Val Ser Ala Ser Asp Ser Ser Leu Asn Ile195 200 205Asn Gly Val Glu Asp Tyr Lys Ser Ile Phe Asp Gly Asp Gly Lys Thr
210 215 220Tyr Gln Asn Val Gln Gln Phe Ile Asp Glu Gly Asn Tyr Ser Ser Gly225 230 235 240Asp Asn His Thr Leu Arg Asp Pro His Tyr Val Glu Asp Lys Gly His245 250 255Lys Tyr Leu Val Phe Glu Ala Asn Thr Gly Thr Glu Asp Gly Tyr Gln260 265 270Gly Glu Glu Ser Leu Phe Asn Lys Ala Tyr Tyr Gly Lys Ser Thr Ser275 280 285Phe Phe Arg Gln Glu Ser Gln Lys Leu Leu Gln Ser Asp Lys Lys Arg290 295 300Thr Ala Glu Leu Ala Asn Gly Ala Leu Gly Met Ile Glu Leu Asn Asp305 310 315 320Asp Tyr Thr Leu Lys Lys Val Met Lys Pro Leu Ile Ala Ser Asn Thr325 330 335Val Thr Asp Glu Ile Glu Arg Ala Asn Val Phe Lys Met Asn Gly Lys340 345 350Trp Tyr Leu Phe Thr Asp Ser Arg Gly Ser Lys Met Thr Ile Asp Gly355 360 365Ile Thr Ser Asn Asp Ile Tyr Met Leu Gly Tyr Val Ser Asn Ser Leu370 375 380Thr Gly Pro Tyr Lys Pro Leu Asn Lys Thr Gly Leu Val Leu Lys Met385 390 395 400Asp Leu Asp Pro Asn Asp Val Thr Phe Thr Tyr Ser His Phe Ala Val405 410 415Pro Gln Ala Lys Gly Asn Asn Val Val Ile Thr Ser Tyr Met Thr Asn420 425 430Arg Gly Phe Tyr Ala Asp Lys Gln Ser Thr Phe Ala Pro Ser Phe Leu435 440 445Leu Asn lle Lys Gly Lys Lys Thr Ser Val Val Lys Asp Ser Ile Leu450 455 460Glu Gln Gly Gln Leu Thr Val Asn Lys465 470<210>42<211>24<212>DNA<213>人工DNA<220>
<223>引物<400>42cgggatcctt tttaacccat caca 24<210>43<211>29<212>DNA<213>人工DNA<220>
<223>引物<400>43gaagatcttc aaaaggttag gaatacggt 29
<210>44<211>23<212>DNA<213>人工DNA<220>
<223>引物<400>44ccttttgaag atcgaccagt tgg23<210>45<211>44<212>DNA<213>人工DNA<220>
<223>引物<400>45tacctggaat gctgttttcc cagggatcgc agtggtgagt aacc 44<210>46<211>28<212>DNA<213>人工DNA<220>
<223>引物<400>46cctgggaaaa cagcattcca ggtattag 28<210>47<211>23<212>DNA<213>人工DNA<220>
<223>引物<400>47tgcaggtcga ctctagagga tcc23<210>48<211>3774<212>DNA<213>谷氨酸棒杆菌<220>
<221>CPS<222>(1)..(3774)<223>sucA
<400>48atg cta caa ctg ggg ctt agg cat aat cag cca acg acc aac gtt aca 48Met Leu Gln Leu Gly Leu Arg His Asn Gln Pro Thr Thr Asn Val Thr1 5 10 15gtg gat aaa aca aag ctc aat aaa ccc tca aga agc aag gaa aag agg 96Val Asp Lys Thr Lys Leu Asn Lys Pro Ser Arg Ser Lys Glu Lys Arg20 25 30cga gta cct gcc gtg agc agc gct agt act ttc ggc cag aat gcg tgg 144Arg Val Pro Ala Val Ser Ser Ala Ser Thr Phe Gly Gln Asn Ala Trp35 40 45ctg gta gac gag atg ttc cag cag ttc cag aag gac ccc aag tcc gtg 192Leu Val Asp Glu Met Phe Gln Gln Phe Gln Lys Asp Pro Lys Ser Val50 55 60gac aag gaa tgg aga gaa ctc ttt gag gcg cag ggg gga cca aat act 240Asp Lys Glu Trp Arg Glu Leu Phe Glu Ala Gln Gly Gly Pro Asn Thr65 70 75 80acc ccc gct aca aca gaa gca cag cct tca gcg ccc aag gag tct gcg 288Thr Pro Ala Thr Thr Glu Ala Gln Pro Ser Ala Pro Lys Glu Ser Ala85 90 95aaa cca gca cca aag gct gcc cct gca gcc aag gca gca ccg cgc gta 336Lys Pro Ala Pro Lys Ala Ala Pro Ala Ala Lys Ala Ala Pro Arg Val100 105 110gaa acc aag ccg gcc gac aag acc gcc cct aag gcc aag gag tcc tca 384Glu Thr Lys Pro Ala Asp Lys Thr Ala Pro Lys Ala Lys Glu Ser Ser115 120 125gtg cca cag caa cct aag ctt ccg gag cca gga caa acc cca atc agg 432Val Pro Gln Gln Pro Lys Leu Pro Glu Pro Gly Gln Thr Pro Ile Arg130 135 140ggt att ttc aag tcc atc gcg aag aac atg gat atc tcc ctg gaa atc 480Gly Ile Phe Lys Ser Ile Ala Lys Asn Met Asp Ile Ser Leu Glu Ile145 150 155 160cca acc gca acc tcg gtt cgc gat atg cca gct cgc ctc atg ttc gaa 528Pro Thr Ala Thr Ser Val Arg Asp Met Pro Ala Arg Leu Met Phe Glu165 170 175aac cgc gcg atg gtc aac gat cag ctc aag cgc acc cgc ggt ggc aag 576Asn Arg Ala Met Val Asn Asp Gln Leu Lys Arg Thr Arg Gly Gly Lys180 185 190atc tcc ttc acc cac atc att ggc tac gcc atg gtg aag gca gtc atg 624Ile Ser Phe Thr His Ile Ile Gly Tyr Ala Met Val Lys Ala Val Met195 200 205gct cac ccg gac atg aac aac tcc tac gac gtc atc gac ggc aag cca 672Ala His Pro Asp Met Asn Asn Ser Tyr Asp Val Ile Asp Gly Lys Pro210 215 220acc ctg atc gtg cct gag cac atc aac ctg ggc ctt gct atc gac ctt 720Thr Leu Ile Val Pro Glu His Ile Asn Leu Gly Leu Ala Ile Asp Leu225 230 235 240cct cag aag gac ggc tcc cgc gca ctt gtc gta gca gcc atc aag gaa 768Pro Gln Lys Asp Gly Ser Arg Ala Leu Val Val Ala Ala lle Lys Glu245 250 255acc gag aag atg aac ttc tcc gag ttc ctc gca gcc tac gaa gac atc 816Thr Glu Lys Met Asn Phe Ser Glu Phe Leu Ala Ala Tyr Glu Asp Ile260 265 270gtg gca cgc tcc cgc aag ggc aag ctc acc atg gat gac tac cag ggc 864Val Ala Arg Ser Arg Lys Gly Lys Leu Thr Met Asp Asp Tyr Gln Gly275 280 285gtt acc gtt tcc ttg acc aac cca ggt ggc atc ggt acc cgc cac tct 912Val Thr Val Ser Leu Thr Asn Pro Gly Gly Ile Gly Thr Arg His Ser
290 295 300gtt cca cgt cta acc aag ggc cag ggc acc atc atc ggt gtc ggt tcc 960Val Pro Arg Leu Thr Lys Gly Gln Gly Thr Ile Ile Gly Val Gly Ser305 310 315 320atg gat tac cca gca gag ttc cag ggc gct tca gaa gac cgc ctt gca1008Met Asp Tyr Pro Ala Glu Phe Gln Gly Ala Ser Glu Asp Arg Leu Ala325 330 335gag ctc ggc gtt ggc aaa ctt gtc acc atc acc tcc acc tac gat cac1056Glu Leu Gly Val Gly Lys Leu Val Thr Ile Thr Ser Thr Tyr Asp His340 345 350cgc gtg atc cag ggt gct gtg tcc ggt gaa ttc ctg cgc acc atg tct1104Arg Val Ile Gln Gly Ala Val Ser Gly Glu Phe Leu Arg Thr Met Ser355 360 365cgc ctg ctc acc gat gat tcc ttc tgg gat gag atc ttc gac gca atg1152Arg Leu Leu Thr Asp Asp Ser Phe Trp Asp Glu Ile Phe Asp Ala Met370 375 380aac gtt cct tac acc cca atg cgt tgg gca cag gac gtt cca aac acc1200Asn Val Pro Tyr Thr Pro Met Arg Trp Ala Gln Asp Val Pro Asn Thr385 390 395 400ggt gtt gat aag aac acc cgc gtc atg cag ctc att gag gca tac cgc1248Gly Val Asp Lys Asn Thr Arg Val Met Gln Leu Ile Glu Ala Tyr Arg405 410 415tcc cgt gga cac ctc atc gct gac acc aac cca ctt tca tgg gtt cag1296Ser Arg Gly His Leu Ile Ala Asp Thr Asn Pro Leu Ser Trp Val Gln420 425 430cct ggc atg cca gtt cca gac cac cgc gac ctc gac atc gag acc cac1344Pro Gly Met Pro Val Pro Asp His Arg Asp Leu Asp Ile Glu Thr His435 440 445aac ctg acc atc tgg gat ctg gac cgt acc ttc aac gtc ggt ggc ttc1392Asn Leu Thr Ile Trp Asp Leu Asp Arg Thr Phe Asn Val Gly Gly Phe450 455 460ggc ggc aag gag acc atg acc ctg cgc gag gta ctg tcc cgc ctc cgc1440Gly Gly Lys Glu Thr Met Thr Leu Arg Glu Val Leu Ser Arg Leu Arg465 470 475 480gct gcg tac acc ctc aag gtc ggc tcc gaa tac acc cac atc ctg gac1488Ala Ala Tyr Thr Leu Lys Val Gly Ser Glu Tyr Thr His Ile Leu Asp485 490 495cgc gac gag cgc acc tgg ctg cag gac cgc ctc gag gcc gga atg cca1536Arg Asp Glu Arg Thr Trp Leu Gln Asp Arg Leu Glu Ala Gly Met Pro500 505 510aag cca acc cag gca gag cag aag tac atc ctg cag aag ctg aac gcc1584Lys Pro Thr Gln Ala Glu Gln Lys Tyr Ile Leu Gln Lys Leu Asn Ala515 520 525gcg gag gct ttc gag aac ttc ctg cag acc aag tac gtc ggc cag aag1632Ala Glu Ala Phe Glu Asn Phe Leu Gln Thr Lys Tyr Val Gly Gln Lys530 535 540cgc ttc tcc ctc gaa ggt gca gaa gca ctt atc cca ctg atg gac tcc1680Arg Phe Ser Leu Glu Gly Ala Glu Ala Leu Ile Pro Leu Met Asp Ser545 550 555 560gcc atc gac acc gcc gca ggc caa ggc ctc gac gaa gtt gtc atc ggt1728Ala Ile Asp Thr Ala Ala Gly Gln Gly Leu Asp Glu Val Val Ile Gly565 570 575atg cca cac cgt ggt cgc ctc aac gtg ctg ttc aac atc gtg ggc aag1776Met Pro His Arg Gly Arg Leu Asn Val Leu Phe Asn Ile Val Gly Lys580 585 590cca ctg gca tcc atc ttc aac gag ttt gaa ggc caa atg gag cag ggc 1824Pro Leu Ala Ser Ile Phe Asn Glu Phe Glu Gly Gln Met Glu Gln Gly595 600 605
cag atc ggt ggc tcc ggt gac gtg aag tac cac ctc ggt tcc gaa ggc1872Gln Ile Gly Gly Ser Gly Asp Val Lys Tyr His Leu Gly Ser Glu Gly610 615 620cag cac ctg cag atg ttc ggc gac ggc gag atc aag gtc tcc ctg act1920Gln His Leu Gln Met Phe Gly Asp Gly Glu Ile Lys Val Ser Leu Thr625 630 635 640gct aac ccg tcc cac ctg gaa gct gtt aac cca gtg atg gaa ggt atc1968Ala Asn Pro Ser His Leu Glu Ala Val Asn Pro Val Met Glu Gly Ile645 650 655gtc cgc gca aag cag gac tac ctg gac aag ggc gta gac ggc aag act2016Val Arg Ala Lys Gln Asp Tyr Leu Asp Lys Gly Val Asp Gly Lys Thr660 665 670gtt gtg cca ctg ctg ctc cac ggt gac gct gca ttc gca ggc ctg ggc2064Val Val Pro Leu Leu Leu His Gly Asp Ala Ala Phe Ala Gly Leu Gly675 680 685atc gtg cca gaa acc atc aac ctg gct aag ctg cgt ggc tac gac gtc2112Ile Val Pro Glu Thr Ile Asn Leu Ala Lys Leu Arg Gly Tyr Asp Val690 695 700ggc ggc acc atc cac atc gtg gtg aac aac cag atc ggc ttc acc acc2160Gly Gly Thr Ile His Ile Val Val Asn Asn Gln Ile Gly Phe Thr Thr705 710 715 720acc cca gac tcc agc cgc tcc atg cac tac gca acc gac tac gcc aag2208Thr Pro Asp Ser Ser Arg Ser Met His Tyr Ala Thr Asp Tyr Ala Lys725 730 735gca ttc ggc tgc cca gtc ttc cac gtc aac ggc gac gac cca gag gca2256Ala Phe Gly Cys Pro Val Phe His Val Asn Gly Asp Asp Pro Glu Ala740 745 750gtt gtc tgg gtt ggc cag ctg gcc acc gag tac cgt cgt cgc ttc ggc2304Val Val Trp Val Gly Gln Leu Ala Thr Glu Tyr Arg Arg Arg Phe Gly755 760 765aag gac gtc ttc atc gac ctc gtc tgc tac cgc ctc cgc ggc cac aac2352Lys Asp Val Phe Ile Asp Leu Val Cys Tyr Arg Leu Arg Gly His Asn770 775 780gaa gct gat gat cct tcc atg acc cag cca aag atg tat gag ctc atc2400Glu Ala Asp Asp Pro Ser Met Thr Gln Pro Lys Met Tyr Glu Leu Ile785 790 795 800acc ggc cgc gag acc gtt cgt gct cag tac acc gaa gac ctg ctc gga2448Thr Gly Arg Glu Thr Val Arg Ala Gln Tyr Thr Glu Asp Leu Leu Gly805 810 815cgt gga gac ctc tcc aac gaa gat gca gaa gca gtc gtc cgc gac ttc2496Arg Gly Asp Leu Ser Asn Glu Asp Ala Glu Ala Val Val Arg Asp Phe820 825 830cac gac cag atg gaa tct gtg ttc aac gaa gtc aag gaa ggc ggc aag2544His Asp Gln Met Glu Ser Val Phe Asn Glu Val Lys Glu Gly Gly Lys835 840 845aag cag gct gag gca cag acc ggc atc acc ggc tcc cag aag ctt cca2592Lys Gln Ala Glu Ala Gln Thr Gly Ile Thr Gly Ser Gln Lys Leu Pro850 855 860cac ggc ctt gag acc aac atc tcc cgt gaa gag ctc ctg gaa ctg gga2640His Gly Leu Glu Thr Asn Ile Ser Arg Glu Glu Leu Leu Glu Leu Gly865 870 875 880cag gct ttc gcc aac acc cca gaa ggc ttc aac tac cac cca cgt gtg2688Gln Ala Phe Ala Asn Thr Pro Glu Gly Phe Asn Tyr His Pro Arg Val885 890 895gct ccc gtt gct aag aag cgc gtc tcc tct gtc acc gaa ggt ggc atc2736Ala Pro Val Ala Lys Lys Arg Val Ser Ser Val Thr Glu Gly Gly Ile900 905 910gac tgg gca tgg ggc gag ctc ctc gcc ttc ggt tcc ctg gct aac tcc2784
Asp Trp Ala Trp Gly Glu Leu Leu Ala Phe Gly Ser Leu Ala Asn Ser915 920 925ggc cgc ttg gtt cgc ctt gca ggt gaa gat tcc cgc cgc ggt acc ttc2832Gly Arg Leu Val Arg Leu Ala Gly Glu Asp Ser Arg Arg Gly Thr Phe930 935 940acc cag cgc cac gca gtt gcc atc gac cca gcg acc gct gaa gag ttc2880Thr Gln Arg His Ala Val Ala Ile Asp Pro Ala Thr Ala Glu Glu Phe945 950 955 960aac cca ctc cac gag ctt gca cag tcc aag ggc aac aac ggt aag ttc2928Asn Pro Leu His Glu Leu Ala Gln Ser Lys Gly Asn Asn Gly Lys Phe965 970 975ctg gtc tac aac tcc gca ctg acc gag tac gca ggc atg ggc ttc gag2976Leu Val Tyr Asn Ser Ala Leu Thr Glu Tyr Ala Gly Met Gly Phe Glu980 985 990tac ggc tac tcc gta gga aac gaa gac tcc atc gtt gca tgg gaa gca3024Tyr Gly Tyr Ser Val Gly Asn Glu Asp Ser Ile Val Ala Trp Glu Ala995 10001005cag ttc ggc gac ttc gcc aac ggc gct cag acc atc atc gat gag3069Gln Phe Gly Asp Phe Ala Asn Gly Ala Gln Thr Ile Ile Asp Glu101010151020tac gtc tcc tca ggc gaa gct aag tgg ggc cag acc tcc aag ctg3114Tyr Val Ser Ser Gly Glu Ala Lys Trp Gly Gln Thr Ser Lys Leu102510301035atc ctt ctg ctg cct cac ggc tac gaa ggc cag ggc cca gac cac3159Ile Leu Leu Leu Pro His Gly Tyr Glu Gly Gln Gly Pro Asp His104010451050tct tcc gca cgt atc gag cgc ttc ctg cag ctg tgc gct gag ggt3204Ser Ser Ala Arg Ile Glu Arg Phe Leu Gln Leu Cys Ala Glu Gly105510601065tcc atg act gtt gct cag cca tcc acc cca gca aac cac ttc cac3249Ser Met Thr Val Ala Gln Pro Ser Thr Pro Ala Asn His Phe His107010751080cta ctg cgt cgt cac gct ctg tcc gac ctg aag cgt cca ctg gtt3294Leu Leu Arg Arg His Ala Leu Ser Asp Leu Lys Arg Pro Leu Vall08510901095atc ttc acc ccg aag tcc atg ctg cgt aac aag gct gct gcc tcc3339Ile Phe Thr Pro Lys Ser Met Leu Arg Asn Lys Ala Ala Ala Ser110011051110gca cca gaa gac ttc act gag gtc acc aag ttc cag tcc gtg atc3384Ala Pro Glu Asp Phe Thr Glu Val Thr Lys Phe Gln Ser Val Ile111511201125aac gat cca aac gtt gca gat gca gcc aag gtg aag aag gtc atg3429Asn Asp Pro Asn Val Ala Asp Ala Ala Lys Val Lys Lys Val Met113011351140ctg gtc tcc ggc aag ctg tac tac gaa ttg gca aag cgc aag gag3474Leu Val Ser Gly Lys Leu Tyr Tyr Glu Leu Ala Lys Arg Lys Glu114511501155aag gac gga cgc gac gac atc gcg atc gtt cgt atc gaa atg ctc3519Lys Asp Gly Arg Asp Asp Ile Ala Ile Val Arg Ile Glu Met Leu116011651170cac cca att ccg ttc aac cgc atc tcc gag gct ctt gcc ggc tac3564His Pro Ile Pro Phe Asn Arg Ile Ser Glu Ala Leu Ala Gly Tyr117511801185cct aac gct gag gaa gtc ctc ttc gtt cag gat gag cca gca aac3609Pro Asn Ala Glu Glu Val Leu Phe Val Gln Asp Glu Pro Ala Asn119011951200cag ggc cca tgg ccg ttc tac cag gag cac ctc cca gag ctg atc3654Gln Gly Pro Trp Pro Phe Tyr Gln Glu His Leu Pro Glu Leu Ile
120512101215ccg aac atg cca aag atg cgc cgc gtt tcc cgc cgc gct cag tcc3699Pro Asn Met Pro Lys Met Arg Arg Val Ser Arg Arg Ala Gln Ser122012251230tcc acc gca act ggt gtt gcc aag gtg cac cag ctg gag gag aag3744Ser Thr Ala Thr Gly Val Ala Lys Val His Gln Leu Glu Glu Lys123512401245cag ctt atc gac gag gct ttc gag gct taa3774Gln Leu Ile Asp Glu Ala Phe Glu Ala12501255<210>49<211>1257<212>PRT<213>谷氨酸棒杆菌<400>49Met Leu Gln Leu Gly Leu Arg Hi Asn Gln ProThr Thr Asn Val Thr1 5 10 15Val Asp Lys Thr Lys Leu Asn Lys Pro Ser Arg Ser Lys Glu Lys Arg20 25 30Arg Val Pro Ala Val Ser Ser Ala Ser Thr Phe Gly Gln Asn Ala Trp35 40 45Leu Val Asp Glu Met Phe Gln Gln Phe Gln Lys Asp Pro Lys Ser Val50 55 60Asp Lys Glu Trp Arg Glu Leu Phe Glu Ala Gln Gly Gly Pro Asn Thr65 70 75 80Thr Pro Ala Thr Thr Glu Ala Gln Pro Ser Ala Pro Lys Glu Ser Ala85 90 95Lys Pro Ala Pro Lys Ala Ala Pro Ala Ala Lys Ala Ala Pro Arg Val100 105 110Glu Thr Lys Pro Ala Asp Lys Thr Ala Pro Lys Ala Lys Glu Ser Ser115 120 125Val Pro Gln Gln Pro Lys Leu Pro Glu Pro Gly Gln Thr Pro Ile Arg130 135 140Gly Ile Phe Lys Ser Ile Ala Lys Asn Met Asp Ile Ser Leu Glu Ile145 150 155 160Pro Thr Ala Thr Ser Val Arg Asp Met Pro Ala Arg Leu Met Phe Glu165 170 175Asn Arg Ala Met Val Asn Asp Gln Leu Lys Arg Thr Arg Gly Gly Lys180 185 190Ile Ser Phe Thr His Ile Ile Gly Tyr Ala Met Val Lys Ala Val Met195 200 205Ala His Pro Asp Met Asn Asn Ser Tyr Asp Val Ile Asp Gly Lys Pro210 215 220Thr Leu Ile Val Pro Glu His Ile Asn Leu Gly Leu Ala Ile Asp Leu225 230 235 240Pro Gln Lys Asp Gly Ser Arg Ala Leu Val Val Ala Ala Ile Lys Glu245 250 255Thr Glu Lys Met Asn Phe Ser Glu Phe Leu Ala Ala Tyr Glu Asp Ile260 265 270Val Ala Arg Ser Arg Lys Gly Lys Leu Thr Met Asp Asp Tyr Gln Gly275 280 285Val Thr Val Ser Leu Thr Asn Pro Gly Gly Ile Gly Thr Arg His Ser290 295 300Val Pro Arg Leu Thr Lys Gly Gln Gly Thr Ile Ile Gly Val Gly Ser305 310 315 320
Met Asp Tyr Pro Ala Glu Phe Gln Gly Ala Ser Glu Asp Arg Leu Ala325 330 335Glu Leu Gly Val Gly Lys Leu Val Thr Ile Thr Ser Thr Tyr Asp His340 345 350Arg Val Ile Gln Gly Ala Val Ser Gly Glu Phe Leu Arg Thr Met Ser355 360 365Arg Leu Leu Thr Asp Asp Ser Phe Trp Asp Glu Ile Phe Asp Ala Met370 375 380Asn Val Pro Tyr Thr Pro Met Arg Trp Ala Gln Asp Val Pro Asn Thr385 390 395 400Gly Val Asp Lys Asn Thr Arg Val Met Gln Leu Ile Glu Ala Tyr Arg405 410 415Ser Arg Gly His Leu Ile Ala Asp Thr Asn Pro Leu Ser Trp Val Gln420 425 430Pro Gly Met Pro Val Pro Asp His Arg Asp Leu Asp Ile Glu Thr His435 440 445Asn Leu Thr Ile Trp Asp Leu Asp Arg Thr Phc Asn Val Gly Gly Phe450 455 460Gly Gly Lys Glu Thr Met Thr Leu Arg Glu Val Leu Ser Arg Leu Arg465 470 475 480Ala Ala Tyr Thr Leu Lys Val Gly Ser Glu Tyr Thr His Ile Leu Asp485 490 495Arg Asp Glu Arg Thr Trp Leu Gln Asp Arg Leu Glu Ala Gly Met Pro500 505 510Lys Pro Thr Gln Ala Glu Gln Lys Tyr Ile Leu Gln Lys Leu Asn Ala515 520 525Ala Glu Ala Phe Glu Asn Phe Leu Gln Thr Lys Tyr Val Gly Gln Lys530 535 540Arg Phe Ser Leu Glu Gly Ala Glu Ala Leu Ile Pro Leu Met Asp Ser545 550 555 560Ala lle Asp Thr Ala Ala Gly Gln Gly Leu Asp Glu Val Val Ile Gly565 570 575Met Pro His Arg Gly Arg Leu Asn Val Leu Phe Asn Ile Val Gly Lys580 585 590Pro Leu Ala Ser Ile Phe Asn Glu Phe Glu Gly Gln Met Glu Gln Gly595 600 605Gln Ile Gly Gly Ser Gly Asp Val Lys Tyr His Leu Gly Ser Glu Gly610 615 620Gln His Leu Gln Met Phe Gly Asp Gly Glu Ile Lys Val Ser Leu Thr625 630 635 640Ala Asn Pro Ser His Leu Glu Ala Val Asn Pro Val Met Glu Gly Ile645 650 655Val Arg Ala Lys Gln Asp Tyr Leu Asp Lys Gly Val Asp Gly Lys Thr660 665 670Val Val Pro Leu Leu Leu His Gly Asp Ala Ala Phe Ala Gly Leu Gly675 680 685Ile Val Pro Glu Thr Ile Asn Leu Ala Lys Leu Arg Gly Tyr Asp Val690 695 700Gly Gly Thr Ile His Ile Val Val Asn Asn Gln Ile Gly Phe Thr Thr705 710 715 720Thr Pro Asp Ser Ser Arg Ser Met His Tyr Ala Thr Asp Tyr Ala Lys725 730 735Ala Phe Gly Cys Pro Val Phe His Val Asn Gly Asp Asp Pro Glu Ala740 745 750Val Val Trp Val Gly Gln Leu Ala Thr Glu Tyr Arg Arg Arg Phe Gly755 760 765Lys Asp Val Phe Ile Asp Leu Val Cys Tyr Arg Leu Arg Gly His Asn770 775 780
Glu Ala Asp Asp Pro Ser Met Thr Gln Pro Lys Met Tyr Glu Leu Ile785 790 795 800Thr Gly Arg Glu Thr Val Arg Ala Gln Tyr Thr Glu Asp Leu Leu Gly805 810 815Arg Gly Asp Leu Ser Asn Glu Asp Ala Glu Ala Val Val Arg Asp Phe820 825 830His Asp Gln Met Glu Ser Val Phe Asn Glu Val Lys Glu Gly Gly Lys835 840 845Lys Gln Ala Glu Ala Gln Thr Gly Ile Thr Gly Ser Gln Lys Leu Pro850 855 860His Gly Leu Glu Thr Asn Ile Ser Arg Glu Glu Leu Leu Glu Leu Gly865 870 875 880Gln Ala Phe Ala Asn Thr Pro Glu Gly Phe Asn Tyr His Pro Arg Val885 890 895Ala Pro Val Ala Lys Lys Arg Val Ser Ser Val Thr Glu Gly Gly Ile900 905 910Asp Trp Ala Trp Gly Glu Leu Leu Ala Phe Gly Ser Leu Ala Asn Ser915 920 925Gly Arg Leu Val Arg Leu Ala Gly Glu Asp Ser Arg Arg Gly Thr Phe930 935 940Thr Gln Arg His Ala Val Ala Ile Asp Pro Ala Thr Ala Glu Glu Phe945 950 955 960Asn Pro Leu His Glu Leu Ala Gln Ser Lys Gly Asn Asn Gly Lys Phe965 970 975Leu Val Tyr Asn Ser Ala Leu Thr Glu Tyr Ala Gly Met Gly Phe Glu980 985 990Tyr Gly Tyr Ser Val Gly Asn Glu Asp Ser Ile Val Ala Trp Glu Ala995 10001005Gln Phe Gly Asp Phe Ala Asn Gly Ala Gln Thr Ile Ile Asp Glu101010151020Tyr Val Ser Ser Gly Glu Ala Lys Trp Gly Gln Thr Ser Lys Leu102510301035Ile Leu Leu Leu Pro His Gly Tyr Glu Gly Gln Gly Pro Asp His104010451050Ser Ser Ala Arg Ile Glu Arg Phe Leu Gln Leu Cys Ala Glu Gly105510601065Ser Met Thr Val Ala Gln Pro Ser Thr Pro Ala Asn His Phe His107010751080Leu Leu Arg Arg His Ala Leu Ser Asp Leu Lys Arg Pro Leu Val108510901095Ile Phe Thr Pro Lys Ser Met Leu Arg Asn Lys Ala Ala Ala Ser110011051110Ala Pro Glu Asp Phe Thr Glu Val Thr Lys Phe Gln Ser Val Ile111511201125Asn Asp Pro Asn Val Ala Asp Ala Ala Lys Val Lys Lys Val Met113011351140Leu Val Ser Gly Lys Leu Tyr Tyr Glu Leu Ala Lys Arg Lys Glu114511501155Lys Asp Gly Arg Asp Asp Ile Ala Ile Val Arg Ile Glu Met Leu116011651170His Pro Ile Pro Phe Asn Arg Ile Ser Glu Ala Leu Ala Gly Tyr117511801185Pro Asn Ala Glu Glu Val Leu Phe Val Gln Asp Glu Pro Ala Asn119011951200Gln Gly Pro Trp Pro Phe Tyr Gln Glu His Leu Pro Glu Leu Ile120512101215Pro Asn Met Pro Lys Met Arg Arg Val Ser Arg Arg Ala Gln Ser122012251230
Ser Thr Ala Thr Gly Val Ala Lys Val His Gln Leu Glu Glu Lys123512401245Gln Leu Ile Asp Glu Ala Phe Glu Ala1255<210>50<211>30<212>DNA<213>人工DNA<220>
<223>引物pfk50<400>50acccgcaatt ttcgcagcct tagaacacct30<210>51<211>49<212>DNA<213>人工DNA<220>
<223>引物pfk51<400>51ctgttgataa aagcccgaaa aactaattaa acccatcaca acacccgcg 49<210>52<211>32<212>DNA<213>人工DNA<220>
<223>引物pfk52<400>52agcttctgca gaatctcaaa cgcacgctta cc 32<210>53<211>49<212>DNA<213>人工DNA<220>
<223>引物pfk53<400>53cgcgggtgtt gtgatgggtt taattagttt ttcgggcttt tatcaacag 49<210>54<211>40<212>DNA<213>人工DNA
<220>
<223>引物pfk54<400>54gcaaggatcc agggcaaggg gttctagaaa gaccaacgga 40<210>55<211>44<212>DNA<213>人工DNA<220>
<223>引物pfk55<400>55ttctggatcc tctcaaacgc acgcttaccg atctcttgta cgtg 44<210>56<211>1041<212>DNA<213>谷氨酸棒杆菌<220>
<221>CDS<222>(1)..(1041)<223>pfk*1<400>56atg gaa gac atg cga att gct act ctc acg tca ggc ggc gac tgc ccc48Met Glu Asp Met Arg Ile Ala Thr Leu Thr Ser Gly Gly Asp Cys Pro1 5 10 15gga cta aat gcc gtc atc cga gga atc gtc cgc aca gcc agc aat gaa96Gly Leu Asn Ala Val Ile Arg Gly Ile Val Arg Thr Ala Ser Asn Glu20 25 30ttt ggc tcc acc gtc gtt ggt tat caa gac ggt tgg gaa gga ctg tta 144Phe Gly Ser Thr Val Val Gly Tyr Gln Asp Gly Trp Glu Gly Leu Leu35 40 45gcc gat cgt cgc gta cag ctg tat gac gat gaa gat att gac cga atc 192Ala Asp Arg Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile Asp Arg Ile50 55 60ctc ctt cga ggc ggc acc att ttg ggc act ggt cgc ctc cat ccg gac 240Leu Leu Arg Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu His Pro Asp65 70 75 80aag ttt aag gcc gga att gat cag att aag gcc aac tta gaa gac gcc 288Lys Phe Lys Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu Glu Asp Ala85 90 95ggc atc gat gcc ctt atc cca atc ggt ggc gaa gga acc ctg aag ggt 336Gly Ile Asp Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr Leu Lys Gly100 105 110gcc aag tgg ctg tct gat aac ggt atc cct gtt gtc ggt gtc cca aag 384Ala Lys Trp Leu Ser Asp Asn Gly Ile Pro Val Val Gly Val Pro Lys115 120 125acc att gac aat gac gtg aat ggc act gac ttc acc ttc ggt ttc gat 432Thr Ile Asp Asn Asp Val Asn Gly Thr Asp Phe Thr Phe Gly Phe Asp130 135 140act gct gtg gca gtg gct acc gac gct gtt gac cgc ctg cac acc acc 480Thr Ala Val Ala Val Ala Thr Asp Ala Val Asp Arg Leu His Thr Thr
145 150 155 160gct gaa tct cac aac cgt gtg atg atc gtg aag gtc atg ggc cgc cac 528Ala Glu Ser His Asn Arg Val Met Ile Val Lys Val Met Gly Arg His165 170 175gtg ggt tgg att gct ctg cac gca ggt atg gcg ggc ggt gct cac tac 576Val Gly Trp Ile Ala Leu His Ala Gly Met Ala Gly Gly Ala His Tyr180 185 190acc gtt att cca gaa gta cct ttc gat att gca gag atc tgc aag gcg 624Thr Val Ile Pro Glu Val Pro Phe Asp Ile Ala Glu Ile Cys Lys Ala195 200 205atg gaa cgt cgc ttc cag atg ggc gag aag tac ggc att atc gtc gtt 672Met Glu Arg Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile Ile Val Val210 215 220gcg gaa ggt gcg ttg cca cgc gaa ggc acc atg gag ctt cgt gaa ggc 720Ala Glu Gly Ala Leu Pro Arg Glu Gly Thr Met Glu Leu Arg Glu Gly225 230 235 240cac att gac cag ttc ggt cac aag acc ttc acg gga att gga cag cag 768His Ile Asp Gln Phe Gly His Lys Thr Phe Thr Gly Ile Gly Gln Gln245 250 255atc gct gat gag atc cac gtg cgc ctc ggc cac gat gtt cgt acg acc 816Ile Ala Asp Glu Ile His Val Arg Leu Gly His Asp Val Arg Thr Thr260 265 270gtt ctt ggc cac att caa cgt ggt gga acc cca act gct ttc gac cgt 864Val Leu Gly His Ile Gln Arg Gly Gly Thr Pro Thr Ala Phe Asp Arg275 280 285gtt ctg gcc act cgt tat ggt gtt cgt gca gct cgt gcg tgc cat gag 912Val Leu Ala Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala Cys His Glu290 295 300gga agc ttt gac aag gtt gtt gct ttg aag ggt gag agc att gag atg 960Gly Ser Phe Asp Lys Val Val Ala Leu Lys Gly Glu Ser Ile Glu Met305 310 315 320tc acc ttt gaa gaa gcc gtc gga acc ttg aag gaa gtc cca ttc gaa 1008Ile Thr Phe Glu Glu Ala Val Gly Thr Leu Lys Glu Val Pro Phe Glu325 330 335cgc tgg gtt act gcc cag gca atg ttt gga tag1041Arg Trp Val Thr Ala Gln Ala Met Phe Gly340 345<210>57<211>346<212>PRT<213>谷氨酸棒杆菌<400>57Met Glu Asp Met Arg Ile Ala Thr Leu Thr Ser Gly Gly Asp Cys Pro1 5 10 15Gly Leu Asn Ala Val Ile Arg Gly Ile Val Arg Thr Ala Ser Asn Glu20 25 30Phe Gly Ser Thr Val Val Gly Tyr Gln Asp Gly Trp Glu Gly Leu Leu35 40 45Ala Asp Arg Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile Asp Arg Ile50 55 60Leu Leu Arg Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu His Pro Asp65 70 75 80Lys Phe Lys Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu Glu Asp Ala85 90 95Gly Ile Asp Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr Leu Lys Gly100 105 110
Ala Lys Trp Leu Ser Asp Asn Gly Ile Pro Val Val Gly Val Pro Lys115 120 125Thr Ile Asp Asn Asp Val Asn Gly Thr Asp Phe Thr Phe Gly Phe Asp130 135 140Thr Ala Val Ala Val Ala Thr Asp Ala Val Asp Arg Leu His Thr Thr145 150 155 160Ala Glu Ser His Asn Arg Val Met Ile Val Lys Val Met Gly Arg His165 170 175Val Gly Trp Ile Ala Leu His Ala Gly Met Ala Gly Gly Ala His Tyr180 185 190Thr Val Ile Pro Glu Val Pro Phe Asp Ile Ala Glu Ile Cys Lys Ala195 200 205Met Glu Arg Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile Ile Val Val210 215 220Ala Glu Gly Ala Leu Pro Arg Glu Gly Thr Met Glu Leu Arg Glu Gly225 230 235 240His Ile Asp Gln Phe Gly His Lys Thr Phe Thr Gly Ile Gly Gln Gln245 250 255Ile Ala Asp Glu Ile His Val Arg Leu Gly His Asp Val Arg Thr Thr260 265 270Val Leu Gly His Ile Gln Arg Gly Gly Thr Pro Thr Ala Phe Asp Arg275 280 285Val Leu Ala Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala Cys His Glu290 295 300Gly Ser Phe Asp Lys Val Val Ala Leu Lys Gly Glu Ser Ile Glu Met305 310 315 320Ile Thr Phe Glu Glu Ala Val Gly Thr Leu Lys Glu Val Pro Phe Glu325 330 335Arg Trp Val Thr Ala Gln Ala Met Phe Gly340 345<210>58<211>1041<212>DNA<213>谷氨酸棒杆菌<220>
<221>CDS<222>(1)..(1041)<223>pfk*2<400>58atg gaa gac atg cga att gct act ctc acg tca ggc ggc gac tgc ccc 48Met Glu Asp Met Arg Ile Ala Thr Leu Thr Ser Gly Gly Asp Cys Pro1 5 10 15gga cta aat gcc gtc atc cga gga ate gtc cgc aca gcc agc aat gaa 96Gly Leu Asn Ala Val Ile Arg Gly Ile Val Arg Thr Ala Ser Asn Glu20 25 30ttt ggc tcc acc gtc gtt ggt tat caa gac ggt tgg gaa gga ctg tta144Phe Gly Ser Thr Val Val Gly Tyr Gln Asp Gly Trp Glu Gly Leu Leu35 40 45gcc gat cgt cgc gta cag ctg tat gac gat gaa gat att gac cga atc192Ala Asp Arg Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile Asp Arg Ile50 55 60ctc ctt cga ggc ggc acc att ttg ggc act ggt cgc ctc cat ccg gac240Leu Leu Arg Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu His Pro Asp65 70 75 80aag ttt aag gcc gga att gat cag att aag gcc aac tta gaa gac gcc288
Lys Phe Lys Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu Glu Asp Ala85 90 95ggc atc gat gcc ctt atc cca atc ggt ggc gaa gga acc ctg aag ggt 336Gly Ile Asp Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr Leu Lys Gly100 105 110gcc aag tgg ctg tct gat aac ggt atc cct gtt gtc ggt gtc cca aag 384Ala Lys Trp Leu Ser Asp Asn Gly Ile Pro Val Val Gly Val Pro Lys115 120 125acc att gac aat gac gtg aat ggc act gac ttc acc ttc ggt ttc gat 432Thr Ile Asp Asn Asp Val Asn Gly Thr Asp Phe Thr Phe Gly Phe Asp130 135 140act gct gtg gca gtg gct acc gac gct gtt gac cgc ctg cac acc acc 480Thr Ala Val Ala Val Ala Thr Asp Ala Val Asp Arg Leu His Thr Thr145 150 155 160gct gaa tct cac aac cgt gtg atg atc gtg agg gtc atg ggc cgc cac 528Ala Glu Ser His Asn Arg Val Met Ile Val Arg Val Met Gly Arg His165 170 175gtg ggt tgg att gct ctg cac gca ggt atg gcg ggc ggt gct cac tac 576Val Gly Trp Ile Ala Leu His Ala Gly Met Ala Gly Gly Ala His Tyr180 185 190acc gtt att cca gaa gta cct ttc gat att gca gag atc tgc aag gcg 624Thr Val Ile Pro Glu Val Pro Phe Asp Ile Ala Glu Ile Cys Lys Ala195 200 205atg gaa cgt cgc ttc cag atg ggc gag aag tac ggc att atc gtc gtt 672Met Glu Arg Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile Ile Val Val210 215 220gcg gaa ggt gcg ttg cca cgc gaa ggc acc atg gag ctt cgt gaa ggc 720Ala Glu Gly Ala Leu Pro Arg Glu Gly Thr Met Glu Leu Arg Glu Gly225 230 235 240cac att gac cag ttc ggt cac aag acc ttc acg gga att gga cag cag 768His Ile Asp Gln Phe Gly His Lys Thr Phe Thr Gly Ile Gly Gln Gln245 250 255atc gct gat gag atc cac gtg cgc ctc ggc cac gat gtt cgt acg acc 816Ile Ala Asp Glu Ile His Val Arg Leu Gly His Asp Val Arg Thr Thr260 265 270gtt ctt ggc cac att caa cgt ggt gga acc cca act gct ttc gac cgt 864Val Leu Gly His Ile Gln Arg Gly Gly Thr Pro Thr Ala Phe Asp Arg275 280 285gtt ctg gcc act cgt tat ggt gtt cgt gca gct cgt gcg tgc cat gag 912Val Leu Ala Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala Cys His Glu290 295 300gga agc ttt gac aag gtt gtt gct ttg aag ggt gag agc att gag atg 960Gly Ser Phe Asp Lys Val Val Ala Leu Lys Gly Glu Ser Ile Glu Met305 310 315 320atc acc ttt gaa gaa gcc gtc gga acc ttg aag gaa gtc cca ttc gaa1008Ile Thr Phe Glu Glu Ala Val Gly Thr Leu Lys Glu Val Pro Phe Glu325 330 335cgc tgg gtt act gcc cag gca atg ttt gga tag1041Arg Trp Val Thr Ala Gln Ala Met Phe Gly340 345<210>59<211>346<212>PRT<213>谷氨酸棒杆菌<400>59Met Glu Asp Met Arg Ile Ala Thr Leu Thr Ser Gly Gly Asp Cys Pro
1 5 10 15Gly Leu Asn Ala Val Ile Arg Gly Ile Val Arg Thr Ala Ser Asn Glu20 25 30Phe Gly Ser Thr Val Val Gly Tyr Gln Asp Gly Trp Glu Gly Leu Leu35 40 45Ala Asp Arg Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile Asp Arg Ile50 55 60Leu Leu Arg Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu His Pro Asp65 70 75 80Lys Phe Lys Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu Glu Asp Ala85 90 95Gly Ile Asp Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr Leu Lys Gly100 105 110Ala Lys Trp Leu Ser Asp Asn Gly Ile Pro Val Val Gly Val Pro Lys115 120 125Thr Ile Asp Asn Asp Val Asn Gly Thr Asp Phe Thr Phe Gly Phe Asp130 135 140Thr Ala Val Ala Val Ala Thr Asp Ala Val Asp Arg Leu His Thr Thr145 150 155 160Ala Glu Ser His Asn Arg Val Met Ile Val Arg Val Met Gly Arg His165 170 175Val Gly Trp Ile Ala Leu His Ala Gly Met Ala Gly Gly Ala His Tyr180 185 190Thr Val Ile Pro Glu Val Pro Phe Asp Ile Ala Glu Ile Cys Lys Ala195 200 205Met Glu Arg Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile Ile Val Val210 215 220Ala Glu Gly Ala Leu Pro Arg Glu Gly Thr Met Glu Leu Arg Glu Gly225 230 235 240His Ile Asp Gln Phe Gly His Lys Thr Phe Thr Gly Ile Gly Gln Gln245 250 255Ile Ala Asp Glu Ile His Val Arg Leu Gly His Asp Val Arg Thr Thr260 265 270Val Leu Gly His Ile Gln Arg Gly Gly Thr Pro Thr Ala Phe Asp Arg275 280 285Val Leu Ala Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala Cys His Glu290 295 300Gly Ser Phe Asp Lys Val Val Ala Leu Lys Gly Glu Ser Ile Glu Met305 310 315 320Ile Thr Phe Glu Glu Ala Val Gly Thr Leu Lys Glu Val Pro Phe Glu325 330 335Arg Trp Val Thr Ala Gln Ala Met Phe Gly340 345<210>60<211>1041<212>DNA<213>谷氨酸棒杆菌<220>
<221>CDS<222>(1)..(1041)<223>pfk<400>60atg gaa gac atg cga att gct act ctc acg tca ggc ggc gac tgc ccc 48Met Glu Asp Met Arg Ile Ala Thr Leu Thr Ser Gly Gly Asp Cys Pro1 5 10 15
gga cta aat gcc gtc atc cga gga atc gtc cgc aca gcc agc aat gaa 96Gly Leu Asn Ala Val Ile Arg Gly Ile Val Arg Thr Ala Ser Asn Glu20 25 30ttt ggc tcc acc gtc gtt ggt tat caa gac ggt tgg gaa gga ctg tta 144Phe Gly Ser Thr Val Val Gly Tyr Gln Asp Gly Trp Glu Gly Leu Leu35 40 45gcc gat cgt cgc gta cag ctg tat gac gat gaa gat att gac cga atc 192Ala Asp Arg Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile Asp Arg Ile50 55 60ctc ctt cga ggc ggc acc att ttg ggc act ggt cgc ctc cat ccg gac 240Leu Leu Arg Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu His Pro Asp65 70 75 80aag ttt aag gcc gga att gat cag att aag gcc aac tta gaa gac gcc 288Lys Phe Lys Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu Glu Asp Ala85 90 95ggc atc gat gcc ctt atc cca atc ggt ggc gaa gga acc ctg aag ggt 336Gly Ile Asp Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr Leu Lys Gly100 105 110gcc aag tgg ctg tct gat aac ggt atc cct gtt gtc ggt gtc cca aag 384Ala Lys Trp Leu Ser Asp Asn Gly Ile Pro Val Val Gly Val Pro Lys115 120 125acc att gac aat gac gtg aat ggc act gac ttc acc ttc ggt ttc gat 432Thr Ile Asp Asn Asp Val Asn Gly Thr Asp Phe Thr Phe Gly Phe Asp130 135 140act gct gtg gca gtg gct acc gac gct gtt gac cgc ctg cac acc acc 480Thr Ala Val Ala Val Ala Thr Asp Ala Val Asp Arg Leu His Thr Thr145 150 155 160gct gaa tct cac aac cgt gtg atg atc gtg gag gtc atg ggc cgc cac 528Ala Glu Ser His Asn Arg Val Met Ile Val Glu Val Met Gly Arg His165 170 175gtg ggt tgg att gct ctg cac gca ggt atg gcg ggc ggt gct cac tac 576Val Gly Trp Ile Ala Leu His Ala Gly Met Ala Gly Gly Ala His Tyr180 185 190acc gtt att cca gaa gta cct ttc gat att gca gag atc tgc aag gcg 624Thr Val Ile Pro Glu Val Pro Phe Asp Ile Ala Glu Ile Cys Lys Ala195 200 205atg gaa cgt cgc ttc cag atg ggc gag aag tac ggc att atc gtc gtt 672Met Glu Arg Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile Ile Val Val210 215 220gcg gaa ggt gcg ttg cca cgc gaa ggc acc atg gag ctt cgt gaa ggc 720Ala Glu Gly Ala Leu Pro Arg Glu Gly Thr Met Glu Leu Arg Glu Gly225 230 235 240cac att gac cag ttc ggt cac aag acc ttc acg gga att gga cag cag 768His Ile Asp Gln Phe Gly His Lys Thr Phe Thr Gly Ile Gly Gln Gln245 250 255atc gct gat gag atc cac gtg cgc ctc ggc cac gat gtt cgt acg acc 816Ile Ala Asp Glu Ile His Val Arg Leu Gly His Asp Val Arg Thr Thr260 265 270gtt ctt ggc cac att caa cgt ggt gga acc cca act gct ttc gac cgt 864Val Leu Gly His Ile Gln Arg Gly Gly Thr Pro Thr Ala Phe Asp Arg275 280 285gtt ctg gcc act cgt tat ggt gtt cgt gca gct cgt gcg tgc cat gag 912Val Leu Ala Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala Cys His Glu290 295 300gga agc ttt gac aag gtt gtt gct ttg aag ggt gag agc att gag atg 960Gly Ser Phe Asp Lys Val Val Ala Leu Lys Gly Glu Ser Ile Glu Met305 310 315 320atc acc ttt gaa gaa gcc gtc gga acc ttg aag gaa gtc cca ttc gaa1008
Ile Thr Phe Glu Glu Ala Val Gly Thr Leu Lys Glu Val Pro Phe Glu325 330 335cgc tgg gtt act gcc cag gca atg ttt gga tag1041Arg Trp Val Thr Ala Gln Ala Met Phe Gly340 345<210>61<211>346<212>PRT<213>谷氨酸棒杆菌<400>61Met Glu Asp Met Arg Ile Ala Thr Leu Thr Ser Gly Gly Asp Cys Pro1 5 10 15Gly Leu Asn Ala Val Ile Arg Gly Ile Val Arg Thr Ala Ser Asn Glu20 25 30Phe Gly Ser Thr Val Val Gly Tyr Gln Asp Gly Trp Glu Gly Leu Leu35 40 45Ala Asp Arg Arg Val Gln Leu Tyr Asp Asp Glu Asp Ile Asp Arg Ile50 55 60Leu Leu Arg Gly Gly Thr Ile Leu Gly Thr Gly Arg Leu His Pro Asp65 70 75 80Lys Phe Lys Ala Gly Ile Asp Gln Ile Lys Ala Asn Leu Glu Asp Ala85 90 95Gly Ile Asp Ala Leu Ile Pro Ile Gly Gly Glu Gly Thr Leu Lys Gly100 105 110Ala Lys Trp Leu Ser Asp Asn Gly Ile Pro Val Val Gly Val Pro Lys115 120 125Thr Ile Asp Asn Asp Val Asn Gly Thr Asp Phe Thr Phe Gly Phe Asp130 135 140Thr Ala Val Ala Val Ala Thr Asp Ala Val Asp Arg Leu His Thr Thr145 150 155 160Ala Glu Ser His Asn Arg Val Met Ile Val Glu Val Met Gly Arg His165 170 175Val Gly Trp Ile Ala Leu His Ala Gly Met Ala Gly Gly Ala His Tyr180 185 190Thr Val Ile Pro Glu Val Pro Phe Asp Ile Ala Glu Ile Cys Lys Ala195 200 205Met Glu Arg Arg Phe Gln Met Gly Glu Lys Tyr Gly Ile Ile Val Val210 215 220Ala Glu Gly Ala Leu Pro Arg Glu Gly Thr Met Glu Leu Arg Glu Gly225 230 235 240His Ile Asp Gln Phe Gly His Lys Thr Phe Thr Gly Ile Gly Gln Gln245 250 255Ile Ala Asp Glu Ile His Val Arg Leu Gly His Asp Val Arg Thr Thr260 265 270Val Leu Gly His Ile Gln Arg Gly Gly Thr Pro Thr Ala Phe Asp Arg275 280 285Val Leu Ala Thr Arg Tyr Gly Val Arg Ala Ala Arg Ala Cys His Glu290 295 300Gly Ser Phe Asp Lys Val Val Ala Leu Lys Gly Glu Ser Ile Glu Met305 310 315 320Ile Thr Phe Glu Glu Ala Val Gly Thr Leu Lys Glu Val Pro Phe Glu325 330 335Arg Trp Val Thr Ala Gln Ala Met Phe Gly345
<210>62<211>2412<212>DNA<213>植物乳杆菌<220>
<221>CDS<222>(1)..(2412)<223>xpk<400>50ttg caa agg agt tgc aaa ata atg agt gaa gca att aaa tcc aaa aca 48Leu Gln Arg Ser Cys Lys Ile Met Ser Glu Ala Ile Lys Ser Lys Thr1 5 10 15gtt gat tac tct tct gat gaa tat cta aaa cgc gtt gat gaa tat tgg 96Val Asp Tyr Ser Ser Asp Glu Tyr Leu Lys Arg Val Asp Glu Tyr Trp20 25 30cgt gct gct aac tac atc tca gtt ggt caa ctc tat cta cta aat aac 144Arg Ala Ala Asn Tyr Ile Ser Val Gly Gln Leu Tyr Leu Leu Asn Asn35 40 45ccg tta ctt cgg gaa cca cta aag gcg acc gac gtg aaa gtt cat cca 192Pro Leu Leu Arg Glu Pro Leu Lys Ala Thr Asp Val Lys Val His Pro50 55 60atc ggc cat tgg ggc acg att gct ggt caa aac ttt att tat gcc cat 240Tle Gly His Trp Gly Thr Ile Ala Gly Gln Asn Phe Ile Tyr Ala His65 70 75 80tta aac cgg gca atc aat aag tat ggc ttg aac atg ttc tac att gaa 288Leu Asn Arg Ala Ile Asn Lys Tyr Gly Leu Asn Met Phe Tyr Ile Glu85 90 95ggc cct ggt cat ggt ggt caa gta atg gtt tct aac tcc tac tta gat 336Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr Leu Asp100 105 110ggc acc tat acg gaa acg tat cct aaa atc acc caa gac aaa gct ggg 384Gly Thr Tyr Thr Glu Thr Tyr Pro Lys Ile Thr Gln Asp Lys Ala Gly115 120 125atg aaa cgc tta ttc aag caa ttc tca ttc cca ggc ggg gtt gct tcc 432Met Lys Arg Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val Ala Ser130 135 140cat gcc gat cot aag acg cct ggt tcg atc cat gaa ggt ggc gaa ctt 480His Ala Asp Pro Lys Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu145 150 155 160ggc tac tca atc ctg cat ggt gct ggt gca gta tta gat aat cca ggt 528Gly Tyr Ser Ile Leu His Gly Ala Gly Ala Val Leu Asp Asn Pro Gly165 170 175tta att gcc gct acc gtt gtt ggt gat ggt gaa tct gaa act ggg cca 576Leu Ile Ala Ala Thr Val Val Gly Asp Gly Glu Ser Glu Thr Gly Pro180 185 190ttg gca act tct tgg caa gtt aac aag ttc ctt aac cca att aca gac 624Leu Ala Thr Ser Trp Gln Val Asn Lys Phe Leu Asn Pro Ile Thr Asp195 200 205ggg aca gtc ttg cca atc ttg aac tta aac ggc ttc aag att tct aat 672Gly Thr Val Leu Pro Ile Leu Asn Leu Asn Gly Phe Lys Ile Ser Asn210 215 220cca aca gtt ctt tca cgt gaa tca cat gaa gaa ctt gaa gac tac ttt 720Pro Thr Val Leu Ser Arg Glu Ser His Glu Glu Leu Glu Asp Tyr Phe225 230 235 240aaa ggt cta ggc tgg gat cca cac ttt gtt gaa ggt aca gac cct gcc 768Lys Gly Leu Gly Trp Asp Pro His Phe Val Glu Gly Thr Asp Pro Ala
245 250 255aag atg cac aaa att atg gct gaa gaa ttg gat aaa gtc att gaa gaa 816Lys Met His Lys Ile Met Ala Glu Glu Leu Asp Lys Val Ile Glu Glu260 265 270atc cac gca att cgt aag aac gcc aag gat aac aat gat gaa tct cgt 864Ile His Ala Ile Arg Lys Asn Ala Lys Asp Asn Asn Asp Glu Ser Arg275 280 285cct aag tgg cca atg att gtt ttc cgg gca cct aag ggc tgg acc ggt 912Pro Lys Trp Pro Met Ile Val Phe Arg Ala Pro Lys Gly Trp Thr Gly290 295 300cct aag agt tgg gac ggc gaa cca att gaa ggt tca ttc cgg gct cac 960Pro Lys Ser Trp Asp Gly Glu Pro Ile Glu Gly Ser Phe Arg Ala His305 310 315 320caa att cca att cct gtc gat cgc aat cac atg gaa cac gcc gac aaa1008Gln Ile Pro Ile Pro Val Asp Arg Asn His Met Glu His Ala Asp Lys325 330 335tta gtt gac tgg ctc aaa tca tac aaa cca gaa gaa tta ttt gat gaa1056Leu Val Asp Trp Leu Lys Ser Tyr Lys Pro Glu Glu Leu Phe Asp Glu340 345 350aat ggt act tta aaa cca gaa att gcc gca atc atc cct gaa ggc caa1104Asn Gly Thr Leu Lys Pro Glu Ile Ala Ala Ile Ile Pro Glu Gly Gln355 360 365gct cgt atg gct gct aac ccc gtt act aac ggc ggt aag tta act aaa1152Ala Arg Met Ala Ala Asn Pro Val Thr Asn Gly Gly Lys Leu Thr Lys370 375 380gac tta att aca cca aat atc gat gat tat gct ttg gac aac aag agt1200Asp Leu Ile Thr Pro Asn Ile Asp Asp Tyr Ala Leu Asp Asn Lys Ser385 390 395 400cac ggt aag gaa gac ggt tca gac atg act gaa ctt ggt aag tat atc1248His Gly Lys Glu Asp Gly Ser Asp Met Thr Glu Leu Gly Lys Tyr Ile405 410 415cgt gat tta att gag ttg aac aaa gac aac aag aac ttc cgt ggc tgg1296Arg Asp Leu Ile Glu Leu Asn Lys Asp Asn Lys Asn Phe Arg Gly Trp420 425 430ggt cct gac gaa acc tta tct aac aaa cta ggc gct gct ttt gaa gat1344Gly Pro Asp Glu Thr Leu Ser Asn Lys Leu Gly Ala Ala Phe Glu Asp435 440 445acc aaa cgt cag tgg atg gaa cca atc cac gaa cct aat gat gct ttg1392Thr Lys Arg Gln Trp Met Glu Pro Ile His Glu Pro Asn Asp Ala Leu450 455 460tta gca cct caa ggc cgg att att gac tcc atg ttg tca gaa cac atg1440Leu Ala Pro Gln Gly Arg Ile Ile Asp Ser Met Leu Ser Glu His Met465 470 475 480gat gaa ggg atg ttg gaa gct tac aat tta acc gga cgt tac ggt ttc1488Asp Glu Gly Met Leu Glu Ala Tyr Asn Leu Thr Gly Arg Tyr Gly Phe485 490 495ttc gca agt tat gaa tca ttc ctg cgc gtt gtg gat tca atg tta acc1536Phe Ala Ser Tyr Glu Ser Phe Leu Arg Val Val Asp Ser Met Leu Thr500 505 510caa cac ttc aag tgg tta cgg aat tct cac gaa gaa acc cct tgg cgg1584Gln His Phe Lys Trp Leu Arg Asn Ser His Glu Glu Thr Pro Trp Arg515 520 525gct gat gta cct tca ctg aat gtg att gca tca tca aca gcc ttc caa1632Ala Asp Val Pro Ser Leu Asn Val Ile Ala Ser Ser Thr Ala Phe Gln530 535 540caa gat cac aat ggt tac tct cac caa gat cca ggt atc att tca cac1680Gln Asp His Asn Gly Tyr Ser His Gln Asp Pro Gly Ile Ile Ser His545 550 555 560
ttg gct gaa aag aag acc gaa tac gtt cgt gcc tat ctt cca ggt gat1728Leu Ala Glu Lys Lys Thr Glu Tyr Val Arg Ala Tyr Leu Pro Gly Asp565 570 575gcc aat act ttg att gca acc ttt gat aag gct atc caa agc aaa caa1776Ala Asn Thr Leu Ile Ala Thr Phe Asp Lys Ala Ile Gln Ser Lys Gln580 585 590ttg att aat tta atc att gcc agc aag cac cct cgt cca caa tgg ttc1824Leu Ile Ash Leu Ile Ile Ala Ser Lys His Pro Arg Pro Gln Trp Phe595 600 605aca atg gac gaa gct aag cgc tta gtt cgt gat ggc ctt ggt gtt gtt1872Thr Met Asp Glu Ala Lys Arg Leu Val Arg Asp Gly Leu Gly Val Val610 615 620gat tgg gca agc act gat cat ggt gaa gaa ccc gac gtt gtc ttc gca1920Asp Trp Ala Ser Thr Asp His Gly Glu Glu Pro Asp Val Val Phe Ala625 630 635 640act gcc ggc tct gaa cca acg act gaa agc tta gct gcc gta tca atc1968Thr Ala Gly Ser Glu Pro Thr Thr Glu Ser Leu Ala Ala Val Ser Ile645 650 655ttg cat gca cgc ttc cct gaa atg aag att cgc ttc att aac gtt gtt2016Leu His Ala Arg Phe Pro Glu Met Lys Ile Arg Phe Ile Asn Val Val660 665 670gat ctt ctg aag ctg aag aaa gac gac cct cgt ggt tta tca gat gct2064Asp Leu Leu Lys Leu Lys Lys Asp Asp Pro Arg Gly Leu Ser Asp Ala675 680 685gaa ttt gat gct ttc ttc act aag gac aaa cca gtt atc ttt gct tat2112Glu Phe Asp Ala Phe Phe Thr Lys Asp Lys Pro Val Ile Phe Ala Tyr690 695 700cat gca tac gac gac tta gta aag acc atc ttc ttc gat cgc cat aac2160His Ala Tyr Asp Asp Leu Val Lys Thr Ile Phe Phe Asp Arg his Asn705 710 715 720cat aac tta cac gtt cat ggt tac cgc gaa gaa ggc gac att aca acg2208His Asn Leu His Val His Gly Tyr Arg Glu Glu Gly Asp Ile Thr Thr725 730 735cca ttc gac atg cgt gtt cgc aac gaa ctc gat cgt ttc cac tta gtc2256Pro Phe Asp Met Arg Val Arg Asn Glu Leu Asp Arg Phe His Leu Val740745 750aaa gct gcc tta tta gca acg cca gct tat gcc gaa aaa ggt gcc cat2304Lys Ala Ala Leu Leu Ala Thr Pro Ala Tyr Ala Glu Lys Gly Ala His755 760 765gtc att caa gag atg aac agc att tta gac aag cat cat gac tat atc2352Val Ile Gln Glu Met Asn Ser Ile Leu Asp Lys His His Asp Tyr Ile770 775 780cgt gct gaa ggt acc gat att cca gaa gtt gaa aac tgg aaa tgg act2400Arg Ala Glu Gly Thr Asp Ile Pro Glu Val Glu Asn Trp Lys Trp Thr785 790 795 800gca ttg aag tag2412Ala Leu Lys<210>63<211>803<212>PRT<213>植物乳杆菌<400>63Leu Gln Arg Ser Cys Lys Ile Met Ser Glu Ala Ile Lys Ser Lys Thr1 5 10 15Val Asp Tyr Ser Ser Asp Glu Tyr Leu Lys Arg Val Asp Glu Tyr Trp
20 25 30Arg Ala Ala Asn Tyr Ile Ser Val Gly Gln Leu Tyr Leu Leu Asn Asn35 40 45Pro Leu Leu Arg Glu Pro Leu Lys Ala Thr Asp Val Lys Val His Pro50 55 60Ile Gly His Trp Gly Thr Ile Ala Gly Gln Asn Phe Ile Tyr Ala His65 70 75 80Leu Asn Arg Ala Ile Asn Lys Tyr Gly Leu Asn Met Phe Tyr Ile Glu85 90 95Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr Leu Asp100 105 110Gly Thr Tyr Thr Glu Thr Tyr Pro Lys Ile Thr Gln Asp Lys Ala Gly115 120 125Met Lys Arg Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val Ala Ser130 135 140His Ala Asp Pro Lys Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu145 150 155 160Gly Tyr Ser Ile Leu His Gly Ala Gly Ala Val Leu Asp Asn Pro Gly165 170 175Leu Ile Ala Ala Thr Val Val Gly Asp Gly Glu Ser Glu Thr Gly Pro180 185 190Leu Ala Thr Ser Trp Gln Val Asn Lys Phe Leu Asn Pro Ile Thr Asp195 200 205Gly Thr Val Leu Pro Ile Leu Asn Leu Asn Gly Phe Lys Ile Ser Asn210 215 220Pro Thr Val Leu Ser Arg Glu Ser His Glu Glu Leu Glu Asp Tyr Phe225 230 235 240Lys Gly Leu Gly Trp Asp Pro His Phe Val Glu Gly Thr Asp Pro Ala245 250 255Lys Met His Lys Ile Met Ala Glu Glu Leu Asp Lys Val Ile Glu Glu260 265 270Ile His Ala Ile Arg Lys Asn Ala Lys Asp Asn Asn Asp Glu Ser Arg275 280 285Pro Lys Trp Pro Met Ile Val Phe Arg Ala Pro Lys Gly Trp Thr Gly290 295 300Pro Lys Ser Trp Asp Gly Glu Pro Ile Glu Gly Ser Phe Arg Ala His305 310 315 320Gln Ile Pro Ile Pro Val Asp Arg Asn His Met Glu His Ala Asp Lys325 330 335Leu Val Asp Trp Leu Lys Ser Tyr Lys Pro Glu Glu Leu Phe Asp Glu340 345 350Asn Gly Thr Leu Lys Pro Glu Ile Ala Ala Ile Ile Pro Glu Gly Gln355 360 365Ala Arg Met Ala Ala Asn Pro Val Thr Asn Gly Gly Lys Leu Thr Lys370 375 380Asp Leu Ile Thr Pro Asn Ile Asp Asp Tyr Ala Leu Asp Asn Lys Ser385 390 395 400His Gly Lys Glu Asp Gly Ser Asp Met Thr Glu Leu Gly Lys Tyr Ile405 410 415Arg Asp Leu Ile Glu Leu Asn Lys Asp Asn Lys Asn Phe Arg Gly Trp420 425 430Gly Pro Asp Glu Thr Leu Ser Asn Lys Leu Gly Ala Ala Phe Glu Asp435 440 445Thr Lys Arg Gln Trp Met Glu Pro Ile His Glu Pro Asn Asp Ala Leu450 455 460Leu Ala Pro Gln Gly Arg Ile Ile Asp Ser Met Leu Ser Glu His Met465 470 475 480Asp Glu Gly Met Leu Glu Ala Tyr Asn Leu Thr Gly Arg Tyr Gly Phe
485 490 495Phe Ala Ser Tyr Glu Ser Phe Leu Arg Val Val Asp Ser Met Leu Thr500 505 510Gln His Phe Lys Trp Leu Arg Asn Ser His Glu Glu Thr Pro Trp Arg515 520 525Ala Asp Val Pro Ser Leu Asn Val Ile Ala Ser Ser Thr Ala Phe Gln530 535 540Gln Asp His Asn Gly Tyr Ser His Gln Asp Pro Gly Ile Ile Ser His545 550 555 560Leu Ala Glu Lys Lys Thr Glu Tyr Val Arg Ala Tyr Leu Pro Gly Asp565 570 575Ala Asn Thr Leu Ile Ala Thr Phe Asp Lys Ala Ile Gln Ser Lys Gln580 585 590Leu Ile Asn Leu Ile Ile Ala Ser Lys His Pro Arg Pro Gln Trp Phe595 600 605Thr Met Asp Glu Ala Lys Arg Leu Val Arg Asp Gly Leu Gly Val Val610 615 620Asp Trp Ala Ser Thr Asp His Gly Glu Glu Pro Asp Val Val Phe Ala625 630 635 640Thr Ala Gly Ser Glu Pro Thr Thr Glu Ser Leu Ala Ala Val Ser Ile645 650 655Leu His Ala Arg Phe Pro Glu Met Lys Ile Arg Phe Ile Asn Val Val660 665 670Asp Leu Leu Lys Leu Lys Lys Asp Asp Pro Arg Gly Leu Ser Asp Ala675 680 685Glu Phe Asp Ala Phe Phe Thr Lys Asp Lys Pro Val Ile Phe Ala Tyr690 695 700His Ala Tyr Asp Asp Leu Val Lys Thr Ile Phe Phe Asp Arg His Asn705 710 715 720His Asn Leu His Val His Gly Tyr Arg Glu Glu Gly Asp Ile Thr Thr725 730 735Pro Phe Asp Met Arg Val Arg Asn Glu Leu Asp Arg Phe His Leu Val740 745 750Lys Ala Ala Leu Leu Ala Thr Pro Ala Tyr Ala Glu Lys Gly Ala His755 760 765Val Ile Gln Glu Met Asn Ser Ile Leu Asp Lys His His Asp Tyr Ile770 775 780Arg Ala Glu Gly Thr Asp Ile Pro Glu Val Glu Asn Trp Lys Trp Thr785 790 795 800Ala Leu Lys<210>64<211>2379<212>DNA<213>无乳链球菌<220>
<221>CDS<222>(1)..(2379)<223>xpk<400>64atg tca gaa ttt gat aca aaa tca tat tta gaa aaa ctt gat gca tgg48Met Ser Glu Phe Asp Thr Lys Ser Tyr Leu Glu Lys Leu Asp Ala Trp1 5 10 15tgg aga gca gct aat tat att tct gca gca caa atg tat cta aag gat96Trp Arg Ala Ala Asn Tyr Ile Ser Ala Ala Gln Met Tyr Leu Lys Asp
20 25 30aat cct ctc ttg aga cga gag cta gtt gaa aac gac tta aag gtt cat 144Asn Pro Leu Leu Arg Arg Glu Leu Val Glu Asn Asp Leu Lys Val His35 40 45cca att ggt cac tgg ggc act gta cct gga caa aac ttt atc tat gct 192Pro Ile Gly His Trp Gly Thr Val Pro Gly Gln Asn Phe Ile Tyr Ala50 55 60cac tta aat cgc gct att aat aaa tac gat tta gac atg ttc tat att 240His Leu Asn Arg Ala Ile Asn Lys Tyr Asp Leu Asp Met Phe Tyr Ile65 70 75 80gaa ggt cca ggt cat ggt ggt caa gtt atg gta tct aat tca tat tta 288Glu Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr Leu85 90 95gat ggt tca tat act gaa tta aac cca aat att gag caa aca gag gat 336Asp Gly Ser Tyr Thr Glu Leu Asn Pro Asn Ile Glu Gln Thr Glu Asp100 l05 110ggc ttt aag cag tta tgt aaa atc ttc tct ttc cca ggt gga att gca 384Gly Phe Lys Gln Leu Cys Lys Ile Phe Ser Phe Pro Gly Gly Ile Ala115 120 125tcc cat gca gca cca gaa aca cca ggg tca att cat gaa ggt ggt gaa 432Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu130 135 140ctt ggt tac gca ctt tct cat gcg aca ggt gcc atc cta gac aat cca 480Leu Gly Tyr Ala Leu Ser His Ala Thr Gly Ala Ile Leu Asp Asn Pro145 150 155 160gat gtt att gct gcg act gtt att ggt gat ggt gaa ggt gaa aca gga 528Asp Val Ile Ala Ala Thr Val Ile Gly Asp Gly Glu Gly Glu Thr Gly165 170 175cca ctt atg gct ggt tgg cta tct aat acc ttt atc aat cca gta aat 576Pro Leu Met Ala Gly Trp Leu Ser Asn Thr Phe Ile Asn Pro Val Asn180 185 190gat ggt gct gtt cta cca atc ttc tat tta aat ggt ggt aaa atc cat 624Asp Gly Ala Val Leu Pro Ile Phe Tyr Leu Asn Gly Gly Lys Ile His195 200 205aac cca act atc ttt gaa cgt aag aca gat gaa gaa tta tct cag ttt 672Asn Pro Thr Ile Phe Glu Arg Lys Thr Asp Glu Glu Leu Ser Gln Phe210 215 220ttt gaa gga tta ggt tgg aaa cct att ttt gca gat gtt gtt gaa ctc 720Phe Glu Gly Leu Gly Trp Lys Pro Ile Phe Ala Asp Val Val Glu Leu225 230 235 240tct gag gat cat gcg gct gct cat gct ttg ttt gca gaa aaa tta gat 768Ser Glu Asp His Ala Ala Ala His Ala Leu Phe Ala Glu Lys Leu Asp245 250 255caa gct att caa gag att aaa acc att caa tca gaa gca cga caa aaa 816Gln Ala Ile Gln Glu Ile Lys Thr Ile Gln Ser Glu Ala Arg Gln Lys260 265 270cca gca gaa gaa gct atc caa gca aaa ttc cct gtc ttg gtt gca cgt 864Pro Ala Glu Glu Ala Ile Gln Ala Lys Phe Pro Val Leu Val Ala Arg275 280 285att cct aag ggg tgg act ggt cca aaa gct tgg gaa gga aca cca att 912Ile Pro Lys Gly Trp Thr Gly Pro Lys Ala Trp Glu Gly Thr Pro Ile290 295 300gaa ggt ggc ttc cgc gct cac caa gta cca att cca gta gat gcc cac 960Glu Gly Gly Phe Arg Ala His Gln Val Pro Ile Pro Val Asp Ala His305 310 315 320cat atg gaa cat gtc gat tct ctt ttg tca tgg ctt caa tca tac cgt1008His Met Glu His Val Asp Ser Leu Leu Ser Trp Leu Gln Ser Tyr Arg325 330 335
cca gaa gaa tta ttt gat gaa agt ggt aaa atc gtt gat gag att gct1056Pro Glu Glu Leu Phe Asp Glu Ser Gly Lys Ile Val Asp Glu Ile Ala340 345 350gct att tca cca aaa ggt gat cgt cgc atg tcc atg aat cca ata acc1104Ala Ile Ser Pro Lys Gly Asp Arg Arg Met Ser Met Asn Pro Ile Thr355 360 365aat gct ggt att gtt aaa gca atg gat aca gca gat tgg aag aaa ttt1152Asn Ala Gly Ile Val Lys Ala Met Asp Thr Ala Asp Trp Lys Lys Phe370 375 380gct ctt gat att aat gtt cca ggt caa att atg gca caa gat atg att1200Ala Leu Asp Ile Asn Val Pro Gly Gln Ile Met Ala Gln Asp Met Ile385 390 395 400gaa ttt gga aaa tat gca gca gat ttg gta gat gct aat cca gat aat1248Glu Phe Gly Lys Tyr Ala Ala Asp Leu Val Asp Ala Asn Pro Asp Asn405 410 415ttc cgt att ttt ggt cca gat gaa acg aaa tca aat cgt ctt caa gaa1296Phe Arg Ile Phe Gly Pro Asp Glu Thr Lys Ser Asn Arg Leu Gln Glu420 425 430gtg ttt aca cgt act agc cgt caa tgg ctt ggt cgt cgt aaa cca gat1344Val Phe Thr Arg Thr Ser Arg Gln Trp Leu Gly Arg Arg Lys Pro Asp435 440 445tat gac gaa gct tta agt cca gct gga cgt gtt att gat tcc caa ttg1392Tyr Asp Glu Ala Leu Ser Pro Ala Gly Arg Val Ile Asp Ser Gln Leu450 455 460tct gaa cat caa gca gaa ggt ttc tta gaa ggt tat gtt tta act ggt1440Ser Glu His Gln Ala Glu Gly Phe Leu Glu Gly Tyr Val Leu Thr Gly465 470 475 480cgt cac ggc ttc ttt gct tca tac gaa tca ttc ctt cgc gtt gta gat1488Arg His Gly Phe Phe Ala Ser Tyr Glu Ser Phe Leu Arg Val Val Asp485 490 495tca atg gta aca caa cat ttc aaa tgg tta cgt aaa tca aaa aca cac1536Ser Met Val Thr Gln His Phe Lys Trp Leu Arg Lys Ser Lys Thr His500 505 510aca aca tgg cgt aaa aat tat cca gcg ctt aac tta att gca gct tca1584Thr Thr Trp Arg Lys Asn Tyr Pro Ala Leu Asn Leu Ile Ala Ala Ser515 520 525act gtt ttc caa caa gat cat aat ggt tac act cac caa gat cca ggt1632Thr Val Phe Gln Gln Asp His Asn Gly Tyr Thr His Gln Asp Pro Gly530 535 540atc tta act cac tta gct gaa aaa aca cct gaa tac att cgt gaa tat1680Ile Leu Thr His Leu Ala Glu Lys Thr Pro Glu Tyr Ile Arg Glu Tyr545 550 555 560tta cca gca gat act aac tca ctt cta gct gtt atg gat aaa gcg ttt1728Leu Pro Ala Asp Thr Asn Ser Leu Leu Ala Val Met Asp Lys Ala Phe565 570 575aaa gct gaa gat aaa att aat tta att gtg aca tct aag cac cct cgt1776Lys Ala Glu Asp Lys Ile Asn Leu Ile Val Thr Ser Lys His Pro Arg580 585 590cca caa ttt tac tct att gct gaa gca gaa gag tta gtg gca gaa gga1824Pro Gln Phe Tyr Ser Ile Ala Glu Ala Glu Glu Leu Val Ala Glu Gly595 600 605tat aag gtg att gac tgg gct tca aat gtg tcg ctt aat caa gag cca1872Tyr Lys Val Ile Asp Trp Ala Ser Asn Val Ser Leu Asn Gln Glu Pro610 615 620gat gtt gtc ttt gct gcg gcg gga aca gaa cct aac tta gaa gct ttg1920Asp Val Val Phe Ala Ala Ala Gly Thr Glu Pro Asn Leu Glu Ala Leu625 630 635 640gca gct att agt att ctt cat aag gct ttc cca gaa ctt aag att aga1968
Ala Ala Ile Ser Ile Leu His Lys Ala Phe Pro Glu Leu Lys Ile Arg645 650 655ttt gtt aat gta tta gac atc cta aaa ctt cgt cat cct tca caa gat2016Phe Val Asn Val Leu Asp Ile Leu Lys Leu Arg His Pro Ser Gln Asp660 665 670gct cgc ggt tta tca gat gaa gag ttt gac aaa gtc ttt acc aca gat2064Ala Arg Gly Leu Ser Asp Glu Glu Phe Asp Lys Val Phe Thr Thr Asp675 680 685aaa cct gtt att ttt get ttc cat agt tat gaa gat atg att cga gat2112Lys Pro Val Ile Phe Ala Phe His Ser Tyr Glu Asp Met Ile Arg Asp690 695 700att ttc ttt agt cgt cac aat cat aat ttg cat acg cat ggt tac cgt2160Ile Phe Phe Ser Arg His Asn His Asn Leu His Thr His Gly Tyr Arg705 710 715 720gaa aat ggt gat atc aca aca cca ttt gat atg cgc gtg atg tca gaa2208Glu Asn Gly Asp Ile Thr Thr Pro Phe Asp Met Arg Val Met Ser Glu725 730 735ttg gac cga ttc cat ctg gca cag gat gca gct ctt gct tct tta gga2256Leu Asp Arg Phe His Leu Ala Gln Asp Ala Ala Leu Ala Ser Leu Gly740 745 750aat gaa gca caa gcc ttc agt gat gaa atg aat caa atg gtt gct tat2304Asn Glu Ala Gln Ala Phe Set Asp Glu Met Asn Gln Met Val Ala Tyr755 760 765cac aaa gac tat att cgt gaa cac gga gac gat att cca gaa gtg caa2352His Lys Asp Tyr Ile Arg Glu His Gly Asp Asp Ile Pro Glu Val Gln770 775 780aat tgg aaa tgg gaa aat atc aag tag2379Asn Trp Lys Trp Glu Asn Ile Lys785 790<210>65<211>792<212>PRT<213>无乳链球菌<400>65Met Ser Glu Phe Asp Thr Lys Ser Tyr Leu Glu Lys Leu Asp Ala Trp1 5 10 15Trp Arg Ala Ala Asn Tyr Ile Ser Ala Ala Gln MetTyr Leu Lys Asp20 25 30Asn Pro Leu Leu Arg Arg Glu Leu Val Glu Asn Asp Leu Lys Val His35 40 45Pro Ile Gly His Trp Gly Thr Val Pro Gly Gln Asn Phe Ile Tyr Ala50 55 60His Leu Asn Arg Ala Ile Asn Lys Tyr Asp Leu Asp Met Phe Tyr Ile65 70 75 80Glu Gly Pro Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr Leu85 90 95Asp Gly Ser Tyr Thr Glu Leu Asn Pro Asn Ile Glu Gln Thr Glu Asp100 105 110Gly Phe Lys Gln Leu Cys Lys Ile Phe Ser Phe Pro Gly Gly Ile Ala115 120 125Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu130 135 140Leu Gly Tyr Ala Leu Ser His Ala Thr Gly Ala Ile Leu Asp Asn Pro145 150 155 160Asp Val Ile Ala Ala Thr Val Ile Gly Asp Gly Glu Gly Glu Thr Gly
165 170 175Pro Leu Met Ala Gly Trp Leu Ser Asn Thr Phe Ile Asn Pro Val Asn180 185 190Asp Gly Ala Val Leu Pro Ile Phe Tyr Leu Asn Gly Gly Lys Ile His195 200 205Asn Pro Thr Ile Phe Glu Arg Lys Thr Asp Glu Glu Leu Ser Gln Phe210 215 220Phe Glu Gly Leu Gly Trp Lys Pro Ile Phe Ala Asp Val Val Glu Leu225 230 235 240Ser Glu Asp His Ala Ala Ala His Ala Leu Phe Ala Glu Lys Leu Asp245 250 255Gln Ala Ile Gln Glu Ile Lys Thr Ile Gln Ser Glu Ala Arg Gln Lys260 265 270Pro Ala Glu Glu Ala Ile Gln Ala Lys Phe Pro Val Leu Val Ala Arg275 280 285Ile Pro Lys Gly Trp Thr Gly Pro Lys Ala Trp Glu Gly Thr Pro Ile290 295 300Glu Gly Gly Phe Arg Ala His Gln Val Pro Ile Pro Val Asp Ala His305 310 315 320His Met Glu His Val Asp Ser Leu Leu Ser Trp Leu Gln Ser Tyr Arg325 330 335Pro Glu Glu Leu Phe Asp Glu Ser Gly Lys Ile Val Asp Glu Ile Ala340 345 350Ala Ile Ser Pro Lys Gly Asp Arg Arg Met Ser Met Asn Pro Ile Thr355 360 365Asn Ala Gly Ile Val Lys Ala Met Asp Thr Ala Asp Trp Lys Lys Phe370 375 380Ala Leu Asp Ile Asn Val Pro Gly Gln Ile Met Ala Gln Asp Met Ile385 390 395 400Glu Phc Gly Lys Tyr Ala Ala Asp Leu Val Asp Ala Asn Pro Asp Asn405 410 415Phe Arg Ile Phe Gly Pro Asp Glu Thr Lys Ser Asn Arg Leu Gln Glu420 425 430Val Phe Thr Arg Thr Ser Arg Gln Trp Leu Gly Arg Arg Lys Pro Asp435 440 445Tyr Asp Glu Ala Leu Ser Pro Ala Gly Arg Val Ile Asp Ser Gln Leu450 455 460Ser Glu His Gln Ala Glu Gly Phe Leu Glu Gly Tyr Val Leu Thr Gly465 470 475 480Arg His Gly Phe Phe Ala Ser Tyr Glu Ser Phe Leu Arg Val Val Asp485 490 495Ser Met Val Thr Gln His Phe Lys Trp Leu Arg Lys Ser Lys Thr His500 505 510Thr Thr Trp Arg Lys Asn Tyr Pro Ala Leu Asn Leu Ile Ala Ala Ser515 520 525Thr Val Phe Gln Gln Asp His Asn Gly Tyr Thr His Gln Asp Pro Gly530 535 540Ile Leu Thr His Leu Ala Glu Lys Thr Pro Glu Tyr Ile Arg Glu Tyr545 550 555 560Leu Pro Ala Asp Thr Asn Ser Leu Leu Ala Val Met Asp Lys Ala Phe565 570 575Lys Ala Glu Asp Lys Ile Asn Leu Ile Val Thr Ser Lys His Pro Arg580 585 590Pro Gln Phe Tyr Ser Ile Ala Glu Ala Glu Glu Leu Val Ala Glu Gly595 600 605Tyr Lys Val Ile Asp Trp Ala Ser Asn Val Ser Leu Asn Gln Glu Pro610 615 620Asp Val Val Phe Ala Ala Ala Gly Thr Glu Pro Asn Leu Glu Ala Leu
625 630 635 640Ala Ala Ile Ser Ile Leu His Lys Ala Phe Pro Glu Leu Lys Ile Arg645 650 655Phe Val Ash Val Leu Asp Ile Leu Lys Leu Arg His Pro Ser Gln Asp660 665 670Ala Arg Gly Leu Ser Asp Glu Glu Phe Asp Lys Val Phe Thr Thr Asp675 680 685Lys Pro Val Ile Phe Ala Phc His Ser Tyr Glu Asp Met Ile Arg Asp690 695 700Ile Phe Phe Ser Arg His Asn His Asn Leu His Thr His Gly Tyr Arg705 710 715 720Glu Asn Gly Asp Ile Thr Thr Pro Phe Asp Met Arg Val Met Ser Glu725 730 735Leu Asp Arg Phe His Leu Ala Gln Asp Ala Ala Leu Ala Ser Leu Gly740 745 750Asn Glu Ala Gln Ala Phe Ser Asp Glu Met Asn Gln Met Val Ala Tyr755 760 765His Lys Asp Tyr Ile Arg Glu His Gly Asp Asp Ile Pro Glu Val Gln770 775 780Asn Trp Lys Trp Glu Asn Ile Lys785 790<210>66<211>2469<212>DNA<213>乳酸乳球菌<220>
<221>CDS<222>(1)..(2469)<223>xpk<400>66atg aca gaa tat aat tca gaa gct tat ttg aaa aag ctt gat aaa tgg 48Met Thr Glu Tyr Asn Ser Glu Ala Tyr Leu Lys Lys Leu Asp Lys Trp1 5 10 15tgg cga gca gca act tat ctt gga gca gga atg atc ttc ttg aaa gaa 96Trp Arg Ala Ala Thr Tyr Leu Gly Ala Gly Met Ile Phe Leu Lys Glu20 25 30aat cca ttg ttc tct gtg aca ggt act cca att aaa gcg gaa aac ctt144Asn Pro Leu Phe Ser Val Thr Gly Thr Pro Ile Lys Ala Glu Asn Leu35 40 45aaa gcc aat cct att ggg cac tgg ggg acg gtt tca gga caa act ttc192Lys Ala Asn Pro Ile Gly His Trp Gly Thr Val Ser Gly Gln Thr Phe50 55 60ctc tat gct cat gct aat cgt cta atc aat aaa tat gat caa aag atg240Leu Tyr Ala His Ala Asn Arg Leu Ile Asn Lys Tyr Asp Gln Lys Met65 70 75 80ttt tac atg ggt ggc ccc gga cat ggt gga caa gct atg gtt gtt cct255Phe Tyr Met Gly Gly Pro Gly His Gly Gly Gln Ala Met Val Val Pro85 90 95tct tat ctt gat ggc tca tat aca gaa gct tat cca gag att acc caa336Ser Tyr Leu Asp Gly Ser Tyr Thr Glu Ala Tyr Pro Glu Ile Thr Gln100 105 110gat ttg gaa gga atg tca cgt ttg ttt aaa cgt ttc tca ttt cct gga384Asp Leu Glu Gly Met Ser Arg Leu Phe Lys Arg Phe Ser Phe Pro Gly115 120 125gga ata ggg tcg cat atg aca gca caa acc cct ggt tca ctt cat gaa432
Gly Ile Gly Ser His Met Thr Ala Gln Thr Pro Gly Ser Leu His Glu130 135 140gga ggt gag ttg ggt tat gtg cta tca cat gca aca ggg gct att ctt 480Gly Gly Glu Leu Gly Tyr Val Leu Ser His Ala Thr Gly Ala Ile Leu145 150 155 160gat caa cct gaa cag att gct ttt gct gtt gtt ggg gat gga gaa gct 528Asp Gln Pro Glu Gln Ile Ala Phe Ala Val Val Gly Asp Gly Glu Ala165 170 175gaa act gga ccg ttg atg aca agt tgg cac tct att aaa ttc att aat 576Glu Thr Gly Pro Leu Met Thr Ser Trp His Ser Ile Lys Phe Ile Asn180 185 190cct aag aat gat ggg gcg att tta cca att ctt gat tta aat ggt ttt 624Pro Lys Asn Asp Gly Ala Ile Leu Pro Ile Leu Asp Leu Asn Gly Phe195 200 205aaa att tca aat cct act ttg ttc gct cga act tca gat gtt gat att 672Lys Ile Ser Asn Pro Thr Leu Phe Ala Arg Thr Ser Asp Val Asp Ile210 215 220cgt aaa ttc ttt gaa gga ctg ggt tac tca cct cgt tat att gaa aat 720Arg Lys Phe Phe Glu Gly Leu Gly Tyr Ser Pro Arg Tyr Ile Glu Asn225 230 235 240gat gat att cat gat tac atg gct tat cat aaa tta gca gct gaa gtt 768Asp Asp Ile His Asp Tyr Met Ala Tyr His Lys Leu Ala Ala Glu Val245 250 255ttt gat aaa gcg att gaa gac att cat caa att cag aaa gat gcg cgt 816Phe Asp Lys Ala Ile Glu Asp Ile His Gln Ile Gln Lys Asp Ala Arg260 265 270gaa gat aat cgt tat caa aat gga gag att cca gct tgg cca att gtt 864Glu Asp Asn Arg Tyr Gln Asn Gly Glu Ile Pro Ala Trp Pro Ile Val275 280 285atc gca cgt tta cca aaa ggt tgg ggt ggt cca cgt tat aat gat tgg 912Ile Ala Arg Leu Pro Lys Gly Trp Gly Gly Pro Arg Tyr Asn Asp Trp290 295 300tca ggt cct aaa ttt gac ggt aag gga atg cca att gaa cat agt ttc 960Ser Gly Pro Lys Phe Asp Gly Lys Gly Met Pro Ile Glu His Ser Phe305 310 315 320cgt gcg cat caa gtt cca ctt ccg tta tct tct aaa aat atg gga act1008Arg Ala His Gln Val Pro Leu Pro Leu Ser Ser Lys Asn Met Gly Thr325 330 335tta cca gaa ttt gta aaa tgg atg act tct tac caa cca gaa act tta1056Leu Pro Glu Phe Val Lys Trp Met Thr Ser Tyr Gln Pro Glu Thr Leu340 345 350ttt aat gct gat gga agt ttg aaa gaa gag ttg cgt gat ttt gca cca1104Phe Asn Ala Asp Gly Ser Leu Lys Glu Glu Leu Arg Asp Phe Ala Pro355 360 365aaa ggt gag atg cga atg gct tca aac cct gta aca aat ggg gga gtt1152Lys Gly Glu Met Arg Met Ala Ser Asn Pro Val Thr Asn Gly Gly Val370 375 380gat tct tct aat ttg gtt tta cca gat tgg caa gaa ttt gca aat cca1200Asp Ser Ser Asn Leu Val Leu Pro Asp Trp Gln Glu Phe Ala Asn Pro385 390 395 400att tctgaa aat aat cga ggg aaa tta ctc cct gat aca aat gac aat1248Ile Ser Glu Asn Asn Arg Gly Lys Leu Leu Pro Asp Thr Asn Asp Asn405 410 415atg gat atg aat gtt ttg tca aaa tat ttt gct gaa ata gtc aaa ctt1296Met Asp Met Asn Val Leu Ser Lys Tyr Phe Ala Glu Ile Val Lys Leu420 425 430aat cct acg cgt ttc cgt ttg ttt ggt cct gat gaa acc atg tct aat1344Asn Pro Thr Arg Phe Arg Leu Phe Gly Pro Asp Glu Thr Met Ser Asn
435 440 445cgt ttt tgg gaa atg ttt aag gtg acg aat cgt cag tgg atg caa gtc1392Arg Phe Trp Glu Met Phe Lys Val Thr Asn Arg Gln Trp Met Gln Val450 455 460ata aaa aat cca aat gat gaa ttt atc tca cct gag ggt cgc att att1440Ile Lys Asn Pro Asn Asp Glu Phe Ile Ser Pro Glu Gly Arg Ile Ile465 470 475 480gat tct caa tta tca gaa cac caa gca gaa ggt tgg ctt gaa ggt tat1488Asp Ser Gln Leu Ser Glu His Gln Ala Glu Gly Trp Leu Glu Gly Tyr485 490 495act tta acc gga cgc aca gga gca ttt gca agt tat gaa tca ttc ttg1536Thr Leu Thr Gly Arg Thr Gly Ala Phe Ala Ser Tyr Glu Ser Phe Leu500 505 510cga gtc gta gat tca atg tta act caa cat ttc aaa tgg att cgt caa1584Arg Val Val Asp Ser Met Leu Thr Gln His Phe Lys Trp Ile Arg Gln515 520 525gct gca gac caa aaa tgg cgc cat gat tat cct tcg ctt aat gtt att1632Ala Ala Asp Gln Lys Trp Arg His Asp Tyr Pro Ser Leu Asn Val Ile530 535 540tcg acc tca acc gtt ttc caa caa gac cat aat ggt tat act cac caa1680Ser Thr Ser Thr Val Phe Gln Gln Asp His Asn Gly Tyr Thr His Gln545 550 555 560gat cct gga atg ttg act cat ttg gct gaa aag aaa tct gat ttt atc1728Asp Pro Gly Met Leu Thr His Leu Ala Glu Lys Lys Ser Asp Phe Ile565 570 575aga caa tac ttg ccg gct gat ggg aat act ttg ctt gcc gta ttt gac1776Arg Gln Tyr Leu Pro Ala Asp Gly Asn Thr Leu Leu Ala Val Phe Asp580 585 590cgt gct ttt caa gat aga agt aaa att aat cat att gta gcc tct aaa1824Arg Ala Phe Gln Asp Arg Ser Lys Ile Asn His Ile Val Ala Ser Lys595 600 605caa cct cgt caa caa tgg ttt act aaa gaa gaa gct gaa aaa ttg gcg1872Gln Pro Arg Gln Gln Trp Phe Thr Lys Glu Glu Ala Glu Lys Leu Ala610 615 620act gac gga att gca aca att gat tgg gct tca acg gct aaa gat gga1920Thr Asp Gly Ile Ala Thr Ile Asp Trp Ala Ser Thr Ala Lys Asp Gly625 630 635 640gaa gca gta gat tta gtt ttt gcc tca gca gga gct gag cct aca att1968Glu Ala Val Asp Leu Val Phe Ala Ser Ala Gly Ala Glu Pro Thr Ile645 650 655gaa aca ctg gca gct tta cat ctt gta aac gaa gtt ttc cca cag gca2016Glu Thr Leu Ala Ala Leu His Leu Val Asn Glu Val Phe Pro Gln Ala660 665 670aaa ttc cgt tat gtg aac gtg gtt gaa ttg ggt cgg ttg caa aag aaa2064Lys Phe Arg Tyr Val Asn Val Val Glu Leu Gly Arg Leu Gln Lys Lys675 680 685aag gga gca ctc aat caa gaa cgt gaa ctc tca gat gaa gaa ttt gaa2112Lys Gly Ala Leu Asn Gln Glu Arg Glu Leu Ser Asp Glu Glu Phe Glu690 695 700aaa tac ttt ggc cct tca ggc act cca gta att ttt gga ttc cat gga2160Lys Tyr Phe Gly Pro Ser Gly Thr Pro Val Ile Phe Gly Phe His Gly705 710 715 720tat gaa gat tta atc gaa tcc att ttc tat caa aga gga cat gat ggt2208Tyr Glu Asp Leu Ile Glu Ser Ile Phe Tyr Gln Arg Gly His Asp Gly725 730 735ttg att gtt cat ggt tac cgt gaa gat ggt gac atc acg acg act tat2256Leu Ile Val His Gly Tyr Arg Glu Asp Gly Asp Ile Thr Thr Thr Tyr740 745 750
gat atg cgg gtt tac tct gag ctt gac cgt ttc cac caa gcg att gat2304Asp Met Arg Val Tyr Ser Glu Leu Asp Arg Phe His Gln Ala Ile Asp755 760 765gcc atg caa gtt cta tat gtc aac cga aaa gtt aat caa ggt cta gcg2352Ala Met Gln Val Leu Tyr Val Asn Arg Lys Val Asn Gln Gly Leu Ala770 775 780aaa gct ttc att gac cga atg aaa cgg aca cta gtt aaa cac ttt gaa2400Lys Ala Phe Ile Asp Arg Met Lys Arg Thr Leu Val Lys His Phe Glu785 790 795 800gtg aca aga aat gaa gga gtt gat att cct gat ttt act gaa tgg gtt2448Val Thr Arg Asn Glu Gly Val Asp Ile Pro Asp Phe Thr Glu Trp Val805 810 815tgg tcg gat tta aag aaa tag2469Trp Ser Asp Leu Lys Lys820<210>67<211>822<212>PRT<213>乳酸乳球菌<400>67Met Thr Glu Tyr Asn Ser Glu Ala Tyr Leu Lys Lys Leu Asp Lys Trp1 5 10 15Trp Arg Ala Ala Thr Tyr Leu Gly Ala Gly Met Ile Phe Leu Lys Glu20 25 30Asn Pro Leu Phe Ser Val Thr Gly Thr Pro Ile Lys Ala Glu Asn Leu35 40 45Lys Ala Asn Pro Ile Gly His Trp Gly Thr Val Ser Gly Gln Thr Phe50 55 60Leu Tyr Ala His Ala Asn Arg Leu Ile Asn Lys Tyr Asp Gln Lys Met65 70 75 80Phe Tyr Met Gly Gly Pro Gly His Gly Gly Gln Ala Met Val Val Pro85 90 95Ser Tyr Leu Asp Gly Ser Tyr Thr Glu Ala Tyr Pro Glu Ile Thr Gln100 105 110Asp Leu Glu Gly Met Ser Arg Leu Phe Lys Arg Phe Ser Phe Pro Gly115 120 125Gly Ile Gly Ser His Met Thr Ala Gln Thr Pro Gly Ser Leu His Glu130 135 140Gly Gly Glu Leu Gly Tyr Val Leu Ser His Ala Thr Gly Ala Ile Leu145 150 155 160Asp Gln Pro Glu Gln Ile Ala Phe Ala Val Val Gly Asp Gly Glu Ala165 170 175Glu Thr Gly Pro Leu Met Thr Ser Trp His Ser Ile Lys Phe Ile Asn180 185 190Pro Lys Asn Asp Gly Ala Ile Leu Pro Ile Leu Asp Leu Asn Gly Phe195 200 205Lys Ile Ser Asn Pro Thr Leu Phe Ala Arg Thr Ser Asp Val Asp Ile210 215 220Arg Lys Phe Phe Glu Gly Leu Gly Tyr Ser Pro Arg Tyr Ile Glu Asn225 230 235 240Asp Asp Ile His Asp Tyr Met Ala Tyr His Lys Leu Ala Ala Glu Val245 250 255Phe Asp Lys Ala Ile Glu Asp Ile His Gln Ile Gln Lys Asp Ala Arg260 265 270Glu Asp Asn Arg Tyr Gln Asn Gly Glu Ile Pro Ala Trp Pro Ile Val275 280 285
Ile Ala Arg Leu Pro Lys Gly Trp Gly Gly Pro Arg Tyr Asn Asp Trp290 295 300Ser Gly Pro Lys Phe Asp Gly Lys Gly Met Pro Ile Glu His Ser Phe305 310 315 320Arg Ala His Gln Val Pro Leu Pro Leu Ser Ser Lys Asn Met Gly Thr325 330 335Leu Pro Glu Phe Val Lys Trp Met Thr Ser Tyr Gln Pro Glu Thr Leu340 345 350Phe Asn Ala Asp Gly Ser Leu Lys Glu Glu Leu Arg Asp Phe Ala Pro355 360 365Lys Gly Glu Met Arg Met Ala Ser Asn Pro Val Thr Asn Gly Gly Val370 375 380Asp Ser Ser Asn Leu Val Leu Pro Asp Trp Gln Glu Phe Ala Asn Pro385 390 395 400Ile Ser Glu Asn Asn Arg Gly Lys Leu Leu Pro Asp Thr Asn Asp Asn405 410 415Met Asp Met Asn Val Leu Ser Lys Tyr Phe Ala Glu Ile Val Lys Leu420 425 430Asn Pro Thr Arg Phe Arg Leu Phe Gly Pro Asp Glu Thr Met Ser Asn435 440 445Arg Phe Trp Glu Met Phe Lys Val Thr Asn Arg Gln Trp Met Gln Val450 455 460Ile Lys Asn Pro Asn Asp Glu Phe Ile Ser Pro Glu Gly Arg Ile Ile465 470 475 480Asp Ser Gln Leu Ser Glu His Gln Ala Glu Gly Trp Leu Glu Gly Tyr485 490 495Thr Leu Thr Gly Arg Thr Gly Ala Phe Ala Ser Tyr Glu Ser Phe Leu500 505 510Arg Val Val Asp Ser Met Leu Thr Gln His Phe Lys Trp Ile Arg Gln515 520 525Ala Ala Asp Gln Lys Trp Arg His Asp Tyr Pro Ser Leu Asn Val Ile530 535 540Ser Thr Ser Thr Val Phe Gln Gln Asp His Asn Gly Tyr Thr His Gln545 550 555 560Asp Pro Gly Met Leu Thr His Leu Ala Glu Lys Lys Ser Asp Phe Ile565 570 575Arg Gln Tyr Leu Pro Ala Asp Gly Asn Thr Leu Leu Ala Val Phe Asp580 585 590Arg Ala Phe Gln Asp Arg Ser Lys Ile Asn His Ile Val Ala Ser Lys595 600 605Gln Pro Arg Gln Gln Trp Phe Thr Lys Glu Glu Ala Glu Lys Leu Ala610 615 620Thr Asp Gly Ile Ala Thr Ile Asp Trp Ala Ser Thr Ala Lys Asp Gly625 630 635 640Glu Ala Val Asp Leu Val Phe Ala Ser Ala Gly Ala Glu Pro Thr Ile645 650 655Glu Thr Leu Ala Ala Leu His Leu Val Asn Glu Val Phe Pro Gln Ala660 665 670Lys Phe Arg Tyr Val Asn Val Val Glu Leu Gly Arg Leu Gln Lys Lys675 680 685Lys Gly Ala Leu Asn Gln Glu Arg Glu Leu Ser Asp Glu Glu Phe Glu690 695 700Lys Tyr Phe Gly Pro Ser Gly Thr Pro Val Ile Phe Gly Phe His Gly705 710 715 720Tyr Glu Asp Leu Ile Glu Ser Ile Phe Tyr Gln Arg Gly His Asp Gly725 730 735Leu Ile Val His Gly Tyr Arg Glu Asp Gly Asp Ile Thr Thr Thr Tyr740 745 750
Asp Met Arg Val Tyr Ser Glu Leu Asp Arg Phe His Gln Ala Ile Asp755 760 765Ala Met Gln Val Leu Tyr Val Asn Arg Lys Val Asn Gln Gly Leu Ala770 775 780Lys Ala Phe Ile Asp Arg Met Lys Arg Thr Leu Val Lys His Phe Glu785 790 795 800Val Thr Arg Asn Glu Gly Val Asp Ile Pro Asp Phe Thr Glu Trp Val805 810 815Trp Ser Asp Leu Lys Lys820<210>68<211>2406<212>DNA<213>约氏乳杆菌<220>
<221>CDS<222>(1)..(2406)<223>xpk<400>68atg gca gta aat tac gat tct caa gaa tac tta aag agt gtt gac gca 48Met Ala Val Asn Tyr Asp Ser Gln Glu Tyr Leu Lys Ser Val Asp Ala1 5 10 15tac tgg cgt gca get aac tac tta tca gtg gga caa tta ttt tta atg 96Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Phe Leu Met20 25 30aat aat cct tta ctt aaa aga gaa tta aag gca gaa gat gta aaa cct144Asn Asn Pro Leu Leu Lys Arg Glu Leu Lys Ala Glu Asp Val Lys Pro35 40 45aag cca att ggt cac tgg ggt aca att gtg ccg caa aac ttt att tat192Lys Pro Ile Gly His Trp Gly Thr Ile Val Pro Gln Asn Phe Ile Tyr50 55 60gga cat tta aat cgt gca att aag aaa tat gat tta aac atg ttc tac240Gly His Leu Asn Arg Ala Ile Lys Lys Tyr Asp Leu Asn Met Phe Tyr65 70 75 80att gaa ggt tct ggt cac ggt ggt caa gta atg gta tcg aac tca tat288Ile Glu Gly Ser Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr85 90 95tta gat ggt tct tat act gaa cgc tac cca gaa att act caa gat gaa336Leu Asp Gly Ser Tyr Thr Glu Arg Tyr Pro Glu Ile Thr Gln Asp Glu100 105 110aag gga atg gct aag tta ttt aag caa ttt agt ttt cca ggt gga gtt384Lys Gly Met Ala Lys Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val115 120 125gcg tct cat gct gct cca gaa act cct gga tca atc cat gaa ggt gga432Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly130 135 140gaa tta ggc tac tca cta tct cat ggt gtt ggt gca att tta gac aat480Glu Leu Gly Tyr Ser Leu Ser His Gly Val Gly Ala Ile Leu Asp Asn145 150 155 160cca gat gta att gct gca gtt gaa att ggt gac ggt gaa tca gag act528Pro Asp Val Ile Ala Ala Val Glu Ile Gly Asp Gly Glu Ser Glu Thr165 170 175ggt cca ctt get act tct tgg ttt tca agt aag ttc att aat cca att576Gly Pro Leu Ala Thr Ser Trp Phe Ser Ser Lys Phe Ile Asn Pro Ile
180 185 190aaa gat ggt gca gtt att cct atc ttg caa atc aat ggc ttt aag att 624Lys Asp Gly Ala Val Ile Pro Ile Leu Gln Ile Asn Gly Phe Lys Ile195 200 205tct aac cca act atc gtt tct aga atg agt gac gaa gac tta act aag 672Ser Asn Pro Thr Ile Val Ser Arg Met Ser Asp Glu Asp Leu Thr Lys210 215 220tac ttt gaa gga atg ggt tgg aag cca tac ttt gtt tct gca tat aaa 720Tyr Phe Glu Gly Met Gly Trp Lys Pro Tyr Phe Val Ser Ala Tyr Lys225 230 235 240gac ggt gaa ttt aat ggg tat aaa gac cac atg gaa gtt cac caa gaa 768Asp Gly Glu Phe Asn Gly Tyr Lys Asp His Met Glu Val His Gln Glu245 250 255atg gca aag aca atg gac gaa gtt gtt gaa gaa att aaa gct att caa 816Met Ala Lys Thr Met Asp Glu Val Val Glu Glu Ile Lys Ala Ile Gln260 265 270aag cat gcg cgt gaa aac aat gat gat tcc tta gtg aag tgg cca atg 864Lys His Ala Arg Glu Asn Asn Asp Asp Ser Leu Val Lys Trp Pro Met275 280 285att gtc ttt aga gta cct aag ggt tgg acg ggt cca aaa ttt gat cta 912Ile Val Phe Arg Val Pro Lys Gly Trp Thr Gly Pro Lys Phe Asp Leu290 295 300gat ggc aat cca att gaa aat agt ttc cgt gct cac caa att cca att 960Asp Gly Asn Pro Ile Glu Asn Ser Phe Arg Ala His Gln Ile Pro Ile305 310 315 320cct gtt gct caa gat gac atg aat cat aaa gaa atg ctt act gat tgg1008Pro Val Ala Gln Asp Asp Met Asn His Lys Glu Met Leu Thr Asp Trp325 330 335atg gaa agt tat aag cca gaa gaa tta ttc aat gaa gat ggc tca cca1056Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asn Glu Asp Gly Ser Pro340 345 350aaa gat atc gtt aaa gaa aat acc tta tca ggc gac caa aga atg gct1104Lys Asp Ile Val Lys Glu Asn Thr Leu Ser Gly Asp Gln Arg Met Ala355 360 365atg aat cca gta act aat ggt ggg att gat cca aaa gtc tta aat atg1152Met Asn Pro Val Thr Asn Gly Gly Ile Asp Pro Lys Val Leu Asn Met370 375 380cct gac tat cgc gac ttt gca att aaa ttt gat aag cct gga tct gtt1200Pro Asp Tyr Arg Asp Phe Ala Ile Lys Phe Asp Lys Pro Gly Ser Val385 390 395 400gaa aaa caa gat atg gcc gaa tgg gca aaa tat tta gac aag atg tct1248Glu Lys Gln Asp Met Ala Glu Trp Ala Lys Tyr Leu Asp Lys Met Ser405 410 415gaa ttg aac cca act aat ttc cgt ggt ttt ggt cct gat gaa act aaa1296Glu Leu Asn Pro Thr Asn Phe Arg Gly Phe Gly Pro Asp Glu Thr Lys420 425 430tct aat cgt tta ttc caa ctt tta gat aat caa aaa cgt caa tgg atg1344Ser Asn Arg Leu Phe Gln Leu Leu Asp Asn Gln Lys Arg Gln Trp Met435 440 445gaa agt att cat act cca aac gat gaa aac ttg gct cac gaa ggt cgt1392Glu Ser Ile His Thr Pro Asn Asp Glu Asn Leu Ala His Glu Gly Arg450 455 460gta att gac tca caa ttg tca gaa cac caa gat gaa ggt tgg ctt gaa1440Val Ile Asp Ser Gln Leu Ser Glu His Gln Asp Glu Gly Trp Leu Glu465 470 475 480gga tat gta tta act ggt cgt cac gga ttc ttt gct act tat gaa gca1488Gly Tyr Val Leu Thr Gly Arg His Gly Phe Phe Ala Thr Tyr Glu Ala485 490 495
ttt ggt cgt gta gtt gat tca atg ctt acg caa cat atg aaa tgg ttg1536Phe Gly Arg Val Val Asp Ser Met Leu Thr Gln His Met Lys Trp Leu500 505 510aga aaa gct aaa gag caa gct tgg aga cat gat tat cca gcc tta aac1584Arg Lys Ala Lys Glu Gln Ala Trp Arg His Asp Tyr Pro Ala Leu Asn515 520 525tta gtt gat act tca act gtt ttc cag caa gat cac aat ggt tat act1632Leu Val Asp Thr Ser Thr Val Phe Gln Gln Asp His Asn Gly Tyr Thr530 535 540cac caa gat cca ggt atg tta act cat ttg tat gaa aag aat cgt cca1680His Gln Asp Pro Gly Met Leu Thr His Leu Tyr Glu Lys Asn Arg Pro545 550 555 560gat tta att cac gaa tac tta cca gca gat act aat tca ctt ctt gct1728Asp Leu Ile His Glu Tyr Leu Pro Ala Asp Thr Asn Ser Leu Leu Ala565 570 575gta tct gat aag gca ttt aga gat cgc gaa tgc atc aat gtt tta gta1776Val Ser Asp Lys Ala Phe Arg Asp Arg Glu Cys Ile Asn Val Leu Val580 585 590act tct aaa caa cct cgt cct cag tgg ttc tca att gaa gaa gct aaa1824Thr Ser Lys Gln Pro Arg Pro Gln Trp Phe Ser Ile Glu Glu Ala Lys595 600 605aaa ttg gtt gat aag ggt ctt ggt tat gtt gac tgg gca tca act gat1872Lys Leu Val Asp Lys Gly Leu Gly Tyr Val Asp Trp Ala Ser Thr Asp610 615 620aag ggc gct aag cca gat gtt gtt ttt gct tca act ggt act gaa cca1920Lys Gly Ala Lys Pro Asp Val Val Phe Ala Ser Thr Gly Thr Glu Pro625 630 635 640aca att gaa tct tta gct gcc att gac tta ctt cat aag aaa ttc cca1968Thr Ile Glu Ser Leu Ala Ala Ile Asp Leu Leu His Lys Lys Phe Pro645 650 655gac tta aag att cgc tat att aat gta att gat gtt atg aag tta atg2016Asp Leu Lys Ile Arg Tyr Ile Asn Val Ile Asp Val Met Lys Leu Met660 665 670tcc cca gaa aag aat cca aat gca atc agt aat gaa gaa ttc aac cgt2064Ser Pro Glu Lys Asn Pro Asn Ala Ile Ser Asn Glu Glu Phe Asn Arg675 680 685ctt ttc cct aaa ggt aca cca gtt atc ttt gca tgg cat gga ttt aag2112Leu Phe Pro Lys Gly Thr Pro Val Ile Phe Ala Trp His Gly Phe Lys690 695 700cca atg atg gaa tca att tgg ttt gat cgc ggc cgt ggt aaa gat gat2160Pro Met Met Glu Ser Ile Trp Phe Asp Arg Gly Arg Gly Lys Asp Asp705 710 715 720gtt cat att cat ggc tat gaa gaa aat ggt gac att act act cct ttt2208Val His Ile His Gly Tyr Glu Glu Asn Gly Asp Ile Thr Thr Pro Phe725 730 735gat atg cgt gtc tta aac cac atg gat cgc tat gac tta gca aaa gat2256Asp Met Arg Val Leu Asn His Met Asp Arg Tyr Asp Leu Ala Lys Asp740 745 750gta gta gaa agt att cct gag cta aat gaa aag aat gcg gat ttc att2304Val Val Glu Ser Ile Pro Glu Leu Asn Glu Lys Asn Ala Asp Phe Ile755 760 765gat gag atg gat agc ttg ctt gct aaa cac cat caa tat atc cgt gat2352Asp Glu Met Asp Ser Leu Leu Ala Lys His His Gln Tyr Ile Arg Asp770 775 780aac ggt aaa gat atg cct gaa gtt act gaa tgg caa tgg aat ggt tta2400Asn Gly Lys Asp Met Pro Glu Val Thr Glu Trp Gln Trp Asn Gly Leu785 790 795 800aaa taa2406
Lys<210>69<211>801<212>PRT<213>约氏乳杆菌<400>69Met Ala Val Asn Tyr Asp Ser Gln Glu Tyr Leu Lys Ser Val Asp Ala1 5 10 15Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Phe Leu Met20 25 30Asn Asn Pro Leu Leu Lys Arg Glu Leu Lys Ala Glu Asp Val Lys Pro35 40 45Lys Pro Ile Gly His Trp Gly Thr Ile Val Pro Gln Asn Phe Ile Tyr50 55 60Gly His Leu Asn Arg Ala Ile Lys Lys Tyr Asp Leu Asn Met Phe Tyr65 70 75 80Ile Glu Gly Ser Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr85 90 95Leu Asp Gly Ser Tyr Thr Glu Arg Tyr Pro Glu Ile Thr Gln Asp Glu100 105 110Lys Gly Met Ala Lys Leu Phe Lys Gln Phe Ser Phe Pro Gly Gly Val115 120 125Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly130 135 140Glu Leu Gly Tyr Ser Leu Ser His Gly Val Gly Ala Ile Leu Asp Asn145 150 155 160Pro Asp Val Ile Ala Ala Val Glu Ile Gly Asp Gly Glu Ser Glu Thr165 170 175Gly Pro Leu Ala Thr Ser Trp Phe Ser Ser Lys Phe Ile Asn Pro Ile180 185 190Lys Asp Gly Ala Val Ile Pro Ile Leu Gln Ile Asn Gly Phe Lys Ile195 200 205Ser Asn Pro Thr Ile Val Ser Arg Met Ser Asp Glu Asp Leu Thr Lys210 215 220Tyr Phe Glu Gly Met Gly Trp Lys Pro Tyr Phe Val Ser Ala Tyr Lys225 230 235 240Asp Gly Glu Phe Asn Gly Tyr Lys Asp His Met Glu Val His Gln Glu245 250 255Met Ala Lys Thr Met Asp Glu Val Val Glu Glu Ile Lys Ala Ile Gln260 265 270Lys His Ala Arg Glu Asn Asn Asp Asp Ser Leu Val Lys Trp Pro Met275 280 285Ile Val Phe Arg Val Pro Lys Gly Trp Thr Gly Pro Lys Phe Asp Leu290 295 300Asp Gly Asn Pro Ile Glu Asn Ser Phe Arg Ala His Gln Ile Pro Ile305 310 315 320Pro Val Ala Gln Asp Asp Met Asn His Lys Glu Met Leu Thr Asp Trp325 330 335Met Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asn Glu Asp Gly Ser Pro340 345 350Lys Asp Ile Val Lys Glu Asn Thr Leu Ser Gly Asp Gln Arg Met Ala355 360 365Met Asn Pro Val Thr Asn Gly Gly Ile Asp Pro Lys Val Leu Asn Met370 375 380
Pro Asp Tyr Arg Asp Phe Ala Ile Lys Phe Asp Lys Pro Gly Ser Val385 390 395 400Glu Lys Gln Asp Met Ala Glu Trp Ala Lys Tyr Leu Asp Lys Met Ser405 410 415Glu Leu Asn Pro Thr Asn Phe Arg Gly Phe Gly Pro Asp Glu Thr Lys420 425 430Ser Asn Arg Leu Phe Gln Leu Leu Asp Asn Gln Lys Arg Gln Trp Met435 440 445Glu Ser Ile His Thr Pro Asn Asp Glu Asn Leu Ala His Glu Gly Arg450 455 460Val Ile Asp Ser Gln Leu Ser Glu His Gln Asp Glu Gly Trp Leu Glu465 470 475 480Gly Tyr Val Leu Thr Gly Arg His Gly Phe Phe Ala Thr Tyr Glu Ala485 490 495Phe Gly Arg Val Val Asp Ser Met Leu Thr Gln His Met Lys Trp Leu500 505 510Arg Lys Ala Lys Glu Gln Ala Trp Arg His Asp Tyr Pro Ala Leu Asn515 520 525Leu Val Asp Thr Ser Thr Val Phe Gln Gln Asp His Asn Gly Tyr Thr530 535 540His Gln Asp Pro Gly Met Leu Thr His Leu Tyr Glu Lys Asn Arg Pro545 550 555 560Asp Leu Ile His Glu Tyr Leu Pro Ala Asp Thr Asn Ser Leu Leu Ala565 570 575Val Ser Asp Lys Ala Phe Arg Asp Arg Glu Cys Ile Asn Val Leu Val580 585 590Thr Ser Lys Gln Pro Arg Pro Gln Trp Phe Ser Ile Glu Glu Ala Lys595 600 605Lys Leu Val Asp Lys Gly Leu Gly Tyr Val Asp Trp Ala Ser Thr Asp610 615 620Lys Gly Ala Lys Pro Asp Val Val Phe Ala Ser Thr Gly Thr Glu Pro625 630 635 640Thr Ile Glu Ser Leu Ala Ala Ile Asp Leu Leu His Lys Lys Phe Pro645 650 655Asp Leu Lys Ile Arg Tyr Ile Asn Val Ile Asp Val Met Lys Leu Met660 665 670Ser Pro Glu Lys Asn Pro Asn Ala Ile Ser Asn Glu Glu Phe Asn Arg675 680 685Leu Phe Pro Lys Gly Thr Pro Val Ile Phe Ala Trp His Gly Phe Lys690 695 700Pro Met Met Glu Ser Ile Trp Phe Asp Arg Gly Arg Gly Lys Asp Asp705 710 715 720Val His Ile His Gly Tyr Glu Glu Asn Gly Asp Ile Thr Thr Pro Phe725 730 735Asp Met Arg Val Leu Asn His Met Asp Arg Tyr Asp Leu Ala Lys Asp740 745 750Val Val Glu Ser Ile Pro Glu Leu Asn Glu Lys Asn Ala Asp Phe Ile755 760 765Asp Glu Met Asp Ser Leu Leu Ala Lys His His Gln Tyr Ile Arg Asp770 775 780Asn Gly Lys Asp Met Pro Glu Val Thr Glu Trp Gln Trp Asn Gly Leu785 790 795 800Lys<210>70<211>2400<212>DNA
<213>嗜酸乳杆菌<220>
<221>CDS<222>(1)..(2400)<223>
<400>70atg aca gtt aat tac gat tcc aaa gat tac tta aag agc gtt gac gca 48Met Thr Val Asn Tyr Asp Ser Lys Asp Tyr Leu Lys Ser Val Asp Ala1 5 10 15tat tgg cgt gca gct aat tat ttg tca gtt gga caa tta ttt tta atg 96Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Phe Leu Met20 25 30aaa aat ccg ttg tta aag aaa cct tta aca gct gaa gat gta aaa cct144Lys Asn Pro Leu Leu Lys Lys Pro Leu Thr Ala Glu Asp Val Lys Pro35 40 45aag cca atc ggt cac tgg ggt act att gct cca caa aac ttt att tat192Lys Pro Ile Gly His Trp Gly Thr Ile Ala Pro Gln Asn Phe Ile Tyr50 55 60gct cac tta aat cgt gcg ctt aaa aaa tat gac ttg gat atg ttc tat240Ala His Leu Asn Arg Ala Leu Lys Lys Tyr Asp Leu Asp Met Phe Tyr65 70 75 80att gaa ggt tca ggt cac ggt ggc caa gtg atg gtt tca aat tca tat288Ile Glu Gly Ser Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr85 90 95ctt gat ggt tca tat act gaa cgt tat cca gaa att acc caa gat gaa336Leu Asp Gly Ser Tyr Thr Glu Arg Tyr Pro Glu Ile Thr Gln Asp Glu100 105 110aag ggt atg gct aaa ttg ttt aag cgc ttt agt ttc cca ggt ggt gta384Lys Gly Met Ala Lys Leu Phe Lys Arg Phe Ser Phe Pro Gly Gly Val115 120 125gct tct cac gct gct cct gaa act cca ggt tct att cat gaa ggt ggg432Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly130 135 140gaa tta gga tac gca ctt tca cat ggg gta ggt gct att tta gac aat480Glu Leu Gly Tyr Ala Leu Ser His Gly Val Gly Ala Ile Leu Asp Asn145 150 155 160cca gat gta att gct gcc gtt gaa att ggt gat ggt gaa gca gaa act528Pro Asp Val Ile Ala Ala Val Glu Ile Gly Asp Gly Glu Ala Glu Thr165 170 175ggt cca ctt gca gct agc tgg ttc agt gac aag ttt att aat cca att576Gly Pro Leu Ala Ala Ser Trp Phe Ser Asp Lys Phe Ile Asn Pro Ile180 185 190aag gat ggt gca gtt tta cca att ctt caa att aat ggt ttc aag att624Lys Asp Gly Ala Val Leu Pro Ile Leu Gln Ile Asn Gly Phe Lys Ile195 200 205tct aac cca act atc gtt tca aga atg agc gat gaa gaa tta act gaa672Ser Asn Pro Thr Ile Val Ser Arg Met Ser Asp Glu Glu Leu Thr Glu210 215 220tac ttc cgt ggc atg ggt tgg gat ccg cac ttt gtt tca gta ttt aag720Tyr Phe Arg Gly Met Gly Trp Asp Pro His Phe Val Ser Val Phe Lys225 230 235 240ggt ggc cgc ttt gac ggt gaa aag gat cca atg caa gtc cac gaa gaa768Gly Gly Arg Phe Asp Gly Glu Lys Asp Pro Met Gln Val His Glu Glu245 250 255atg gct aaa acc atg gac gaa gta att gaa gaa att aag gct att caa816Met Ala Lys Thr Met Asp Glu Val Ile Glu Glu Ile Lys Ala Ile Gln
260 265 270aag cat gct cgt gaa aat aat gat gct act ttg cca cat tgg cca ttg 864Lys His Ala Arg Glu Asn Asn Asp Ala Thr Leu Pro His Trp Pro Leu275 280 285att atc ttc caa tgt cca aag ggc tgg acc ggt cca aag aag gat ctt 912Ile Ile Phe Gln Cys Pro Lys Gly Trp Thr Gly Pro Lys Lys Asp Leu290 295 300gac ggc aat cca att gaa aac tca ttt aga gca cac caa att cca att 960Asp Gly Asn Pro Ile Glu Asn Ser Phe Arg Ala His Gln Ile Pro Ile305 310 315 320cct gtc tca caa tac gat atg aaa cat gtt gat atg ttg act gat tgg1008Pro Val Ser Gln Tyr Asp Met Lys His Val Asp Met Leu Thr Asp Trp325 330 335ctt gaa agt tat aag cca aac gaa tta ttc aac gaa gat ggt tca cca1056Leu Glu Ser Tyr Lys Pro Asn Glu Leu Phe Asn Glu Asp Gly Ser Pro340 345 350aag gaa att gtt act gaa aac act gct aag ggt gat caa cgt atg gca1104Lys Glu Ile Val Thr Glu Asn Thr Ala Lys Gly Asp Gln Arg Met Ala355 360 365atg aat ccg atc act aat ggt ggt aag gat cct aaa cga ttg aac cta1l52Met Asn Pro Ile Thr Asn Gly Gly Lys Asp Pro Lys Arg Leu Asn Leu370 375 380cca gat tat cgc aac ttt gca ctt aag ttt gac aag cca ggt tca gtt1200Pro Asp Tyr Arg Asn Phe Ala Leu Lys Phe Asp Lys Pro Gly Ser Val385 390 395 400gaa gca caa gac atg gtt gaa tgg gct aaa tat tta aac gaa gtt gct1248Glu Ala Gln Asp Met Val Glu Trp Ala Lys Tyr Leu Asn Glu Val Ala405 410 415aaa ctt aac cca act act ttc cgt ggc ttt ggt cct gat gaa tct aaa1296Lys Leu Asn Pro Thr Thr Phe Arg Gly Phe Gly Pro Asp Glu Ser Lys420 425 430tca aac cgt tta ttt aaa ctt tta gat gat caa aag cgt caa tgg gaa1344Ser Asn Arg Leu Phe Lys Leu Leu Asp Asp Gln Lys Arg Gln Trp Glu435 440 445cct gaa gtt cat gaa cca aat gat gaa aac ttg gca cca agt ggc cgc1392Pro Glu Val His Glu Pro Asn Asp Glu Asn Leu Ala Pro Ser Gly Arg450 455 460gtt atc gat tca caa tta tca gaa cac caa gac gaa ggc ttc ctt gaa1440Val Ile Asp Ser Gln Leu Ser Glu His Gln Asp Glu Gly Phe Leu Glu465 470 475 480ggc tac gtt tta act ggt cgt cac ggc ttc ttt gca acc tac gaa gca1488Gly Tyr Val Leu Thr Gly Arg His Gly Phe Phe Ala Thr Tyr Glu Ala485 490 495ttt ggt cgt gta gta gat tcg atg ctt act caa cat atg aag tgg ctt1536Phe Gly Arg Val Val Asp Ser Met Leu Thr Gln His Met Lys Trp Leu500 505 510aga aaa gct aaa gaa caa tat tgg cgt cat gat tat cca tca ctt aac1584Arg Lys Ala Lys Glu Gln Tyr Trp Arg His Asp Tyr Pro Ser Leu Asn515 520 525ttt gtt gct act tca aca gta ttc caa caa gat cac aat ggt tac act1632Phe Val Ala Thr Ser Thr Val Phe Gln Gln Asp His Asn Gly Tyr Thr530 535 540cac caa gat cca ggc att tta act cac tta tat gaa aag aat cgt cca1680His Gln Asp Pro Gly Ile Leu Thr His Leu Tyr Glu Lys Asn Arg Pro545 550 555 560gat tta gtt cat gaa tac ttg cca tca gat act aat act tta ctt gct1728Asp Leu Val His Glu Tyr Leu Pro Ser Asp Thr Asn Thr Leu Leu Ala565 570 575
gta ggt aac aag gca ttt act gat cgt gaa tgt att aat gtt tta gta1776Val Gly Asn Lys Ala Phe Thr Asp Arg Glu Cys Ile Asn Val Leu Val580 585 590act tca aag caa cct cgt cca caa tgg ttc tca att gag gaa gca caa1824Thr Ser Lys Gln Pro Arg Pro Gln Trp Phe Ser Ile Glu Glu Ala Gln595 600 605aag tta gtt gat aaa ggt tta agt tac att gat tgg gct tca act gat1872Lys Leu Val Asp Lys Gly Leu Ser Tyr Ile Asp Trp Ala Ser Thr Asp610 615 620aaa ggt gta aaa cca gat att gtc ttt gct tca aca gaa act gaa cca1920Lys Gly Val Lys Pro Asp Ile Val Phe Ala Ser Thr Glu Thr Glu Pro625 630 635 640aca att gaa act ttg gca gca att gat att ttg cat gac aag ttc cca1968Thr Ile Glu Thr Leu Ala Ala Ile Asp Ile Leu His Asp Lys Phe Pro645 650 655gat ctt aag att cgc tac att aac gta att gat gtg atg aaa tta atg2016Asp Leu Lys Ile Arg Tyr Ile Asn Val Ile Asp Val Met Lys Leu Met660 665 670tca cca aag gac aat aag aat ggt att tct gat gaa gaa ttt gat cgc2064Ser Pro Lys Asp Asn Lys Asn Gly Ile Ser Asp Glu Glu Phe Asp Arg675 680 685tta ttc cca aag gac gtt cct gta ate ttt gca tgg cac ggc tac aag2112Leu Phe Pro Lys Asp Val Pro Val Ile Phe Ala Trp His Gly Tyr Lys690 695 700agt atg atg gaa tca att tgg ttt gca cgt aac cgt cat aat gta cat2160Ser Met Met Glu Ser Ile Trp Phe Ala Arg Asn Arg His Asn Val His705 710 715 720att cac tgc tac gaa gaa aac ggt gat att act acc cca ttt gat atg2208Ile His Cys Tyr Glu Glu Asn Gly Asp Ile Thr Thr Pro Phe Asp Met725 730 735cgt gtt ttg aac cac ctt gac aga ttt gat ctt gcc aaa gat gct gtt2256Arg Val Leu Asn His Leu Asp Arg Phe Asp Leu Ala Lys Asp Ala Val740 745 750gaa agt gtt gat aaa ttg aag ggc aag aac gct gac ttt ate agt cat2304Glu Ser Val Asp Lys Leu Lys Gly Lys Asn Ala Asp Phe Ile Ser His755 760 765atg gat gac ttg ctt gaa aag cac cac caa tac att cgt gat aat ggt2352Met Asp Asp Leu Leu Glu Lys His His Gln Tyr Ile Arg Asp Asn Gly770 775 780aaa gat atg cca gaa gtt act gaa tgg aag tgg aag ggc ttg aag taa2400Lys Asp Met Pro Glu Val Thr Glu Trp Lys Trp Lys Gly Leu Lys785 790 795<210>71<211>799<212>PRT<213>嗜酸乳杆菌<400>71Met Thr Val Asn Tyr Asp Ser Lys Asp Tyr Leu Lys Ser Val Asp Ala1 5 10 15Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Leu Phe Leu Met20 25 30Lys Asn Pro Leu Leu Lys Lys Pro Leu Thr Ala Glu Asp Val Lys Pro35 40 45Lys Pro Ile Gly His Trp Gly Thr Ile Ala Pro Gln Asn Phe Ile Tyr50 55 60Ala His Leu Asn Arg Ala Leu Lys Lys Tyr Asp Leu Asp Met Phe Tyr
65 70 75 80Ile Glu Gly Ser Gly His Gly Gly Gln Val Met Val Ser Asn Ser Tyr85 90 95Leu Asp Gly Ser Tyr Thr Glu Arg Tyr Pro Glu Ile Thr Gln Asp Glu100 105 110Lys Gly Met Ala Lys Leu Phe Lys Arg Phe Ser Phe Pro Gly Gly Val115 120 125Ala Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly130 135 140Glu Leu Gly Tyr Ala Leu Ser His Gly Val Gly Ala Ile Leu Asp Asn145 150 155 160Pro Asp Val Ile Ala Ala Val Glu Ile Gly Asp Gly Glu Ala Glu Thr165 170 175Gly Pro Leu Ala Ala Ser Trp Phe Ser Asp Lys Phe Ile Asn Pro Ile180 185 190Lys Asp Gly Ala Val Leu Pro Ile Leu Gln Ile Asn Gly Phe Lys Ile195 200 205Ser Asn Pro Thr Ile Val Ser Arg Met Ser Asp Glu Glu Leu Thr Glu210 215 220Tyr Phe Arg Gly Met Gly Trp Asp Pro His Phe Val Ser Val Phe Lys225 230 235 240Gly Gly Arg Phe Asp Gly Glu Lys Asp Pro Met Gln Val His Glu Glu245 250 255Met Ala Lys Thr Met Asp Glu Val Ile Glu Glu Ile Lys Ala Ile Gln260 265 270Lys His Ala Arg Glu Asn Asn Asp Ala Thr Leu Pro His Trp Pro Leu275 280 285Ile Ile Phe Gln Cys Pro Lys Gly Trp Thr Gly Pro Lys Lys Asp Leu290 295 300Asp Gly Asn Pro Ile Glu Asn Ser Phe Arg Ala His Gln Ile Pro Ile305 310 315 320Pro Val Ser Gln Tyr Asp Met Lys His Val Asp Met Leu Thr Asp Trp325 330 335Leu Glu Ser Tyr Lys Pro Asn Glu Leu Phe Asn Glu Asp Gly Ser Pro340 345 350Lys Glu Ile Val Thr Glu Asn Thr Ala Lys Gly Asp Gln Arg Met Ala355 360 365Met Asn Pro Ile Thr Asn Gly Gly Lys Asp Pro Lys Arg Leu Asn Leu370 375 380Pro Asp Tyr Arg Asn Phe Ala Leu Lys Phe Asp Lys Pro Gly Ser Val385 390 395 400Glu Ala Gln Asp Met Val Glu Trp Ala Lys Tyr Leu Asn Glu Val Ala405 410 415Lys Leu Asn Pro Thr Thr Phe Arg Gly Phe Gly Pro Asp Glu Ser Lys420 425 430Ser Asn Arg Leu Phe Lys Leu Leu Asp Asp Gln Lys Arg Gln Trp Glu435 440 445Pro Glu Val His Glu Pro Asn Asp Glu Asn Leu Ala Pro Ser Gly Arg450 455 460Val Ile Asp Ser Gln Leu Ser Glu His Gln Asp Glu Gly Phe Leu Glu465 470 475 480Gly Tyr Val Leu Thr Gly Arg His Gly Phe Phe Ala Thr Tyr Glu Ala485 490 495Phe Gly Arg Val Val Asp Ser Met Leu Thr Gln His Met Lys Trp Leu500 505 510Arg Lys Ala Lys Glu Gln Tyr Trp Arg His Asp Tyr Pro Ser Leu Asn515 520 525Phe Val Ala Thr Ser Thr Val Phe Gln Gln Asp His Asn Gly Tyr Thr
530 535 540His Gln Asp Pro Gly Ile Leu Thr His Leu Tyr Glu Lys Asn Arg Pro545 550 555 560Asp Leu Val His Glu Tyr Leu Pro Ser Asp Thr Asn Thr Leu Leu Ala565 570 575Val Gly Asn Lys Ala Phe Thr Asp Arg Glu Cys Ile Asn Val Leu Val580 585 590Thr Ser Lys Gln Pro Arg Pro Gln Trp Phe Ser Ile Glu Glu Ala Gln595 600 605Lys Leu Val Asp Lys Gly Leu Ser Tyr Ile Asp Trp Ala Ser Thr Asp610 615 620Lys Gly Val Lys Pro Asp Ile Val Phe Ala Ser Thr Glu Thr Glu Pro625 630 635 640Thr Ile Glu Thr Leu Ala Ala Ile Asp Ile Leu His Asp Lys Phe Pro645 650 655Asp Leu Lys Ile Arg Tyr Ile Asn Val Ile Asp Val Met Lys Leu Met660 665 670Ser Pro Lys Asp Asn Lys Asn Gly Ile Ser Asp Glu Glu Phe Asp Arg675 680 685Leu Phe Pro Lys Asp Val Pro Val Ile Phe Ala Trp His Gly Tyr Lys690 695 700Ser Met Met Glu Ser Ile Trp Phe Ala Arg Asn Arg His Asn Val His705 710 715 720Ile His Cys Tyr Glu Glu Asn Gly Asp Ile Thr Thr Pro Phe Asp Met725 730 735Arg Val Leu Asn His Leu Asp Arg Phe Asp Leu Ala Lys Asp Ala Val740 745 750Glu Ser Val Asp Lys Leu Lys Gly Lys Asn Ala Asp Phe Ile Ser His755 760 765Met Asp Asp Leu Leu Glu Lys His His Gln Tyr Ile Arg Asp Asn Gly770 775 780Lys Asp Met Pro Glu Val Thr Glu Trp Lys Trp Lys Gly Leu Lys785 790 795<210>72<211>2478<212>DNA<213>长双歧杆菌<220>
<221>CDS<222>(1)..(2478)<223>
<400>72atg acg agt cct gtt att ggc acc cct tgg aag aag ctc aac gct ccg 48Met Thr Ser Pro Val Ile Gly Thr Pro Trp Lys Lys Leu Ash Ala Pro1 5 10 15gtt tcc gag gaa gcc ctc gaa ggc gtt gac aag tac tgg cgc gtt gcc 96Val Ser Glu Glu Ala Leu Glu Gly Val Asp Lys Tyr Trp Arg Val Ala20 25 30aac tac ctt tcc atc ggc cag att tat ctg cgt tcc aac ccg ctg atg144Asn Tyr Leu Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met35 40 45aag gag ccc ttc acc cgc gaa gat gtg aag cac cgt ctg gtg ggc cac192Lys Glu Pro Phe Thr Arg Glu Asp Val Lys His Arg Leu Val Gly His50 55 60
tgg ggc act acc cct ggc ctg aac ttc ctc atc ggc cac atc aac cgt 240Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Ile Gly His Ile Asn Arg65 70 75 80ttc att gct gac cac ggc cag aac acc gtg atc atc atg ggc ccg ggc 288Phe Ile Ala Asp His Gly Gln Asn Thr Val Ile Ile Met Gly Pro Gly85 90 95cac ggt ggc ccg gcc ggt acc tcc cag tcc tac ctg gac ggc acc tac 336His Gly Gly Pro Ala Gly Thr Ser Gln Ser Tyr Leu Asp Gly Thr Tyr100 105 110acc gag acc ttc ccg aag atc acc aag gac gaa gct ggt ctg cag aag 384Thr Glu Thr Phe Pro Lys Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys115 120 125ttc ttc cgt cag ttc tct tac ccg ggc ggc att ccg tcc cac ttc gct 432Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala130 135 140ccg gag acc ccg ggc tcc atc cac gag ggt ggt gag ctg ggt tac gct 480Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala145 150 155 160ctg tcc cac gct tac ggc gcc atc atg gac aac ccg agc ctg ttt gtc 528Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu Phe Val165 170 175ccg gcc atc gtc ggc gac ggc gag gct gag acc ggc ccg ctg gct acc 576Pro Ala Ile Val Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr180 185 190ggc tgg cag tcc aac aag ctc gtg aac ccg cgc acc gac ggt atc gtg 624Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val195 200 205ctg ccg atc ctg cac ctc aac ggc tac aag atc gcc aac ccg acc atc 672Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile210 215 220ctg tcc cgc atc tcc gac gaa gag ctc cac gag ttc ttc cac ggc atg 720Leu Ser Arg Ile Ser Asp Glu Glu Leu His Glu Phe Phe His Gly Met225 230 235 240ggt tac gag ccc tac gag ttc gtc gct ggc ttc gat gat gag gac cac 768Gly Tyr Glu Pro Tyr Glu Phe Val Ala Gly Phe Asp Asp Glu Asp His245 250 255atg tcc atc cac cgt cgc ttc gcc gag ctg tgg gag acc atc tgg gac 816Met Ser Ile His Arg Arg Phe Ala Glu Leu Trp Glu Thr Ile Trp Asp260 265 270gag atc tgc gac atc aag gcc acc gct cag acc gac aac gtg cac cgt 864Glu Ile Cys Asp Ile Lys Ala Thr Ala Gln Thr Asp Asn Val His Arg275 280 285ccg ttc tac ccg atg ctg atc ttc cgc acc ccg aag ggc tgg acc tgc 912Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys290 295 300ccg aag tac atc gac ggc aag aag acc gag ggc tcc tgg cgt tcc cac 960Pro Lys Tyr Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ser His305 310 315 320cag gtg ccg ctg gct tcc gcc cgc gac acc gag gcc cac ttc gag gtt1008Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val325 330 335ctc aag aac tgg ctc gag tcc tac aag ccg gaa gag ctg ttc gac gcc1056Leu Lys Asn Trp Leu Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asp Ala340 345 350aac ggt gct gtc aag gac gac gtc ctt gcc ttc atg ccg aag ggc gag1104Asn Gly Ala Val Lys Asp Asp Val Leu Ala Phe Met Pro Lys Gly Glu355 360 365ctg cgt atc ggt gcc aac ccg aac gcc aac ggt ggt gtg atc cgc aac1152
Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Val Ile Arg Asn370 375 380gac ctg aag ctg ccg aac ctc gag gac tac gag gtc aag gaa gtg gct1200Asp Leu Lys Leu Pro Asn Leu Glu Asp Tyr Glu Val Lys Glu Val Ala385 390 395 400gag tac ggc cac ggc tgg ggc cag ctc gag gcc acc cgt acc ctg ggt1248Glu Tyr Gly His Gly Trp Gly Gln Leu Glu Ala Thr Arg Thr Leu Gly405 410 415gcc tac act cgc gac atc atc aag aac aac ccg cgc gac ttc cgc atc1296Ala Tyr Thr Arg Asp Ile Ile Lys Asn Asn Pro Arg Asp Phe Arg Ile420 425 430ttc gga ccg gat gag acc gct tcc aac cgt ctg cag gct tcc tac gaa1344Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gln Ala Ser Tyr Glu435 440 445gtc acc aac aag cag tgg gat gcc ggc tac atc tcc gac gag gtc gac1392Val Thr Asn Lys Gln Trp Asp Ala Gly Tyr Ile Ser Asp Glu Val Asp450 455 460gag cac atg cac gtc tcc ggc cag gtc gtt gag cag ctg tcc gag cac1440Glu His Met His Val Ser Gly Gln Val Val Glu Gln Leu Ser Glu His465 470 475 480cag atg gaa ggc ttc ctc gag gct tac ctg ctg acc ggt cgt cac ggc1488Gln Met Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly485 490 495atc tgg agc tcc tac gag tcc ttc gtc cac gtg atc gac tcc atg ctg1536Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu500 505 510aac cag cac gcc aag tgg ctt gag gct acc gtc cgc gag att ccg tgg1584Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp515 520 525cgc aag ccg att gcc tcc atg aac ctg ctg gtc tcc tcc cac gtt tgg1632Arg Lys Pro Ile Ala Ser Met Asn Leu Leu Val Ser Ser His Val Trp530 535 540cgt cag gac cac aac ggc ttc tcc cac cag gat ccg ggt gtc acc tcc1680Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser545 550 555 560gtc ctg ctg aac aag tgc ttc cac aac gac cac gtc atc ggc atc tac1728Val Leu Leu Asn Lys Cys Phe His Asn Asp His Val Ile Gly Ile Tyr565 570 575ttc gcc acc gat gcg aac atg ctg ctg gcc atc gcc gag aag tgc tac1776Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Tyr580 585 590aag tcc acc aac aag atc aac gcc atc atc gct ggt aag cag cct gct1824Lys Ser Thr Asn Lys Ile Asn Ala Ile Ile Ala Gly Lys Gln Pro Ala595 600 605gcc acc tgg ctg acc ctg gac gag gct cgt gcc gag ctc gag aag ggt1872Ala Thr Trp Leu Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Lys Gly610 615 620gcc gcc gct tgg gat tgg gct tcc acc gcc aag aac aac gat gag gcc1920Ala Ala Ala Trp Asp Trp Ala Ser Thr Ala Lys Asn Asn Asp Glu Ala625 630 635 640gag gtc gtg ctt gcc gcc gcc ggc gat gtc ccg act cag gag atc atg1968Glu Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met645 650 655gct gct tcc gac aag ctg aag gaa ctg ggc atc aag ttc aag gtt gtg2016Ala Ala Ser Asp Lys Leu Lys Glu Leu Gly Ile Lys Phe Lys Val Val660 665 670aac gtt gcc gac ctg ctc tcc ctg cag tcc gcc aag gag aac gac gag2064Asn Val Ala Asp Leu Leu Ser Leu Gln Ser Ala Lys Glu Asn Asp Glu
675 680 685gct ctg acc gac gag gag ttc gcc gac atc ttc acc gcc gac aag ccg2112Ala Leu Thr Asp Glu Glu Phe Ala Asp Ile Phe Thr Ala Asp Lys Pro690 695 700gtg ctg ttc gcg tac cac tcc tac gct cac gac gtg cgt ggc ctg atc2160Val Leu Phe Ala Tyr His Ser Tyr Ala His Asp Val Arg Gly Leu Ile705 710 715 720tac gac cgt ccg aac cac gac aac ttc aac gtc cac ggc tac gag gag2208Tyr Asp Arg Pro Asn His Asp Asn Phe Asn Val His Gly Tyr Glu Glu725 730 735gag ggc tcc acc acc acc ccg tac gac atg gtt cgt gtc aac cgc atc2256Glu Gly Ser Thr Thr Thr Pro Tyr Asp Met Val Arg Val Asn Arg Ile740 745 750gac cgc tac gag ctg acc gct gag gct ctg cgc atg atc gac gcc gac2304Asp Arg Tyr Glu Leu Thr Ala Glu Ala Leu Arg Met Ile Asp Ala Asp755 760 765aag tac gcc gac aag atc gac gag ctc gag aag ttc cgt gat gag gcc2352Lys Tyr Ala Asp Lys Ile Asp Glu Leu Glu Lys Phe Arg Asp Glu Ala770 775 780ttc cag ttc gcc gtc gac aac ggc tac gat cac ccg gac tac acc gac2400Pho Gln Phe Ala Val Asp Asn Gly Tyr Asp His Pro Asp Tyr Thr Asp785 790 795 800tgg gtg tac tcc ggc gtg aac acc gac aag aag ggt gcc gtc acc gct2448Trp Val Tyr Ser Gly Val Asn Thr Asp Lys Lys Gly Ala Val Thr Ala805 810 815acc gcc gct acc gct ggc gac aac gag tga2478Thr Ala Ala Thr Ala Gly Asp Asn Glu820 825<210>73<211>825<212>PRT<213>长双歧杆菌<400>73Met Thr Ser Pro Val Ile Gly Thr Pro Trp Lys Lys Leu Asn Ala Pro1 5 10 15Val Ser Glu Glu Ala Leu Glu Gly Val Asp Lys Tyr Trp Arg Val Ala20 25 30Asn Tyr Leu Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met35 40 45Lys Glu Pro Phe Thr Arg Glu Asp Val Lys His Arg Leu Val Gly His50 55 60Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Ile Gly His Ile Asn Arg65 70 75 80Phe Ile Ala Asp His Gly Gln Asn Thr Val Ile Ile Met Gly Pro Gly85 90 95His Gly Gly Pro Ala Gly Thr Ser Gln Ser Tyr Leu Asp Gly Thr Tyr100 105 110Thr Glu Thr Phe Pro Lys Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys115 120 125Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Phe Ala130 135 140Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala145 150 155 160Leu Ser His Ala Tyr Gly Ala Ile Met Asp Asn Pro Ser Leu Phe Val165 170 175
Pro Ala Ile Val Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr180 185 190Gly Trp Gln Ser Asn Lys Leu Val Asn Pro Arg Thr Asp Gly Ile Val195 200 205Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile210 215 220Leu Ser Arg Ile Ser Asp Glu Glu Leu His Glu Phe Phe His Gly Met225 230 235 240Gly Tyr Glu Pro Tyr Glu Phe Val Ala Gly Phe Asp Asp Glu Asp His245 250 255Met Ser Ile His Arg Arg Phe Ala Glu Leu Trp Glu Thr Ile Trp Asp260 265 270Glu Ile Cys Asp Ile Lys Ala Thr Ala Gln Thr Asp Asn Val His Arg275 280 285Pro Phe Tyr Pro Met Leu Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys290 295 300Pro Lys Tyr Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ser His305 310 315 320Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val325 330 335Leu Lys Asn Trp Leu Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asp Ala340 345 350Asn Gly Ala Val Lys Asp Asp Val Leu Ala Phe Met Pro Lys Gly Glu355 360 365Leu Arg Ile Gly Ala Asn Pro Asn Ala Asn Gly Gly Val Ile Arg Asn370 375 380Asp Leu Lys Leu Pro Asn Leu Glu Asp Tyr Glu Val Lys Glu Val Ala385 390 395 400Glu Tyr Gly His Gly Trp Gly Gln Leu Glu Ala Thr Arg Thr Leu Gly405 410 415Ala Tyr Thr Arg Asp Ile Ile Lys Asn Asn Pro Arg Asp Phe Arg Ile420 425 430Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gln Ala Ser Tyr Glu435 440 445Val Thr Asn Lys Gln Trp Asp Ala Gly Tyr Ile Ser Asp Glu Val Asp450 455 460Glu His Met His Val Ser Gly Gln Val Val Glu Gln Leu Ser Glu His465 470 475 480Gln Met Glu Gly Phe Leu Glu Ala Tyr Leu Leu Thr Gly Arg His Gly485 490 495Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu500 505 510Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp515 520 525Arg Lys Pro Ile Ala Ser Met Asn Leu Leu Val Ser Ser His Val Trp530 535 540Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser545 550 555 560Val Leu Leu Asn Lys Cys Phe His Asn Asp His Val Ile Gly Ile Tyr565 570 575Phe Ala Thr Asp Ala Asn Met Leu Leu Ala Ile Ala Glu Lys Cys Tyr580 585 590Lys Ser Thr Asn Lys Ile Asn Ala Ile Ile Ala Gly Lys Gln Pro Ala595 600 605Ala Thr Trp Leu Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Lys Gly610 615 620Ala Ala Ala Trp Asp Trp Ala Ser Thr Ala Lys Asn Asn Asp Glu Ala625 630 635 640
Glu Val Val Leu Ala Ala Ala Gly Asp Val Pro Thr Gln Glu Ile Met645 650 655Ala Ala Ser Asp Lys Leu Lys Glu Leu Gly Ile Lys Phe Lys Val Val660 665 670Asn Val Ala Asp Leu Leu Ser Leu Gln Ser Ala Lys Glu Asn Asp Glu675 680 685Ala Leu Thr Asp Glu Glu Phe Ala Asp Ile Phe Thr Ala Asp Lys Pro690 695 700Val Leu Phe Ala Tyr His Ser Tyr Ala His Asp Val Arg Gly Leu Ile705 710 715 720Tyr Asp Arg Pro Asn His Asp Asn Phe Asn Val His Gly Tyr Glu Glu725 730 735Glu Gly Ser Thr Thr Thr Pro Tyr Asp Met Val Arg Val Asn Arg Ile740 745 750Asp Arg Tyr Glu Leu Thr Ala Glu Ala Leu Arg Met Ile Asp Ala Asp755 760 765Lys Tyr Ala Asp Lys Ile Asp Glu Leu Glu Lys Phe Arg Asp Glu Ala770 775 780Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp His Pro Asp Tyr Thr Asp785 790 795 800Trp Val Tyr Ser Gly Val Asn Thr Asp Lys Lys Gly Ala Val Thr Ala805 810 815Thr Ala Ala Thr Ala Gly Asp Asn Glu820 825<210>74<211>2454<212>DNA<213>Chlorobium tepidum<220>
<221>CDS<222>(1)..(2454)<223>
<400>74ttg att gta aga tcg gaa aat aga gtt ccg gat gaa ctc cgg act cag 48Leu Ile Val Arg Ser Glu Asn Arg Val Pro Asp Glu Leu Arg Thr Gln1 5 10 15cat aca aca act atc aaa tca aca ata ctg atg act gaa atg acg act 96His Thr Thr Thr Ile Lys Ser Thr Ile Leu Met Thr Glu Met Thr Thr20 25 30ccg ctt tcc cct cgc gaa ctc gat ctg atg aac gct tac tgg cga gcg144Pro Leu Ser Pro Arg Glu Leu Asp Leu Met Asn Ala Tyr Trp Arg Ala35 40 45gcg aac tac ctt tcc gtc ggc cag att tat ctg atg gac aat cca ctc192Ala Asn Tyr Leu Ser Val Gly Gln Ile Tyr Leu Met Asp Asn Pro Leu50 55 60ctg aaa gag ccg ctc tcc aaa gaa cat atc aag ccc cgc ctg ctc ggc240Leu Lys Glu Pro Leu Ser Lys Glu His Ile Lys Pro Arg Leu Leu Gly65 70 75 80cac tgg ggc acc act ccc ggt ctc aat ttc ctt tac gtg cac ctg aac288His Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Tyr Val His Leu Asn85 90 95cgc atc atc cgc aac cgc gat ctt gac att atc tat atc gcc ggg ccg336Arg Ile Ile Arg Asn Arg Asp Leu Asp Ile Ile Tyr Ile Ala Gly Pro100 105 110
gga cat ggc ggg cct gcg ctg gtg gcg aac gta tgg ctg gag ggt acc 384Gly His Gly Gly Pro Ala Leu Val Ala Asn Val Trp Leu Glu Gly Thr115 120 125tac agt gag tac tat ccc gat gtg tcg ttc gac gag gcg ggc atg aag 432Tyr Ser Glu Tyr Tyr Pro Asp Val Ser Phe Asp Glu Ala Gly Met Lys130 135 140cgg ctg ttc cgg cag ttc tcg ttt ccg ggc ggt att ccg agc cac gtg 480Arg Leu Phe Arg Gln Phe Ser Phe Pro Gly Gly Ile Pro Ser His Val145 150 155 160gct ccc gca acg ccg gga tcg atc cat gag ggc gga gag ctg ggc tat 528Ala Pro Ala Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr165 170 175gcg ttg tcg cac gct tac ggc gcg gtg ttc gac aat ccc gat ctt gtc 576Ala Leu Ser His Ala Tyr Gly Ala Val Phe Asp Asn Pro Asp Leu Val180 185 190gca gcc tgt gtc atc ggc gat ggc gag gca gag acg ggg cca ctg gcg 624Ala Ala Cys Val Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala195 200 205acg gcg tgg cac agc aac aag ttc ctg aac ccg aag cgc gac ggg gcg 672Thr Ala Trp His Ser Asn Lys Phe Leu Asn Pro Lys Arg Asp Gly Ala210 215 220gtg ctg ccg gtt ctg cac ctg aac ggc tac aag atc gcc aac cca acg 720Val Leu Pro Val Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr225 230 235 240gtg ctg gcg cgc att tcg cac gag gag ctg gag cag ctc atg att ggc 768Val Leu Ala Arg Ile Ser His Glu Glu Leu Glu Gln Leu Met Ile Gly245 250 255tac ggc tac aaa ccg tac ttc gtt gag ggg gat gac ccg gcg acg atg 816Tyr Gly Tyr Lys Pro Tyr Phe Val Glu Gly Asp Asp Pro Ala Thr Met260 265 270cac cag atg atg gcg gcg acg atg gac cgc tgc ttc gat gag atc gcc 864His Gln Met Met Ala Ala Thr Met Asp Arg Cys Phe Asp Glu Ile Ala275 280 285gaa atc cag cgc cgg gcg agg gtc gat ggc gtg acc gag cga ccg atg 912Glu Ile Gln Arg Arg Ala Arg Val Asp Gly Val Thr Glu Arg Pro Met290 295 300tgg ccg atg atc gtg ttt cgc tct ccg aaa ggg tgg acg ggg ccg aag 960Trp Pro Met Ile Val Phe Arg Ser Pro Lys Gly Trp Thr Gly Pro Lys305 310 315 320gtg gtt gac ggc aaa ccc gcc gag ggg agc tgg cgt tca cac cag gtg1008Val Val Asp Gly Lys Pro Ala Glu Gly Ser Trp Arg Ser His Gln Val325 330 335ccg ttc agc acg gtg cga gac aat ccg gag cac atg gcg ctg ctc gaa1056Pro Phe Ser Thr Val Arg Asp Asn Pro Glu His Met Ala Leu Leu Glu340 345 350acg tgg ctg aaa agc tac cgt gcc gaa gag ctg ttc aca gcg gat ggc1104Thr Trp Leu Lys Ser Tyr Arg Ala Glu Glu Leu Phe Thr Ala Asp Gly355 360 365gtt ctt ctt cct gag ttg cag gag ctg gct ccg cgt ggc aag aag cgc1152Val Leu Leu Pro Glu Leu Gln Glu Leu Ala Pro Arg Gly Lys Lys Arg370 375 380atg ggc gat att cca cac gcc aac ggc ggc ctg ttg ctc aag gag ttg1200Met Gly Asp Ile Pro His Ala Asn Gly Gly Leu Leu Leu Lys Glu Leu385 390 395 400cgg atg ccg gac ttt cgg gaa tat gga atc gat gta ccg aag ccc gga1248Arg Met Pro Asp Phe Arg Glu Tyr Gly Ile Asp Val Pro Lys Pro Gly405 410 415tcg gtg gag gcc gaa gcg cct aag ccg atg gcc cgc ttt ctg cgc gac1296
Ser Val Glu Ala Glu Ala Pro Lys Pro Met Ala Arg Phe Leu Arg Asp420 425 430atc atg aag atg aac gag aag gcg gcc aac ttc cgc gtt ttc ggg ccg1344Ile Met Lys Met Asn Glu Lys Ala Ala Asn Phe Arg Val Phe Gly Pro435 440 445gac gag acc gca tca aac cgc ctt ggc gag ctg ttc gaa gag acc gac1392Asp Glu Thr Ala Ser Asn Arg Leu Gly Glu Leu Phe Glu Glu Thr Asp450 455 460cgc acg tgg atg gcc ggg atg ctg ccg acc gac gat cat ctg tcg cgc1440Arg Thr Trp Met Ala Gly Met Leu Pro Thr Asp Asp Hi5 Leu Ser Arg465 470 475 480gat ggc cgc gtg atg gaa att ctc tcg gag cac acc tgc cag ggg tgg1488Asp Gly Arg Val Met Glu Ile Leu Ser Glu His Thr Cys Gln Gly Trp485 490 495ctt gaa gga tac ctt ttg acc gga cgc cac ggc ttc ttc tca tgc tac1536Leu Glu Gly Tyr Leu Leu Thr Gly Arg His Gly Phe Phe Ser Cys Tyr500 505 510gag gcg ttc atc cac atc atc gac tcg atg ttc aac cag cac gcc aag1584Glu Ala Phe Ile His Ile Ile Asp Ser Met Phe Asn Gln His Ala Lys515 520 525tgg ctg aag gtg act ggt gcc gaa att ccc tgg cgc cgg ccc atc gct1632Trp Leu Lys Val Thr Gly Ala Glu Ile Pro Trp Arg Arg Pro Ile Ala530 535 540tcg ctg aac tat ttc ctg act tcg cac gtg tgg cgg cag gat cac aac1680Ser Leu Asn Tyr Phe Leu Thr Ser His Val Trp Arg Gln Asp His Asn545 550 555 560ggc ttt tcg cat cag gat ccc ggt ttt att gac cat gtg gtc aac aag1728Gly Phe Ser His Gln Asp Pro Gly Phe Ile Asp His Val Val Asn Lys565 570 575aag tcg agc gtg att cgt gtt tat ctg ccg cca gac gcc aat tcg ctg1776Lys Ser Ser Val Ile Arg Val Tyr Leu Pro Pro Asp Ala Asn Ser Leu580 585 590ctg tcg gtc aca aac cat tgc ctg cgc tcc cgc aat tac atc aat gtg1824Leu Ser Val Thr Asn His Cys Leu Arg Ser Arg Asn Tyr Ile Asn Val595 600 605att gtg gcg ggt aaa cag cca gcg tgg cag tgg ctt gac atg gag tcc1872Ile Val Ala Gly Lys Gln Pro Ala Trp Gln Trp Leu Asp Met Glu Ser610 615 620gcc gtg cgg cac tgt acc agc ggc atc ggc atc tgg gag tgg gcc tcg1920Ala Val Arg His Cys Thr Ser Gly Ile Gly Ile Trp Glu Trp Ala Ser625 630 635 640aat gac gcg aat gag ggc gag ccg gac gtg gtg atg gct tgt gcg ggc1968Asn Asp Ala Asn Glu Gly Glu Pro Asp Val Val Met Ala Cys Ala Gly645 650 655gac gtg ccg acg ctt gaa acg ctg gcc gcc gtc aag att ctg cgc aaa2016Asp Val Pro Thr Leu Glu Thr Leu Ala Ala Val Lys Ile Leu Arg Lys660 665 670ctc gcg ccg gag ttg aag atc agg gtg gtc aac gtg gtc gat ctc atg2064Leu Ala Pro Glu Leu Lys Ile Arg Val Val Asn Val Val Asp Leu Met675 680 685act ctc cag ccg aaa gag gag cac ccg cac ggc ctt gcc gat cgc gat2112Thr Leu Gln Pro Lys Glu Glu His Pro His Gly Leu Ala Asp Arg Asp690 695 700ttc gac gac atg ttt act aca gac aag ccg atc atc ttc gcc tat cac2160Phe Asp Asp Met Phe Thr Thr Asp Lys Pro Ile Ile Phe Ala Tyr His705 710 715 720ggt tac ccg tgg ctg atc cac cgg ctg acc tac cgc cgc acg aac cac2208Gly Tyr Pro Trp Leu Ile His Arg Leu Thr Tyr Arg Arg Thr Asn His
725 730 735cac aac ctg cac gtg cgt ggc tac aag gag gag ggc acc acg acg acg2256His Asn Leu His Val Arg Gly Tyr Lys Glu Glu Gly Thr Thr Thr Thr740 745 750ccg ttc gac atg gtg gtg atg aac gag ctc gac cgc ttc cac ctg gta2304Pro Phe Asp Met Val Val Met Asn Glu Leu Asp Arg Phe His Leu Val755 760 765gcc gac gta gcc aac cgc gtg gag agc ctc agg cca caa gct gcc tac2352Ala Asp Val Ala Asn Arg Val Glu Ser Leu Arg Pro Gln Ala Ala Tyr770 775 780atc aaa caa tac gtc cgc gac cgc ctg atc gag cac aag gag tac atc2400Ile Lys Gln Tyr Val Arg Asp Arg Leu Ile Glu His Lys Glu Tyr Ile785 790 795 800acg aaa tat ggc gag gat atg ccg gaa gtg agg gat tgg cgc tgg gag2448Thr Lys Tyr Gly Glu Asp MPt Pro Glu Val Arg Asp Trp Arg Trp Glu805 810 815gat tga2454Asp<210>75<211>817<212>PRT<213>Chlorobium tepidum<400>61Leu Ile Val Arg Ser Glu Asn Arg Val Pro Asp Glu Leu Arg Thr Gln1 5 10 15His Thr Thr Thr Ile Lys Ser Thr Ile Leu Met Thr Glu Met Thr Thr20 25 30Pro Leu Ser Pro Arg Glu Leu Asp Leu Met Asn Ala Tyr Trp Arg Ala35 40 45Ala Asn Tyr Leu Ser Val Gly Gln Ile Tyr Leu Met Asp Asn Pro Leu50 55 60Leu Lys Glu Pro Leu Ser Lys Glu His Ile Lys Pro Arg Leu Leu Gly65 70 75 80His Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Tyr Val His Leu Asn85 90 95Arg Ile Ile Arg Asn Arg Asp Leu Asp Ile Ile Tyr Ile Ala Gly Pro100 105 110Gly His Gly Gly Pro Ala Leu Val Ala Asn Val Trp Leu Glu Gly Thr115 120 125Tyr Ser Glu Tyr Tyr Pro Asp yal Ser Phe Asp Glu Ala Gly Met Lys130 135 140Arg Leu Phe Arg Gln Phe Ser Phe Pro Gly Gly Ile Pro Ser His Val145 150 155 160Ala Pro Ala Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr165 170 175Ala Leu Ser His Ala Tyr Gly Ala Val Phe Asp Asn Pro Asp Leu Val180 185 190Ala Ala Cys Val Ile Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala195 200 205Thr Ala Trp His Ser Asn Lys Phe Leu Asn Pro Lys Arg Asp Gly Ala210 215 220Val Leu Pro Val Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr225 230 235 240Val Leu Ala Arg Ile Ser His Glu Glu Leu Glu Gln Leu Met Ile Gly
245 250 255Tyr Gly Tyr Lys Pro Tyr Phe Val Glu Gly Asp Asp Pro Ala Thr Met260 265 270His Gln Met Met Ala Ala Thr Met Asp Arg Cys Phe Asp Glu Ile Ala275 280 285Glu Ile Gln Arg Arg Ala Arg Val Asp Gly Val Thr Glu Arg Pro Met290 295 300Trp Pro Met Ile Val Phe Arg Ser Pro Lys Gly Trp Thr Gly Pro Lys305 310 315 320Val Val Asp Gly Lys Pro Ala Glu Gly Ser Trp Arg Ser His Gln Val325 330 335Pro Phe Ser Thr Val Arg Asp Asn Pro Glu His Met Ala Leu Leu Glu340 345 350Thr Trp Leu Lys Ser Tyr Arg Ala Glu Glu Leu Phe Thr Ala Asp Gly355 360 365Val Leu Leu Pro Glu Leu Gln Glu Leu Ala Pro Arg Gly Lys Lys Arg370 375 380Met Gly Asp Ile Pro Hi s Ala Asn Gly Gly Leu Leu Leu Lys Glu Leu385 390 395 400Arg Met Pro Asp Phe Arg Glu Tyr Gly Ile Asp Val Pro Lys Pro Gly405 410 415Ser Val Glu Ala Glu Ala Pro Lys Pro Met Ala Arg Phe Leu Arg Asp420 425 430Ile Met Lys Met Asn Glu Lys Ala Ala Asn Phe Arg Val Phe Gly Pro435 440 445Asp Glu Thr Ala Ser Asn Arg Leu Gly Glu Leu Phe Glu Glu Thr Asp450 455 460Arg Thr Trp Met Ala Gly Met Leu Pro Thr Asp Asp His Leu Ser Arg465 470 475 480Asp Gly Arg Val Met Glu Ile Leu Ser Glu His Thr Cys Gln Gly Trp485 490 495Leu Glu Gly Tyr Leu Leu Thr Gly Arg His Gly Phe Phe Ser Cys Tyr500 505 510Gll Ala Phe Ile His Ile Ile Asp Ser Met Phe Asn Gln His Ala Lys515 520 525Trp Leu Lys Val Thr Gly Ala Glu Ile Pro Trp Arg Arg Pro Ile Ala530 535 540Ser Leu Asn Tyr Phe Leu Thr Ser His Val Trp Arg Gln Asp His Asn545 550 555 560Gly Phe Ser His Gln Asp Pro Gly Phe Ile Asp His Val Val Asn Lys565 570 575Lys Ser Ser Val Ile Arg Val Tyr Leu Pro Pro Asp Ala Asn Ser Leu580 585 590Leu Ser Val Thr Asn His Cys Leu Arg Ser Arg Asn Tyr Ile Asn Val595 600 605Ile Val Ala Gly Lys Gln Pro Ala Trp Gln Trp Leu Asp Met Glu Ser610 615 620Ala Val Arg His Cys Thr Ser Gly Ile Gly Ile Trp Glu Trp Ala Ser625 630 635 640Asn Asp Ala Asn Glu Gly Glu Pro Asp Val Val Met Ala Cys Ala Gly645 650 655Asp Val Pro Thr Leu Glu Thr Leu Ala Ala Val Lys Ile Leu Arg Lys660 665 670Leu Ala Pro Glu Leu Lys Ile Arg Val Val Asn Val Val Asp Leu Met675 680 685Thr Leu Gln Pro Lys Glu Glu His Pro His Gly Leu Ala Asp Arg Asp690 695 700Phe Asp Asp Met Phe Thr Thr Asp Lys Pro Ile Ile Phe Ala Tyr His
705 710 715 720Gly Tyr Pro Trp Leu Ile His Arg Leu Thr Tyr Arg Arg Thr Asn His725 730 735His Asn Leu His Val Arg Gly Tyr Lys Glu Glu Gly Thr Thr Thr Thr740 745 750Pro Phe Asp Met Val Val Met Asn Glu Leu Asp Arg Phe His Leu Val755 760 765Ala Asp Val Ala Asn Arg Val Glu Ser Leu Arg Pro Gln Ala Ala Tyr770 775 780Ile Lys Gln Tyr Val Arg Asp Arg Leu Ile Glu His Lys Glu Tyr Ile785 790 795 800Thr Lys Tyr Gly Glu Asp Met Pro Glu Val Arg Asp Trp Arg Trp Glu805 810 815Asp<210>76<211>2370<212>DNA<213>猪布鲁氏菌<220>
<221>CDS<222>(1)..(2370)<223>
<400>76gtg cca gca aaa ggg cct ctc aca ccg cag cag crt tca ctc atc aac 48Val Pro Ala Lys Gly Pro Leu Thr Pro Gln Gln Leu Ser Leu Ile Asn1 5 10 15cgt tac tgg cgc gcg gcg aat tat ctt tcc gtc ggc cag att tat ctg 96Arg Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Ile Tyr Leu20 25 30atg aaa aat ccc ctg ttg cgc gaa ccg ctc cag cct gag cac atc aag144Met Lys Asn Pro Leu Leu Arg Glu Pro Leu Gln Pro Glu His Ile Lys35 40 45ccg cgg ctt ctc ggc cat tgg ggc acg aca ccc ggc ctc aat ttc atc192Pro Arg Leu Leu Gly His Trp Gly Thr Thr Pro Gly Leu Asn Phe Ile50 55 60tat gcg cat ctc aac cgc att att cag cag cgc aac gcc aat gtg ate240Tyr Ala His Leu Asn Arg Ile Ile Gln Gln Arg Asn Ala Asn Val Ile65 70 75 80tat att tgc ggc ccc ggc cat ggc ggg ecg ggc atg gtg gcc aac acc288Tyr Ile Cys Gly Pro Gly His Gly Gly Pro Gly Met Val Ala Asn Thr85 90 95tat ctg gag ggc acc tat tcc gaa atc tat ccc gca atc agc gaa gat336Tyr Leu Glu Gly Thr Tyr Ser Glu Ile Tyr Pro Ala Ile Ser Glu Asp100 105 1l0gaa gcg ggc atg gaa agg ctc ttc cgc cag ttt tcc ttc ccc ggc gga384Glu Ala Gly Met Glu Arg Leu Phe Arg Gln Phe Ser Phe Pro Gly Gly115 120 125ata cca agc cat gcc gcg ccg gaa aca ccg ggc tct arc cac gaa ggg432Ile Pro Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly130 135 140ggc gaa ctg ggt tat gcg ctc gtc cac gcc tat ggt gcg gcc ttc gac480Gly Glu Leu Gly Tyr Ala Leu Val His Ala Tyr Gly Ala Ala Phe Asp145 150 155 160
aat ccc gat ctg gtg gtg gcc tgc gtc gtg ggc gac gga gaa gcg gaa 528Asn Pro Asp Leu Val Val Ala Cys Val Val Gly Asp Gly Glu Ala Glu165 170 175acc ggc gcg ctt gca act tcg tgg cac tcc aac aaa ttc ctc aat ccg 576Thr Gly Ala Leu Ala Thr Ser Trp His Ser Asn Lys Phe Leu Asn Pro180 185 190gcg cgc gat ggt gcg gtt ctg ccg atc ctg cat ctc aat ggc tac aag 624Ala Arg Asp Gly Ala Val Leu Pro Ile Leu His Leu Asn Gly Tyr Lys195 200 205atc gcc aac ccc acc gtg ctg gcc cgc ctt tcg gat gat gat ctg gac 672Ile Ala Asn Pro Thr Val Leu Ala Arg Leu Ser Asp Asp Asp Leu Asp210 215 220aat ctt ttc cgt ggc tac ggt tat gag cct ttc ttt gtt gaa ggc agc 720Asn Leu Phe Arg Gly Tyr Gly Tyr Glu Pro Phe Phe Val Glu Gly Ser225 230 235 240gag cct gcc gac atg cac cag aag atg gcc gca aca ctg gac acg att 768Glu Pro Ala Asp Met His Gln Lys Met Ala Ala Thr Leu Asp Thr Ile245 250 255ttc cag cgc att cag gac atc aag aaa aat gcc gat gtg cac tcg ccc 816Phe Gln Arg Ile Gln Asp Ile Lys Lys Asn Ala Asp Val His Ser Pro260 265 270gag cgc ccg cgc tgg ccg atg att att ctc aga agc ccg aag ggc tgg 864Glu Arg Pro Arg Trp Pro Met Ile Ile Leu Arg Ser Pro Lys Gly Trp275 280 285acc ggc cca aaa acc gtg gac ggt ctg gtt gtt gaa aac tat tgg cgc 912Thr Gly Pro Lys Thr Val Asp Gly Leu Val Val Glu Asn Tyr Trp Arg290 295 300gcc cat cag gtg ccg gtt gcc aat tgc cgc gaa aac gat gcc cat cgc 960Ala His Gln Val Pro Val Ala Asn Cys Arg Glu Asn Asp Ala His Arg305 310 315 320aaa atc ctc gaa gac tgg atg aag agt tac gac ccg tcc gat ctg ttc1008Lys Ile Leu Glu Asp Trp Met Lys Ser Tyr Asp Pro Ser Asp Leu Phe325 330 335gac gaa aaa ggg gcg ctg aag cct gaa ttg cgg gcg ctg gcg cca aag1056Asp Glu Lys Gly Ala Leu Lys Pro Glu Leu Arg Ala Leu Ala Pro Lys340 345 350ggt gaa gcg cgc ata ggg gcc aat ccg cat gcc aat ggc ggg ctc ttg1104Gly Glu Ala Arg Ile Gly Ala Asn Pro His Ala Asn Gly Gly Leu Leu355 360 365cgt aaa gag ctt cac atg ccg gat ttc cgc caa tat gcg gtc aat gtc1152Arg Lys Glu Leu His Met Pro Asp Phe Arg Gln Tyr Ala Val Asn Val370 375 380acc gaa ccg gga gcg ata gaa gcg caa tca acc aaa ata ttg ggc gat1200Thr Glu Pro Gly Ala Ile Glu Ala Gln Ser Thr Lys Ile Leu Gly Asp385 390 395 400ttt ctg cgc gat gtg atg aaa ctg aac gag acg gaa aag aac ttc cgc1248Phe Leu Arg Asp Val Met Lys Leu Asn Glu Thr Glu Lys Asn Phe Arg405 410 415atc ttc ggc ccc gac gaa acc gcc tcc aac agg ctc ggc agc gta tta1296Ile Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gly Ser Val Leu420 425 430gag gcg acg aac cgg gtc tgg atg gcc gaa acg ctg gac atg gat gac1344Glu Ala Thr Asn Arg Val Trp Met Ala Glu Thr Leu Asp Met Asp Asp435 440 445cac ctc gcc gcc gac ggg cgt gta atg gag gtt ctc agc gaa cat ctc1392His Leu Ala Ala Asp Gly Arg Val Met Glu Val Leu Ser Glu His Leu450 455 460tgc cag ggc tgg ctt gaa ggc tat ctt ctc agt ggc cgg cac ggc ttc1440
Cys Gln Gly Trp Leu Glu Gly Tyr Leu Leu Ser Gly Arg His Gly Phe465 470 475 480ttt tcc tgc tac gaa gcc ttc atc cac att atc gat tcc atg ttc aac1488Phe Ser Cys Tyr Glu Ala Phe Ile His Ile Ile Asp Ser Met Phe Asn485 490 495caa cat gcc aaa tgg ttg cag gtt gcg cgc gaa ctg gaa tgg cgc aag1536Gln His Ala Lys Trp Leu Gln Val Ala Arg Glu Leu Glu Trp Arg Lys500 505 510ccc atc tca tcg ctc aat tat ctg ctg acc tcc cat gtc tgg cgg cag1584Pro Ile Ser Ser Leu Asn Tyr Leu Leu Thr Ser His Val Trp Arg Gln515 520 525gac cat aac ggc ttc tcc cat cag gat ccc ggc ttc gtc gat ctc gtc1632Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Phe Val Asp Leu Val530 535 540gcc aac aaa agc gcc gat atc gtg cgt gtc tat ttc ccg ccg gat gcc1680Ala Asn Lys Ser Ala Asp Ile Val Arg Val Tyr Phe Pro Pro Asp Ala545 550 555 560aac acc ctt ttg tgg gtg gga gat cac tgc ctg aaa acc tgg aac cgc1728Asn Thr Leu Leu Trp Val Gly Asp His Cys Leu Lys Thr Trp Asn Arg565 570 575gtg aat gtc atc gtg gcg ggc aag cag ccg gaa ccg caa tgg ctg acc1776Val Asn Val Ile Val Ala Gly Lys Gln Pro Glu Pro Gln Trp Leu Thr580 585 590atg gcg gag gct gag aaa cat tgc gaa gca ggt ctc ggc ata tgg gaa1824Met Ala Glu Ala Glu Lys His Cys Glu Ala Gly Leu Gly Ile Trp Glu595 600 605tgg gcg ggc acg gaa gac ggg ctg gag ccg gat atc gtc atg gcc tgc1872Trp Ala Gly Thr Glu Asp Gly Leu Glu Pro Asp Ile Val Met Ala Cys610 615 620gcg ggc gat gtg ccg acc atg gaa acg ctt gcc gcg gtt gat ctt ctg1920Ala Gly Asp Val Pro Thr Met Glu Thr Leu Ala Ala Val Asp Leu Leu625 630 635 640cgc cag tcc ctg ccg cat ctg cgc att cgc gtg gtg aat gtg gtt gac1968Arg Gln Ser Leu Pro His Leu Arg Ile Arg Val Val Asn Val Val Asp645 650 655ctc atg gtg ctg caa tcg ccg cat cag cac ccg cat ggc att tcc gat2016Leu Met Val Leu Gln Ser Pro His Gln His Pro His Gly Ile Ser Asp660 665 670gag gaa ttc gac cgg atg ttc acc acc aac agg ccc gtc atc ttc gcc2064Glu Glu Phe Asp Arg Met Phe Thr Thr Asn Arg Pro Val Ile Phe Ala675 680 685tat cac ggc tat cct tat ctc atc cat cga ctg gtc tat aag cgc acc2112Tyr His Gly Tyr Pro Tyr Leu Ile His Arg Leu Val Tyr Lys Arg Thr690 695 700aac cac tcc aat ttc cac gtt cgt ggt ttt atc gag cag gga acg acc2160Asn His Ser Asn Phe His Val Arg Gly Phe Ile Glu Gln Gly Thr Thr705 710 715 720aca acg cct ttc gac atg acc gtg ctg aac gag ctg gat cgt ttc cac2208Thr Thr Pro Phe Asp Met Thr Val Leu Asn Glu Leu Asp Arg Phe His725 730 735ctc gcg atg gaa gcc gtc gag cgc ctg ccg ctg ggc gaa agc gtt gcc2256Leu Ala Met Glu Ala Val Glu Arg Leu Pro Leu Gly Glu Ser Val Ala740 745 750aaa ccc ctg atc gac aat ttc acg gag aaa ctt gcg ctg cat aag gac2304Lys Pro Leu Ile Asp Asn Phe Thr Glu Lys Leu Ala Leu His Lys Asp755 760 765tat atc cga cag cat ggt gag gac atg ccg gaa atc cgc gac tgg aaa2352Tyr Ile Arg Gln His Gly Glu Asp Met Pro Glu Ile Arg Asp Trp Lys
770 775 780tgg aca tgg ccg cga taa 2370Trp Thr Trp Pro Arg785<210>77<211>789<212>PRT<213>猪布鲁氏菌<400>77Val Pro Ala Lys Gly Pro Leu Thr Pro Gln Gln Leu Ser Leu Ile Asn1 5 10 15Arg Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln Ile Tyr Leu20 25 30Met Lys Asn Pro Leu Leu Arg Glu Pro Leu Gln Pro Glu His Ile Lys35 40 45Pro Arg Leu Leu Gly His Trp Gly Thr Thr Pro Gly Leu Asn Phe Ile50 55 60Tyr Ala His Leu Asn Arg Ile Ile Gln Gln Arg Asn Ala Asn Val Ile65 70 75 80Tyr Ile Cys Gly Pro Gly His Gly Gly Pro Gly Met Val Ala Asn Thr85 90 95Tyr Leu Glu Gly Thr Tyr Ser Glu Ile Tyr Pro Ala Ile Ser Glu Asp100 105 110Glu Ala Gly Met Glu Arg Leu Phe Arg Gln Phe Ser Phe Pro Gly Gly115 120 125Ile Pro Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile His Glu Gly130 135 140Gly Glu Leu Gly Tyr Ala Leu Val His Ala Tyr Gly Ala Ala Phe Asp145 150 155 160Asn Pro Asp Leu Val Val Ala Cys Val Val Gly Asp Gly Glu Ala Glu165 170 175Thr Gly Ala Leu Ala Thr Ser Trp His Ser Asn Lys Phe Leu Asn Pro180 185 190Ala Arg Asp Gly Ala Val Leu Pro Ile Leu His Leu Asn Gly Tyr Lys195 200 205Ile Ala Asn Pro Thr Val Leu Ala Arg Leu Ser Asp Asp Asp Leu Asp210 215 220Asn Leu Phe Arg Gly Tyr Gly Tyr Glu Pro Phe Phe Val Glu Gly Ser225 230 235 240Glu Pro Ala Asp Met His Gln Lys Met Ala Ala Thr Leu Asp Thr Ile245 250 255Phe Gln Arg Ile Gln Asp Ile Lys Lys Asn Ala Asp Val His Ser Pro260 265 270Glu Arg Pro Arg Trp Pro Met Ile Ile Leu Arg Ser Pro Lys Gly Trp275 280 285Thr Gly Pro Lys Thr Val Asp Gly Leu Val Val Glu Asn Tyr Trp Arg290 295 300Ala His Gln Val Pro Val Ala Asn Cys Arg Glu Asn Asp Ala His Arg305 310 315 320Lys Ile Leu Glu Asp Trp Met Lys Ser Tyr Asp Pro Ser Asp Leu Phe325 330 335Asp Glu Lys Gly Ala Leu Lys Pro Glu Leu Arg Ala Leu Ala Pro Lys340 345 350Gly Glu Ala Arg Ile Gly Ala Asn Pro His Ala Asn Gly Gly Leu Leu355 360 365
Arg Lys Glu Leu His Met Pro Asp Phe Arg Gln Tyr Ala Val Asn Val370 375 380Thr Glu Pro Gly Ala Ile Glu Ala Gln Ser Thr Lys Ile Leu Gly Asp385 390 395 400Phe Leu Arg Asp Val Met Lys Leu Asn Glu Thr Glu Lys Asn Phe Arg405 410 415Ile Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gly Ser Val Leu420 425 430Glu Ala Thr Asn Arg Val Trp Met Ala Glu Thr Leu Asp Met Asp Asp435 440 445His Leu Ala Ala Asp Gly Arg Val Met Glu Val Leu Ser Glu His Leu450 455 460Cys Gln Gly Trp Leu Glu Gly Tyr Leu Leu Ser Gly Arg His Gly Phe465 470 475 480Phe Ser Cys Tyr Glu Ala Phe Ile His Ile Ile Asp Ser Met Phe Asn485 490 495Gln His Ala Lys Trp Leu Gln Val Ala Arg Glu Leu Glu Trp Arg Lys500 505 510Pro Ile Ser Ser Leu Asn Tyr Leu Leu Thr Ser His Val Trp Arg Gln515 520 525Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Phe Val Asp Leu Val530 535 540Ala Asn Lys Ser Ala Asp Ile Val Arg Val Tyr Phe Pro Pro Asp Ala545 550 555 560Asn Thr Leu Leu Trp Val Gly Asp His Cys Leu Lys Thr Trp Asn Arg565 570 575Val Asn Val Ile Val Ala Gly Lys Gln Pro Glu Pro Gln Trp Leu Thr580 585 590Met Ala Glu Ala Glu Lys His Cys Glu Ala Gly Leu Gly Ile Trp Glu595 600 605Trp Ala Gly Thr Glu Asp Gly Leu Glu Pro Asp Ile Val Met Ala Cys610 615 620Ala Gly Asp Val Pro Thr Met Glu Thr Leu Ala Ala Val Asp Leu Leu625 630 635 640Arg Gln Ser Leu Pro His Leu Arg Ile Arg Val Val Asn Val Val Asp645 650 655Leu Met Val Leu Gln Ser Pro His Gln His Pro His Gly Ile Ser Asp660 665 670Glu Glu Phe Asp Arg Met Phe Thr Thr Asn Arg Pro Val Ile Phe Ala675 680 685Tyr His Gly Tyr Pro Tyr Leu Ile His Arg Leu Val Tyr Lys Arg Thr690 695 700Asn His Ser Asn Phe His Val Arg Gly Phe Ile Glu Gln Gly Thr Thr705 710 715 720Thr Thr Pro Phe Asp Met Thr Val Leu Asn Glu Leu Asp Arg Phe His725 730 735Leu Ala Met Glu Ala Val Glu Arg Leu Pro Leu Gly Glu Ser Val Ala740 745 750Lys Pro Leu Ile Asp Asn Phe Thr Glu Lys Leu Ala Leu His Lys Asp755 760 765Tyr Ile Arg Gln His Gly Glu Asp Met Pro Glu Ile Arg Asp Trp Lys770 775 780Trp Thr Trp Pro Arg785<210>78<211>2379
<212>DNA<213>流产布鲁氏菌<220>
<221>CDS<222>(1)..(2379)<223>
<400>78atg agc act gtg cca gca aaa ggg cct ctc aca ccg cag cag ctt tca 48Met Ser Thr Val Pro Ala Lys Gly Pro Leu Thr Pro Gln Gln Leu Ser1 5 10 15ctc atc aac cgt tac tgg cgc gcg gcg aat tat ctt tcc gtc ggc cag 96Leu Ile Asn Arg Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln20 25 30att tat ctg atg aaa aat ccc ctg ttg cgc gaa ccg ctc cag cct gag144Ile Tyr Leu Met Lys Asn Pro Leu Leu Arg Glu Pro Leu Gln Pro Glu35 40 45cac atc aag ccg cgg ctt ctc ggc cat tgg ggc acg aca ccc ggc ctc192His Ile Lys Pro Arg Leu Leu Gly His Trp Gly Thr Thr Pro Gly Leu50 55 60aat ttc atc tat gcg cat ctc aac cgc attatt cag cag cgc aac gcc 240Asn Phe Ile Tyr Ala His Leu Asn Arg Ile Ile Gln Gln Arg Asn Ala65 70 75 80aat gtg atc tat att tgc ggc ccc ggc cat ggc ggg ccg ggc atg gtg288Asn Val Ile Tyr Ile Cys Gly Pro Gly His Gly Gly Pro Gly Met Val85 90 95gcc aac acc tat ctg gag ggc acc tat tcc gaa atc tat ccc gca atc336Ala Asn Thr Tyr Leu Glu Gly Thr Tyr Ser Glu Ile Tyr Pro Ala Ile100 105 110agc gaa gat gaa gcg ggc atg gaa agg ctc ttc cgc cag ttt tcc ttc384Ser Glu Asp Glu Ala Gly Met Glu Arg Leu Phe Arg Gln Phe Ser Phe115 120 125ccc ggc gga ata cca agc cat gcc gcg ccg gaa aca ccg ggc tct atc432Pro Gly Gly Ile Pro Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile130 135 140cac gaa ggg ggc gaa ctg ggt tat gcg ctc gtc cac gcc tat ggt gcg480His Glu Gly Gly Glu Leu Gly Tyr Ala Leu Val His Ala Tyr Gly Ala145 150 155 160gcc ttc gac aat ccc gat ctg gtg gtg gcc tgc gtc gtg ggc gac gga528Ala Phe Asp Asn Pro Asp Leu Val Mal Ala Cys Val Val Gly Asp Gly165 170 175gaa gcg gaa acc ggc gcg ctt gca act tcg tgg cac tcc aac aaa ttc576Glu Ala Glu Thr Gly Ala Leu Ala Thr Ser Trp His Ser Asn Lys Phe180 185 190ctc aat ccg gcg cgc gat ggt gcg gtt ctg ccg atc ctg cat ctc aat624Leu Asn Pro Ala Arg Asp Gly Ala Val Leu Pro Ile Leu His Leu Asn195 200 205ggc tac aag atc gcc aac ccc acc gtg ctg gcc cgc ctt tcg gat gat672Gly Tyr Lys Ile Ala Asn Pro Thr Val Leu Ala Arg Leu Ser Asp Asp210 215 220gat ctg gac aat ctt ttc cgt ggc tac ggt tat gag cct ttc ttt gtt720Asp Leu Asp Asn Leu Phe Arg Gly Tyr Gly Tyr Glu Pro Phe Phe Val225 230 235 240gaa ggc agc gag cct gcc gac atg cac cag aag atg gcc gca aca ctg768Glu Gly Ser Glu Pro Ala Asp Met His Gln Lys Met Ala Ala Thr Leu245 250 255gac acg att ttc cag cgc att cag gac atc aag aaa aat gcc gat gtg816
Asp Thr Ile Phe Gln Arg Ile Gln Asp Ile Lys Lys Asn Ala Asp Val260 265 270cac tcg ccc gag cgc ccg cgc tgg ccg atg att att ctc aga agc ccg 864His Ser Pro Glu Arg Pro Arg Trp Pro Met Ile Ile Leu Arg Ser Pro275 280 285aag ggc tgg acc ggc cca aaa acc gtg gac ggt ctg gtt gtt gaa aac 912Lys Gly Trp Thr Gly Pro Lys Thr Val Asp Gly Leu Val Val Glu Asn290 295 300tat tgg cgc gcc cat gag gtg ccg gtt gcc aat tgc cgc gaa aac gat 960Tyr Trp Arg Ala His Glu Val Pro Val Ala Asn Cys Arg Glu Asn Asp305 310 315 320gcc cat cgc aaa atc ctc gaa gac tgg atg aag agt tac gac ccg tcc1008Ala His Arg Lys Ile Leu Glu Asp Trp Met Lys Ser Tyr Asp Pro Ser325 330 335gat ctg ttc gac gaa aaa ggg gcg ctg aag cct gaa ttg cgg gcg ctg1056Asp Leu Phe Asp Glu Lys Gly Ala Leu Lys Pro Glu Leu Arg Ala Leu340 345 350gcg cca aag ggt gaa gcg cgc ata ggg gcc aat ccg cat gcc aat ggc1104Ala Pro Lys Gly Glu Ala Arg Ile Gly Ala Asn Pro His Ala Asn Gly355 360 365ggg ctc ttg cgt aaa gag ctt cac atg ccg gat ttc cgc caa tat gcg1152Gly Leu Leu Arg Lys Glu Leu His Met Pro Asp Phe Arg Gln Tyr Ala370 375 380gtc aat gtc acc gaa ccg gga gcg ata gaa gcg caa tca acc aaa ata1200Val Asn Val Thr Glu Pro Gly Ala Ile Glu Ala Gln Ser Thr Lys Ile385 390 395 400ttg ggc gat ttt ctg cgc gat gtg atg aaa ctg aac gag acg gaa aag1248Leu Gly Asp Phe Leu Arg Asp Val Met Lys Leu Asn Glu Thr Glu Lys405 410 415aac ttc cgc atc ttc ggc ccc gac gaa acc gcc tcc aac agg ctc ggc1296Asn Phe Arg Ile Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gly420 425 430agc gta tta gag gcg acg aac cgg gtc tgg atg gcc gaa acg ctg gac1344Ser Val Leu Glu Ala Thr Asn Arg Val Trp Met Ala Glu Thr Leu Asp435 440 445atg gat gac cac ctc gcc gcc gac ggg cgt gta atg gag gtt ctc agc1392Met Asp Asp His Leu Ala Ala Asp Gly Arg Val Met Glu Val Leu Ser450 455 460gaa cat ctc tgc cag ggc tgg ctt gaa ggc tat ctt ctc agt ggc cgg1440Glu His Leu Cys Gln Gly Trp Leu Glu Gly Tyr Leu Leu Ser Gly Arg465 470 475 480cac ggc ttc ttt tcc tgc tac gaa gcc ttc atc cac att atc gat tcc1488His Gly Phe Phe Ser Cys Tyr Glu Ala Phe Ile His Ile Ile Asp Ser485 490 495atg ttc aac caa cat gcc aaa tgg ttg cag gtt gcg cgc gaa ctg gaa1536Met Phe Asn Gln His Ala Lys Trp Leu Gln Val Ala Arg Glu Leu Glu500 505 510tgg cgc aag ccc atc tca tcg ctc aat tat ctg ctg acc tcc cat gtc1584Trp Arg Lys Pro Ile Ser Ser Leu Asn Tyr Leu Leu Thr Ser His Val515 520 525tgg cgg cag gac cat aac ggc ttc tcc cat cag gat ccc ggc ttc gtcl632Trp Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Phe Val530 535 540gat ctc gtc gcc aac aaa agc gcc gat atc gcg cgt gtc tat ttc ccg1680Asp Leu Val Ala Asn Lys Ser Ala Asp Ile Ala Arg Val Tyr Phe Pro545 550 555 560ccg gat gcc aac acc ctt ttg tgg gtg gga gat cac tgc ctg aaa acc1728Pro Asp Ala Asn Thr Leu Leu Trp Val Gly Asp His Cys Leu Lys Thr
565 570 575tgg aac cgc gtg aat gtc atc gtg gcg ggc aag cag ccg gaa ccg caa1776Trp Asn Arg Val Asn Val Ile Val Ala Gly Lys Gln Pro Glu Pro Gln580 585 590tgg ctg acc atg gcg gag gct gag aaa cat tgc gaa gca ggt ctc ggc1824Trp Leu Thr Met Ala Glu Ala Glu Lys His Cys Glu Ala Gly Leu Gly595 600 605ata tgg gaa tgg gcg ggt acg gaa gac ggg ctg gag ccg gat atc gtc1872Ile Trp Glu Trp Ala Gly Thr Glu Asp Gly Leu Glu Pro Asp Ile Val610 615 620atg gcc tgc gcg ggc gat gtg ccg acc atg gaa acg ctt gcc gcg gtt1920Met Ala Cys Ala Gly Asp Val Pro Thr Met Glu Thr Leu Ala Ala Val625 630 635 640gat ctt ctg cgc cag tcc ctg ccg cat ctg cgc att cgc gtg gtg aat1968Asp Leu Leu Arg Gln Ser Leu Pro His Leu Arg Ile Arg Val Val Asn645 650 655gtg gtt gac ctc atg gtg ctg caa tcg ccg cat cag cac ccg cat ggc2016Val Val Asp Leu Met Val Leu Gln Ser Pro His Gln His Pro His Gly660 665 670att tcc gat gag gaa ttc gac cgg atg ttc acc acc aac agg ccc gtc2064Ile Ser Asp Glu Glu Phe Asp Arg Met Phe Thr Thr Asn Arg Pro Val675 680 685atc ttc gcc tat cac ggc tat cct tat ctc atc cat cga ctg gtc tat2112Ile Phe Ala Tyr His Gly Tyr Pro Tyr Leu Ile His Arg Leu Val Tyr690 695 700aag cgc acc aac cac tcc aat ttc cac gtt cgt ggt ttt atc gag cag2160Lys Arg Thr Asn His Ser Asn Phe His Val Arg Gly Phe Ile Glu Gln705 710 715 720gga acg acc aca acg cct ttc gac atg acc gtg ctg aac gag ctg gat2208Gly Thr Thr Thr Thr Pro Phe Asp Met Thr Val Leu Asn Glu Leu Asp725 730 735cgt ttc cac ctc gcg atg gaa gcc gtc gag cgc ctg ccg ctg ggc gaa2256Arg Phe His Leu Ala Met Glu Ala Val Glu Arg Leu Pro Leu Gly Glu740 745 750agc gtt gcc aaa ccc ctg atc gac aat ttc acg gag aaa ctt gcg ctg2304Ser Val Ala Lys Pro Leu Ile Asp Asn Phe Thr Glu Lys Leu Ala Leu755 760 765cat aag gac tat atc cga cag cat ggt gag gac atg ccg gaa atc cgc2352His Lys Asp Tyr Ile Arg Gln His Gly Glu Asp Met Pro Glu Ile Arg770 775 780gac tgg aaa tgg aca tgg ccg cga taa2379Asp Trp Lys Trp Thr Trp Pro Arg785 790<210>79<211>792<212>PRT<213>流产布鲁氏菌<400>79Met Ser Thr Val Pro Ala Lys Gly Pro Leu Thr Pro Gln Gln Leu Ser1 5 10 15Leu Ile Asn Arg Tyr Trp Arg Ala Ala Asn Tyr Leu Ser Val Gly Gln20 25 30Ile Tyr Leu Met Lys Asn Pro Leu Leu Arg Glu Pro Leu Gln Pro Glu35 40 45His Ile Lys Pro Arg Leu Leu Gly His Trp Gly Trr Thr Pro Gly Leu
50 55 60Asn Phe Ile Tyr Ala His Leu Asn Arg Ile Ile Gln Gln Arg Asn Ala65 70 75 80Asn Val Ile Tyr Ile Cys Gly Pro Gly His Gly Gly Pro Gly Met Val85 90 95Ala Asn Thr Tyr Leu Glu Gly Thr Tyr Ser Glu Ile Tyr Pro Ala Ile100 105 110Ser Glu Asp Glu Ala Gly Met Glu Arg Leu Phe Arg Gln Phe Ser Phe115 120 125Pro Gly Gly Ile Pro Ser His Ala Ala Pro Glu Thr Pro Gly Ser Ile130 135 140His Glu Gly Gly Glu Leu Gly Tyr Ala Leu Val His Ala Tyr Gly Ala145 150 155 160Ala Phe Asp Asn Pro Asp Leu Val Val Ala Cys Val Val Gly Asp Gly165 170 175Glu Ala Glu Thr Gly Ala Leu Ala Thr Ser Trp His Ser Asn Lys Phe180 185 190Leu Asn Pro Ala Arg Asp Gly Ala Val Leu Pro Ile Leu His Leu Asn195 200 205Gly Tyr Lys Ile Ala Asn Pro Thr Val Leu Ala Arg Leu Ser Asp Asp210 215 220Asp Leu Asp Asn Leu Phe Arg Gly Tyr Gly Tyr Glu Pro Phe Phe Val225 230 235 240Glu Gly Ser Glu Pro Ala Asp Met His Gln Lys Met Ala Ala Thr Leu245 250 255Asp Thr Ile Phe Gln Arg Ile Gln Asp Ile Lys Lys Asn Ala Asp Val260 265 270His Ser Pro Glu Arg Pro Arg Trp Pro Met Ile Ile Leu Arg Ser Pro275 280 285Lys Gly Trp Thr Gly Pro Lys Thr Val Asp Gly Leu Val Val Glu Asn290 295 300Tyr Trp Arg Ala His Glu Val Pro Val Ala Asn Cys Arg Glu Asn Asp305 310 315 320Ala His Arg Lys Ile Leu Glu Asp Trp Met Lys Ser Tyr Asp Pro Ser325 330 335Asp Leu Phe Asp Glu Lys Gly Ala Leu Lys Pro Glu Leu Arg Ala Leu340 345 350Ala Pro Lys Gly Glu Ala Arg Ile Gly Ala Asn Pro His Ala Asn Gly355 360 365Gly Leu Leu Arg Lys Glu Leu His Met Pro Asp Phe Arg Gln Tyr Ala370 375 380Val Asn Val Thr Glu Pro Gly Ala Ile Glu Ala Gln Ser Thr Lys Ile385 390 395 400Leu Gly Asp Phe Leu Arg Asp Val Met Lys Leu Asn Glu Thr Glu Lys405 410 415Asn Phe Arg Ile Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gly420 425 430Ser Val Leu Glu Ala Thr Asn Arg Val Trp Met Ala Glu Thr Leu Asp435 440 445Met Asp Asp His Leu Ala Ala Asp Gly Arg Val Met Glu Val Leu Ser450 455 460Glu His Leu Cys Gln Gly Trp Leu Glu Gly Tyr Leu Leu Ser Gly Arg465 470 475 480His Gly Phe Phe Ser Cys Tyr Glu Ala Phe Ile His Ile Ile Asp Ser485 490 495Met Phe Asn Gln His Ala Lys Trp Leu Gln Val Ala Arg Glu Leu Glu500 505 510Trp Arg Lys Pro Ile Ser Ser Leu Asn Tyr Leu Leu Thr Ser His Val
515 520 525Trp Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Phe Val530 535 540Asp Leu Val Ala Asn Lys Ser Ala Asp Ile Ala Arg Val Tyr Phe Pro545 550 555 560Pro Asp Ala Asn Thr Leu Leu Trp Val Gly Asp His Cys Leu Lys Thr565 570 575Trp Asn Arg Val Asn Val Ile Val Ala Gly Lys Gln Pro Glu Pro Gln580 585 590Trp Leu Thr Met Ala Glu Ala Glu Lys His Cys Glu Ala Gly Leu Gly595 600 605Ile Trp Glu Trp Ala Gly Thr Glu Asp Gly Leu Glu Pro Asp Ile Val610 615 620Met Ala Cys Ala Gly Asp Val Pro Thr Met Glu Thr Leu Ala Ala Val625 630 635 640Asp Leu Leu Arg Gln Ser Leu Pro His Leu Arg Ile Arg Val Val Asn645 650 655Val Val Asp Leu Met Val Leu Gln Ser Pro His Gln His Pro His Gly660 665 670Ile Ser Asp Glu Glu Phe Asp Arg Met Phe Thr Thr Asn Arg Pro Val675 680 685Ile Phe Ala Tyr His Gly Tyr Pro Tyr Leu Ile His Arg Leu Val Tyr690 695 700Lys Arg Thr Asn His Ser Asn Phe His Val Arg Gly Phe Ile Glu Gln705 710 715 720Gly Thr Thr Thr Thr Pro Phe Asp Met Thr Val Leu Asn Glu Leu Asp725 730 735Arg Phe His Leu Ala Met Glu Ala Val Glu Arg Leu Pro Leu Gly Glu740 745 750Ser Val Ala Lys Pro Leu Ile Asp Asn Phe Thr Glu Lys Leu Ala Leu755 760 765His Lys Asp Tyr Ile Arg Gln His Gly Glu Asp Met Pro Glu Ile Arg770 775 780Asp Trp Lys Trp Thr Trp Pro Arg785 790<210>80<211>2436<212>DNA<213>荚膜甲基球菌<220>
<221>CDS<222>(1)..(2436)<223>
<400>80atg gca acc caa ttc ccc ctg tcc tcc gaa ttc gaa cgg ctc acc gtc 48Met Ala Thr Gln Phe Pro Leu Ser Ser Glu Phe Glu Arg Leu Thr Val1 5 10 15tac ggg ccg acc cgc gcc act gtc agc gga aca ccg ctg gat gcc gaa 96Tyr Gly Pro Thr Arg Ala Thr Val Ser Gly Thr Pro Leu Asp Ala Glu20 25 30gaa gtc cgc aaa atc cat gcg ttc tgg cga gcc tgc aat tac ctg gcg144Glu Val Arg Lys Ile His Ala Phe Trp Arg Ala Cys Asn Tyr Leu Ala35 40 45ctg ggc atg atc tac ctg cgg ggg aat ccg ctg ctg cgt gaa ccg ctc192
Leu Gly Met Ile Tyr Leu Arg Gly Asn Pro Leu Leu Arg Glu Pro Leu50 55 60aaa ccc gag cac atc aag cac cgt ctg ctc ggc cat tgg ggc tcc agc 240Lys Pro Glu His Ile Lys His Arg Leu Leu Gly His Trp Gly Ser Ser65 70 75 80ccc aac ctt gcg ttc gtc tac acc cat ctg aac cgg gcc atc cgg aaa 288Pro Asn Leu Ala Phe Val Tyr Thr His Leu Asn Arg Ala Ile Arg Lys85 90 95cac gac ctc gac atg atc ttc atg gcc ggc ccc ggc cac ggc gcc ccc 336His Asp Leu Asp Met Ile Phe Met Ala Gly Pro Gly His Gly Ala Pro100 105 110ggc gtg ctg ggc ccg ctc tac ctg gaa ggc agc tac tcc gaa atc tac 384Gly Val Leu Gly Pro Leu Tyr Leu Glu Gly Ser Tyr Ser Glu Ile Tyr115 120 125ccc gac aag gac ctg agc gaa gaa ggc ctg ctg aac ttc ttc aag cag 432Pro Asp Lys Asp Leu Ser Glu Glu Gly Leu Leu Asn Phe Phe Lys Gln130 135 140ttc tcc ttc ccc ggc gga atc ggc agc cat tgc acc ccc gag aca ccg 480Phe Ser Phe Pro Gly Gly Ile Gly Ser His Cys Thr Pro Glu Thr Pro145 150 155 160ggc tcg atc cac gag ggc ggc gag ctg ggc tac gtg ctg tcg cac gcc 528Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Val Leu Ser His Ala165 170 175tgc ggc gca gcc ttc gac aac ccg gac ctg atc gtg gcc gcc gtg gtc 576Cys Gly Ala Ala Phe Asp Asn Pro Asp Leu Ile Val Ala Ala Val Val180 185 190ggc gac ggc gag gcg gaa acc ggc ccc ctg gcc act tcc tgg cac atc 624Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr Ser Trp His Ile195 200 205aac aaa ttt ctc aac ccg atc cgc gac ggc gcg gtg ctg ccg atc ctc 672Asn Lys Phe Leu Asn Pro Ile Arg Asp Gly Ala Val Leu Pro Ile Leu210 215 220aac ctc aac ggc tac aag atc aac aac ccc acc ctg ctg gcg cgc atc 720Asn Leu Asn Gly Tyr Lys Ile Asn Asn Pro Thr Leu Leu Ala Arg Ile225 230 235 240agc cac gaa gaa ttg gaa aat ctg ctc agg ggt tac ggc tac acg cct 768Ser His Glu Glu Leu Glu Asn Leu Leu Arg Gly Tyr Gly Tyr Thr Pro245 250 255tat ttc gtg gag ggc tcg gaa ccg gaa agc atg cac cag gcg atg gcc 816Tyr Phe Val Glu Gly Ser Glu Pro Glu Ser Met His Gln Ala Met Ala260 265 270gcg acg gtg gac cgc agc atc gaa gac atc cgc gcc gcc cag acc gag 864Ala Thr Val Asp Arg Ser Ile Glu Asp Ile Arg Ala Ala Gln Thr Glu275 280 285gcc cgc gcc agc ggc atc gcc cga cgg ccg cgc tgg ccg atg atc gtg 912Ala Arg Ala Ser Gly Ile Ala Arg Arg Pro Arg Trp Pro Met Ile Val290 295 300ctg cgc tcg ccc aag ggc tgg acc gcg cca cgc cag atc gac ggc cac 960Leu Arg Ser Pro Lys Gly Trp Thr Ala Pro Arg Gln Ile Asp Gly His305 310 315 320aac gtc gag ggc ttc tgg cgc gct cat cag gtg ccg gta gcc gac gtc1008Asn Val Glu Gly Phe Trp Arg Ala His Gln Val Pro Val Ala Asp Val325 330 335gcg aaa aat ccc gaa cac ctg aag ctg ctg gaa ggc tgg atg cgc agc1056Ala Lys Asn Pro Glu His Leu Lys Leu Leu Glu Gly Trp Met Arg Ser340 345 350tac aag ccg gaa gaa ctg ttc gac gcc gaa ggc tgc ccc gtc gcc gaa1104Tyr Lys Pro Glu Glu Leu Phe Asp Ala Glu Gly Cys Pro Val Ala Glu
355 360 365atc cgg gaa atg gcg ccg gcc ggt ctc cgc cgc atg ggt ctc aac ccc1152Ile Arg Glu Met Ala Pro Ala Gly Leu Arg Arg Met Gly Leu Asn Pro370 375 380cac gcc aac ggc ggc cat ctg aaa aag gcc ctg cgc atc cct aat ttc1200His Ala Asn Gly Gly His Leu Lys Lys Ala Leu Arg Ile Pro Asn Phe385 390 395 400cgc aac tac ggc atc gaa gtc gcc aaa ccg ggc cag atc gaa gcg ccg1248Arg Asn Tyr Gly Ile Glu Val Ala Lys Pro Gly Gln lle Glu Ala Pro405 410 415aac acc cag ccg ctg ggc gtg ttc ctg cgc gac gtg atg aag gag aac1296Asn Thr Gln Pro Leu Gly Val Phe Leu Arg Asp Val Met Lys Glu Asn420 425 430gcg cac aac ttc cgg ctg ttc ggc cct gac gag aat acc tcc aac aag1344Ala His Asn Phe Arg Leu Phe Gly Pro Asp Glu Asn Thr Ser Asn Lys435 440 445ctg gac gcc gtc tac gcc gct gcc aag aaa ttc tgg atc gcc gag tat1392Leu Asp Ala Val Tyr Ala Ala Ala Lys Lys Phe Trp Ile Ala Glu Tyr450 455 460ttc ccc gaa gac cag gat ggc ggc gaa ctg gcc ccc gac ggc cgg gtc1440Phe Pro Glu Asp Gln Asp Gly Gly Glu Leu Ala Pro Asp Gly Arg Val465 470 475 480atg gaa atg ctc agc gag cat acc ctg gaa ggc atg ctg gaa ggc tac1488Met Glu Met Leu Ser Glu His Thr Leu Glu Gly Met Leu Glu Gly Tyr485 490 495ctt ctg acc ggg cgc cac ggc ttc ctc tcg acc tac gaa gcc ttc gtc1536Leu Leu Thr Gly Arg His Gly Phe Leu Ser Thr Tyr Glu Ala Phe Val500 505 510cac gtc atc gat tcg atg ttc aac cag cac gcc aag tgg ctg tcc atc1584His Val Ile Asp Ser Met Phe Asn Gln His Ala Lys Trp Leu Ser Ile515 520 525tgc aac cag ctg tcc tgg cgc cag gac gtg gcc tcg ctc aac ctg ctc1632Cys Asn Gln Leu Ser Trp Arg Gln Asp Val Ala Ser Leu Asn Leu Leu530 535 540atc act tcg acg gtc tgg cga cag gac cac aac ggc ttc acc cac cag1680Ile Thr Ser Thr Val Trp Arg Gln Asp His Asn Gly Phe Thr His Gln545 550 555 560gac ccc ggt ttc ctg gac gtc gtc gtg aac aag agc gcc gac gtc acc1728Asp Pro Gly Phe Leu Asp Val Val Val Asn Lys Ser Ala Asp Val Thr565 570 575cgc atc tac ctg ccg ccc gac gtc aac agc ctg ctg tcg gtc gcc gac1776Arg Ile Tyr Leu Pro Pro Asp Val Asn Ser Leu Leu Ser Val Ala Asp580 585 590cac tgc ctg cgc agc cag aac tac atc aac gtg atc gtg tcg gac aaa1824His Cys Leu Arg Ser Gln Asn Tyr Ile Asn Val Ile Val Ser Asp Lys595 600 605cag ttg cat ctg cag ttc atg gac atg gac gcc gcg atc gcc cat tgc1872Gln Leu His Leu Gln Phe Met Asp Met Asp Ala Ala Ile Ala His Cys610 615 620acg gag ggc ttg ggc atc tgg gaa tgg gcc agc aac gac gag ggc cag1920Thr Glu Gly Leu Gly Ile Trp Glu Trp Ala Ser Asn Asp Glu Gly Gln625 630 635 640gag ccc gac gtg gtc atg gcc tgc gcc ggc gac atc ccg acc ctg gaa1968Glu Pro Asp Val Val Met Ala Cys Ala Gly Asp Ile Pro Thr Leu Glu645 650 655gcc ctg gcc gcc acc gcc ctg ctg cgc gag gag ttc ccg gaa ctg aag2016Ala Leu Ala Ala Thr Ala Leu Leu Arg Glu Glu Phe Pro Glu Leu Lys660 665 670
atc cgc ttc atc aac gtg gtg gat ctg ttc aag ctg cag ccc gag tcc2064Ile Arg Phe Ile Asn Val Val Asp Leu Phe Lys Leu Gln Pro Glu Ser675 680 685gag cat ccg cac ggt ctc agc gac aag gat ttc gac agc ctg ttc acc2112Glu His Pro His Gly Leu Ser Asp Lys Asp Phe Asp Ser Leu Phe Thr690 695 700agg gac aag ccg gtc atc ttc aac ttc cac ggc tat ccc tgg ctg atc2160Arg Asp Lys Pro Val Ile Phe Asn Phe His Gly Tyr Pro Trp Leu Ile705 710 715 720cac cgc ctc gcc tac cgg cgc acc aac cac gcc aac atg cac gtg cgc2208His Arg Leu Ala Tyr Arg Arg Thr Asn His Ala Asn Met His Val Arg725 730 735ggc tac aag gag aaa ggc aac atc aac acc ccg ctg gaa ctg gcg atc2256Gly Tyr Lys Glu Lys Gly Asn Ile Asn Thr Pro Leu Glu Leu Ala Ile740 745 750aac aac cag atc gac cgc ttc agc ctg gcc atc gac gtc atc gac cgg2304Asn Asn Gln Ile Asp Arg Phe Ser Leu Ala Ile Asp Val Ile Asp Arg755 760 765att ccg gag atc gcg gtc tcc ggc gcc cac gcc aag gcc cgg ttc cgc2352Ile Pro Glu Ile Ala Val Ser Gly Ala His Ala Lys Ala Arg Phe Arg770 775 780aaa cag cag atc gcc tgc cgc cag tat gcc tac gag cac ggc gtc gac2400Lys Gln Gln Ile Ala Cys Arg Gln Tyr Ala Tyr Glu His Gly Val Asp785 790 795 800atg ccg gag gtc gcg ggc tgg cgc tgg ccg ggc tga2436Met Pro Glu Val Ala Gly Trp Arg Trp Pro Gly805 810<210>81<211>811<212>PRT<213>荚膜甲基球菌<400>81Met Ala Tht Gln Phe Pro Leu Ser Ser Glu Phe Glu Arg Leu Thr Val1 5 10 15Tyr Gly Pro Thr Arg Ala Thr Val Ser Gly Thr Pro Leu Asp Ala Glu20 25 30Glu Val Arg Lys Ile His Ala Phe Trp Arg Ala Cys Asn Tyr Leu Ala35 40 45Leu Gly Met Ile Tyr Leu Arg Gly Asn Pro Leu Leu Arg Glu Pro Leu50 55 60Lys Pro Glu His Ile Lys His Arg Leu Leu Gly His Trp Gly Ser Ser65 70 75 80Pro Asn Leu Ala Phe Val Tyr Thr His Leu Asn Arg Ala Ile Arg Lys85 90 95His Asp Leu Asp Met Ile Phe Met Ala Gly Pro Gly His Gly Ala Pro100 105 110Gly Val Leu Gly Pro Leu Tyr Leu Glu Gly Ser Tyr Ser Glu Ile Tyr115 120 125Pro Asp Lys Asp Leu Ser Glu Glu Gly Leu Leu Asn Phe Phe Lys Gln130 135 140Phe Ser Phe Pro Gly Gly Ile Gly Ser His Cys Thr Pro Glu Thr Pro145 150 155 160Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Val Leu Ser His Ala165 170 175Cys Gly Ala Ala Phe Asp Asn Pro Asp Leu Ile Val Ala Ala Val Val
180 185 190Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr Ser Trp His Ile195 200 205Asn Lys Phe Leu Asn Pro Ile Arg Asp Gly Ala Val Leu Pro Ile Leu210 215 220Asn Leu Asn Gly Tyr Lys Ile Asn Asn Pro Thr Leu Leu Ala Arg Ile225 230 235 240Ser His Glu Glu Leu Glu Asn Leu Leu Arg Gly Tyr Gly Tyr Thr Pro245 250 255Tyr Phe Val Glu Gly Ser Glu Pro Glu Ser Met His Gln Ala Met Ala260 265 270Ala Thr Val Asp Arg Ser Ile Glu Asp Ile Arg Ala Ala Gln Thr Glu275 280 285Ala Arg Ala Ser Gly Ile Ala Arg Arg Pro Arg Trp Pro Met Ile Val290 295 300Leu Arg Ser Pro Lys Gly Trp Thr Ala Pro Arg Gln Ile Asp Gly His305 310 315 320Asn Val Glu Gly Phe Trp Arg Ala His Gln Val Pro Val Ala Asp Val325 330 335Ala Lys Asn Pro Glu His Leu Lys Leu Leu Glu Gly Trp Met Arg Ser340 345 350Tyr Lys Pro Glu Glu Leu Phe Asp Ala Glu Gly Cys Pro Val Ala Glu355 360 365Ile Arg Glu Met Ala Pro Ala Gly Leu Arg Arg Met Gly Leu Asn Pro370 375 380His Ala Asn Gly Gly His Leu Lys Lys Ala Leu Arg Ile Pro Asn Phe385 390 395 400Arg Asn Tyr Gly Ile Glu Val Ala Lys Pro Gly Gln Ile Glu Ala Pro405 410 415Asn Thr Gln Pro Leu Gly Val Phe Leu Arg Asp Val Met Lys Glu Asn420 425 430Ala His Asn Phe Arg Leu Phe Gly Pro Asp Glu Asn Thr Ser Asn Lys435 440 445Leu Asp Ala Val Tyr Ala Ala Ala Lys Lys Phe Trp Ile Ala Glu Tyr450 455 460Phe Pro Glu Asp Gln Asp Gly Gly Glu Leu Ala Pro Asp Gly Arg Val465 470 475 480Met Glu Met Leu Ser Glu His Thr Leu Glu Gly Met Leu Glu Gly Tyr485 490 495Leu Leu Thr Gly Arg His Gly Phe Leu Ser Thr Tyr Glu Ala Phe Val500 505 510His Val Ile Asp Ser Met Phe Asn Gln His Ala Lys Trp Leu Ser Ile515 520 525Cys Asn Gln Leu Ser Trp Arg Gln Asp Val Ala Ser Leu Asn Leu Leu530 535 540Ile Thr Ser Thr Val Trp Arg Gln Asp His Asn Gly Phe Thr His Gln545 550 555 560Asp Pro Gly Phe Leu Asp Val Val Val Asn Lys Ser Ala Asp Val Thr565 570 575Arg Ile Tyr Leu Pro Pro Asp Val Asn Ser Leu Leu Ser Val Ala Asp580 585 590His Cys Leu Arg Ser Gln Asn Tyr Ile Asn Val Ile Val Ser Asp Lys595 600 605Gln Leu His Leu Gln Phe Met Asp Met Asp Ala Ala Ile Ala His Cys610 615 620Thr Glu Gly Leu Gly Ile Trp Glu Trp Ala Ser Asn Asp Glu Gly Gln625 630 635 640Glu Pro Asp Val Val Met Ala Cys Ala Gly Asp Ile Pro Thr Leu Glu
645 650 655Ala Leu Ala Ala Thr Ala Leu Leu Arg Glu Glu Phe Pro Glu Leu Lys660 665 670Ile Arg Phe Ile Asn Val Val Asp Leu Phe Lys Leu Gln Pro Glu Ser675 680 685Glu His Pro His Gly Leu Ser Asp Lys Asp Phe Asp Ser Leu Phe Thr690 695 700Arg Asp Lys Pro Val Ile Phe Asn Phe His Gly Tyr Pro Trp Leu Ile705 710 715 720His Arg Leu Ala Tyr Arg Arg Thr Asn His Ala Asn Met His Val Arg725 730 735Gly Tyr Lys Glu Lys Gly Asn Ile Asn Thr Pro Leu Glu Leu Ala Ile740 745 750Asn Asn Gln Ile Asp Arg Phe Ser Leu Ala Ile Asp Val Ile Asp Arg755 760 765Ile Pro Glu Ile Ala Val Ser Gly Ala His Ala Lys Ala Arg Phe Arg770 775 780Lys Gln Gln Ile Ala Cys Arg Gln Tyr Ala Tyr Glu His Gly Val Asp785 790 795 800Met Pro Glu Val Ala Gly Trp Arg Trp Pro Gly805 810
权利要求
1.具有生产有用代谢物的能力的细菌，其中所述细菌被修饰以具有增加的D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶活性。
2.根据权利要求1的细菌，其中所述有用代谢物源自乙酰辅酶A。
3.根据权利要求1的细菌，其中所述有用代谢物选自由L-谷氨酸、L-谷氨酰胺、L-脯氨酸、L-精氨酸、L-亮氨酸、L-半胱氨酸、琥珀酸酯和聚羟基丁酸酯构成的组。
4.根据权利要求1的细菌，其中所述细菌天然不具有D-木酮糖-5-磷酸磷酸转酮酶和果糖-6-磷酸磷酸转酮酶的活性，并且其中所述细菌用编码D-木酮糖-5-磷酸磷酸转酮酶和/或果糖-6-磷酸磷酸转酮酶的DNA片段转化。
5.根据权利要求1的细菌，其中所述细菌被进一步修饰以具有减少的6-磷酸果糖激酶的活性。
6.根据权利要求1的细菌，其中所述细菌选自由肠杆菌科、棒状杆菌型细菌和芽孢杆菌属细菌构成的组。
7.根据权利要求1的细菌，其中所述细菌属于埃希氏菌属或泛菌属。
8.生产有用代谢物的方法，包括在培养基中培养根据权利要求1至7中任一项的细菌，并从所述培养基和/或所述细菌收集所述有用代谢物。
9.根据权利要求8的方法，其中所述有用代谢物源自乙酰辅酶A。
10.根据权利要求9的方法，其中所述有用代谢物选自由L-谷氨酸、L-谷氨酰胺、L-脯氨酸、L-精氨酸、L-亮氨酸、L-半胱氨酸、琥珀酸酯和聚羟基丁酸酯构成的组。
全文摘要
本发明提供了具有生产有用代谢物的能力的细菌，所述有用代谢物源自乙酰辅酶A，如L－谷氨酸、L－谷氨酰胺、L－脯氨酸、L－精氨酸、L－亮氨酸、L－半胱氨酸、琥珀酸酯和聚羟基丁酸酯，其中所述细菌被修饰从而增强D－木酮糖－5－磷酸磷酸转酮酶和/或果糖－6－磷酸磷酸转酮酶的活性。本发明还提供了使用所述细菌生产有用代谢物的方法。
文档编号C12P13/12GK101090964SQ200580027259
公开日2007年12月19日 申请日期2005年8月10日 优先权日2004年8月10日
发明者尤里·I·科兹洛夫, 知念秋人, 泉井裕, 原吉彦, 安枝寿, 康斯坦廷·V·赖巴克, 埃卡特里纳·A·斯利文斯卡亚, 乔安娜·Y·凯塔什基纳 申请人:味之素株式会社