專利名稱:改進的酶的制作方法
技術領域:
本發(fā)明提供了經(jīng)修飾的酶,其具有比相應的野生型酶更高的II型GTP環(huán)化水解酶活性。所述經(jīng)修飾的酶和編碼它的多核苷酸可被用于生產核黃素、核黃素前體、黃素單核苷酸(FMN)、黃素腺嘌呤二核苷酸(FAD)及其衍生物。
所有植物及很多微生物都合成核黃素(維生素B2),但其不被高等動物生產。因為其是碳水化合物酶促氧化所需的輔酶(例如黃素單核苷酸和黃素腺嘌呤二核苷酸)的前體,因此核黃素對于基礎代謝來說是必不可少的。在高等動物中,核黃素不足會導致脫發(fā)、皮膚炎癥、視力退化以及生長障礙。
過去已經(jīng)以多種不同的方式獲得了對具有提高的核黃素比例(rate)和產量的核黃素生產菌株的工程化。例如,(1)經(jīng)典誘變被用于產生在選用生物的基因組中具有隨機突變的變體,接著通過針對嘌呤類似物的更高抗性進行選擇和/或針對核黃素生產增加進行篩選。(2)或者,核黃素生物合成的終點酶,即催化鳥苷三磷酸酯(GTP)和核酮糖-5-磷酸酯向核黃素轉化的酶,被過量表達,也導致向目標產物的更高通量。但是,在后一種手段中,核黃素生物合成蛋白的強烈過量表達對宿主細胞造成了額外的代謝負擔,其可能隨之誘導脅迫應答反應以及其它不利的對細胞生理的副作用。
催化從鳥苷三磷酸酯(GTP)和核酮糖-5-磷酸酯生物合成核黃素所需的酶在B.substilis中由四個基因(ribG、ribB、ribA和ribH)編碼。這些酶定位于操縱子中,其基因順序不同于這些酶催化的酶反應的順序。例如,催化核黃素生物合成中第一個步驟的II型GTP環(huán)狀水解酶,由操縱子中的第三個基因ribA編碼。ribA基因還編碼第二種酶活性,即,3,4-二羥基-2-丁酮-4-磷酸酯合酶(DHBPS),其催化核酮糖-5-磷酸酯向四碳單元3,4-二羥基-2-丁酮-4-磷酸酯(DHBP)的轉化。脫氨酶和還原酶是操縱子的第一個基因ribG所編碼的。核黃素生物合成中的倒數(shù)第二個步驟是2,4-二氧四氫蝶啶合酶催化的,該酶是最后一個rib基因ribH的產物??刂圃撏緩阶詈笠徊降暮它S素合酶由操縱子的第二個基因ribB所編碼。位于rib操縱子3’末端的基因的功能目前還不清楚;但是該基因的產物不是核黃素合成所需要的。
核黃素操縱子從ribPl啟動子的轉錄由弱化機制所控制,該機制涉及位于ribPl和ribG之間的調控引導區(qū)域。該引導區(qū)域中ribO突變導致核黃素操縱子的表達被負調(deregulate)。在含有ribC基因中的錯義突變的菌株中也能觀察到被負調的表達。ribC基因還顯示出能編碼B.subtilis的黃素激酶/FAD合酶(Mack,M.,et al.,J.Bacteriol.,180950-955,1998)。負調突變降低了ribC基因產物的黃素激酶活性,導致黃素單核苷酸(FMN)(核黃素調控體系的效應器分子)的細胞內濃度減少。
近來,Bacillus subtilis被遺傳改造為能在短發(fā)酵周期生產高產量的核黃素(美國專利5,837,528)。該手段將對經(jīng)典遺傳突變選擇和發(fā)酵的改進與通過負調和增加基因表達水平對核黃素生物合成基因進行的遺傳工程組合起來。在該系統(tǒng)中,通過突變黃素激酶編碼ribC基因、通過將rib基因與強且組成型的啟動子連接以及通過增加rib基因的拷貝數(shù)來增加rib基因的表達。
如已討論的,rib基因的過量表達對生產菌株施加了額外的負擔,其可能對核黃素前體、核黃素、FMN、FAD或它們的衍生物的生產造成負面影響。為了避免該缺點,本發(fā)明的一個目的是提供具有提高的比活性的II型GTP環(huán)化水解酶突變體。在生產菌株中單獨使用這些突變酶或將其與其它Rib蛋白的改進突變體組合使用,將允許更高的通量率,同時對細胞代謝的額外負擔更少或沒有。
本文中使用的術語“II型GTP環(huán)化水解酶”可包括能催化GTP向2,5-二氨基-6-核糖氨-4(3H)-嘧啶酮-5’-磷酸酯(DRAPP)的轉化的任何酶。該酶是否能催化進一步的反應,例如核酮糖-5-磷酸酯向DHBP的轉化是不相干的?!癐I型GTP環(huán)化水解酶”可與
圖1或表4所示的氨基酸序列的一種或多種酶同源?!巴础敝概c圖1或表4所示的氨基酸序列中的一種或多種至少大約50%相同,優(yōu)選至少大約60%相同,更優(yōu)選至少大約70%相同,進一步更優(yōu)選至少大約80%相同,進一步更優(yōu)選至少大約85%相同,進一步更優(yōu)選至少大約90%或95%相同,以及最優(yōu)選至少大約98%相同的II型GTP環(huán)化水解酶。
術語“%相同”如在本領域已知的一樣,表示多肽或多核苷酸序列之間的相關性,可以是通過對此類序列的鏈之間進行匹配來測定的?!跋嗤浴笨捎靡阎椒ㄈ菀椎販y定,例如,用程序BESTFIT(GCG WisconsinPackage,version 10.2,Accelrys Inc.,9685 Scranton Road,San Diego,CA92121-3752,USA)來進行,其中使用下述參數(shù)缺口產生懲罰8,缺口延伸懲罰2(缺省參數(shù))。
“野生型酶”或“野生型II型GTP環(huán)化水解酶”可以包括與圖l或表4所示的任意一種酶同源的任何II型GTP環(huán)化水解酶,其可用作為起始點,用于設計根據(jù)本發(fā)明的具有提高的活性的菌株。本發(fā)明上下文中的“野生型”可以包括可從自然界獲得的II型GTP環(huán)化水解酶序列,以及合成的II型GTP環(huán)化水解酶變體(只要它們與圖1或表4所示序列中任意一條同源),如果它們可通過本發(fā)明的任何教導具有更高活性的話。術語“野生型II型GTP環(huán)化水解酶”和“未經(jīng)修飾的II型GTP環(huán)化水解酶”在本文中可以替代使用。
“突變體”、“突變體酶”或“突變體GTP環(huán)化水解酶II”可以包括可根據(jù)本發(fā)明的教導從給定的野生型酶/II型GTP環(huán)化水解酶(根據(jù)上文定義)獲得的、且比相應的野生型酶具有更高活性的任何變體。就本發(fā)明的范圍而言,其與突變體是如何獲得的并不相關;此類突變體可例如通過定點誘變、飽和誘變、隨機誘變/定向進化、對整個細胞/生物體的化學或UV誘變和本領域已知的其它方法來獲得。這些突變體還可以例如通過設計合成基因來產生和/或通過體外(不含細胞的)翻譯來生產。為測試比活性,可通過本領域技術人員已知的方法來表達突變體。術語“突變體II型GTP環(huán)化水解酶”和“經(jīng)修飾的II型GTP環(huán)化水解酶”在本文中可互換使用。這還應用于術語“突變體酶”和“經(jīng)修飾的酶”。
“核黃素前體”和“核黃素衍生物、FMN或FAD”在本專利申請的上下文中可以包括在它們的(生物)合成中需要II型GTP環(huán)化水解酶作為中間產物酶的任何和所有代謝物。在本專利申請的上下文中,此類(生物)合成途徑是天然的或非天然的(即,不是天然存在的途徑,而是通過生物技術工程改造的)并不相干。優(yōu)選地,該合成途徑是自然界中生物化學途徑。核黃素前體和核黃素衍生物、FMN或FAD包括但不限于DRAPP;5-氨基-6-核糖氨-2,4(1H,3H)-嘧啶二酮-5’-磷酸酯;2,5-二氨基-6-核糖醇氨基(ribitylamino)-4-(3H)-嘧啶酮-5’-磷酸酯;5-氨基-6-核糖醇氨基-2,4(1H,3H)-嘧啶二酮-5’-磷酸酯;5-氨基-6-核糖醇氨基-2,4(1H,3H)-嘧啶二酮;6,7-二甲基-8-核糖醇二氧四氫蝶啶(DMRL);以及黃素蛋白。術語“核黃素”還包括核黃素衍生物,例如,核黃素-5-磷酸酯及其鹽,例如,核黃素-5-磷酸鈉。
一般而言,本發(fā)明的一個目的是提供具有II型GTP環(huán)化水解酶活性的酶,所述酶被修飾為其催化性質較之未經(jīng)修飾的II型GTP環(huán)化水解酶更有利(即,展示出更高的比活性)。
本發(fā)明涉及一種經(jīng)修飾的II型GTP環(huán)化水解酶,其較之相應的未經(jīng)修飾的II型GTP環(huán)化水解酶展示出更高的(比)活性,其中,(i)經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列較之相應未經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列含有至少一處突變,以及(ii)所述至少一處突變位于下述位置,所述位置選自由對應于SEQID NO2所示的Bacillus subtilis II型GTP環(huán)化水解酶氨基酸序列的第261、270、276、279、308和347位的氨基酸位置構成的組。
因此,本發(fā)明的一個目的是提供一種經(jīng)修飾的II型GTP環(huán)化水解酶,其中(i)所述經(jīng)修飾的酶較之相應未經(jīng)修飾的酶的比活性增加,以及(ii)所述經(jīng)修飾的酶的氨基酸序列包含一處或多處突變,所述突變包括在對應于SEQ ID NO2的261、270、276、279、308和/或347位的氨基酸位置上的1、2、3、4、5或6處突變。
術語“至少一處突變”指在上文定義的位置的、導致產生較之未經(jīng)修飾的酶具有提高的比活性的經(jīng)修飾II型GTP環(huán)化水解酶II的一處或多處突變。上文所述的經(jīng)修飾的酶可由在上文所定義的位置的、導致產生較之未經(jīng)修飾的酶的提高比活性的僅1、2、3、4、5或6處突變構成,但是也可包括在其它位置的其它氨基酸突變,只要得到的經(jīng)修飾的酶具有增加的比活性即可。因此,經(jīng)修飾的酶包括一處或多處突變,所述突變包括對應于SEQ ID NO2所示的Bacillus subtilis II型GTP環(huán)化水解酶氨基酸序列的第261、270、276、279、308和/或347位的氨基酸位置上的1、2、3、4、5或6處突變。在上文定義的這些位置之外的位置上的突變的例子是對應于SEQ ID NO2的196、282和/或325位氨基酸位置的位置上的氨基酸突變。
本文中使用的術語“比活性”指在適宜給定的反應條件下,例如Ritzet al.(J.Biol.Chem.276,22273-22277,2001)、Koh et al.(Mol.Gen.Genet.251,591-598,1996)或Schramek et al.(J.Biol.Chem.276,44157-44162,2001)中所述的,或在實施例2中詳細描述的反應條件下,野生型和突變體II型GTP環(huán)化水解酶的反應速率?!氨然钚浴倍x為給定溫度下每給定量的蛋白在給定的時間段產生的產物和/或消耗的底物的量。典型地,“比活性”表示為每分鐘每mg蛋白形成的μmol產物或消耗的μmol底物。典型地,μmol/分鐘被簡寫為U(=Unit)。因此,針對μmol/分鐘/(mg蛋白)或U/(mg蛋白)的比活性的單位定義在本文中可互換使用。
應當理解,在本發(fā)明的上下文中,比活性應當在相似,或者優(yōu)選相同長度的多肽鏈的基礎上進行比較。增加給定的野生型酶的長度,例如形成融合蛋白,由此降低總體酶的表觀比活性,并不能繞開本發(fā)明。
根據(jù)本發(fā)明,經(jīng)修飾的II型GTP環(huán)化水解酶展示出比對應的未經(jīng)修飾的酶要高的比活性。優(yōu)選地,較之對應的未經(jīng)修飾的II型GTP環(huán)化水解酶,本發(fā)明的經(jīng)修飾的II型GTP環(huán)化水解酶的比活性增加了至少大約5、10、25、40、60、70、80、85、90%,更優(yōu)選地,至少大約70%(關于比活性測量,見下文)。優(yōu)選地,比活性的增加對應本申請實施例1所述的實驗條件。試驗混合物中存在大約0.004-0.02U/ml(對應大約40μg/ml Bacillus subtilis II型GTP環(huán)化水解酶或20μg/ml本文所述的最優(yōu)突變體),優(yōu)選大約0.004U/ml II型GTP環(huán)化水解酶活性,反應在37℃進行。
較之對應的未經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列,本發(fā)明的經(jīng)修飾II型GTP環(huán)化水解酶的氨基酸序列具有上文定義的至少一處突變。所述突變可以是一處或多處添加、缺失和/或取代,優(yōu)選地,一處或多處取代,其中,未經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列中存在的給定氨基酸在本發(fā)明的經(jīng)修飾的II型GTP環(huán)化水解酶中被不同的氨基酸替換。經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列較之對應的未經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列可以含有至少一處氨基酸取代,即,可以包含一處或多處突變,包括對應于SEQ ID NO2的第261、270、276、279、308和/或347位的氨基酸位置上的1、2、3、4、5或6處氨基酸取代,優(yōu)選地,2、3、4或5處氨基酸取代。因此,經(jīng)修飾的酶較之對應的未經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列優(yōu)選含有至少2、至少3、至少4或至少5處取代。
在一種實施方式中,提供了可從Bacillus(優(yōu)選Bacillus subtilis)獲得的經(jīng)修飾的II型GTP環(huán)化水解酶,其中(i)所述經(jīng)修飾的酶較之相應未經(jīng)修飾的酶的比活性增加,以及(ii)所述經(jīng)修飾的酶的氨基酸序列包含一處或多處突變,所述突變包括在對應于SEQ ID NO2的261、270、276、279、308和/或347位的氨基酸位置上的1、2、3、4、5或6處突變。
在一種實施方式中,未經(jīng)修飾的酶對應SEQ ID NO2所示的Bacillussubtilis II型GTP環(huán)化水解酶。因此,較之野生型酶具有增加的比活性的經(jīng)修飾的酶包含一處或多處突變,所述突變包括在對應于SEQ ID NO2的261、270、276、279、308和/或347位的氨基酸位置上的1、2、3、4、5或6處突變。在另一種實施方式中,具有增加的上文定義的比活性的經(jīng)修飾的酶除了上述氨基酸位置之外,還含有其它氨基酸突變,所述其它突變在選自由第196、282、235位或其任意組合組成的組的位置上,優(yōu)選地,含有氨基酸取代,更優(yōu)選地,所述取代是Y196C(半胱氨酸代替酪氨酸)、A282T(蘇氨酸代替丙氨酸)或F325Y(酪氨酸代替苯丙氨酸)。
未經(jīng)修飾的II型GTP環(huán)化水解酶可以是任何這樣的II型GTP環(huán)化水解酶,對其來說增加比活性是人們所想要的。未經(jīng)修飾的II型GTP環(huán)化水解酶包括但不限于從自然界獲得的II型GTP環(huán)化水解酶,例如真核或原核來源的酶,優(yōu)選地,真菌或細菌來源的。更優(yōu)選地,未經(jīng)修飾的酶選自圖1或表4所示的那些或者與圖1或表4所示的氨基酸序列中任意一條同源,尤其選自Ashbya、Saccharomyces、Eremothecium、Candida、Neurospora、Schizosaccharomyces、Archeoglobus、Streptomyces、Helicobacter、Escherichia、Corynebacterium、Thermotoga、Arabidopsis、Lycopersicum、Oryza、Alcaligenes、Pseudomonas、Dinococcus、Lactobacillus、Photobacterium和Bacillus構成的組,優(yōu)選選自Candidaguilliermondii、Ashbya gossypii(Eremothecium ashbyii)(SEQ IDNO33)、Saccharomyces cerevisiae、Neurospora crassa、Schizosaccharomyces pombe、Archeoglobus fulgidus、Streptomycescoelicolor、Helicobacter pylori J99、Escherichia coli(SEQ ID NO35)、Corynebacterium glutamicum(SEQ ID NO37)、Bacillus amyloliquefaciens(SEQ ID NO39)、Bacillus cereus(SEQ ID NO41)、Bacillushalodurans(SEQ ID NO43)、Thermotoga maritima、Arabidopsisthaliana、Lycopersicum exculentum、Oryza sativum、Alcaligeneseutrophus、Pseudomonas putida菌株KT2440、Corynebacterium efficiens、Deinococcus radiodurans、Lactobacillus plantarum、Photobacteriumphosphoreum、Pseudomonas putida菌株KT2440(第二個基因)和Bacillussubtilis(SEQ ID NO2)構成的組。最優(yōu)選的未經(jīng)修飾的酶可從Bacillussubtilis獲得。
本發(fā)明的經(jīng)修飾的II型GTP環(huán)化水解酶可通過對對應的未經(jīng)修飾的II型GTP環(huán)化水解酶進行突變來獲得。在一種實施方式中,未經(jīng)修飾的酶對應SEQ ID NO2所示的B.subtilis II型GTP環(huán)化水解酶,經(jīng)修飾的酶包含一處或多處突變,所述突變包括在對應于SEQ ID NO2的261、270、276、279、308和/或347位氨基酸位置上的1、2、3、4、5或6處突變,其中,所述經(jīng)修飾的酶的比活性較之未經(jīng)修飾的酶有所增加。
優(yōu)選地,所述至少一處突變位于下述一處或多處氨基酸位置,所述位置選自由對應于SEQ ID NO2所示的Bacillus subtilis II型GTP環(huán)化水解酶氨基酸序列的第261、279、308和347位的氨基酸位置構成的組。因此,在一種實施方式中,經(jīng)修飾的II型GTP環(huán)化水解酶包含一處或多處突變,所述突變包括在對應于SEQ ID NO2的261、279、308和/或347位氨基酸位置上的1、2、3或4處突變。在一種優(yōu)選的實施方式中,經(jīng)修飾的酶可從Bacillus subtilis獲得,其包含突變的SEQ ID NO2所示的第261、279、308和/或347位氨基酸,分別對應氨基酸V261、Q279、K308和M374。
在另一種優(yōu)選的實施方式中,所述至少一處突變位于下述一處或多處氨基酸位置,所述位置選自由對應于SEQ ID NO2所示的Bacillussubtilis II型GTP環(huán)化水解酶氨基酸序列的第270、279、308和347位的氨基酸位置構成的組。因此,在一種實施方式中,經(jīng)修飾的II型GTP環(huán)化水解酶包含一處或多處突變,所述突變包括在對應于SEQ ID NO2的270、279、308和/或347位氨基酸位置上的1、2、3或4處突變。優(yōu)選地,經(jīng)修飾的酶可從Bacillus subtilis獲得,其包含突變的SEQ ID NO2所示的第270、279、308和/或347位氨基酸,分別對應氨基酸G270、Q279、K308和M374。
在另一種優(yōu)選的實施方式中,所述至少一處突變位于下述一處或多處氨基酸位置,所述位置選自由對應于SEQ ID NO2所示的Bacillussubtilis II型GTP環(huán)化水解酶氨基酸序列的第276、279、308和347位的氨基酸位置構成的組。因此,在一種實施方式中,經(jīng)修飾的II型GTP環(huán)化水解酶包含一處或多處突變,所述突變包括在對應于SEQ ID NO2的276、279、308和/或347位的氨基酸位置上的1、2、3或4處突變。優(yōu)選地,經(jīng)修飾的酶可從Bacillus subtilis獲得,其包含突變的SEQ ID NO2所示的第276、79、308和/或347氨基酸,分別對應氨基酸A276、Q279、K308和M374。
優(yōu)選地,經(jīng)修飾的II型GTP環(huán)化水解酶的一處或多處氨基酸突變是一處或多處氨基酸取代。
經(jīng)修飾的II型GTP環(huán)化水解酶可以包含一處或多處突變,所述突變包括上文定義的氨基酸位置上的僅一處突變,此類突變(特別是氨基酸取代)可以包括在對應于SEQ ID NO2所示的Bacillus subtilis II型GTP環(huán)化水解酶氨基酸序列的第261、270、276、279、308或347位的氨基酸位置上的一處突變。存在于未經(jīng)修飾的II型GTP環(huán)化水解酶中對應于261位的氨基酸可以是纈氨酸,存在于未經(jīng)修飾的II型GTP環(huán)化水解酶中對應于270位的氨基酸可以是甘氨酸,存在于未經(jīng)修飾的II型GTP環(huán)化水解酶中對應于276位的氨基酸可以是丙氨酸,存在于未經(jīng)修飾的II型GTP環(huán)化水解酶中對應于279位的氨基酸可以是谷氨酰胺,存在于未經(jīng)修飾的II型GTP環(huán)化水解酶中對應于308位的氨基酸可以是賴氨酸,存在于未經(jīng)修飾的II型GTP環(huán)化水解酶中對應于347位的氨基酸可以是甲硫氨酸。
未經(jīng)修飾的II型GTP環(huán)化水解酶中的氨基酸可以改變,從而,對應于261位的氨基酸可變?yōu)楸彼?例如,V261A),對應于270位的氨基酸可變?yōu)楸彼峄蚓彼?例如,G270A和G270R),對應于276位的氨基酸可變?yōu)樘K氨酸(例如,A276T),對應于279位的氨基酸可變?yōu)榫彼?例如,Q279A),對應于308位的氨基酸可變?yōu)榫彼?例如,K308R),對應于347位的氨基酸可變?yōu)楫惲涟彼?例如,M347I)。在一種實施方式中,經(jīng)修飾的酶可從Bacillus subtilis獲得,其包含SEQ IDNO2的下述位置的氨基酸取代,所述位置選自由261、270、276、279、308和347位構成的組。優(yōu)選地,所述取代是V261A、G270A、G270R、A276T、Q279R、K308R或M347I。
經(jīng)修飾的II型GTP環(huán)化水解酶可以包含一處或多處突變,所述突變包括在上文定義的氨基酸位置上的兩處突變,此類突變(特別是氨基酸取代)可以包括對應于上文定義的位置中兩處的氨基酸位置上的突變,例如,對應于SEQ ID NO2所示的261/270、261/276、261/279、261/308、261/347、270/276、270/279、270/308、270/347、276/279、276/308、276/347、279/308、279/347或308/347的位置的組合。優(yōu)選的是氨基酸取代,例如,V261A/A276T、V261A/Q279R、V261A/K308R、V261A/M347I、G270A/Q279R、G270A/K308R、G270A/M347I、A276T/Q279R、A276T/K308R或A276T/M347I,其中,這些位置對應于SEQ ID NO2的氨基酸位置。在一種實施方式中,此類優(yōu)選取代包括在可從Bacillus subtilis獲得的經(jīng)修飾的II型GTP環(huán)化水解酶中,其中,未經(jīng)修飾的酶對應于SEQ ID NO2。優(yōu)選地,SEQ ID NO2的經(jīng)修飾的II型GTP環(huán)化水解酶包含取代V261A/A276T或A276T/M347I。
經(jīng)修飾的II型GTP環(huán)化水解酶可以包含一處或多處突變,所述突變包括在上文定義的氨基酸位置上的三處突變,此類突變(特別是氨基酸取代)可以包括對應于上文定義的位置中三處的氨基酸位置上的突變,例如,對應于SEQ ID NO2所示的261/279/308、261/279/347、261/308/347、270/279/308、270/279/347、270/308/347、276/279/308、276/308/347或276/279/347的位置的組合。優(yōu)選的是氨基酸取代,例如,V261A/Q279R/K308R、V261A/K308R/M347I、V261A/Q279R/M347I、G270A/Q279R/K308R、G270A/K308R/M347I、G270A/Q279R/M347I、A276T/Q279R/K308R、A276T/K308R/M347I或A276T/Q279R/M347I,其中,這些位置對應于SEQ ID NO2的氨基酸位置。在一種實施方式中,此類優(yōu)選取代包括在可從Bacillus subtilis獲得的經(jīng)修飾的II型GTP環(huán)化水解酶中,其中,未經(jīng)修飾的酶對應于SEQ ID NO2。優(yōu)選地,SEQ IDNO2的經(jīng)修飾的II型GTP環(huán)化水解酶包含取代A276T/Q279R/M347I。
經(jīng)修飾的II型GTP環(huán)化水解酶可以包含一處或多處突變,所述突變包括在上文定義的氨基酸位置上的四處突變,此類突變(特別是氨基酸取代)可以包括對應于上文定義的位置中四處的氨基酸位置上的突變,例如,對應于SEQ ID NO2所示的261/279/308/347、270/279/308/347或276/279/308/347的位置的組合。優(yōu)選的是氨基酸取代,例如,V261A/Q279R/K308R/M347I、G270A/Q279R/K308R/M347I或A276T/Q279R/K308R/M347I,其中,這些位置對應于SEQ ID NO2的氨基酸位置。在一種實施方式中,此類優(yōu)選取代包括在可從Bacillus subtilis獲得的經(jīng)修飾的II型GTP環(huán)化水解酶中,其中,未經(jīng)修飾的酶對應于SEQ ID NO2。優(yōu)選地,SEQ ID NO2的經(jīng)修飾的II型GTP環(huán)化水解酶包含取代A276T/Q279R/K308R/M347I。
最優(yōu)選的是表1或2公開的突變的組合(見下文)。這些實施例中鑒定出的氨基酸位置可被轉入不同來源的II型GTP環(huán)化水解酶,例如,圖1或表4所示的那些。
本發(fā)明的經(jīng)修飾的II型GTP環(huán)化水解酶可以包含外源氨基酸,優(yōu)選地,在其N或C末端包含?!巴庠窗被帷北硎驹谔烊?天然存在)的II型GTP環(huán)化水解酶中不存在的氨基酸,優(yōu)選地,在天然II型GTP環(huán)化水解酶中不存在的至少大約3、至少大約5或至少大約7個連續(xù)氨基酸的小段。外源氨基酸的優(yōu)選小段包括但不限于促進重組產生的經(jīng)修飾II型GTP環(huán)化水解酶的提純的“標簽”。此類標簽的例子包括但不限于“His6”標簽、FLAG標簽、myc標簽等。為計算比活性,需要針對這些額外的氨基酸對值進行校正(還見上文)。
在另一種實施方式中,經(jīng)修飾的II型GTP環(huán)化水解酶較之對應的未經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列可含有一處或多處,例如兩處缺失。優(yōu)選地,缺失影響對應的未經(jīng)修飾II型GTP環(huán)化水解酶的N或C末端氨基酸,而不會顯著降低酶的功能屬性,例如比活性。
本發(fā)明的多肽和多核苷酸,包括經(jīng)修飾的II型GTP環(huán)化水解酶,可以以經(jīng)分離的形式提供,優(yōu)選地,被純化至同質(homogeneity)。本文中使用的術語“經(jīng)分離的”指物質被從其原始環(huán)境(例如,如果是天然存在的話,天然環(huán)境)移出。例如,存在于活體微生物中的天然存在的多核苷酸或多肽是未經(jīng)分離的,但是從天然系統(tǒng)中共存在的物質中的一些或全部分離出的同樣的多核苷酸或多肽就是經(jīng)分離的。此類多核苷酸可以是載體的一部分和/或此類多核苷酸或多肽可以是組合物的一部分,但仍是經(jīng)分離的,因為此類載體或組合物并非其天然環(huán)境的一部分。經(jīng)分離的多肽優(yōu)選超過80%純,更優(yōu)選超過90%純,進一步更優(yōu)選超過95%純,最優(yōu)選超過99%純。純度可以通過本領域已知的方法來測定,例如,通過SDS-PAGE以及隨后的蛋白染色。然后可以通過密度測定來對蛋白條帶加以定量。用于測定純度的其它方法都是技術人員已知的。
本發(fā)明還涉及包含下述核苷酸序列的多核苷酸,所述序列編碼根據(jù)本發(fā)明的經(jīng)修飾II型GTP環(huán)化水解酶。本文中使用的“多核苷酸”指多聚核糖核苷酸或者多聚脫氧核糖核苷酸,其可以是未經(jīng)修飾的RNA或DNA或經(jīng)修飾的RNA或DNA。多核苷酸包括但不限于單鏈和雙鏈DNA,是單雙鏈區(qū)域混合物的DNA,單鏈或雙鏈RNA,是單雙鏈區(qū)域混合物的RNA,雜交分子(其包括可以是單鏈或更典型地雙鏈或單雙鏈區(qū)域混合物的DNA和RNA)。術語“多核苷酸”包括下述DNA或RNA,其中包含一個或多個不常見堿基,例如,肌苷,或者一個或多個經(jīng)修飾的堿基,例如,三苯甲基化堿基。
可通過對編碼未經(jīng)修飾的II型GTP環(huán)化水解酶的多核苷酸序列加以修飾來獲得本發(fā)明的多核苷酸。此類編碼未經(jīng)修飾的II型GTP環(huán)化水解酶的多核苷酸序列的例子包括但不限于圖1或表4的氨基酸序列,特別是SEQ ID NOs2、33、35、37、39、41和43。編碼根據(jù)本發(fā)明的經(jīng)修飾II型GTP環(huán)化水解酶的多核苷酸的非限制性例子是SEQ ID NOs6、8、10、12、14、16、18、20、22、24和26所示的。
用于向編碼未經(jīng)修飾的II型GTP環(huán)化水解酶的核苷酸序列引入突變(例如添加、缺失和/或取代)的方法包括但不限于定點誘變和基于PCR的方法。
可通過體外誘變的方法(見,例如Sambrook et al.,Molecular Cloning,Cold Spring Harbor Laboratory Press,New York),從編碼本領域已知的II型GTP環(huán)化水解酶的基因組或cDNA序列開始來構建本發(fā)明的DNA序列,如可從例如Genbank(Intelligenetics,California,USA)、EuropeanBioinformatics Institute(Hinston Hall,Cambridge,GB)、NBRF(Georgetown University,Medical Centre,Washington DC,USA)和Vecbase(University of Wisconsin,Biotechnology Centre,Madison,Wisconsin,USA)或從圖1或表4公開的序列信息獲得的。對本發(fā)明實踐而言也是優(yōu)選的、對給定DNA序列進行突變的另一種方法是通過聚合酶鏈式反應(PCR)進行誘變??捎帽绢I域已知的、例如在in Sambrook et al.(MolecularCloning)中描述的方法從各種菌株/生物來分離作為起始材料的DNA。但是,應當理解,編碼待根據(jù)本發(fā)明構建/突變的II型GTP環(huán)化水解酶的DNA還可基于已知DNA序列來制備,例如,通過本領域已知的方法構建合成基因來制備(例如,EP 747483所述)。
本發(fā)明的多核苷酸可以是經(jīng)分離的多核苷酸,即,基本不含其它核酸序列(例如但不限于其它染色體和染色體外DNA和RNA)的多核苷酸。本領域技術人員已知的傳統(tǒng)核酸純化方法可用于獲得經(jīng)分離的多核苷酸。該術語還包括重組多核苷酸和化學合成的多核苷酸。
在又一種實施方式中,本發(fā)明涉及功能性多核苷酸,其中,啟動子、核糖體結合位點(如果必要的話,在細菌細胞的情況下)和終止子與根據(jù)本發(fā)明的多核苷酸可操作地連接。在另一種實施方式中,本發(fā)明涉及包含此類多核苷酸的載體或質粒。載體或質粒優(yōu)選包含至少一種標記基因。術語“可操作地連接”在本文中被用來表示核酸序列在單條核酸片段上的締合,使得一條序列的功能受到另一條影響。例如,當能影響編碼序列表達的時候,即,編碼序列處于啟動子的轉錄控制之下時,啟動子就是與該編碼序列可操作地連接的。編碼序列可以與調控序列以正義或反義定向可操作地連接。術語“表達”指DNA序列轉錄為mRNA和/或mRNA翻譯為氨基酸序列,術語“過量表達”表示經(jīng)修飾的生物(例如,通過轉化或轉染修飾的)中基因產物的生產超過了對應的未經(jīng)修飾的生物中的生產水平,這是通過對基因表達進行負調和/或通過在生物體內增加基因自身來實現(xiàn)的。
一旦獲得了本發(fā)明的完整DNA序列,它們即可被整合進載體或者直接引入宿主生物的基因組,這通過本領域已知的、在Sambrook et al.(s.a.)中描述的方法來進行,以在合適的宿主系統(tǒng)中(過量)表達被編碼的多肽。但是,本領域技術人員知道,DNA序列自身也可用于轉化本發(fā)明的合適宿主系統(tǒng),以獲得被編碼多肽的(過量)表達。
合適的宿主細胞可以是真核或原核細胞。合適的宿主細胞的例子包括但不限于,細菌細胞,例如藻青菌、鏈球菌、葡萄狀球菌、腸球菌,例如,Bacillus,例如,Bacillus subtilis或Streptomyces,例如,Streptomyceslividans或Streptococcus pneumoniae,E.coli,例如,E.coli K12菌株,例如M15 or HB 10l。宿主細胞可以是真菌細胞,包括酵母細胞,例如Aspergilli的細胞,例如Aspergillus niger或Aspergillus oryzae,Trichoderma,例如Trichoderma reesei,Ashbya,例如Ashbya gossypii,Eremothecium,例如Eremothecium ashbyii,Saccharomyces,例如,Saccharomyces cerevisiae,Candida,例如Candida flareri,Pichia,例如Pichia pastoris,Hansenula polymorpha,例如H.polymorpha(DSM 5215)和Kluyveromyces。合適的宿主細胞還可以選自動物細胞,包括哺乳動物細胞,例如,CHO、COS、HeLa、3T3、BHK、293、CV-1和昆蟲細胞,例如Drosophila S2和Spodoptera Sf9細胞以及植物細胞,例如,裸子或被子植物細胞。
可用于在真菌中表達的載體是本領域已知的,其被描述于例如EP420358中或由Cullen et al.(Bio/Technology 5,369-376,1987)、Ward(inMolecular Industrial Mycology,Systems and Applications for FilamentousFungi,Marcel Dekker,New York,1991)、Upshall et al.(Bio/Technology 5,1301-1304,1987)、Gwynne et al.(Bio/Technology 5,71-79,1987)或Puntet al.(J.Biotechnol.17,19-34,1991)所述,關于酵母的,由Sreekrishna etal.(J.Basic Microbiol.28,265-278,1988;Biochemistry 28,4117-4125,1989)、Hitzemann et al.(Nature 293,717-722,1981)所述或描述于EP183070、EP 183071、EP 248227或EP 263311中。可用于在E.coli中表達的合適的載體是本領域已知的,其被描述于Sambrook et al.(s.a.)中??捎糜谠跅U菌(Bacilli)中表達的合適的載體是本領域已知的,其被描述于例如EP 207459或EP 405370中,或由Yansura and Henner在Proc.Natl.Acad.Sci.USA 81,439-443(1984)或由Henner,Le Grice和Nagarajan在Meth.Enzymol.185,199-228,1990中所述。可用于在H.polymorpha中表達的合適的載體是本領域已知的,其被描述于例如Gellissen et al.,Biotechnology 9,291-295,1991中。
此類載體已經(jīng)攜帶有調控元件,例如啟動子,或者本發(fā)明的多核苷酸可被工程化,以含有此類元件??墒褂玫暮线m啟動子元件是本領域已知的,其例如是對Trichoderma reesei而言,cbh1-或pki1-啟動子,對Aspergillus oryzae而言,amy-啟動子,對Aspergillus nige而言,glaA-、alcA-、aphA-、tpiA-、gpdA-和pkiA-啟動子??捎糜谠诮湍钢斜磉_的合適的啟動子元件是本領域已知的,例如它們是對在Sacsharomycescerevisiae中表達而言,pho5-或gap-啟動子,以及例如,對Pichia pastoris而言,aox1-啟動子,對H.polymorpha而言,F(xiàn)MD-或MOX啟動子。
用于細菌表達的合適啟動子和載體包括,例如,Giacomini et al.(Gene 144,17-24,1994)所述的合成啟動子,來自Bacillus subtilis的vegI啟動子或強細菌噬菌體T5啟動子。關于通過合適質?;蛲ㄟ^編碼II型GTP環(huán)化水解酶的DNA序列來向染色體DNA中的整合,從而在細菌中表達所要求的(突變體)II型GTP環(huán)化水解酶的合適教導可在例如美國專利6,322,995中找到。
因此,包含本發(fā)明多核苷酸的載體,優(yōu)選地,用于在細菌、真菌、動物或植物宿主中表達所述多核苷酸的載體,以及此類經(jīng)過轉化的細菌或真菌、動物或植物宿主也是本發(fā)明的目的。
本發(fā)明還涉及用于生產核黃素、核黃素前體、FMN、FAD或其一種或多種衍生物的方法,所述方法包括(a)在合適的培養(yǎng)基中,于允許經(jīng)修飾的II型GTP環(huán)化水解酶在合適宿主中表達的條件下培養(yǎng)所述宿主細胞;以及(b)可選地,從培養(yǎng)基中分離出產物(核黃素、核黃素前體、FMN、FAD或其一種或多種衍生物)。
此類方法可用于對下述產品中的一種或多種進行生物技術生產核黃素、核黃素前體、FMN、FAD或其一種或多種衍生物。此類衍生物可包括黃素蛋白。
對根據(jù)本發(fā)明的合適宿主細胞進行遺傳和代謝工程改造的方法是本領域技術人員已知的。類似地,針對核黃素、核黃素前體、FMN、FAD或其一種或多種衍生物的(可能)合適的純化方法是精細化學生物合成和生產領域所公知的。
應當理解,根據(jù)本發(fā)明用于對核黃素、核黃素前體、FMN、FAD或其一種或多種衍生物進行生物技術生產的方法不限于如上所述的全細胞發(fā)酵工藝,其還可使用,例如,透過的(permabilized)宿主細胞、細胞粗提取物、從細胞殘余物(例如通過離心或過濾)純化的細胞提取物,或者甚至用經(jīng)過分離的酶重構的反應途徑。此類工藝的組合也在本發(fā)明的范圍內。
在不含細胞的生物合成的情況(例如,用重構的反應途徑)下,經(jīng)分離的酶是否從宿主細胞制備以及是否從宿主細胞分離(通過體外轉錄/翻譯或其它方法)都是不相干的。
本發(fā)明還涉及一種方法,用于生產本發(fā)明的經(jīng)修飾的II型GTP環(huán)化水解酶,所述方法包括(a)在允許本發(fā)明的經(jīng)修飾的II型GTP環(huán)化水解酶表達的條件下培養(yǎng)本發(fā)明的宿主細胞;以及(b)從細胞或從培養(yǎng)基中回收經(jīng)修飾的II型GTP環(huán)化水解酶。
本發(fā)明的經(jīng)修飾的II型GTP環(huán)化水解酶可從經(jīng)過遺傳工程改造的宿主細胞制備,所述細胞包含合適的表達系統(tǒng)。
為重組生產本發(fā)明的多肽,可對宿主細胞進行遺傳工程改造,以包括進本發(fā)明的多核苷酸或載體或質粒。將多核苷酸或載體引入宿主細胞可用本領域已知的標準方法來進行,例如,磷酸鈣轉染、DEAE-右旋糖苷介導的轉染、微注射、陽離子脂類介導的轉染、電穿孔、轉導、轟擊引入和感染。
大量表達系統(tǒng)可用于生產本發(fā)明的經(jīng)修飾的II型GTP環(huán)化水解酶。此類載體還包括上文提到的那些。通常,適合在宿主中保持、增殖或表達多核苷酸和/或表達多肽的任何系統(tǒng)或載體都可用于這方面的表達。
在真核生物重組表達系統(tǒng)中,為將翻譯的蛋白分泌進內質網(wǎng)內腔、分泌進周質空間或分泌進細胞外環(huán)境,可將合適的分泌信號包括進被表達的多肽。這些信號對多肽來說可以是內源,或者它們可以是異源信號。
可通過公知方法從重組細胞培養(yǎng)物中回收及純化本發(fā)明的多肽,所述方法包括硫酸銨或乙醇沉淀、酸提取、陰離子或陽離子交換色譜、磷酸纖維素色譜、疏水相互作用色譜、親和色譜、羥磷灰石色譜和高效液相色譜。當多肽在分離和/或純化期間變性的情況下,可使用用于蛋白重折疊的公知技術來重建活性構象。
本發(fā)明的II型GTP環(huán)化水解酶還可根據(jù)例如Pen et al.在Bio/Technology 11,811-814,1994中或EP 449375中所述的方法在植物中表達,優(yōu)選按照例如EP 449376所述在種子中表達。啟動子和終止子的一些合適的例子包括來自胭脂堿合酶(nos)、章魚堿合酶(ocs)和花菜花葉病毒(CaMV)基因的那些??墒褂玫囊活愑行У闹参飭幼邮歉咚街参飭幼?。此類啟動子與本發(fā)明的遺傳序列可操作地相連,其應當能促進本發(fā)明的基因產物的表達。可在本發(fā)明中使用的高水平植物啟動子包括核酮糖-1,5-二磷酸羧化酶小亞基啟動子(例如來自大豆的)以及葉綠體a/b結合蛋白的啟動子。
對本發(fā)明蛋白的商業(yè)化生產是人們想要的,可以應用多種培養(yǎng)方法。例如,可以通過分批或連續(xù)培養(yǎng)方法獲得從重組微生物宿主過量表達的特定基因產物的大規(guī)模生產。分批和補料分批培養(yǎng)方法是本領域內常用且公知的,其例子已由Thomas D.Brock在BiotechnologyA Textbook ofIndustrial Microbiology,Second Edition(1989),Sinauer Associates,Inc.,Sunderland,Mass.,中或由Deshpande,Appl.Biochem.Biotechnol.36,227-234,1992描述過。用于連續(xù)發(fā)酵工藝調節(jié)營養(yǎng)物和生長因子的方法以及用于最大化產物形成速率的方法是工業(yè)微生物學領域公知的,前述Brock詳細描述了大量方法。
發(fā)酵培養(yǎng)基還可含有合適的碳底物。合適的底物可包括但不限于單糖,例如葡萄糖和果糖;寡糖,例如乳糖或蔗糖;多糖,例如淀粉或纖維素或其混合物;以及來自可更新進料的未經(jīng)純化的混合物。應當認識到,在本發(fā)明中利用的碳源可包括多種不同的含碳底物,其僅受對微生物選擇的限制。
本發(fā)明還涉及一種方法,用于制備具有提高的比活性的II型GTP環(huán)化水解酶,所述方法包括下述步驟(a)提供編碼第一種II型GTP環(huán)化水解酶的多核苷酸,所述酶具有希望被提高的比活性;(b)將一處或多處突變引入所述多核苷酸序列,使得經(jīng)突變的多核苷酸序列編碼經(jīng)修飾的II型GTP環(huán)化水解酶,所述酶較之第一種II型GTP環(huán)化水解酶包含一處或多處突變,其中,所述一處或多處突變包括對應于SEQ ID NO2的261、270、276、279、308和/或347位的氨基酸位置上的1、2、3、4、5或6處突變;
(c)可選地,將所述經(jīng)突變的多核苷酸插入載體或質粒;(d)將所述多核苷酸或所述載體或質粒引入合適的宿主細胞;以及(e)在允許所述經(jīng)修飾的II型GTP環(huán)化水解酶表達的條件下培養(yǎng)所述宿主細胞。
本發(fā)明還包括提供一種方法,用于制備具有提高的比活性的II型GTP環(huán)化水解酶,所述方法包括下述步驟(a)提供編碼第一種II型GTP環(huán)化水解酶的多核苷酸,所述酶具有希望被提高的比活性;(b)提供對于比活性有影響的位置;(c)確定用于替換(b)中確定的、野生型II型GTP環(huán)化水解酶的給定氨基酸的最佳氨基酸,以及,將一處或多處突變引入(a)多核苷酸序列中(b)確定的位置,使得經(jīng)突變的多核苷酸序列編碼新的II型GTP環(huán)化水解酶;(d)可選地,將所述經(jīng)突變的多核苷酸插入載體或質粒;(d)將所述多核苷酸或所述載體或質粒引入合適的宿主細胞;以及(e)在允許所述經(jīng)修飾的II型GTP環(huán)化水解酶表達的條件下培養(yǎng)所述宿主細胞。
在一種實施方式中,上述方法或步驟(c)通過飽和誘變來進行。但是應當理解,這并非用于確定將替換野生型II型GTP環(huán)化水解酶給定位置的氨基酸的氨基酸,以獲得具有提高的比活性的經(jīng)過修飾的II型GTP環(huán)化水解酶的唯一一種方法。
按照上文所述,例如通過飽和誘變,從未經(jīng)修飾的II型GTP環(huán)化水解酶來制備具有提高的比活性的經(jīng)修飾的II型GTP環(huán)化水解酶包括但不限于從圖1或標4所示的未經(jīng)修飾的蛋白來制備突變的II型GTP環(huán)化水解酶蛋白,特別是從SEQ ID NOs2、33、35、37、39、41和43所代表的那些,例如,Bacillus subtilis或Ashbya gossypii的未經(jīng)修飾的II型GTP環(huán)化水解酶蛋白。用于PCR反應的引物是使得一條引物(例如正義引物)可含有經(jīng)突變的核苷酸,以及另一條引物(例如反義引物)可含有野生型核苷酸序列。用這些引物對兒和野生型ribA的基因組DNA進行的PCR可獲得在給定位置攜帶有特定突變的PCR產物,這取決于所用的引物的經(jīng)突變核苷酸序列。使用標準方法,例如QIAquick PCR純化試劑盒(Qiagen)對得到的PCR產物進行純化之后,可用限制性酶(例如BamHI和EcoRI)對DNA進行切割,連接進合適的載體(例如,pQE60),并轉化進II型GTP環(huán)化水解酶陰性的菌株。此類菌株的例子是含有質粒pREP4的E.coli菌株Rib7(Richter et al.,J.Bacteriol.175,4045-4051,1993)。通過DNA測序驗證了正確的序列之后,可按照上文所述對經(jīng)突變的RibA進行純化和分析。如果Ashbya gossypii被用于生產具有提高的比活性的II型GTP環(huán)化水解酶,則飽和誘變必須在T126、G135、A141、L144、N182和I221氨基酸殘基/位置進行,這些位置分別對應于SEQ IDNO2所示的Bacillus subtilis II型GTP環(huán)化水解酶的V261、G270、A276、Q279、K308和M347殘基,這些位置顯示出對后一種酶的比活性有影響(見表4)。
該方法的優(yōu)選實施方式對應于經(jīng)修飾的II型GTP環(huán)化水解酶、編碼它們的多核苷酸、載體和質粒、宿主細胞以及本文所述的方法的優(yōu)選實施方式。第一種和第二種II型GTP環(huán)化水解酶分別對應于未經(jīng)修飾的和經(jīng)修飾的II型GTP環(huán)化水解酶(見上文)。
本發(fā)明的一個目的是提供一種多核苷酸,其中包含編碼上文所述經(jīng)修飾的II型GTP環(huán)化水解酶的核酸序列;提供一種載體(優(yōu)選地,表達載體),其中包含此類多核苷酸;提供一種宿主細胞,其已經(jīng)過此類多核苷酸或載體的轉化;提供一種方法,用于制備本發(fā)明的II型GTP環(huán)化水解酶,其中在合適的培養(yǎng)條件下培養(yǎng)前述宿主細胞,通過本領域已知的方法從此類宿主細胞或培養(yǎng)基分離出II型GTP環(huán)化水解酶;以及提供一種方法,用于對核黃素、核黃素前體、FMN、FAD或其一種或多種衍生物進行生物技術生產,這基于已被此類多核苷酸或載體轉化過的和/或此類多核苷酸可穩(wěn)定整合進其染色體的宿主細胞來進行。
本發(fā)明還有一個目的是提供(i)一種DNA序列,其編碼攜帶有本發(fā)明的至少一種特定突變的II型GTP環(huán)化水解酶,并且能在標準條件下與本發(fā)明的特定經(jīng)修飾的II型GTP環(huán)化水解酶的DNA序列中的任何一種雜交,或(ii)一種DNA序列,其編碼攜帶有本發(fā)明的至少一種特定突變的II型GTP環(huán)化水解酶,但是因為遺傳密碼的簡并性其不能雜交,但與能在標準條件下與本發(fā)明的特定經(jīng)修飾的II型GTP環(huán)化水解酶的DNA序列中的任何一種雜交的DNA序列編碼具有完全相同氨基酸序列的多肽,或(iii)一種DNA序列,其是上述DNA序列的片段,并保持有該片段來源的多肽的活性。
用于雜交的“標準條件”在本發(fā)明的上下文中表示本領域技術人員通常用來探測特定雜交信號的條件,其被描述于例如Sambrook et al.,″Molecular Cloning″,second edition,Cold Spring Harbor Laboratory Press1989,New York中,優(yōu)選地,本領域技術人員熟悉的所謂嚴謹雜交和非嚴謹洗滌條件或者更優(yōu)選地所謂嚴謹雜交和嚴謹洗滌條件,其被描述于例如Sambrook et al.(s.a.)中。嚴謹雜交條件的一個具體例子是在42℃于包含50%甲酰胺、5 x SSC(150mM NaCl、15mM檸檬酸三鈉)、50mM磷酸鈉(pH7.6)、5 x Denhardt′s溶液、10%硫酸右旋糖苷和20μg/ml變性剪切鮭魚靜DNA的溶液中進行過夜溫育,接著在0.1 x SSC中于大約65℃下洗雜交支持物。
此外,本發(fā)明的一個目的是提供一種DNA序列,其可通過基于特別描述的本發(fā)明的DNA序列設計的PCR引物,通過所謂的聚合酶鏈式反應(“PCR”)獲得。應當理解,由此獲得的DNA序列編碼下述II型GTP環(huán)化水解酶,所述酶具有與對其進行設計的酶至少相同的突變,并且顯示出相當?shù)幕钚浴?br>
本文所述的本發(fā)明的多種實施方式可交叉組合。
圖1通過GCG程序包的PILEUP程序,對使用標準數(shù)據(jù)庫如SWISS-PROT和TrEMBL通過BLASTN程序發(fā)現(xiàn)的92條II型GTP環(huán)化水解酶計算的多條序列比對(Candida guilliermondii、Ashbya gossypii、Saccharomyces cerevisiae、Neurospora crassa、Schizosaccharomycespombe、Archaeoglobus fulgidus、Streptomyces coelicolor、Helicobacterpylori J99、Helicobacter pylori、Pyrococcus furiosus、Thermotogamaritima、Chlamydia muridarum、Chlamydia trachomatis、Chlamydiacaviae GPIC、Arabidopsis thaliana、Lycopersicum esculentum、Oryzasativa、Alcaligenes eutrophus、Neisseria meningitidis (血清組A)、Neisseria meningitidis(血清組B,兩種II型GTP環(huán)化水解酶)、Pseudomonas putida(兩種II型GTP環(huán)化水解酶)、Pseudomonas syringae(兩種II型GTP環(huán)化水解酶)、Actinobacillus actinomycetemcomitans(Haemophilus actinomycetemcomitans)、Haemophilus influenzae、Pasteurella multocida、Escherichia coli、Escherichia coli O6、Salmonellatyphimurium、Yersinia pestis、Buchnera aphidicola (subsp.Acyrthosiphonpisum) (Acyrthosiphor pisum共生細菌)、Buchnera aphidicola(subsp.Schizaphis graminum)、Wigglesworthia glossinidia brevipalpis、Buchneraaphidicola (subsp.Baizongia pistaciae)、Mycobacterium leprae、Mycobacterium tuberculosis、Corynebacterium efficiens、Corynebacteriumglutamicum、Corynebacterium ammoniagenes(Brevibacteriumammoniagenes)、Staphylococcus aureus、Staphylococcus epidermidid、Actinobacillus pleuropneuumoniae、Lactococcus lactis(Streptococcuslactis)、Streptococcus agalactiae、Streptococcus pneumoniae、Clostridiumacetobutylicum、Fusobacterium nucleatum、Anabaena spec.、Synechocystisspec.、Synechococcus elongatus (Thermosynechococcus elongatus)、Bacillus amyloliquefaciens、Bacillus subtilis、Bacillus cereus、Bacillushalodurans、Clostridium Perfringens、Clostridium tetani、Chlorobiumtepidum、Aquifex aeolicus、Leptospira interrogans、Deinococcusradiodurans、Bacteroides thetaiotaomicron、Caulobacter crescentus、Coxiella burnetii、Rhizobium etli、Lactobacillus plantarum、Pseudomonasglumae、Streptomyces avermitilis、Photobacterium phosphoreum、Azospirillum brasilense、Agrobacterium tumefaciens、Rhizobium meliloti(Sinorhizobium meliloti)、Brucella melitensis、Brucella suis、Rhizobiumloti(Mesorhizobium loti)、Nitrosomonas europaea、Ralstoniasolanacearum(Pseudomonas solanacearum)、Xanthomonas axonopodis、Xanthomonas campestris、Vibrio parahaemolyticus、Vibrio vulnificus、Vibriocholerae、Vibrio fischeri、Shewanella oneidensis、Photobacteriumphosphoreum、Photobacterium leiognathi、Pseudomonas aeruginosa、Dehalospirillum muhivorans、Xylella fastidiosa)。編號方式涉及所進行的比對。一些氨基酸序列編碼僅具有II型GTP環(huán)化水解酶活性的酶,例如,來自Ashbya gossypii、Streptomyces coelicolor、Helicobacter pyloriJ99、Heliobacter pylori、Arabidopsis thaliana、Alcaligenes eutrophus、Neisseria meningitidis(血清組A)、Neisseria meningitidis(血清組B)、Pseudomonas putida、Pseudomonas syringae、Actinobacillusactinomycetemcomitans (Haemophilus actinomycetemcomitans)、Haemophilus influenzae、Pasteurella multocida、Escherichia coli、Escherichia coli O6、Salmonella typhimurium、Yersinia pestis、Buchneraaphidicola(subsp.Acyrthosiphon pisum)(Acyrthosiphon pisum共生細菌)、Buchnera aphidicola(subsp.Schizaphis graminum)、Wigglesworthiaglossinidia brevipalpis、Buchnera aphidicola(subsp.Baizongia pistaciae)、Pseudomonas glumae、Streptomyces avermitilis或Photobacteriumphosphoreum的酶。其它酶,例如來自B.subtilis的RibA酶除此之外還含有具有DHBP合酶活性的結構域。來自B.subtilis的RibA的氨基酸序列被下劃線示出。與發(fā)現(xiàn)對比活性具有正面影響的氨基酸殘基(261、270、276、279、308、347位氨基酸殘基)以及對來自B.subtilis的RibA的蛋白酶敏感性具有正面影響的氨基酸殘基(196)同源或等價的、并在下述實施例之一被討論的位置以加粗字母表示。用于這些位置的編號按照B.subtilis野生型氨基酸序列來進行。圖的開始是所用序列的名稱、數(shù)據(jù)庫編號以及括號中的序列來源生物。
下述非限制性的實施例將進一步闡述本發(fā)明。
實施例1測量II型GTP環(huán)化水解酶活性以及測定比活性用于測量II型GTP環(huán)化水解酶活性的酶促試驗改良自Ritz et al.(J.Biol.Chem.276,22273-22277,2001)。最終試驗緩沖液含有50mMTris/HCl,pH 8.5,10mM MgCl2、7.5mM巰基乙醇、2.5mM GTP和0.1mg/ml牛血清清蛋白。純化(見實施例5)之后,將酶保持于含有50mMTris/HCl,pH8.5,10mM MgCl2、7.5 mM巰基乙醇和10%甘油的緩沖液中。將底物加入酶,測量在310nm處的吸光度,此時GTP不顯示吸光度,接著進行超過20-30分鐘。最終反應混和物含有0.02至0.04mg/ml之間的、來自B.subtilis的II型GTP環(huán)化水解酶或表1或2所示的突變體之一。針對DRAPP的吸光系數(shù)6.28[mM-1cm-1]被用于計算活性。蛋白測定使用來自Bio-Rad(Cat.No.500-0002,Bio-Rad Laboratories AG,Nenzlingerweg 2,CH-4153 Reinach,Switzerland)的Protein Assay來進行。
根據(jù)上文給出的“比活性”的定義,一個單位是在上文描述的條件下每分鐘催化1μmol DRAPP形成的RibA的量。比活性是在上文描述的條件下每分鐘通過1mg RibA形成的DRAPP的量。使用上述定義,His6-標簽化的B.subtilis RibA蛋白的II型GTP環(huán)化水解酶比活性是0.115U/mg。
實施例2測試酶試驗的性能最優(yōu)的試驗應滿足多種要求,例如隨酶濃度的線性和隨時間的線性。使用實施例l所述的條件和22μg酶,在310nm處吸光度的增加進行25分鐘。為測試在何種范圍內試驗對酶濃度是線性的,測試該試驗對逐漸增加的酶濃度(0-40μg His6-標簽化的RibA)的相關性。該試驗被證明在25分鐘內和0至40μg His6-標簽化的來自B.subtilis的RibA的情況下是線性的。
這之后,測試His6-標簽化的來自B.subtilis的RibA的II型GTP環(huán)化水解酶活性對GTP濃度的相關性。使用如實施例1所述的條件。但是,GTP濃度在0.05至0.25mM終濃度之間變動。數(shù)據(jù)顯示了針對GTP的Km值為0.07mM,以及對His6-標簽化的來自B.subtilis的RibA的II型GTP環(huán)化水解酶活性而言37℃的比活性為大約115mU/mg蛋白。本實施例的實驗顯示,II型GTP環(huán)化水解酶試驗實際上隨時間和酶(II型GTP環(huán)化水解酶)濃度成線性,在給定的條件下,對Bacillus subtilis的II型GTP環(huán)化水解酶而言,2.5mM的GTP濃度可能是允許對酶的比活性進行可靠測量而言最優(yōu)的。
實施例3 從Bacillus subtilis分離基因組DNA在Veal Infusion Broth(Becton Dickinson,Sparks,MD 21152,USA)中于30℃對B.subtilis進行過夜培養(yǎng)。將1.5ml培養(yǎng)物轉移進1.5ml小管并離心。將細胞沉淀重新懸浮于0.5ml懸浮緩沖液(50mM Tris/HCl,pH7.5,50mM Na2EDTA,15%蔗糖和1mg/ml新鮮加入的裂解酶)中。室溫下溫育10分鐘后,加入1μl二乙氧基二甲酸酯。然后加入10μl的10%SDS溶液,顛倒小管數(shù)次。將小管在70℃溫育5分鐘,以釋放細菌DNA。加入50μl 5M乙酸鉀,在冰上冷卻小管,在其上放置45分鐘。之后在4℃對樣品進行30分鐘的離心。將上清液轉移進新的1.5ml小管,在室溫下向其中裝入乙醇(裝至1.5ml)。5分鐘離心之后,棄去上清液,干燥DNA沉淀。然后用70%和96%的乙醇洗DNA,將其溶解于10mM Tris/HCl,pH7.5,1mM EDTA和10μg/ml RNase A中。
實施例4 構建表達載體用于表達ribA及其突變體,ribA編碼來自B.substilis的II型GTP環(huán)化水解酶和DHBP合酶通過PCR來克隆B.subtilis的ribA基因(SEQ ID NO1),其編碼II型GTP環(huán)化水解酶和DHBP合酶。根據(jù)實施例3分離B.subtilis的基因組DNA。將100ng該DNA或編碼ribA基因的經(jīng)突變形式的模板用于PCR,其中使用引物RibA 1S(SEQ ID NO27)和RibA lAS(SEQ IDNO28)。使用下述PCR條件每種引物2μM,每種核苷酸0.2mM,2.5U校正(proof-reading)DNA聚合酶(Stratagene,Gebouw California,1101CB Amsterdam Zuidoost,The Netherlands)以及100ng基因組DNA,它們處于與DNA聚合酶一起提供的合適緩沖液中。
溫度調控如下所示步驟13分鐘,95℃步驟230秒,95℃步驟330秒,52℃步驟460秒,72℃
步驟2至4重復30次。
1.3 kb的PCR產物用作為PCR2的模板,其中,用RibA 2S(SEQ IDNO2)替換引物RibA 1S。該反應的PCR產物(SEQ ID NO3)編碼B.subtilis ribA的N末端His6標簽化版本(SEQ ID NO4),通過瓊脂糖電泳凝膠對其進行分離,從凝膠上洗脫,用EcoRI和BamHI消化,連接進用EcoRI和BamHI消化過的載體pQE60(Qiagen AG,Hilden,Germany)。該質粒被稱為pQE60ribANhis。
實施例5分析野生型酶和突變體酶使用上文所述的方法和技術人員已知的方法來產生經(jīng)突變的酶。被進一步分析的來自B.subtilis的RibA突變體展示于表1中。按照實施例4中所述,將所有突變體基因克隆進pQE60載體。所有最終的構建體都含有N末端的His6標簽。
表1較之B.subtilis的野生型RibA蛋白的氨基酸交換定義的突變體(數(shù)字代表SEQ ID NO2中相應的氨基酸位置)。
從實施例4的質粒來表達RibA突變體酶,按照″The QiaExpressionist″,Qiagen,Hilden,Germany,March 2001,edition 5所述進行純化。按照實施例1和實施例2所述對經(jīng)純化的酶(RibA突變體)的酶性能加以分析。表2比較了RibA突變體(見表1)的II型GTP環(huán)化水解酶比活性與B.subtilis的野生型RibA的II型GTP環(huán)化水解酶比活性。按照實施例4所述,使用RibA的N末端His6標簽化的酶版本來測量活性。數(shù)字代表SEQ ID NO2中的相應的氨基酸位置。
表2比較經(jīng)突變的和野生型(WT)B.subtilis RibA(全部都是N末端His6標簽化的)的II型GTP環(huán)化水解酶比活性
括號中的氨基酸替換最可能不影響突變體的II型GTP環(huán)化水解酶活性。氨基酸交換Y196C降低RibA的蛋白酶敏感性。
實施例6 構建過量表達RibA突變體的重組B.subtilis菌株,所述突變體顯示出更高的II型GTP環(huán)化水解酶比活性在下述實施例中,首先將經(jīng)突變的ribA多核苷酸序列RibA Y196C,A276T,A282T(PCR III),RibA Y196C,A276T,Q279R,A282T,K308R,M347I(構建體C)和RibA Y196C,A276T,Q279R,A282T,K308R,F(xiàn)325Y,M347I(構建體E)引入到含有強組成型啟動子PvegI的載體中,然后在E.coli中進行進一步操作。用多核苷酸序列和側翼載體序列對天然感受態(tài)B.subtilis微生物的轉化得到過量表達經(jīng)突變ribA的B.subtilis菌株。用標準重組DNA技術來構建多核苷酸序列和B.subtilis菌株。見,例如,Sambrook et al.,Molecular Cloning.A Laboratory Manual(2ndEd.),ColdSpring Harbor Laboratory Press(1989)和Harwood and Cutting,MolecularBiology Methods for Bacillus,John Wiley and Sons(1990)。
為擴增經(jīng)突變的ribA,使用來自含有突變體PCRIII、構建體C或構建體E的DNA,以及RibANde+l(SEQ ID NO30)and RibA4AS(SEQ IDNO31)作為引物,通過PCR來擴增含有整條ribA編碼序列的1.2kb的DNA片段。
用于PCR反應的反應條件由下述30個循環(huán)構成95℃變性1分鐘,52℃退火1分鐘,72℃延伸2分鐘。用Pfu Turbo DNA聚合酶(Stratagene,Gebouw California,1101 CB Amsterdam Zuidoost,TheNetherlands)使得PCR產生的錯誤最小化。使用QIAquick PCR純化試劑盒(Qiagen)來純化PCR產物,使用NdeI和BamHI進行雙消化。將經(jīng)過消化的PCR產物克隆進pXI16載體(Huembelin et al.,J.Ind.Microbiol.Biotechnol.22,1-7,1999),其包括合適的限制性位點,適合用于克隆來自B.subtilis的強組成型PvegI啟動子下游緊接著的多核苷酸序列。pXI16載體還含有來自B.thuringiensis的cryT轉錄終止子,用于通過雙交換事件同源重組進B.subtilis基因組的sacB側翼序列,以及紅霉素抗性標記。通過DNA測序來驗證經(jīng)突變的ribA含有的每種質粒。
用ApaI消化每種質粒,除去來自PvegI啟動子的空白區(qū)域,重新連接,再用FspI進行消化,轉入天然感受態(tài)B.subtilis 1012細胞。在含有終濃度為2μg/ml紅霉素的TBAB平板(Tryptose Blood Agar Base,BectonDickinson,Sparks,MD 21152,USA)上選擇轉化子。DNA測序確證了這些菌株中的經(jīng)突變ribA多核苷酸序列是正確的。根據(jù)實施例7來測試核黃素的的過量生產。
通過一般化的轉導,將PvegI啟動子驅動的經(jīng)突變ribA多核苷酸序列引入過量生產核黃素的B.subtilis菌株RB50∷(pRF69)n∷(pRF93)m,其在Perkins et al.,J.Ind.Microbiol.Biotechnol.228-18(1999)中已被描述。根據(jù)Harwood and Cutting,Molecular Biology Methods for Bacillus,John Wiley andSons(1990)來使用細菌噬菌體PBS1的標準技術。在含有終濃度為2μg/ml紅霉素的TBAB平板上選擇轉導產物。通過PCR分析和DNA測序來檢查轉化子,以驗證經(jīng)突變ribA多核苷酸序列的正確插入。
實施例7使用具有提高的比活性的II型GTP環(huán)化水解酶改進的核黃素的生產為測試突變影響II型GTP環(huán)化水解酶比活性的體內效果,將Bacillussubtilis II型環(huán)化水解酶(RibA)突變體PCR III、構建體C或構建體E引入到過量生產核黃素的B.subtilis菌株RB50∷(pRF69)n∷(pRF93)m(Perkinset al,J.Ind.Microbiol.Biotechnol.22,8-18,1999)中,例如,sacB基因座。在僅因為存在或不存在ribA基因的突變而有所不同的兩種B.subtilis重組菌株中直接比較核黃素的生產。按照實施例8所述來培養(yǎng)Bacillus菌株。
實施例8用于評估核黃素生產的培養(yǎng)條件在對過量生產核黃素的B.subtilis菌株RB50∷(pRF69)n∷(pRF93)m的補料分批培養(yǎng)中測試核黃素的生產,其中,PvegI啟動子驅動的II型環(huán)化水解酶突變體PCR III、構建體C或構建體E整合進sacB基因座(見實施例6)。菌株發(fā)酵按照EP 405370來進行。
實施例9用于測定核黃素的分析方法為測定核黃素,可以使用下述分析方法(Bretzel et al.,J.Ind.Microbiol.Biotechnol.22,19-26,1999)。
色譜系統(tǒng)是裝備有二元泵、柱熱穩(wěn)定儀和冷卻的自動上樣儀的Hewlett-Packard 1100系統(tǒng)。二極管陣列探測器和熒光探測器串聯(lián)使用。記錄兩種信號,280nm處的UV和激發(fā)446nm、發(fā)射520nm處的熒光痕跡。
使用帶保護柱(guard cartridge)的不銹鋼Supelcosil LC-8-DB柱(150x 4.6mm,3μm顆粒大小)。移動相為100mM乙酸(A)和甲醇(B)。使用根據(jù)下述安排的梯度洗脫
時間[分鐘]%A %B0 9826 982155050255050柱溫設定為20℃,流速為1.0ml/分鐘。運行時間為25分鐘。
不進行進一步處理,就對發(fā)酵樣品進行稀釋、過濾和分析。通過與外部標準物比較來定量核黃素?;?80nm處的UV信號來進行計算。從Fluka(9471 Buchs,Switzerland)購買的核黃素被用作為標準物質(純度≥99.0%)。
實施例10 在與Bacillus subtilis II型GTP環(huán)化水解酶同源的II型GTP環(huán)化水解酶中鑒定對應的殘基采用下述參數(shù)缺口產生懲罰8,缺口延伸懲罰2以及blosum62.cmp矩陣(缺省參數(shù)),用程序“PILEUP”(GCG Wisconsin Package,version10.2,Accelrys Inc.,9685 Scranton Road,San Diego,CA 92121-3752,USA)來計算使用標準數(shù)據(jù)庫(例如SWISS-PROT和TrEMBL,見圖1)通過BLASTN程序發(fā)現(xiàn)的92條不同II型GTP環(huán)化水解酶的多條氨基酸序列比對。
在本發(fā)明上下文中的同源II型GTP環(huán)化水解酶可展示出與圖1所示的II型GTP環(huán)化水解酶氨基酸序列中的任意一條的相似性。圖1提供了對92條II型GTP環(huán)化水解酶氨基酸序列進行的多序列比對的例子,其中來自Bacillus subtilis的II型GTP環(huán)化水解酶被下劃線示出。圖1作為例子提供,并不表示其是所有已知II型GTP環(huán)化水解酶的全集。同源殘基,即位于氨基酸序列比對中相同位置(即,位于例如圖1中的同一列)上的不同II型GTP環(huán)化水解酶的殘基被預計為相似地定位于每種蛋白的3D結構中,在每種蛋白中滿足結構和功能方面的差不多的功能。實施例中討論的、與來自B.subtilis的II型GTP環(huán)化水解酶的氨基酸殘基同源的氨基酸殘基在圖1中以加粗表示,B.subtilis的氨基酸序列中的相應位置被添加到比對的每列之上。
92種不同生物中對應于特定氨基酸位置(即與已發(fā)現(xiàn)對Bacillussubtilis的II型GTP環(huán)化水解酶的氨基酸序列(SEQ ID NO2)比活性具有正面影響的氨基酸殘基(261、270、276、279、308、347位氨基酸殘基)同源/等價的位置)的氨基酸殘基被概括于表4中,其中,左列的編碼代表生物(根據(jù)圖1),由所用的序列名稱,數(shù)據(jù)庫編號和括號中的序列來源生物開始(1) gch2_bacsuSWISS-PROTgch2_bacsu(Bacillus subtilis)(2) gch2_cangugeneseqpaay69776(Candida guilliermondii)(3) gch2_ashgoTrEMBLCAA02912(Ashbya gossypii(Eremotheciumgosypii))(4) gch2_yeastSWISS-PROTgch2_yeast(Saccharomyces cerevisiae)(5) gch2_neucrTrEMBLQ871B3(Neurospora crassa)(6) gch2_schpoTrEMBLQ9P7M9(Schizosaccharomyces pombe)(7) gch2_arcfuSWISS-PROTgch2_arcfu(Archaeoglobus fulgidus)(8) gch2_strcoSWISS-PROTgch2_strco(Streptomyces coelicolor)(9) gch2_helpjSWISS-PROTgch2_helpj(Helicobacter pylori J99)(10)gch2_helpySWISS-PROTgch2_helpy(Heliobacter pylori)(11)gch2_pyrfuTrEMBLQ8U4L7(Pyrococcus furiosus)(12)gch2_themaSWISS-PROTgch2_thema(Thermotoga maritima)(13)gch2_chlmuSWISS-PROTgch2_chlmu(Chlamydia muridarum)(14)gch2_chltrSWISS-PROTgch2_chltr(Chlamydia trachomatis)(15)gch2_chlcaTrEMBLAAP0563 5(Chlamydia caviae GPIC)(16)gch2_chlpnSWISS-PROTgch2_chlpn(Chlamydia pneumoniae)(17)gch2_arathSWIS S-PROTgch2_arath(Arabidopsis thaliana)(18)gch2_lycesTrEMBLCAC09119(Lycopersicum esculentum)(19)gch2_orysaTrEMBLAAO72560(Oryza sativum)(20)gch2_alceuTrEMBLQ9F184(Alcaligenes eutrophus)(21)gch2_neimaSWISS-PROTgch2_neima(Neisseria meningitidis(血清組A))(22) gch2_neimbSWISS-PROTgch2_neimb(Neisseria meningitidis(血清組B))(23) gch2_psepkSWISS-PROTgch2_psepk(Pseudomonas putida(菌株KT2440))(24) gch2_psesmSWISS-PROTgch2_psesm(Pseudomonas syringae(pv.tomato))(25) gch2_actacTrEMBLQ9JRR0(Actinobacillus actinomycetemcomitans(Haemophilus actinomycetemcomitans))(26) gch2_haeinSWISS-PROTgch2_pasmu gch2_haein(Haemophilusinfluenzae)(27)gch2_pasmuSWISS-PROT(Pasteurella multocida)(28) gch2_ecO6TrEMBLQ8FHU5(Escherichia coli O6)(29) gch2_ecoliSWISS-PROTgch2_ecoli(Escherichia coli)(30) gch2_saltyTrEMBLQ8XFY7(Salmonella tYPhimurium)(31) gch2_yerpeTrEMBLQ8ZEF0(Yersinia pestis)(32) gch2_bucaiSWISS-PROTgch2_bucai(Buchnera aphidicola(subsp.Acyrthosiphon pisum)(Acyrthosiphon pisum共生細菌))(33) gch2_bucapSWISS-PROTgch2_bucap(Buchnera aphidicola(subsp.Schizaphis graminum))(34) gch2_wigbrSWISS-PROTgch2_wigbr(Wigglesworthia glossinidiabrevipalpis)(35) gch2_bucbpSWISS-PROTgch2_wigbr(Buchhera aphidicola(subsp.Baizongia pistaciae))(36) gch2_mycleTrEMBLQ9CCP4(Mycobacterium leprae)(37) gch2_myctuSWISS-PROTgch2_myctu(Mycobacterium tuberculosis)(38) gch2_corefTrEMBLQ8FT57(Corynebacterium efficiens)(39) gch2_corglGENESEQPAAB79913(Corynebacterium glutamicum)(40) gch2_coramSWISS-PROTgch2_coram(Corynebacteriumammoniagenes(Brevibacterium ammoniagenes))(41) gch2_staauTrEMBLQ8NW14(Staphylococcus aureus(菌株MW2))(42) gch2_staepGENESEQPABP40248(Staphylococcus epidermidis)(43) gch2_actplSWISS-PROTgch2_actpl(Actinobacillus pleuropneumoniae)(44) gch2_laclaTrEMBLQ9CGU7(Lactococcus lactis(subsp.lactis)(Streptocoecus lactis))(45) gch2_stcagTrEMBLQ8E658(Streptococcus agalactiae(血清型III))(46) gch2_stcpnTrEMBLQ8DRF1(Streptococcus pneumoniae(菌株ATCC BAA-255/R6))(47) gch2_cloacTrEMBLQ97LG9(Clostridium acetobutylicum)(48) gch2_fusnuTrEMBLQ8RIR1(Fusobacterium nucleatum(subsp.nucleatum))(49) gch2_anaspTrEMBLQ8RIR1(Anabaena sp.(菌株PCC7120))(50) gch2_syny3SWISS-PROTgch2_syny3(Synechocystis sp.(菌株PCC6803))(51) gch2_synelTrEMBLQ8DI64 Synechococcus elongatus(Thermosynechococcus elongatus)(52) gch2_bacamSWISS-PROTgch2_bacam(Bacillus amyloliquefaciens)(53) gch2_bacceTrEMBLAAP11030(Bacillus cereus ATCC 14579)(54) gch2_bachaTrEMBLQ9KCL5(Bacillus halodurans)(55) gch2_clopeTrEMBLQ8XMX0(Clostridium perfringens)(56) gch2_cloteTrEMBLQ897Q8(Clostridium tetani)(57) gch2_chlteTrEMBLQ8KC35(Chlorobium tepidum)(58) gch2_aquaeSWISS-PROTgch2_aquae(Aquifex aeolicus)(59) gch2_lepinTrEMBLQ8F701(Leptospira interrogans)(60) gch2_deiraTrEMBLQ9RXZ9(Deinococcus radiodurans)(61) gch2_bacthTrEMBLQ8A528(Bacteroides thetaiotaomicron)(62) gch2_caucrTrEMBLQ9A9S5(Caulobacter crescentus)(63) gch2_coxbuTrEMBLAAO90191(CoxieUa burnetii RSA 493)
(64) gch2_rhietTrEMBLQ8KL38(Rhizobium etli)(65) gch2_lacplTrEMBLQ88X17(Lactobacillus plantarum)(66) gch2_pseglTrEMBLQ8RS38(Pseudomonas glumae)(67) gch2_StravTrEMBLBAC71833(Streptomyces avermitilis)(68) gch2_phopoSWISS-PROTgch2_phopo(Photobacterium phosphoreum)(69) gch2_azobrSWISS-PROTgch2_azobr(Azospirillum brasilense)(70) gch2_agrtuTrEMBLQ8UHC9(Agrobacterium tumefaciens(菌株C58/ATCC 33970))(71) gch2_rhimeTrEMBLQ92RH2(Rhizobium meliloti(Sinorhizobiummeliloti))(72) gch2_brumeTrEMBLQ8YFL5(Brucella melitensis)(73) gch2_brusuTrEMBLQ8G298(Brucella suis)(74) gch2_rhiloTrEMBLQ985Z3(Rhizobium 1oti(Mesorhizobium loti))(75) gch2_brajaTrEMBLQ89RZ7(Bradyrhizobium japonicum)(76) gch2_niteuTrEMBLCAD86468(Nitrosomonas europaea ATCC 19718)(77) gch2_ralsoTrEMBLQ8Y1H7(Ralstonia solanacearum(Pseudomonassolanacearum))(78) gch2_neimeTrEMBLQ9JZ77(Neisseria meningitidis(血清組B,發(fā)現(xiàn)了第二種酶))(79) gch2_xanaxTrEMBLQ8PPD7(Xanthomonas axonopodis(pv.citri))(80) gch2_xancaTrEMBLQ8PCM8(Xanthomonas campestris(pV.campestris))(81) gch2_vibpaTrEMBLQ87RU5(Vibrio parahaemolyticus)(82) gch2_vibvuTrEMBLQ8DF98(Vibrio Vulnificus)(83) gch2_vibchTrEMBLQ9KPU3(Vibrio cholerae)(84) gch2_vibfiTrEMBLQ8G9G5(Vibrio fischeri)(85) gch2_sheonTrEMBLQ8EBP2(Shewanella oneidensis)(86) gch2_phophTrEMBLQ8G9H7(Photobacterium phosphoreum)(87) ribb_pholeTrEMBLQ93E93(Photobacterium leiognathi)
(88) gch2_psepuTrEMBLQ88GB1(Pseudomonas putida(菌株KT2440,發(fā)現(xiàn)了第二種酶))(89) gch2_psesyTrEMBLQ882G0(Pseudomonas syringae(pv.Tomato,發(fā)現(xiàn)了第二種酶))(90) gch2_pseaeTrEMBLQ9HWX4(Pseudomonas aeruginosa)(91) rib_dehmuSWISS-PROTribb_dehmu(Dehalospirillum multivorans)(92) gch2_xylfaTrEMBLQ87D69(Xylella fastidiosa(菌株Temeculal/ATCC 700964))表4對應于SEQ ID NO2的B.subtili RibA的V261,G270,A276,Q279,K308和M347位置的位置/氨基酸殘基。左列數(shù)字代表不同生物(如上所述)。
表4所示的例子被用于闡述??舍槍εc圖1或表4所示的序列中任意一條同源的所有其它II型GTP環(huán)化水解酶確定對應的殘基。
序列表<110>帝斯曼知識產權資產管理有限公司<120>改進的酶<130>22257<160> 43<170>PatentIn version 3.2<210>1<211>1197<212>DNA<213>Bacillus subtilis<400>1atgtttcatc cgatagaaga agcactggac gctttaaaaa aaggcgaagt catcatcgtt 60gtagatgatg aagacagaga aaatgaagga gactttgtgg ctcttgccga gcatgcaacg 120ccggaagtca ttaactttat ggcgacacat gggagaggac tgatctgcac gccgctcagt 180gaggaaatcg cagacaggct tgatcttcac cctatggttg agcataatac agactctcac 240cacactgcat ttaccgtaag catagaccat cgtgaaacga agacaggtat cagcgctcaa 300gaaagatctt ttaccgttca agcattgctg gacagcaaat ccgtgccatc tgattttcag 360cgtccggggc acatttttcc actgattgcg aaaaaaggag gtgtcctgaa aagagcgggc 420catacagaag ctgctgttga tcttgctgaa gcttgtggat ctccaggagc cggcgtcatt 480tgtgaaatta tgaatgaaga cggaacgatg gcgagagtgc ctgagctcat tgaaattgcg 540aaaaagcatc aattaaaaat aatcaccatt aaggatttga ttcaataccg ttacaatctg 600acaacacttg tcgagcgtga agttgacatt acgctgccta ctgattttgg gacatttaag 660gtttatggat acacaaatga ggtagatgga aaagagcatg tcgcatttgt gatgggagat 720gtgccgttcg gagaagaacc ggtattggtc cgggtgcatt cagaatgtct cacaggtgac 780gtgtttggct ctcatcgctg tgattgcgga ccgcagctgc acgccgcgct gaaccaaatt 840gccgcagaag gccgtggagt gctcctgtac ttgcgccaag aaggacgagg catcggttta 900atcaataaat taaaagctta taagcttcag gaacaaggct atgacaccgt agaagccaat 960gaggcgcttg gattcttgcc ggatcttcgc aactatggca tcggagcaca aattttacgc1020gacctcggtg tccggaatat gaagcttttg acgaataatc cgcgaaaaat cgcaggcctt1080gaaggctacg gactcagtat ttcagaaaga gtgccgcttc aaatggaggc gaaagaacac1140aataaaaaat atttgcaaac caaaatgaac aagctaggtc atttacttca tttctaa 1197
<210>2<211>398<212>PRT<213>Bacillus subtilis<400>2Met Phe His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu1 5 10 15Val Ile Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe20 25 30Val Ala Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala35 40 45Thr His Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala50 55 60Asp Arg Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His65 70 75 80His Thr Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly85 90 95Ile Ser Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser100 105 110Lys Ser Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu115 120 125Ile Ala Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala130 135 140Ala Val Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile145 150 155 160Cys Glu Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu165 170 175Ile Glu Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp180 185 190Leu Ile Gln Tyr Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val195 200 205
Asp Ile Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr210 215 220Thr Asn Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp225 230 235 240Val Pro Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys245 250 255Leu Thr Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln260 265 270Leu His Ala Ala Leu Asn Gln Ile Ala Ala Glu Gly Arg Gly Val Leu275 280 285Leu Tyr Leu Arg Gin Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu290 295 300Lys Ala Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn305 310 315 320Glu Ala Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala325 330 335Gln Ile Leu Arg Asp Leu Gly Val Arg Asn Met Lys Leu Leu Thr Asn340 345 350Asn Pro Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser355 360 365Glu Arg Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr370 375 380Leu Gln Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe385 390 395<210>3<211>1239<212>DNA<213>Bacillus subtilis<400>3atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180
atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatac cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccgcg ctgaaccaaa ttgccgcaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080atgaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctaa 1239<210>4<211>412<212>PRT<213>Bacillus subtilis<400>4Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60
Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met VaI Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Ash Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Tyr Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275280 285Ala Ala Leu Asn Gln Ile Ala Ala Glu Gly Arg Gly Val Leu Leu Tyr
290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Met Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>5<211>1239<212>DNA<213>Bacillus subtilis<400>5atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatgc cgttacaatc tgacaacact tgtcgagcgt 660
gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccacg ctgaaccaaa ttgccgcaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080atgaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctaa 1239<210>6<211>412<212>PRT<213>Bacillus subtilis<400>6Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp ghe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125
Val Pro Ser Asp Phe Gln Arg Pro Gly His lle Phe Pro Leu lle Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Cys Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 27Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275280 285Ala Thr Leu Asn Gln Ile Ala Ala Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Met Lys Leu Leu Thr Asn Asn Pro355 360 365
Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Mer Glu Ala Lys Glu His Ash Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>7<211>1239<212>DNA<213>Bacillus subtilis<400>7atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatgc cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgcgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccgcg ctgaaccaaa ttgccgcaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080atgaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200
accaaaatga acaagctagg tcatttactt catttctaa 1239<210>8<211>412<212>PRT<213>Bacillus subtilis<400>8Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu65 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190
Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Cys Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Ala Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275 280 285Ala Ala Leu Asn Gln Ile Ala Ala Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Met Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>9<211>1239<212>DNA
<213>Bacillus subtilis<400>9atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccgDaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acaDactctc accacactgc atttaccgta 300agcatagacc atcDtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatac cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccgcg ctgaaccaaa ttgccgcaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080ataaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctaa 1239<210>10<211>412<212>PRT<213>Bacillus subtilis<400>10Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30
Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Ash Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Tyr Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225 230 235240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr
260 265 270Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275 280 285Ala Ala Leu Asn Gln Ile Ala Ala Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Ile LysLeu Leu Thr Asn Asn Pro355 360365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>11<211>1239<212>DNA<213>Bacillus subtilis<400>11atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420
ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatgc cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccacg ctgaaccaaa ttgccacaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080atgaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctaa1239<210>12<211>412<212>PRT<213>Bacillus subtilis<400>12Met Arg Gly Ser His His His His His His Gly Ile AsP His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95
Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Cys Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly T yr Thr Asn225 230235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275280 285Ala Thr Leu Asn Gln Ile Ala Thr Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335
Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Iie340 345 350Leu Arg Asp Leu Gly Val Arg Asn Met Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>13<211>1239<212>DNA<213>Bacillus subtilis<400>13atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatgc cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccacg ctgaaccaaa ttgccacaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960
tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg 1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat 1080ataaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt 1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa 1200accaaaatga acaagctagg tcatttactt catttctaa 1239<210>14<211>412<212>PRT<213>Bacillus subtilis<400>14Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phel 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160
Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Cys Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Mer Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275 280 285Ala Thr Leu Asn Gln Ile Ala Thr Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Cly Tle Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Ash Ile Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400
Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>15<211>1239<212>DNA<213>Bacillus subtilis<400>15atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatgc cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccacg ctgaaccgaa ttgccacaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080ataaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctaa 1239<210>16<211>412<212>PRT<213>Bacillus subtilis
<400>16Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 1015His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Va1 Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Va1 Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Cys Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn
225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275 280 285Ala Thr Leu Asn Arg Ile Ala Thr Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Ile Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>17<211>1239<212>DNA<213>Bacillus subtilis<400>17atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt180
atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatgc cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccact ctgaaccgaa ttgccacaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataggcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080ataaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctaa1239<210>18<211>412<212>PRT<213>Bacillus subtilis<400>18Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60
Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu A la Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Cys Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275 280 285Ala Thr Leu Asn Arg Ile Ala Thr Glu Gly Arg Gly Val Leu Leu Tyr290 295 300
Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Arg Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Ile Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>19<211>1239<212>DNA<213>Bacillus subtilis<400>19atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatac cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacatttg aggtttatgg atacacaaat 720
gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg caccgcagct gcacgccgcg ctgaaccaaa ttgccgcaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080atgaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctaa 1239<210>20<211>412<212>PRT<213>Bacillus subtilis<400>20Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met ValGlu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125
Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Tyr Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Glu Val Tyr Gly Tyr Thr Asn225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Val Phe Gly Ser Hig Arg Cys Asp Cys Ala Pro Gln Leu His275 280 285Ala Ala Leu Asn Gln Ile Ala Ala Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Met Lys Leu Leu Thr Asn Asn Pro355 360 365
Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Va1 Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>21<211>1239<212>DNA<213>Bacillus subtilis<400>21atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatgc cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccacg ctgaaccgaa ttgccacaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataggcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggatacttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080ataaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200
accaaaatga acaagctagg tcatttactt catttctaa<210>22<211>412<212>PRT<213>Bacillus subtilis<400>22Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile
195 200 205Gln Cya Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275 280 285Ala Thr Leu Asn Arg Ile Ala Thr Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Arg Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Tyr Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Ile Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 95 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>23<211>1239<212>DNA<213>Bacillus subtilis
<400>23atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatac cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgtgtttgg ctctcatcgc 840tgtgattgca gaccgcagct gcacgccgcg ctgaaccaaa ttgccgcaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctegg tgtccggaat1080atgaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctaa1239<210>24<211>412<212>PRT<213>Bacillus subtilis<400>24Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30
Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr val Gln A la Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg A la Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly A la Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Tyr Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225 230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270
Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Arg Pro Gln Leu His275 280 285Ala Ala Leu Asn Gln Ile Ala Ala Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315 320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Met Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>25<211>1239<212>DNA<213>Bacillus subtilis<400>25atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttca tccgatagaa 60gaagcactgg acgctttaaa aaaaggcgaa gtcatcatcg ttgtagatga tgaagacaga 120gaaaatgaag gagactttgt ggctcttgcc gagcatgcaa cgccggaagt cattaacttt 180atggcgacac atgggagagg actgatctgc acgccgctca gtgaggaaat cgcagacagg 240cttgatcttc accctatggt tgagcataat acagactctc accacactgc atttaccgta 300agcatagacc atcgtgaaac gaagacaggt atcagcgctc aagaaagatc ttttaccgtt 360caagcattgc tggacagcaa atccgtgcca tctgattttc agcgtccggg gcacattttt 420ccactgattg cgaaaaaagg aggtgtcctg aaaagagcgg gccatacaga agctgctgtt 480
gatcttgctg aagcttgtgg atctccagga gccggcgtca tttgtgaaat tatgaatgaa 540gacggaacga tggcgagagt gcctgagctc attgaaattg cgaaaaagca tcaattaaaa 600atgatcacca ttaaggattt gattcaatgc cgttacaatc tgacaacact tgtcgagcgt 660gaagttgaca ttacgctgcc tactgatttt gggacattta aggtttatgg atacacaaat 720gaggtagatg gaaaagagca tgtcgcattt gtgatgggag atgtgccgtt cggagaagaa 780ccggtattgg tccgggtgca ttcagaatgt ctcacaggtg acgcgtttgg ctctcatcgc 840tgtgattgcg gaccgcagct gcacgccacg ctgaaccaaa ttgccgcaga aggccgtgga 900gtgctcctgt acttgcgcca agaaggacga ggcatcggtt taatcaataa attaaaagct 960tataagcttc aggaacaagg ctatgacacc gtagaagcca atgaggcgct tggattcttg1020ccggatcttc gcaactatgg catcggagca caaattttac gcgacctcgg tgtccggaat1080atgaagcttt tgacgaataa tccgcgaaaa atcgcaggcc ttgaaggcta cggactcagt1140atttcagaaa gagtgccgct tcaaatggag gcgaaagaac acaataaaaa atatttgcaa1200accaaaatga acaagctagg tcatttactt catttctga 1239<210>26<211>412<212>PRT<213>Bacillus subtilis<400>26Met Arg Gly Ser His His His His His His Gly Ile Asp His Met Phe1 5 10 15His Pro Ile Glu Glu Ala Leu Asp Ala Leu Lys Lys Gly Glu Val Ile20 25 30Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe Val Ala35 40 45Leu Ala Glu His Ala Thr Pro Glu Val Ile Asn Phe Met Ala Thr His50 55 60Gly Arg Gly Leu Ile Cys Thr Pro Leu Ser Glu Glu Ile Ala Asp Arg65 70 75 80Leu Asp Leu His Pro Met Val Glu His Asn Thr Asp Ser His His Thr85 90 95
Ala Phe Thr Val Ser Ile Asp His Arg Glu Thr Lys Thr Gly Ile Ser100 105 110Ala Gln Glu Arg Ser Phe Thr Val Gln Ala Leu Leu Asp Ser Lys Ser115 120 125Val Pro Ser Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu Ile Ala130 135 140Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala Ala Val145 150 155 160Asp Leu Ala Glu Ala Cys Gly Ser Pro Gly Ala Gly Val Ile Cys Glu165 170 175Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Leu Ile Glu180 185 190Ile Ala Lys Lys His Gln Leu Lys Met Ile Thr Ile Lys Asp Leu Ile195 200 205Gln Cys Arg Tyr Asn Leu Thr Thr Leu Val Glu Arg Glu Val Asp Ile210 215 220Thr Leu Pro Thr Asp Phe Gly Thr Phe Lys Val Tyr Gly Tyr Thr Asn225230 235 240Glu Val Asp Gly Lys Glu His Val Ala Phe Val Met Gly Asp Val Pro245 250 255Phe Gly Glu Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu Thr260 265 270Gly Asp Ala Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu His275 280 285Ala Thr Leu Asn Gln Ile Ala Ala Glu Gly Arg Gly Val Leu Leu Tyr290 295 300Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Ile Asn Lys Leu Lys Ala305 310 315320Tyr Lys Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn Glu Ala325 330 335
Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala Gln Ile340 345 350Leu Arg Asp Leu Gly Val Arg Asn Met Lys Leu Leu Thr Asn Asn Pro355 360 365Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser Glu Arg370 375 380Val Pro Leu Gln Met Glu Ala Lys Glu His Asn Lys Lys Tyr Leu Gln385 390 395 400Thr Lys Met Asn Lys Leu Gly His Leu Leu His Phe405 410<210>27<211>61<212>DNA<213>人工<220>
<223>引物RibA 1s<400>27atgagaggat ctcaccatca ccatcaccat gggatcgatc atatgtttcatccgatagaa60g 6l<210>28<211>38<212>DNA<213>人工<220>
<223>引物RibA lAs<400>28tataattgga tccttagaaa tgaagtaaat gacctagc38<210>29<211>54<212>DNA<213>人工<220>
<223>引物RibA 2s<400>29attaatgaat tcattaaaga ggagaaatta actatgagag gatctcacca tcac54
<210>30<211>31<212>DNA<213>人工<220>
<223>引物RibANde+1<400>30ggagggtttc atatgtttca tccgatagaa g31<210>31<211>2l<212>DNA<213>人工<220>
<223>引物RibA4As<400>31taattaagct tggatcctta g21<210>32<211>906<212>DNA<213>Ashbya gossypii<400>32atgactgaat acacagtgcc agaagtgagg tgtgtcgcac gcgcgcgcat accgacggta 60cagggcaccg atgtcttcct ccatctatac cacaactcga tcgacagcaa ggaacaccta 120gcgattgtct tcggcgagaa catacgctcg cggagtctgt tccggtaccg gaaagacgac 180acgcagcagg cgcggatggt gcggggcgcc tacgtgggcc agctgtaccc cgggcggacc 240gaggcagacg cggatcggcg tcagggcctg gagctgcggt ttgatgagac agggcagctg 300gtggtggagc gggcgacgac gtggaccagg gagccgacac tggtgcggct gcactcggag 360tgttacacgg gcgagacggc gtggagcgcg cggtgcgact gcggggagca gttcgaccag 420gcgggtaagc tgatggctgc ggcgacagag ggcgaggtgg ttggcggtgc ggggcacggc 480gtgatcgtgt acctgcggca ggagggccgc ggcatcgggc taggcgagaa gctgaaggcg 540tacaacctgc aggacctggg cgcggacacg gtgcaggcga acgagctgct caaccaccct 600gcggacgcgc gcgacttctc gttggggcgc gcaatcctac tggacctcgg tatcgaggac 660atccggttgc tcacgaataa ccccgacaag gtgcagcagg tgcactgtcc gccggcgcta 720cgctgcatcg agcgggtgcc catggtgccg ctttcatgga ctcagcccac acagggcgtg 780cgctcgcgcg agctggacgg ctacctgcgc gccaaggtcg agcgcatggg gcacatgctg 840cagcggccgc tggtgctgca cacgtctgcg gcggccgagc tcccccgcgc caacacacac 900
atataa 906<210>33<211>301<212>PRT<213>Ashbya gossypii<400>33Met Thr Glg Tyr Thr Val Pro Glu Val Arg Cys Val Ala Arg Ala Arg1 5 10 15Ile Pro Thr Val Gln Gly Thr Asp Val Phe Leu His Leu Tyr His Asn20 25 30Ser Ile Asp Ser Lys Glu His Leu Ala Ile Val Phe Gly Glu Asn Ile35 40 45Arg Ser Arg Ser Leu Phe Arg Tyr Arg Lys Asp Asp Thr Gln Gln Ala50 55 60Arg Met Val Arg Gly Ala Tyr Val Gly Gln Leu Tyr Pro Gly Arg Thr65 70 75 80Glu Ala Asp Ala Asp Arg Arg Gln Gly Leu Glu Leu Arg Phe Asp Glu85 90 95Thr Gly Gln Leu Val Val Glu Arg Ala Thr Thr Trp Thr Arg Glu Pro100 105 110Thr Leu Val Arg Leu His Ser Glu Cys Tyr Thr Gly Glu Thr Ala Trp115 120 125Ser Ala Arg Cys Asp Cys Gly Glu Gln Phe Asp Gln Ala Gly Lys Leu130 135 140Met Ala Ala Ala Thr Glu Gly Glu Val Val Gly Gly Ala Gly His Gly145 150 155160Val Ile Val Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Gly Glu165 170 175Lys Leu Lys Ala Tyr Asn Leu Gln Asp Leu Gly Ala Asp Thr Val Gln180 185 190
Ala Asn Glu Leu Leu Asn His Pro Ala Asp Ala Arg Asp Phe Ser Leu195 200 205Gly Arg Ala Ile Leu Leu Asp Leu Gly Ile Glu Asp IleArg Leu Leu210 215 220Thr Asn Asn Pro Asp Lys Val Gln Gln Val His Cys Pro Pro Ala Leu225 230 235 240Arg Cys Ile Glu Arg Val Pro Met Val Pro Leu Ser Trp Thr Gln Pro245 250 255Thr Gln Gly Val Arg Ser Arg Glu Leu Asp Gly Tyr Leu Arg Ala Lys260 265 270Val Glu Arg Met Gly His Met Leu Gln Arg Pro Leu Val Leu His Thr275 280 285Ser Ala Ala Ala Glu Leu Pro Arg Ala Asn Thr His Ile290 295 300<210>34<211>591<212>DNA<213>Escherichia coli<400>34atgcagctta aacgtgtggc agaagccaaa ctgccaaccc catggggcga tttcctgatg 60gtgggatttg aagaattggc aaccggacac gatcatgtcg cgctagtcta tggcgatatt 120tccgggcata ccccggtact tgcgcgcgtc cattccgaat gtctgaccgg tgacgccctg 180ttcagcttgc gctgcgattg tggcttccag ctcgaagcgg cattgacgca aattgccgag 240gaaggccgtg gtattttgct gtatcaccgt caggaaggtc gtaacattgg tctgctgaat 300aaaatccgcg cttacgcact gcaggatcaa ggttacgata ccgtagaggc taaccaccag 360ttaggcttcg ccgctgatga gcgcgacttc actctttgcg ctgatatgtt caaactcctt 420ggcgtcaatg aagtccgctt gttaaccaat aacccgaaaa aagtcgaaat tctgaccgaa 480gcagggatta atattgttga acgcgtacca ttgattgtag gtcgtaaccc caataacgaa 540cattatctcg ataccaaagc cgagaaaatg ggccatttgc tgaacaaata a 591<210>35<211>196<212>PRT<213>Escherichia coli
<400>35Met Gln Leu Lys Arg Val Ala Glu Ala Lys Leu Pro Thr Pro Trp Gly1 5 10 15Asp Phe Leu Met Va1 Gly Phe Glu Glu Leu Ala Thr Gly His Asp His20 25 30Val Ala Leu Val Tyr Gly Asp Ile Ser Gly His Thr Pro Val Leu Ala35 40 45Arg Val His Ser Glu Cys Leu Thr Gly Asp Ala Leu Phe Ser Leu Arg50 55 60Cys Asp Cys Gly Phe Gln Leu Glu Ala Ala Leu Thr Gln Ile Ala Glu65 70 75 80Glu Gly Arg Gly Ile Leu Leu Tyr His Arg Gln Glu Gly Arg Asn Ile85 90 95Gly Leu Leu Asn Lys Ile Arg Ala Tyr Ala Leu Gln Asp Gln Gly Tyr100 105 110Asp Thr Val Glu Ala Asn His Gln Leu Gly Phe Ala Ala Asp Glu Arg115 120 125Asp Phe Thr Leu Cys Ala Asp Met Phe Lys Leu Leu Gly Val Asn Glu130 135 140Val Arg Leu Leu Thr Asn Asn Pro Lys Lys Val Glu Ile Leu Thr Glu145 150 155 160Ala Gly Ile Asn Ile Val Glu Arg Val Pro Leu Ile Val Gly Arg Asn165 170 175Pro Asn Asn Glu His Tyr Leu Asp Thr Lys Ala Glu Lys Met Gly His180 185 190Leu Leu Asn Lys195<210>36<211>1269<212>DNA<213>Corynebacterium glutamicum
<400>36gtgagtgaac atgagcaggc acacagccaa ttagattctg ttgaagaggc catcgctgac 60atcgctgcgg gtaaagccgt cgtggtggta gatgatgaag atcgtgaaaa tgaaggcgac 120atcatctttg ccgccgaatt agccactcca gaattagtcg ctttcatggt gcgttattcc 180tcgggataca tctgtgcgcc attaaccgca aaggatgcag atcgtcttga tctgcctccg 240atgaccgcgc acaatcagga tgcccgcggc accgcttaca ccgtgaccgt tgatgccaac 300accggcacca caggcatttc tgcaacagac cgcgcccaca ctttgcgctt gcttgctgat 360ccagaagccg accgcacgga tttcacccgt cccggacacg ttgtgccact gcgtgctcgt 420gaaggtggcg tcttggtgcg cgctggacac accgaagcag ctgtcgattt ggctcgcgct 480gcaggcctgc gcccagcagg tgttatctgc gaagtggtca gtgaagagga ccccaccggc 540atggctcggg ttcctgagct gcgccgcttc tgcgatgagc acgatctgaa gctgatctct 600attgagcagc tcattgagtg gcgtcgcaag aatgaaattt tggtggagcg ccaggtggaa 660actgtgctgc ctaccgattt cggcacgttc aaggctgttg gttaccgttc catcatcgat 720ggcaccgagc ttgttgccat tgttgccggc gacgtggcat ccgacggtgg cgaaaacgtc 780ctggttcgag tccactctga gtgcttgact ggtgatgttt ttggatcccg gcgctgcgac 840tgtggacagc agctgcacga gtctttgcgc ctgatccagg aagctggtcg gggagtagtg 900gtgtacatgc gtgggcatga gggacgaggc attggtctgc tcgccaagct acgcgcctac 960caactccagg atgaaggtgc cgacaccgtc gatgccaacc tcgcacttgg tcttccagcc1020gatgcccgcg aatttggcac csgcgcccag attctctacg acttgggtgt gcgctcgctc1080aacttgatca gcaacaaccc agccaagaag gtgggacttg aaggccacgg catttccatt1140gccagccgaa cccccatccc tgttgctgtt catgaagaca atgttcgata cctgaaaacc1200aagcgtgacc gcatgggaca tgacctccca gatgtcgcac tgtgggaaca agagcaccca1260gaaaactaa1269<210>37<211>422<212>PRT<213>Corynebacterium glutamicum<400>37Val Ser Glu His Glu Gln Ala His Ser Gln Leu Asp Ser Val Glu Glu1 5 10 15Ala Ile Ala Asp Ile Ala Ala Gly Lys Ala Val Val Val Val Asp Asp
20 25 30Glu Asp Arg Glu Asn Glu Gly Asp Ile Ile Phe Ala Ala Glu Leu Ala35 40 45Thr Pro Glu Leu Val Ala Phe Met Val Arg Tyr Ser Ser Gly Tyr Ile50 55 60Cys Ala Pro Leu Thr Ala Lys Asp Ala Asp Arg Leu Asp Leu Pro Pro65 70 75 80Met Thr Ala His Asn Gln Asp Ala Arg Gly T hr Ala Tyr Thr Val Thr85 90 95Val Asp Ala Asn Thr Gly Thr Thr Gly Ile Ser Ala Thr Asp Arg Ala100 105 110His Thr Leu Arg Leu Leu Ala Asp Pro Glu A la Asp Arg Thr Asp Phe115 120 125Thr Arg Pro Gly His Val Val Pro Leu Arg Ala Arg Glu Gly Gly Val130 135 140Leu Val Arg Ala Gly His Thr Glu A la Ala Val Asp Leu A la Arg Ala145 150 155 160Ala Gly Leu Arg Pro Ala Gly Val Ile Cys Glu Val Val Ser Glu Glu165 170 175Asp Pro Thr Gly Met Ala Arg Val Pro Glu Leu Arg Arg Phe Cys Asp180 185 190Glu His Asp Leu Lys Leu Ile Ser Ile Glu Gln Leu Ile Glu Trp Arg195 200 205Arg Lys Asn Glu Ile Leu Val Glu Arg Gln Val Glu Thr Val Leu Pro210 215 220Thr Asp Phe Gly Thr Phe Lys Ala Val Gly Tyr Arg Ser Ile Ile Asp225 230 235 240Gly Thr Glu Leu Val Ala Ile Val Ala Gly Asp Val Ala Ser Asp Gly245 250 255
Gly Glu Asn Val Leu Val Arg Val His Ser Glu Cys Leu Thr Gly Asp260 265 270Val Phe Gly Ser Arg Arg Cys Asp Cys Gly Gln Gln Leu His Glu Ser275 280 285Leu Arg Leu Ile Gln Glu Ala Gly Arg Gly Val Val Val Tyr Met Arg290 295 300Gly His Glu Gly Arg Gly Ile Gly Leu Leu Ala Lys Leu Arg Ala Tyr305 310 315 320Gln Leu Gln Asp Glu Gly Ala Asp Thr Val Asp Ala Asn Leu Ala Leu325 330 335Gly Leu Pro Ala Asp Ala Arg Glu Phe Gly Thr Ser Ala Gln Ile Leu340 345 350Tyr Asp Leu Gly Val Arg Ser Leu Asn Leu Ile Ser Asn Asn Pro A la355 360 365Lys Lys Val Gly Leu Glu Gly His Gly Ile Ser Ile Ala Ser Arg Thr370 375 380Pro Ile Pro Val Ala Val His Glu Asp Asn Val Arg Tyr Leu Lys Thr385 390 395 400Lys Arg Asp Arg Met Gly His Asp Leu Pro Asp Val Ala Leu Trp Glu405 410 415Gln Glu His Pro Glu Asn420<210>38<211>1197<212>pNA<213>Bacillus amyloliguefaciens<400>38atgtttcatc cgatagaaga ggcattagaa gcgctgaaaa aaggtgaagt catcatcgtt 60gtcgatgatg aagacagaga aaacgaagga gatttcgtag cgctcgctga gcatgctacg120cctgaagtgg tgaattttat ggcgacccac gggagaggcc tgatctgcac gccgctttct180gaagacatcg ccggccggct ggatcttcat ccaatggtcg atcataatac agactcgcat240gagaccgcgt ttacagtcag cattgaccac aagctgacaa aaacgggaat cagcgctcag300
gaacgttcct ttacgattca ggcgcttttg gacgaagaat ctgtgcctgg cgattttcag360cgtccgggtc atatttttcc cttaatagca aaaaaaggag gcgtcctgaa gcgggcgggc420cacacggaag cagccgttga cctggcaaaa gcatgcggtt ctcaaggagc ggacgtcatt480tgtgaaatta tgaatgaaga cggcacaatg gcgagagtgc ctgagattag cgagattgcg540aaaagccacc agctgaaaat gattacgata aaagacttaa tagaataccg ctacaacatt600acaacacttg tgaacagaga agttgacatt acgctgccga ctgacttcgg cacgttccgg660gtttacggat atacaaacga ggtggacgga aaagaacatc tcgcctttgt catgggcgat720gtcccgttta acagcggacc cgttcttgtc agagtgcact cagaatgcct gaccggcgat780gtgtttgcat cccaccgctg tgattgcggg cctcagcttc atgccgcgtt gcgccaaatt840gccgaagaag gccgcggcgt tctattgtat ttgcgtcagg aaggcagagg aatcggtctc900atcaataagc tgaaagcgta tcgattgcag gaacaagggt acgacacggt tgaagcgaac960gaagcgctcg gctttctgcc tgacttgcgc aactatggca tcggcgccca gattctccgc 1020gatttagggg ttcagcatat gaaactttta accaataacc cccggaaaat cgccggcctt 1080gaagggtacg gactaagcat ttcagatcgg gtgccgcttc aaatggaagc gagtgagcac 1140aacaagcagt atttacaaac caaaatgaaa aaactcggac acttgcttca tttctaa 1197<210>39<10>39<211>398<212>PRT<213>Bacillus amyloliquefaciens<400>39Met Phe His Pro Ile Glu Glu A la Leu Glu Ala Leu Lys Lys Gly Glu1 510 15Val Ile Ile Val Val Asp Asp Glu Asp Arg Glu Asn Glu Gly Asp Phe20 25 30Val Ala Leu Ala Glu His Ala Thr Pro Glu Val Val Asn Phe Met Ala35 40 45Thr His Gly Arg Gly Leu Ile Cys Thr Pro Leu Set Glu Asp Ile Ala50 55 60Gly Arg Leu Asp Leu His Pro Met Val Asp His Asn Thr Asp Ser His65 70 75 80
Glu Thr Ala Phe Thr Val Ser Ile Asp His Lys Leu Thr Lys Thr Gly85 90 95Ile Ser Ala Gln Glu Arg Ser Phe Thr Ile Gln Ala Leu Leu Asp Glu100 105 110Glu Ser Val Pro Gly Asp Phe Gln Arg Pro Gly His Ile Phe Pro Leu115 120 125Ile Ala Lys Lys Gly Gly Val Leu Lys Arg Ala Gly His Thr Glu Ala130 135 140Ala Val Asp Leu Ala Lys Ala Cys Gly Ser Gln Gly Ala Asp Val Ile145 150 155 160Cys Glu Ile Met Asn Glu Asp Gly Thr Met Ala Arg Val Pro Glu Ile165 170 175Ser Glu lle Ala Lys Ser His Gln Leu Lys Met Ile Thr Ile Lys Asp180 185 190Leu Ile Glu Tyr Arg Tyr Asn Ile Thr Thr Leu Val Asn ArG Glu Val195 200 205Asp lle Thr Leu Pro Thr Asp Phe Gly Thr Phe Arg Val Tyr Gly Tyr210 215 220Thr Asn Glu Val Asp Gly Lys Glu His Leu Ala Phe Val Met Gly Asp225 230 235 240Val Pro Phe Asn Ser Gly Pro Val Leu Val Arg Val His Ser Glu Cys245 250 255Leu Thr Gly Asp Val Phe Ala Ser His Arg Cys Asp Cys Gly Pro Gln260 265 270Leu His Ala Ala Leu Arg Gln Ile Ala Glu Glu Gly Arg Gly Val Leu275 280 285Leu Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu IleAsn Lys Leu290 295 300Lys Ala Tyr Arg Leu Gln Glu Gln Gly Tyr Asp Thr Val Glu Ala Asn305 310 315 320
Glu Ala Leu Gly Phe Leu Pro Asp Leu Arg Asn Tyr Gly Ile Gly Ala325 330335Gln Ile Leu Arg Asp Leu Gly Val Gln His Met LysLeu Leu Thr Asn340 345350Asn Pro Arg Lys Ile Ala Gly Leu Glu Gly Tyr Gly Leu Ser Ile Ser355 360 365Asp Arg Val Pro Leu Gln Met Glu A la Ser Glu His Asn Lys Gln Tyr370 375 380Leu Gln Thr Lys Met Lys Lys Leu Gly His Leu Leu His Phe385 390 395<210>40<211>1194<212>DNA<213>Bacillus cereus<400>40atgtttcatc gtattgaaga agctctagaa gatttaaaaa aaggtaaagt cgttatcgta 60tgtgatgatg aaaaccgaga aaatgaaggc gattttattg ctttagcaga gtacattaca 120ccagaaacaa taaattttat gattacacat ggccgtggtc tcgtttgtgt accgattacg 180gaaggatacg cagaacgtct acaattagaa ccaatggtat ctcataatac agattcacat 240catactgcgt ttacagtgag cattgaccat gtctctacaa caacagggat tagcgctcac 300gaacgtgcaa ctacgataca agaattgtta aaccccgcat caaaaggtgc tgatttcaat 360cgacctggac atatctttcc attaattgcg aaagaaggcg gtgtcctgcg tcgtgcaggt 420catacagaag ctgctgttga tttagcaaag ctatgcggtg ccgaaccagc tggagttatt 480tgcgagatta taaatgagga cggcacgatg gcacgtgtac ctgatttaat agaatgcgca 540aaacaatttg atataaaaat gattacaata gaagatttaa ttgcttaccg ccgccatcat 600gaaacacttg tgacgagaga agcggaaatt acattaccta cagatttcgg tactttccac 660gcaattggct attctaactc attagatacg aaagaacata tcgcacttgt aaaaggtgat 720atttcaacag gtgaaccggt acttgtacgt gttcattctg aatgcttaac aggagatgta 780ttcggttcac atcgctgcga ttgcggacca caactccatg cagcacttgc tcaaattgag 840cgtgaaggaa aaggtgttct tctttatatg aggcaagaag gaagaggcat tgggcttctt 900aataagcttc gtgcttataa attacaagaa gaaggattcg atactgtaga agcaaatgaa 960
aaactcggct tccctgctga tcttcgtgat tacggtatcg gtgctcaaat attaaaagat1020ttaggtttac agagtttacg attattaacg aataacccaa gaaaaattgc tggcttacaa1080ggttacgatt tagaagtagt cgagcgtgta ccgttgcaaa tgccagcaaa agaagagaat1140aaatcgtatt tacaaacgaa agtaaacaaa ttaggacact tactaaactt ataa 1194<210>41<211>397<212>PRT<213>Bacillus cereus<400>41Met Phe His Arg Ile Glu Glu Ala Leu Glu Asp Leu Lys Lys Gly Lys1 5 10 15Val Val Ile Val Cys Asp Asp Glu Asn Arg Glu Asn Glu Gly Asp Phe20 25 30Ile Ala Leu Ala Glu Tyr Ile Thr Pro Glu Thr Ile Asn Phe Met Ile35 40 45Thr His Gly Arg Gly Leu Val Cys Val Pro Ile Thr Glu Gly Tyr Ala50 55 60Glu Arg Leu Gln Leu Glu Pro Met Val Sar His Asn Thr Asp Sar His65 70 75 80His Thr Ala Phe Thr Val Sar Ile Asp His yal Sar Thr Thr Thr Gly85 90 95Ile Sar Ala His Glu Arg Ala Thr Thr Ile Gln Glu Leu Leu Asn Pro100 105 110Ala Ser Lys Gly Ala Asp Phe Asn Arg Pro Gly His Ile Phe Pro Leu115 120 125Ile Ala Lys Glu Gly Gly Val Leu Arg Arg Ala Gly His Thr Glu Ala130 135 140Ala Val Asp Leu Ala Lys Leu Cys Gly Ala Glu Pro Ala Gly Val Ile145 150 155 160Cys Glu Ile Ile Asn Glu Asp Gly Thr Met Ala Arg Val Pro Asp Leu165 170 175
Ile Glu Cys Ala Lys Gln Phe Asp Ile Lys Met Ile Thr Ile Glu Asp180 185 190Leu Ile Ala Tyr Arg Arg His His Glu Thr Leu Val Thr Arg Glu Ala195 200 205Glu Ile Thr Leu Pro Thr Asp Phe Gly Thr Phe His Ala Ile Gly Tyr210 215 220Ser Asn Ser Leu Asp Thr Lys Glu His Ile Ala Leu Val Lys Gly Asp225 230 235 240Ile Ser Thr Gly Glu Pro Val Leu Val Arg Val His Ser Glu Cys Leu245 250 255Thr Gly Asp Val Phe Gly Ser His Arg Cys Asp Cys Gly Pro Gln Leu260 265 270His Ala Ala Leu A la Gln Ile Glu Arg Glu Gly Lys Gly Val Leu Leu275 280 285Tyr Met Arg Gln Glu Gly Arg Gly Ile Gly Leu Leu Asn Lys Leu Arg290 295 300Ala Tyr Lys Leu Gln Glu Glu Gly Phe Asp Thr Val Glu Ala Asn Glu305 310 315 320Lys Leu Gly Phe Pro Ala Asp Leu Arg Asp Tyr Gly Ile Gly Ala Gln325 330 335Ile Leu Lys Asp Leu Gly Leu Gln Ser Leu Arg Leu Leu Thr Asn Asn340 345 350Pro Arg Lys Ile Ala Gly Leu Gln Gly Tyr Asp Leu Glu Val Val Glu355 360 365Arg Val Pro Leu Gln Met Pro Ala Lys Glu Glu Asn Lys Ser Tyr Leu370 375 380Gln Thr Lys Val Asn Lys Leu Gly His Leu Leu Asn Leu385 390 395<210>42<211>1215
<212>DNA<213>Bacillus halodurans<400>42atggacaaaa agctatttga tccgattgaa gaagcaatat atgaattaat gcaaggtcga 60gtcgtgatcg tttgtgatga tgaggatcgg gaaaacgaag gggattttgt agcccttgct 120gaaaaagcaa caccagaagt gattaacttc atgatcacgc atggccgtgg tctcgtttgc 180acgccaatca cggaagagcg ggcaaaggaa ttagatcttg tccccatggt ggaccataat 240accgatcccc atggtacggc gtttaccgtc agcattgatc atcaaatgac gaccacagga 300atttctgccc atgaacgggc tatgacgatt caggcgttaa ttgataagaa aacgaaaaag 360caccacttca aacgaccagg tcacattttc cccctaatag cgaaaaacgg aggagtactc 420cgacgggccg gtcatacaga agcggccgtt gatctagctc gtttgtcagg cgctgagccg 480gcaggggtta tttgtgaaat cattaaagaa gatggttcaa tggcacgagt tcctgatttg 540cgaaaaatcg ccgatcagtt tgaactgaag atgatcacaa ttaaagattt aatcgaatat 600cgtcaccgta aagacaagct tgtcaagcgt gaagtagata tttccttacc gacggatttc 660ggctcattcc gtgcaatcgg ttatacagat gtcattgatg gaaaagagag tgtcgcttta 720gtgaaaggac agattgttga aggtgaacca acactcgttc gtgttcactc cgaatgttta 780acaggtgatg tgttcggttc tcaccgttgc gattgtggcc cacaactcca ggcagctctc 840acacaaatcg agcaacaagg caaagggata ctcctttata tgcgtcaaga gggtcgtggt 900atcggtctca tgaataagtt gaaggcatac aagcttcaag aagaaggcta tgatactgta 960gaagcaaatg agaaattagg ctttcctgct gatcttcggg actatggaat gggcgcgcaa1020attttacgcg acttaggtgt gtcaaaaatg cgcctcctta caaacaatcc gcgaaaaatt1080acgggcttga aagggtatgg ccttgaagtg gttgaacggg tgccgctcca attacctcat1140aacaaagata atgagcgcta tttgaaaaca aagcacgaaa agttaggaca tctgctaaat1200tttactcatt cgtaa1215<210>43<211>404<212>PRT<213>Bacillus halodurans<400>43Met Asp Lys Lys Leu Phe Asp Pro Ile Glu Glu Ala Ile Tyr Glu Leu1 5 10 15Met Gln Gly Arg Val Val Ile Val Cys Asp Asp Glu Asp Arg Glu Asn
20 25 30Glu Gly Asp Phe Val Ala Leu Ala Glu Lys Ala The Pro Glu Val Ile35 40 45Asn Phe Met Ile Thr His Gly Arg Gly Leu Val Cys Thr Pro Ile Thr50 55 60Glu Glu Arg Ala Lya Glu Leu Asp Leu val pro Met Val Asp His Asn65 70 75 80Thr Asp Pro His Gly Thr Ala Phe Thr Val Ser Ile Asp His Gln Met85 90 95Thr Thr Thr Gly Ile Ser Ala His Glu Arg Ala Met Thr Ile Gln Ala100 105 110Leu Ile Asp Lys Lys Thr Lys Lys His His Phe Lys Arg Pro Gly His1l5 l20 125Ile Phe Pro Leu Ile Ala Lys Asn Gly Gly Val Leu Arg Arg Ala Gly130 135 140His THr Glu Ala Ala Val Asp Leu Ala Arg Leu Ser Gly Ala Glu Pro145 150 155 160Ala Gly Val Ile Cys Glu Ile Ile Lys Glu Asp Gly Ser Met Ala Arg165 170 175Val Pro Asp Leu Arg Lys Ile Ala Asp Gln phe Glu Leu Lys Met Ile180 185 190Thr Ile Lys Asp Leu Ile Glu Tyr Arg His Arg Lys Asp Lys Leu Val195 200 205Lys Arg Glu Val Asp Ile Ser Leu Pro Thr Asp phe Gly Ser Phe Arg210 215 220Ala Ile Gly Tyr Thr Asp Val Ile Asp Gly Lys Glu Ser Val Ala Leu225 230 235 240Val Lys Gly Gln Ile val Glu Gly Glu Pro Thr Leu Val Arg Val Ris245 250 255
ser Glu Cys Leu Thr Gly Asp Val Phe Gly 5er His Arg Cys Asp Cys260 265 270Gly Pro Gln Leu Gln Ala Ala Leu Thr Gln Ile Glu Gln Gln Gly Lys275 280 285Gly Ile Leu Leu Tyr Met Arg Gln Glu Gly Arg Gly lle Gly Leu Met290 295 300Asn Lys Leu Lys Ala Tyr Lys Lou Gln Glu Glu Gly Tyr Asp Thr Val305 310 315 320Glu Ala Asn Glu Lys Leu Gly Phe Pro Ala Asp Leu Arg Asp Tyr Gly325 330 335Met Gly Ala Gln Ile Leu Arg Asp Leu Gly Val Ser Lys Het Arg Leu340 345 350Leu Thr Asn As Pro Arg Lys Ile Thr Gly Leu Lys Gly Tyr Gly Leu355360 365Glu Val Val Glu Arg Val pro Leu Gln Leu Pro His Asn Lys Asp Asn370 375 380Glu Arg Tyr Leu Lys Thr Lys His Glu Lys Leu Gly His Leu Leu Asn385 390 395 400Phe Thr His Ser
權利要求
1.一種經(jīng)修飾的II型GTP環(huán)化水解酶,其中(i)所述經(jīng)修飾的酶較之對應的未經(jīng)修飾的酶的比活性增加,以及(ii)所述經(jīng)修飾的酶的氨基酸序列包含一處或多處突變,所述突變包括在對應于SEQ ID NO2的261、270、276、279、308和/或347位的氨基酸位置上的1、2、3、4、5或6處突變。
2.如權利要求1所述的經(jīng)修飾的II型GTP環(huán)化水解酶,其中,所述經(jīng)修飾的II型GTP環(huán)化水解酶展示出比對應的未經(jīng)修飾的II型GTP環(huán)化水解酶高至少大約10%的比活性。
3.如權利要求1或2所述的經(jīng)修飾的II型GTP環(huán)化水解酶,其中所述一處或多處突變是一處或多處取代。
4.如權利要求1至3中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶,其中所述對應的未經(jīng)修飾的II型GTP環(huán)化水解酶的氨基酸序列選自由圖1所列出的序列,特別是來自Ashbya,Saccharomyces,Eremothecium,Candida,Neurospora,Schizosaccharomyces,Archeoglobus,Streptomyces,Helicobacter,Escherichia,Corynebacterium,Thermotoga,Arabidopsis,Lycopersicum,Oryza,Alcaligenes,Pseudomonas,Dinococcus,Lactobacillus,Photobacterium或Bacillus的。
5.如權利要求4所述的經(jīng)修飾的II型GTP環(huán)化水解酶,其中所述未經(jīng)修飾的II型GTP環(huán)化水解酶的序列選自由SEQ ID NOs2、33、35、37、39、41和43構成的組。
6.如權利要求1至5中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶,包含在對應于SEQ ID NO2的261位的氨基酸位置上的突變,優(yōu)選地,是丙氨酸對纈氨酸的取代。
7.如權利要求1至5中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶,包含在對應于SEQ ID NO2的270位的氨基酸位置上的突變,優(yōu)選地,是丙氨酸或精氨酸對甘氨酸的取代。
8.如權利要求1至5中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶,包含在對應于SEQ ID NO2的276位的氨基酸位置上的突變,優(yōu)選地,是蘇氨酸對丙氨酸的取代。
9.如權利要求1至5中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶,包含在對應于SEQ ID NO2的279位的氨基酸位置上的突變,優(yōu)選地,是精氨酸對谷氨酰胺的取代。
10.如權利要求1至5中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶,包含在對應于SEQ ID NO2的308位的氨基酸位置上的突變,優(yōu)選地,是精氨酸對賴氨酸的取代。
11.如權利要求1至5中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶,包含在對應于SEQ ID NO2的347位的氨基酸位置上的突變,優(yōu)選地,是異亮氨酸對甲硫氨酸的取代。
12.一種多核苷酸,包含編碼根據(jù)權利要求1至11中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶的核苷酸序列。
13.如權利要求12所述的多核苷酸序列,其中,所述編碼經(jīng)修飾的II型GTP環(huán)化水解酶的核苷酸序列選自由SEQ ID NOs6、8、10、12、14、16、18、20、22、24和26構成的組。
14.如權利要求12或13所述的多核苷酸,其與啟動子、核糖體結合位點和終止子序列可操作地連接,所述多核苷酸是轉錄功能性的。
15.一種載體或質粒,其包含如權利要求12至14中任意一項所述的多核苷酸。
16.如權利要求15所述的載體或質粒,其還包含至少一種標記基因。
17.一種宿主細胞,其包含如權利要求12至14中任意一項所述的多核苷酸。
18.如權利要求17所述的宿主細胞,其選自由細菌細胞、真菌細胞、動物細胞和植物細胞構成的組。
19.如權利要求18所述的宿主細胞,其選自由Bacillus subtilis,Candidaflareri,Eremothecium ashbyii,Ashbya gossypii和Saccharomycescerevisiae構成的組。
20.一種生產核黃素、核黃素前體、FMN、FAD或其衍生物的方法,所述方法包含(a)在合適的培養(yǎng)基中培養(yǎng)根據(jù)權利要求17至19中任意一項所述的宿主細胞;以及(b)可選地,從培養(yǎng)基中分離核黃素、核黃素前體、FMN、FAD或其衍生物。
21.一種方法,用于生產如權利要求1至11中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶的方法,所述方法包括(a)在合適的培養(yǎng)基中培養(yǎng)根據(jù)權利要求17至19中任意一項所述的宿主細胞的群體;以及(b)可選地,從細胞或從培養(yǎng)基中回收所述經(jīng)修飾的II型GTP環(huán)化水解酶。
22.一種方法,用于生產具有提高的比活性的II型GTP環(huán)化水解酶,所述方法包括如下步驟(a)提供編碼II型GTP環(huán)化水解酶的多核苷酸;(b)將一處或多處突變引入所述多核苷酸序列,使得經(jīng)突變的多核苷酸序列編碼經(jīng)修飾的II型GTP環(huán)化水解酶,所述酶較之第一種II型GTP環(huán)化水解酶具有更高的比活性并包含一處或多處突變,其中,所述一處或多處突變包括對應于SEQ ID NO2的261、270、276、279、308和/或347位的氨基酸位置上的1、2、3、4、5或6處突變;(c)可選地,將所述經(jīng)突變的多核苷酸與啟動子、核糖體結合位點和終止子可操作地相連,或將經(jīng)突變的多核苷酸插入載體或質粒;(d)將所述多核苷酸、轉錄功能性多核苷酸或所述載體或質粒引入合適的宿主細胞;以及(e)在允許所述經(jīng)修飾的II型GTP環(huán)化水解酶表達的條件下培養(yǎng)所述宿主細胞。
23.如權利要求1至11中任意一項所述的經(jīng)修飾的II型GTP環(huán)化水解酶或如權利要求12至14中任意一項所述的多核苷酸的用途,用于提高核黃素、核黃素前體、FMN、FAD或其衍生物的生產。
24.一種方法,用于生產具有提高的比活性的II型GTP環(huán)化水解酶,所述方法包括如下步驟(a)提供編碼第一種II型GTP環(huán)化水解酶的多核苷酸,所述酶具有希望被提高的比活性;(b)提供對于比活性有影響的位置;(c)確定用于替換(b)中確定的、野生型II型GTP環(huán)化水解酶的給定氨基酸的最佳氨基酸,以及,將一處或多處突變引入(a)多核苷酸序列中(b)確定的位置,使得經(jīng)突變的多核苷酸序列編碼新的II型GTP環(huán)化水解酶;(d)可選地,將所述經(jīng)突變的多核苷酸插入載體或質粒;(d)將所述多核苷酸或所述載體或質粒引入合適的宿主細胞;以及(e)在允許所述經(jīng)修飾的II型GTP環(huán)化水解酶表達的條件下培養(yǎng)所述宿主細胞。
全文摘要
本發(fā)明涉及經(jīng)修飾的II型GTP環(huán)化水解酶及編碼它們的多核苷酸,所述酶展示出增加的比活性。本發(fā)明還涉及包含這些多核苷酸的載體和含有此類載體的宿主細胞。本發(fā)明提供了用于生產所述經(jīng)修飾的酶的方法和用于生產核黃素、核黃素前體、FMN、FAD或其衍生物的方法。
文檔編號C12N15/55GK101014705SQ200580022895
公開日2007年8月8日 申請日期2005年7月7日 優(yōu)先權日2004年7月7日
發(fā)明者斯比勒·埃伯特, 漢斯-彼得·霍曼, 馬丁·萊瑪恩, 奈杰爾·約翰·芒希亞, 馬卡斯·維斯 申請人:帝斯曼知識產權資產管理有限公司