使用編碼metH的棒桿菌細菌發酵生產含硫精細化學品的方法

2023-07-06 13:13:21

專利名稱：使用編碼metH的棒桿菌細菌發酵生產含硫精細化學品的方法
技術領域：
本發明涉及通過使用表達編碼甲硫氨酸合酶(metH)基因的核苷酸序列的細菌發酵生產含硫精細化學品，尤其是L-甲硫氨酸的方法。
背景技術：
含硫精細化學品如，甲硫氨酸、高半胱氨酸、S-腺苷甲硫氨酸、穀胱甘肽、半胱氨酸、生物素、硫胺、硫辛酸在細胞中通過天然代謝過程產生並用於許多的工業分支中，包括食品、動物飼料、化妝品和製藥工業。這些總稱為「含硫精細化學品」的物質包括有機酸、蛋白原性(Proteinogenic)胺基酸和非蛋白原性胺基酸、維生素和輔因子。在每種情況下通過培養被開發以產生並分泌大量所需物質的細菌的方法，可以大規模、最便利地生產這些含硫精細化學品。尤其適於該目標的生物體為棒桿菌細菌，一種革蘭氏陽性非致病性細菌。
公知可以通過棒桿菌細菌，尤其是穀氨酸棒狀桿菌(corynebacteriumglutamicum)株系發酵生產胺基酸。由於巨大的重要性，生產方法不斷被改進。方法改進可涉及與發酵的技術方面有關的措施，例如，攪拌和氧氣提供，或者涉及營養培養基組成例如，發酵過程中糖濃度，或者涉及給出產物的後處理，例如通過離子交換層析後處理，或者涉及微生物自身的內在性能特點。
已經通過株系選擇開發了從一組含硫精細化學品選擇性組合產生期望的化合物的多種突變株系。通過應用誘變、選擇和突變選擇的方法，所述微生物的性能特點在特定分子的生產方面得到提高。然而，這是一種費時且困難的方法。以這種方法可以得到各種菌株，例如對於抗代謝物或者抑制劑例如，甲硫氨酸類似物α-甲基甲硫氨酸、乙硫氨酸、正亮氨酸、N-乙醯基正亮氨酸、S-三氟甲基高半胱氨酸、2-氨基-5-heprenoitic acid、硒代甲硫氨酸、甲硫氨酸磺醯亞胺、methoxine、1-氨基環戊烷甲酸具有抗性的菌株或者對於在調節中起重要作用的代謝物是營養缺陷型的菌株和產生含硫精細化學品，例如，L-甲硫氨酸的菌株。
重組DNA技術也被使用了一些年以通過擴增各胺基酸生物合成基因和研究對胺基酸生產的作用來改善生產L-胺基酸的棒狀桿菌菌株。
WO-A-02/10209描述了使用生產L-甲硫氨酸的棒桿菌細菌發酵生產L-甲硫氨酸的方法，該細菌中至少metH基因被過表達並且此metH編碼序列來自穀氨酸棒狀桿菌ATCC 13032。
發明概述本發明的一個目的是提供含硫精細化學品，尤其是L-甲硫氨酸的新的改進的發酵生產方法。
我們已經發現通過提供一種含硫精細化學品的發酵生產方法可以實現該目的，該方法包括在棒桿菌細菌中表達編碼具有metH活性的蛋白的異源核苷酸序列。
本發明首先涉及至少一種含硫精細化學品的發酵生產方法，該方法包括下面的步驟a)發酵產生期望的含硫精細化學品的棒桿菌細菌培養物，該棒桿菌細菌表達至少一種編碼具有甲硫氨酸合酶(metH)活性的蛋白的異源核苷酸序列；b)在培養基或細菌細胞中富集含硫精細化學品，和c)分離優選含有L-甲硫氨酸的含硫精細化學品。
上面的metH-編碼核苷酸序列與來自穀氨酸棒狀桿菌ATCC 13032的metH-編碼序列的同源性優選小於70％。metH-編碼序列優選來自下列表I的生物體中的任一種
列表I

ATCC美國典型培養物保藏中心，Rockville，MD，USAPCC藍細菌巴斯德培養物保藏中心，法國巴黎根據本發明使用的metH編碼序列優選含有根據SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49和51的編碼序列或者編碼具有metH活性的蛋白的與上面序列同源的核苷酸序列。
此外，根據本發明使用的metH編碼序列優選編碼具有metH活性的蛋白，所述蛋白含有根據SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50和52的胺基酸序列或者代表具有metH活性的蛋白的與上面序列同源的胺基酸序列。
metH編碼序列優選為可以在棒桿菌細菌中複製或者穩定整合到染色體的DNA或RNA。
根據優選的實施方案，本發明方法通過如下方式實施a)使用質粒載體轉化的細菌菌株，其中所述質粒載體攜帶至少一份處於調節序列控制下的metH編碼序列的拷貝或者b)使用一種菌株，該菌株中編碼metH的序列已經被整合到細菌染色體中。
此外，優選為發酵而過表達編碼metH的序列。
此外，還可能希望發酵這樣的細菌，在該細菌中期望的含硫精細化學品的生物合成途徑或者與之相關的生物合成途徑或其他代謝途徑中的至少另一基因也已經被擴增；和/或者至少一種減少期望的含硫精細化學品的生產的代謝途徑至少部分地被關閉。
還可能希望發酵這樣細菌，其中期望的含硫精細化學品的生物合成途徑中的至少另一種基因的活性不受到代謝物的不希望的影響。
因此，根據本發明方法的另一個實施方案，發酵棒桿菌細菌，其中同時，至少一種選自a)天冬氨酸激酶-編碼基因lysC，b)天冬氨酸-半醛脫氫酶-編碼基因asd，c)甘油醛-3-磷酸脫氫酶-編碼基因gap，
d)3-磷酸甘油酸激酶-編碼基因pgk，e)丙酮酸羧化酶-編碼基因pyc，f)丙糖磷酸異構酶-編碼基因tpi，g)高絲氨酸O-乙醯轉移酶-編碼基因metA，h)胱硫醚γ-合酶-編碼基因metB，i)胱硫醚γ-裂合酶-編碼基因metC，j)絲氨酸羥甲基轉移酶-編碼基因glyA，k)O-乙醯高絲氨酸硫化氫解酶-編碼基因metY，l)亞甲基四氫葉酸還原酶-編碼基因metF，m)磷酸絲氨酸氨基轉移酶-編碼基因serC，n)磷酸絲氨酸磷酸酶-編碼基因serB，o)絲氨酸乙醯基轉移酶-編碼基因cysE，p)高絲氨酸脫氫酶-編碼基因hom的基因被過表達。
根據本發明的另一個實施方案，發酵棒桿菌，其中，同時，至少一種選自上面提到的a)到p)的基因被突變，從而尤其使得與未突變的蛋白相比，相應突變的蛋白的活性受代謝物的影響程度較小(如果存在)，並且尤其是精細化學品的本發明生產沒有受到不利影響。由於突變，該蛋白也可能具有更高活性(底物轉化)和/或底物特異性，從而增強期望的精細化學品的生產。
根據本發明的另一個實施方案，發酵棒桿菌，其中，同時，至少一種選自q)高絲氨酸激酶-編碼基因thrB，r)蘇氨酸脫水酶-編碼基因ilvA，s)蘇氨酸合酶-編碼基因thrC，t)內消旋-二氨基庚二酸D-脫氫酶-編碼基因ddh，u)磷酸烯醇丙酮酸羧基激酶-編碼基因pck，v)葡萄糖-6-磷酸6-異構酶-編碼基因pgi，
w)丙酮酸氧化酶-編碼基因poxB，x)二氫吡啶二羧酸合酶-編碼基因dapA，y)二氫吡啶二羧酸還原酶-編碼基因dapB；或z)二氨基吡啶甲酸脫羧酶-編碼基因lysA的基因被弱化，尤其是通過減小相應基因的表達速率，或者通過表達具有較低活性(底物轉化)的蛋白來實現此目的。
根據本發明方法的另一個實施方案，發酵棒桿菌細菌，其中，同時，至少一種選自上面q)到z)的基因被突變，從而使得相應蛋白的酶活性被部分或完全減小。
優選在本發明方法中使用微生物物種穀氨酸棒狀桿菌。
在該方法的另一個實施方案中，使用抗至少一種甲硫氨酸生物合成抑制劑的微生物。這些抑制劑為甲硫氨酸類似物，如α-甲基甲硫氨酸、乙硫氨酸、正亮氨酸、N-乙醯基正亮氨酸、S-三氟甲基高半胱氨酸、2-氨基-5-heprenoic acid、硒代甲硫氨酸、甲硫氨酸磺醯亞胺、methoxine、1-氨基環戊烷甲酸。
本發明還涉及從發酵液生產含L-甲硫氨酸的動物飼料添加劑的方法，其包括下面的步驟a)在發酵培養基中培養和發酵產生L-甲硫氨酸的微生物；b)從含有L-甲硫氨酸的發酵液除去水；c)除去按重量計發酵過程中形成的生物量的0到100％；和d)乾燥根據b)和/或c)得到的發酵液，以便得到期望的粉末或顆粒形式的動物飼料添加劑。
本發明還涉及第一次從上面的微生物分離的編碼metH的序列，涉及該序列編碼的甲硫氨酸合酶，以及這些多核苷酸和蛋白質的相應功能同系物。
尤其，本發明還涉及實施上面的方法所需要的表達構建體和微生物。
因此，本發明還涉及如下方面-編碼lysC thr311ile的質粒pCIS lysC thr311ile或其功能等價物，即具有比野生型更大的相應天冬氨酸激酶活性的lysC突變體；-用質粒pCIS lysC thr311ile轉化的，尤其是選自棒狀桿菌屬微生物，尤其是穀氨酸棒狀桿菌種的宿主生物體，如轉化菌株LU 1479 lysC 311ile；-編碼天藍色鏈黴菌(Streptomyces coelicolor)metH的質粒pC PhsdhmetH；-如上定義的宿主生物體，其被編碼外源metH的質粒轉化；尤其是用質粒pC Phsdh metH Sc轉化；-具有抗至少一種甲硫氨酸生物合成抑制劑的如上定義的宿主生物體，如轉化菌株LU 1479 lysC 311ile ET-16，其任選用外源編碼metH的序列轉化，如轉化菌株LU 1479 lysC 311ile ET-16 pC Phsdh metH Sc。
發明詳述a)一般術語具有甲硫氨酸合酶(簡寫為metH(系統名5-甲基四氫葉酸高半胱氨酸S-甲基轉移酶；EC 2.1.1.13))生物活性的蛋白質指能夠使用輔因子5-甲基四氫葉酸(MTHF)、鈷胺素(維生素B12)和S-腺苷甲硫氨酸將高半胱氨酸轉化成甲硫氨酸和四氫葉酸的那些蛋白。儘管輔因子5-甲基四氫葉酸按化學劑量進入反應(1mol MTHF/1 mol形成的甲硫氨酸)，但是S-腺苷甲硫氨酸是按亞化學計量轉變，如文獻所描述的。另一方面，鈷胺素在轉化中起催化作用。metH蛋白的其他細節是技術人員公知的。(Banerjee R.V.，Matthews R.G.，FASEB J.，41450-1459，1990，Ludwig ML.，MatthewesRG.，Annual Review of Biochemistry.66269-313，1997，Drennan CL.，Matthews RG.，Ludwig ML.，Current Opinion in Structural Biology.4919-29，1994)。技術人員可區分依賴鈷胺素的5-甲基四氫葉酸高半胱氨酸S-甲基轉移酶的活性和獨立於鈷胺素的5-甲基四氫蝶醯基三穀氨酸高半胱氨酸S-甲基轉移酶(EC 2.1.1.14)，也稱為metE的活性。技術人員可使用酶測定法檢測metH的酶活性，測定法方案可以是Jarrett JT.，GouldingCW.，Fluhr K.，Huang S.，Matthews RG.，Methods in Enzymology.281196-213，1997。
在本發明的範圍內，術語「含硫精細化學品」包括含有至少一個共價結合的硫原子並且可通過本發明的發酵方法得到的任何化學化合物。其非限制性實例為甲硫氨酸、高半胱氨酸、S-腺苷甲硫氨酸，尤其是甲硫氨酸和S-腺苷甲硫氨酸。
在本發明的範圍內，術語「L-甲硫氨酸」、「甲硫氨酸」、高半胱氨酸和S-腺苷甲硫氨酸也包括相應的鹽如，甲硫氨酸鹽酸鹽或甲硫氨酸硫酸鹽。
「多核苷酸」通常指多聚核糖核苷酸(RNA)和多聚脫氧核糖核苷酸(DNA)，其可以分別是未修飾的RNA和DNA，或者分別是修飾的RNA和DNA。
根據本發明，「多肽」指含有通過肽鍵相連的兩個或多個胺基酸的肽或蛋白質。
術語「代謝物」指在生物體的代謝中作為中間物或者終產物產生並且，除了作為化學構件之外，也可能對酶和它們的催化活性具有調節作用的化學化合物。從文獻已知這種代謝物可以以抑制和刺激的方式作用於酶的活性(Biochemistry，Stryer，Lubert，1995 W.H.Freeman Company，NewYork，New York)。文獻中也已有報導稱可以在生物體中生產所受到的代謝物的影響已經被改變的酶，所述改變通過如用UV輻射、離子化輻射或誘變物質突變基因組DNA，並隨後選擇特定表型來實現(Sahm H.，EggelingL.，de Graaf AA.，Biological Chemistry 381(9-10)899-910，2000；Eikmanns BJ.，Eggeling L.，Sahm.H.，Antonie van Leeuwenhoek.，64145-63，1993-94)。這些改變的性質也可以通過特定措施實現。技術人員知道怎樣在酶基因中特異地修飾編碼蛋白的DNA的特定核苷酸使得從表達的DNA序列得到的蛋白具有某種新的性質，例如改變代謝物對未修飾的蛋白的調節作用。
可以影響酶活性，從而減小反應速度或者改變對底物的親和性或者改變多個反應速率。
術語「表達」和「擴增」或「過表達」在本發明的上下文中描述微生物中相應DNA編碼的一種或多種酶的產生或其胞內活性的增加。為此，例如，可以向生物體中導入基因、將現有基因替代為另一個基因、增加一種或多種基因的拷貝數，使用強啟動子或者使用編碼具有高活性的相應酶的基因，並且適宜時，這些措施可以組合使用。
b)本發明的metH蛋白本發明還包括上面列表I中特別公開的生物體的metH酶的「功能等價物」。
在本發明範圍內，具體公開的多肽的「功能等價物」或類似物為與具體公開的多肽不同的多肽，這些多肽還具有期望的生物學活性如，底物特異性。
根據本發明，「功能等價物」尤其指在上面提到的序列位置的至少一個位置具有不同於具體提到的胺基酸的胺基酸，但是仍然具有一種上面提到的生物學活性的突變體。「功能等價物」從而還包括可以通過一個或多個胺基酸添加、置換、缺失和/或倒位得到的突變體，所述修飾可以在序列的任何位置發生，只要它們導致具有本發明的性質譜的突變體即可。尤其是當突變的和未修飾的多肽的反應模式在質上相匹配，即例如，相同的底物以不同速度被轉化時，存在功能等價物。
「功能等價物」自然還包括可以從其他生物體得到的多肽，以及天然發生的變體。例如，可通過序列比較發現同源序列區，並且可以按照本發明的具體指導方案確立等價酶。
「功能等價物」還包括例如具有期望的生物學功能的本發明多肽的片段，優選各結構域或序列基序。
「功能等價物」還包括融合蛋白，其具有一個上面提到的多肽序列或衍生自該多肽序列的功能等價物和以功能性方式(即融合蛋白各部分的功能受到可忽略的功能削弱)N-或C-連接的至少一個功能上不同的異源序列。這種異源序列的非限制性實例為，例如，信號肽、酶、免疫球蛋白、表面抗原、受體或受體配體。
根據本發明，「功能等價物」包括具體公開的蛋白的同系物。這些同系物具有，例如在全長上，與具體公開的序列之一至少30％，或者約40％、50％，優選至少約60％、65％、70％或75％，尤其是至少85％，例如，90％、95％或99％的同源性，該同源性通過Pearson和Lipman，Proc.Natl.Acad.，Sci.(USA)85(8)，1988，2444-2448的算法計算。同源性程度尤其反映了修飾的和未修飾的序列之間的同一性程度。
本發明的蛋白或多肽的同系物可通過誘變，例如，通過蛋白的點突變或截短產生。如此處所用的術語「同系物」，也涉及蛋白的變體形式，其可以作為蛋白活性的激動劑或拮抗劑。
本發明蛋白的同系物可通過篩選突變體，例如，截短突變體的組合文庫鑑定。可以，例如，通過核酸水平上的組合誘變，例如，通過合成的寡核苷酸混合物的酶促連接產生蛋白質變體的多樣化文庫。有許多方法可用於從簡併寡核苷酸序列製備潛在同系物的文庫。簡併基因序列的化學合成可以在自動DNA合成儀上進行，然後合成的基因可被連接到適宜的表達載體中。一組簡併基因的使用使得可以在一個混合物中提供編碼期望的一組潛在蛋白質序列的全部序列。合成簡併寡核苷酸的方法是技術人員公知的(例如，Narang，S.A.，(1983)Tetrahedron 393；Itakura等，(1984)Annu.Rev.Biochem.53323；Itakura等，(1984)Science 1981056；Ike等，(1983)Nucleic acid Res.11477)。
此外，具有蛋白密碼子的片段的文庫可用於產生蛋白質片段的多樣化群體以備篩選和隨後選擇本發明蛋白的同系物。在一個實施方案中，可通過將編碼序列的雙鏈PCR片段用核酸酶在每個分子僅發生約1次切割的條件下處理，變性雙鏈DNA，再次退火DNA以形成可含有不同帶切口產物的有義/反義對的雙鏈DNA，用S1核酸酶處理從新形成的雙鏈體除去單鏈部分並將所得片段文庫連接到表達載體，從而產生編碼序列片段的文庫。可以通過此方法設計編碼本發明蛋白質的不同大小的N-末端、C-末端和內部片段的表達文庫。
在現有技術中公知一些技術可用於從通過點突變或截短產生的組合文庫篩選基因產物和從cDNA文庫篩選具有選擇的性質的基因產物。這些技術可經改變以適於快速篩選通過本發明的同系物的組合誘變產生的基因文庫。高通量分析篩選大基因文庫最經常使用的技術包括將基因文庫克隆到可複製的表達載體中，用所得載體文庫轉化適宜的細胞並在一定條件下表達組合基因，在該條件下期望活性的檢測方便了編碼該基因(其產物已經被檢測)的載體的分離。遞歸整體誘變(REM)——一種增加文庫中功能突變體的頻率的技術——可與篩選試驗組合使用以鑑定同系物(Arkin undYourvan(1992)PNAS 887811-7815；Delgrave等(1993)Protein Engineering6(3)327-331)。
c)本發明的多核苷酸本發明還涉及編碼上面的metH酶和可通過例如使用人工核苷酸類似物得到的該metH酶的功能等價物的核酸序列(單-和雙鏈DNA和RNA序列，例如cDNA和mRNA)。
本發明不僅涉及編碼本發明多肽或蛋白質或者其生物活性部分的分離的核酸分子，還涉及可用作例如，用以鑑定或擴增本發明的編碼核酸的雜交探針或引物的核酸片段。
此外，本發明的核酸分子可含有來自該基因的編碼區的3』和/或5』末端的非翻譯序列。
「分離的」核酸分子與存在於該核酸的天然來源中的其他核酸分子分離並且如果其通過重組技術製備那麼還可以基本上沒有其他細胞物質或培養基，或者如果其通過化學合成那麼還可以基本上沒有化學前體或其他化學物質。
本發明還包括與具體描述的核苷酸序列或其部分互補的核酸分子。
本發明的核苷酸序列使得可以產生可用於鑑定和/或克隆其他細胞類型和生物體中的同源序列的探針和引物。這些探針和引物通常互補於一個核苷酸序列區，該序列區在嚴緊條件下雜交本發明核酸序列的有義鏈或相應的反義鏈的至少約12個，優選至少約25個，例如40、50或75個連續核苷酸。
本發明的其他核酸序列衍生自SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49或51並且通過加入、置換、插入或缺失一個或多個核苷酸而不同，但是仍然編碼具有期望的性質譜的多肽。這些可以是與上面序列，例如在全長範圍內，在至少約50％、55％、60％、65％、70％、80％或90％，優選至少約95％、96％、97％、98％或99％的序列位置相同的多核苷酸。
本發明還包括按照特定來源或宿主生物體的密碼子使用與具體提到的序列相比含有「沉默」突變或被修飾的那些核酸序列，以及天然發生的變體，例如，剪接變體或等位基因變體。本發明還涉及可通過保守核苷酸置換(即相關胺基酸被相同電荷、大小、極性和/或溶解性的胺基酸代替)得到的序列。
本發明還涉及通過序列多態性從具體公開的核酸衍生的分子。因為群體內個體間的天然變異而可能存在這些遺傳多態性。這些天然變異通常導致基因的核苷酸序列中1到5％的差異。
本發明還包括與上面提到的編碼序列雜交或與其互補的核酸序列。這些多核苷酸可以在基因組或cDNA文庫的篩選時被發現，並且適宜時，可通過PCR，使用適宜的引物從這些文庫擴增，然後，例如，用適宜的探針分離。另一個可能方案是用本發明的多核苷酸或載體轉化適宜的微生物，繁殖微生物從而擴增多核苷酸，然後分離它們。再一個可能方案是通過化學途徑合成本發明的多核苷酸。
能夠與多核苷酸「雜交」的性質指多核苷酸或寡核苷酸能夠在嚴緊條件下結合幾乎互補的序列，而在這些條件下不存在非互補序列之間的非特異性結合。為此，序列應該70-100％，優選90-100％互補。互補序列能夠相互特異結合的性質可以例如在Northern或Southern印跡技術或者，對於引物結合，在PCR或在RT-PCR中被利用。具有30個或更多個鹼基對長度的寡核苷酸通常用於此目的。嚴緊條件指，例如，在Northern印跡技術中在50-70℃，優選60-65℃使用洗滌溶液，例如，具有0.1％SDS的0.1xSSC緩衝液(20×SSC；3M NaCl，0.3M檸檬酸鈉，pH7.0)，用於洗脫非特異雜交的cDNA探針或寡核苷酸。在該情況下，如上面所提到的，僅僅具有高度互補性的核酸保持相互結合。嚴緊條件的設置是技術人員公知的並且在例如Ausubel等，Current Protocols in Molecular Biology，John WileySons，N.Y.(1989)，6.3.1-6.3.6.beschrieben中描述。
d)metH編碼基因的分離編碼酶甲硫氨酸合酶(EC 2.1.1.13)的metH基因可以以本身已知的方式從上面列表I的生物體分離。
為了分離上面列表I的生物體的metH基因或者其他基因，首先在大腸桿菌(E.Coli)中產生該生物體的基因文庫。基因文庫的產生在通常已知的教科書和手冊中有詳細描述。可提及的的實例為Winnacker的教科書Gene und Klone，Eine Einführung in die Gentechnologie(Verlag Chemie，Weinheim，Germany，1990)，和Sambrook等的手冊Molecular Cloning，ALaboratory Manual(Cold Spring Harbor Laboratory Press，1989)。一個非常熟知的基因文庫是大腸桿菌K-12株系W3110的基因文庫，其通過Kohara等(Cell 50，495-508(198))在λ載體中產生。
為了在大腸桿菌中產生列表I的生物體的基因文庫，可以使用粘粒載體SuperCos1(Wahl等，1987，Proceedings of the National Academy ofSciences USA，842160-2164)，或者質粒如pBR322(BoliVal；Life Sciences，25，807-818(1979))或pUC9(Vieira等，1982，Gene，19259-268)。適宜的宿主尤其是限制性和重組缺陷的那些大腸桿菌菌株。其一個實例是Grant等(Proceedings of the National Academy of Sciences USA，87(1990)4645-4649)描述的菌株DH5αmcr。通過粘粒幫助克隆的長DNA片段又可以被亞克隆到適於測序的通用載體中並隨後被測序，測序方法如在例如Sanger等(Proceedings of the National Academy of Sciences of theUnited States of America，745463-5467，1977)中所描述的。
所得DNA序列可以使用已知的算法或序列分析程序例如，Staden的程序(Nucleic Acids Research 14，217-232(1986))、Marck的程序(NucleicAcids Research 16，1829-1836(1988))或Butler的GCG程序(Methods ofBiochemical Analysis 39，74-97(1998))進行研究。
發現了來自根據上面的表I的生物體的編碼metH的DNA序列。具體地，發現了根據SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49和51的DNA序列。此外，使用上面描述的方法，從存在的所述DNA序列得到相應蛋白質的胺基酸序列。SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50和52描述了得到的metH基因產物的胺基酸序列。
由於遺傳密碼簡併性從根據SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49和51的序列得到的DNA編碼序列也是本發明的主題。同樣，本發明也涉及與所述序列或者來自於其的序列部分雜交的DNA序列。
通過雜交鑑定DNA序列的教導可由技術人員在例如手冊BoehringerMannheim GmbH的「The DIG System Users Guide für FilterHybridization」(Mannheim，Germany，1993)和在Leibl等(InternationalJournal of Systematic Bacteriology(1991)41255-260)中發現。利用聚合酶鏈式反應(PCR)擴增DNA序列的教導可由技術人員在例如手冊GaitOligonucleotide synthesisA Practical Approach(IRL Press，Oxford，UK，1984)和在Newton和GrahamPCR(Spektrum Akademischer Verlag，Heidelberg，德國，1994)中發現。
還公知蛋白的N-和/或C-末端的變化不會實質上損害其功能或者甚至可能穩定所述功能。相關信息可由技術人員在Ben-Bassat等(Journal ofBacteriology 169751-757(1987))、O』Regan等(Gene 77237-251(1989))、Sahin-Toth等(Protein Sciences 3240-247(1994))、Hochuli等(Biotechnology 61321-1325(1988))和遺傳學和分子生物學的已知的教科書中發現。
相應從SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50和52得到的胺基酸序列也同樣是本發明的部分。
e)根據本發明使用的宿主細胞本發明還涉及作為宿主細胞的微生物，尤其是棒桿菌細菌，其中，該微生物含有載體，尤其是穿梭載體或質粒載體，載體上攜帶至少一個如本發明所定義的metH基因，或者在該微生物中本發明的metH基因被表達或擴增。
這些微生物可以從葡萄糖、蔗糖、乳糖、果糖、麥芽糖、糖蜜、澱粉、纖維素或從甘油和乙醇產生含硫精細化學品，尤其是L-甲硫氨酸。所述微生物優選棒桿菌細菌，尤其是棒狀桿菌屬。對於棒狀桿菌屬，必須提到尤其是現有技術中公知能夠產生L-胺基酸的穀氨酸棒狀桿菌菌株。
可以提及的適宜的棒桿菌細菌菌株的實例為棒狀桿菌屬菌株，尤其是穀氨酸棒狀桿菌種，如穀氨酸棒狀桿菌ATCC13032，醋谷棒狀桿菌(Corynebacterium acetoglutamicum)ATCC 15806，嗜乙醯乙酸棒狀桿菌(Corynebacterium acetoacidophilum)ATCC13870，Corynebacterium thermoaminogenes FERM BP-1539，棲糖蜜棒狀桿菌(Corynebacterium melassecola)ATCC 17965或者短桿菌屬(Brevibacterium)，如黃色短桿菌(brevibacterium flavum)ATCC 14067乳發酵短桿菌(Brevibacterium lactofermentum)ATCC 13869和擴展短桿菌(Brevibacterium divaricatum)ATCC 14020；或者同樣產生期望的精細化學品或其前體的衍生的菌株如穀氨酸棒狀桿菌KFCC10065穀氨酸棒狀桿菌ATCC21608縮寫KFCC指韓國培養物保藏中心聯盟(Korean Federatioin ofCulture Collection)，縮寫ATCC指美國典型培養物保藏中心(AmericanType Strain Culture Collection)，縮寫FERM指日本工業科學技術機構國立生命科學和人體技術研究所保藏中心(National Institute of Bioscienceand Human Technology)。
f)實施本發明的發酵根據本發明，發現棒桿菌細菌在過表達來自表I的生物體的metH基因後，以有利的方式產生含硫精細化學品，尤其是L-甲硫氨酸。
為了實現過表達，技術人員可以採取單獨的或組合的不同措施。因此，可以增加合適的基因的拷貝數或者突變位於結構基因上遊的啟動子和調節區或者核糖體結合位點。整合到結構基因的上遊的表達盒以相同方式起作用。誘導型啟動子使得還可以在發酵的L-甲硫氨酸生產過程中增加表達。表達還可以通過延長mRNA的壽命來提高。此外，通過防止酶蛋白的降解還可以增強酶活性。基因或基因構建體可以以變化的拷貝數存在於質粒中或者被整合到染色體上並在其中擴增。另一個可能的備選方案是通過改變培養基組分和培養的操作來實現相關基因的過表達。
用於此目的的教導可由技術人員在例如，Martin等(Biotechnology5，137-146(1987))、Guerrero等(Gene 138，35-41(1994))，Tsuchiya和Morinaga(Bio/Technology 6，428-430(1988))、Eikmanns等(Gene 102，93-98(1991))、歐洲專利0472869、美國專利4,601,893、Schwarzer和Pühler(Biotechnology 9，84-87(1991)、Remscheid等(Applied andEnvironmental Microbiology 60，126-132(1994))、Labarre等(Journal ofBacteriology 175，1001-1007(1993))、專利申請WO 96/15246、Malumbres等(Gene134，15-24(1993))、日本公開說明書JP-A-10-229891、Jensen undHammer(Biotechnology and Bioengineering 58，191-195(1998))、Makrides(Microbiological Reviews 60512-538(1996)和遺傳學和分子生物學的已知的教科書中發現。
本發明因此還涉及含有處於調節核酸序列的遺傳控制下的編碼本發明多肽的核酸序列的表達構建體；涉及含有至少一個所述表達構建體的載體。本發明的這種構建體優選包括特定編碼序列的5』上遊的啟動子和3』下遊的終止子以及，適宜時，其它調節元件，在每種情況下這些調節元件可操作地連接到編碼序列上。「可操作地連接」指啟動子、編碼序列、終止子和適宜時，其它調節元件的順序排列，從而每個調節元件可在編碼序列表達中正確執行其功能。可操作連接的序列的實例為活化序列和增強子，等等。其他調節元件包括可選擇標記、擴增信號、複製起點，等等。適宜的調節序列在例如Goeddel，基因表達技術酶學方法185，Academic Press，SanDiego，CA(1990)中描述。
除了人工調節序列，天然調節序列仍然可存在於實際結構基因的上遊。遺傳修飾可以，適宜時，關閉該天然調節並增加或減少基因的表達。然而，基因構建體也可以具有更簡單的設計，即沒有額外的調節信號被插入到結構基因的上遊並且天然啟動子及其調節作用沒有被除去。相反，可以突變天然調節序列使調節作用不再發生並且基因表達得到增加或減少。基因構建體可含有核酸序列的一份或多份拷貝。
有用的啟動子的實例為來自穀氨酸棒狀桿菌啟動子的ddh、amy、lysC、dapA、lysA，以及革蘭氏陽性細菌啟動子SPO2(見「枯草芽孢桿菌及其近親」，Sonenshein，Abraham L.，Hoch，James A.，Losick，Richard；ASM Press，華盛頓哥倫比亞特區，和Patek M.Eikmanns BJ.，Patek J.，Sahm H.，Microbiology.142 1297-309，1996)或者優選在革蘭氏陰性細菌中應用的cos、tac、trp、tet、trp-tet、lpp、lac、lpp-lac、laclq、T7、T5、T3、gal、trc、ara、SP6、λ-PR和λ-PL啟動子。還優選使用誘導型啟動子，例如光可誘導的啟動子以及尤其是溫度可誘導的啟動子，如PrPl啟動子。原則上可以使用所有天然啟動子及其調節序列。此外，還可以有利地使用合成啟動子。
所提及的調節序列旨在使得核酸序列可以特異表達。取決於宿主生物，這可能意味著，例如，基因僅僅在誘導後表達或過表達，或者其立即表達和/或過表達。
關於這一點，調節序列和因子可以優選對表達具有有益作用，從而增加或減少表達。因此，可以有利地通過使用強轉錄信號如啟動子和/或增強子在轉錄水平增強調節元件。然而，除此之外還可以通過例如提高mRNA的穩定性增強翻譯。
通過將適宜的啟動子、適宜的Shine-Dalgarno序列融合到metH核苷酸序列和適宜的終止信號上製備表達盒。為此，使用一般重組和克隆技術，例如在Current Protocols in Molecular Biology，1993，John Wiley Sons，Incorporated，New York，New York，PCR Methods，Gelfand，David H.，Innis，Michael A.，Sninsky，John J.，1999，Academic Press，Incorporated，California，San Diego，PCR Cloning Protocols，Methods in MolecularBiology，Ser.，Vol.192，第二版，Humana Press，New Jersey，Totowa.T.Maniatis，E.F.Fritsch和J.Sambrook，分子克隆實驗室指南，冷泉港實驗室，冷泉港，NY(1989)和T.J.Silhavy，M.L.Berman und L.W.Enquist，基因融合實驗，冷泉港實驗室，冷泉港，NY(1984)和Ausubel，F.M.等，Current Protocols in Molecular Biology，Greene Publishing Assoc.andWiley Interscience(1987)中描述的技術。
重組核酸構建體或基因構建體在適宜的宿主生物體中表達，這可通過將構建體有利地插入宿主特異性載體中實現，該載體使得這些基因在宿主中的最佳表達成為可能。載體是技術人員熟知的並且可在例如，「克隆載體」(Pouwels P.H.等，Hrsg，Elsevier，Amsterdam-New York-Oxford，1985)中發現。術語「載體」指質粒和技術人員公知的所有其他載體，例如，噬菌體、轉座子、IS元件、質粒、粘粒和線性或環狀DNA。這些載體可在宿主生物體中自主複製或者隨染色體複製。
通過例如使用游離型質粒過表達本發明的metH基因而擴增這些基因。適宜的質粒為在棒桿菌細菌中複製的那些質粒。許多公知的質粒載體，例如，pZ1(Menkel等，Applied and Environmental Microloiotogy(應用和環境微生物學)(1989)64549-554)、pEKEx1(Eikmanns等，Gene 10293-98(1991))或pHS2-1(Sonnen等，Gene 10769-74(1991))以隱蔽性質粒pHM1519、pBL1或pGA1為基礎。其他質粒載體，例如，pCLiK5MCS，或者那些基於pCG4(US-A4,489,160)或pNG2(Serwold-Davis等，FEMSMicrobiology Letters 66，119-124(1990))或pAG1(US-A5,158,891)的質粒可以以相同的方式使用。
適宜的質粒載體還包括在其幫助下可以應用基因擴增方法(見例如Remscheid等(Applied and Environmental Microbiology 60，126-132(1994))通過整合到染色體中來複製和擴增hom-thrB操縱子的質粒。在該方法中，完整基因被克隆到可以在宿主(通常大腸桿菌)中複製但是不能在穀氨酸棒狀桿菌中複製的質粒載體中。合適的載體為，例如，pSUP301(Sirnon等，Bio/Technology 1，784-791(1983))，pK 18mob或pK 19mob(Sch_fer等，Gene 145，69-73(1994))，Bernard等，Journal of Molecular Biology，234534-541(1993))、pEM1(Schrumpf等，1991，Journal of Bacteriology 1734510-4516)或pBGS8(Spratt等，1986，Gene 41337-342)。然後將含有將要擴增的基因的質粒通過轉化轉移到期望的穀氨酸棒狀桿菌菌株中。轉化方法例如在Thierbach等(Applied Microbiology and Biotechnology 29，356-362(1988))、Dunican和Shivnan(Biotechnology 7，1067-1070(1989))和Tauch等(FEMS Microbiological Letters 123，343-347(1994))中描述。
酶的活性可以通過相應基因中的突變來影響，從而使酶反應的速度部分或完全地減小。這種突變的實例是技術人員公知的(Motoyama H.，YanoH.，Terasaki Y.，Anazawa H.，Applied Environmental Microbiology.673064-70，2001，Eikmanns BJ.，Eggeling L.，Sahm H.，Antonie vanLeeuwenhoek.64145-63，1993-94)。
此外，對於生產含硫精細化學品，尤其是L-甲硫氨酸，除了表達和擴增本發明的metH基因，可能有利的是，還擴增參與甲硫氨酸生物合成途徑或者與其相關(即與其功能上相聯繫)的生物合成途徑或其他代謝途徑，如半胱氨酸、賴氨酸或蘇氨酸代謝途徑，如尤其是天冬氨酸半醛合成、糖酵解、糖回補、磷酸戊糖代謝、檸檬酸循環或胺基酸輸出的一種或多種酶。
從而，可以擴增一種或多種下面的基因以產生含硫精細化學品，尤其是L-甲硫氨酸(即，例如，以更高拷貝數存在或者編碼具有更高活性或特異性的酶)-天冬氨酸激酶-編碼基因lysC(EP 1 108 790 A2；DNA-SEQ NO.281)，-天冬氨酸-半醛脫氫酶-編碼基因asd(EP 1 108 790 A2；DNA-SEQ NO.282)，-甘油醛-3-磷酸脫氫酶-編碼基因gap(Eikmanns(1992)，Journalof Bacteriology 1746076-6086)，-3-磷酸甘油酸激酶-編碼基因pgk(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-丙酮酸羧化酶-編碼基因pyc(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-丙糖磷酸異構酶-編碼基因tpi(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-高絲氨酸O-乙醯轉移酶-編碼基因metA(EP 1 108 790 A2；DNA-SEQ NO.725)，-胱硫醚γ-合酶-編碼基因metB(EP 1 108 790 A2；DNA-SEQ NO.3491)，-胱硫醚γ-裂合酶-編碼基因metC(EP 1 108 790 A2；DNA-SEQNO.3061)，-絲氨酸羥甲基轉移酶-編碼基因glyA(EP 1 108 790 A2；DNA-SEQ NO.1110)，-O-乙醯高絲氨酸硫化氫解酶-編碼基因metY(EP 1 108 790 A2；DNA-SEQ NO.726)，-亞甲基四氫葉酸還原酶-編碼基因metF(EP 1 108 790 A2；DNA-SEQ NO.2379)，-磷酸絲氨酸氨基轉移酶-編碼基因serC(EP 1 108 790 A2；DNA-SEQ NO.928)，-磷酸絲氨酸磷酸酶-編碼基因serB(EP 1 108 790 A2；DNA-SEQNO.334，DNA-SEQ NO.467，DNA-SEQ NO.2767)，-絲氨酸乙醯基轉移酶-編碼基因cysE(EP 1 108 790 A2；DNA-SEQ NO.2818)，-高絲氨酸脫氫酶-編碼基因hom(EP 1 108 790 A2；DNA-SEQNO.1306)。
從而，對棒桿菌中含硫精細化學品，尤其是L-甲硫氨酸的生產，可能有利的是同時突變至少一種下面的基因，尤其是使得相應蛋白的活性與未突變蛋白的相比受到代謝物的影響較小或者根本不受影響-天冬氨酸激酶-編碼基因lysC(EP 1 108 790 A2；DNA-SEQ NO.281)，-丙酮酸羧化酶-編碼基因pyc(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-高絲氨酸O-乙醯轉移酶-編碼基因metA(EP 1 108 790 A2；DNA-SEQ NO.725)，-胱硫醚γ-合酶-編碼基因metB(EP 1 108 790 A2；DNA-SEQ NO.3491)，-胱硫醚γ-裂合酶-編碼基因metC(EP 1 108 790 A2；DNA-SEQNO.3061)，-絲氨酸羥甲基轉移酶-編碼基因glyA(EP 1 108 790 A2；DNA-SEQ NO.1110)，-O-乙醯高絲氨酸硫化氫解酶-編碼基因metY(EP 1 108 790 A2；DNA-SEQ NO.726)，-亞甲基四氫葉酸還原酶-編碼基因metF(EP 1 108 790 A2；DNA-SEQ NO.2379)，
-磷酸絲氨酸氨基轉移酶-編碼基因serC(EP 1 108 790 A2；DNA-SEQ NO.928)，-磷酸絲氨酸磷酸酶-編碼基因serB(EP 1 108 790 A2；DNA-SEQNO.334，DNA-SEQ NO.467，DNA-SEQ NO.2767)，-絲氨酸乙醯基轉移酶-編碼基因cysE(EP 1 108 790 A2；DNA-SEQ NO.2818)，-高絲氨酸脫氫酶-編碼基因hom(EP 1 108 790 A2；DNA-SEQNO.1306)。
對含硫精細化學品，尤其是L-甲硫氨酸生產，可能還有利的是除了表達和擴增本發明的metH基因之一，還弱化一種或多種下面的基因，尤其是減少其表達，或者將其關閉-高絲氨酸激酶-編碼基因thrB(EP 1 108 790 A2；DNA-SEQ NO.3453)，-蘇氨酸脫水酶-編碼基因ilvA(EP 1 108 790 A2；DNA-SEQ NO.2328)，-蘇氨酸合酶-編碼基因thrC(EP 1 108 790 A2；DNA-SEQ NO.3486)，-內消旋-二氨基庚二酸D-脫氫酶-編碼基因ddh(EP 1 108 790 A2；DNA-SEQ NO.3494)，-磷酸烯醇丙酮酸羧基激酶-編碼基因pck(EP 1 108 790 A2；DNA-SEQ NO.3157)，-葡萄糖-6-磷酸6-異構酶-編碼基因pgi(EP 1 108 790 A2；DNA-SEQ NO.950)，-丙酮酸氧化酶-編碼基因poxB(EP 1 108 790 A2；DNA-SEQ NO.2873)，-二氫吡啶二羧酸合酶-編碼基因dapA(EP 1 108 790 A2；DNA-SEQ NO.3476)，-二氫吡啶二羧酸還原酶-編碼基因dapB(EP 1 108 790 A2；DNA-SEQ NO.3477)；-二氨基吡啶甲酸脫羧酶-編碼基因lysA(EP 1 108 790 A2；DNA-SEQ NO.3451)。
對含硫精細化學品，尤其是L-甲硫氨酸生產還有利的是除了在棒桿菌中表達和擴增本發明的metH基因之一，同時還突變至少一種下面的基因使得相應蛋白質的酶促活性部分或全部減小-高絲氨酸激酶-編碼基因thrB(EP 1 108 790 A2；DNA-SEQ NO.3453)，-蘇氨酸脫水酶-編碼基因ilvA(EP 1 108 790 A2；DNA-SEQ NO.2328)，-蘇氨酸合酶-編碼基因thrC(EP 1 108 790 A2；DNA-SEQ NO.3486)，-內消旋-二氨基庚二酸D-脫氫酶-編碼基因ddh(EP 1 108 790 A2；DNA-SEQ NO.3494)，-磷酸烯醇丙酮酸羧基激酶-編碼基因pck(EP 1 108 790 A2；DNA-SEQ NO.3157)，-葡萄糖-6-磷酸6-異構酶-編碼基因pgi(EP 1 108 790 A2；DNA-SEQ NO.950)，-丙酮酸氧化酶-編碼基因poxB(EP 1 108 790 A2；DNA-SEQ NO.2873)，-二氫吡啶二羧酸合酶-編碼基因dapA(EP 1 108 790 A2；DNA-SEQ NO.3476)，-二氫吡啶二羧酸還原酶-編碼基因dapB(EP 1 108 790 A2；DNA-SEQ NO.3477)；-二氨基吡啶甲酸脫羧酶-編碼基因lysA(EP 1 108 790 A2；DNA-SEQ NO.3451)。
對含硫精細化學品，尤其是L-甲硫氨酸生產還有利的是除了表達和擴增本發明的metH基因，還消除不想要的例如減少精細化學品產率的次級反應(Nakayama「胺基酸生產微生物的培養」，微生物產品的過量生產(Overproduction of Microbial Products)，Krumphanzl，Sikyta，Vanek(編者)，Academic Press，倫敦，英國，1982)。
根據本發明產生的微生物可以連續的或者分批的或者以補料分批或者反覆補料分批方法培養以產生含硫精細化學品，尤其是L-甲硫氨酸。公知的培養方法的綜述可在Chmiel的教科書(Bioproze β technik 1.Einführungin die Bioverfahrenstechnik(Gustav Fischer Verlag，Stuttgart，1991))或者在Storhas的教科書(Bioreaktoren und periphere Einrichtungen(ViewegVerlag，Braunschweig/Weisbaden，1994))中找到。
使用的培養基必須以適宜的方式滿足具體菌株的要求。美國細菌學學會的教科書「Manual of Methods for General Bacteriology」(WashingtonD.C.，USA，1981)包含各種微生物培養基的描述。
可根據本發明使用的所述培養基通常含有一種或多種碳源、氮源、無機鹽、維生素和/或微量元素。
優選的碳源為糖，如單糖、二糖或多糖。非常好的碳源的實例為葡萄糖、果糖、甘露糖、半乳糖、核糖、山梨糖、核酮糖、乳糖、麥芽糖、蔗糖、棉籽糖、澱粉和纖維素。也可以通過複雜化合物如糖蜜或糖精練的其他副產物將糖加入培養基。也可能有利的是加入不同碳源的混合物。其他可能的碳源為油和脂肪，例如，大豆油、向日葵油、花生油和椰油，脂肪酸，例如，棕櫚酸、硬脂酸和亞油酸，醇，例如，甘油、甲醇和乙醇，以及有機酸，例如，乙酸和乳酸。
氮源通常為有機或無機氮化合物或含有所述化合物的物質。氮源的實例包括氨氣和銨鹽，如硫酸銨、氯化銨、磷酸銨、碳酸銨和硝酸銨、硝酸鹽、尿素、胺基酸和複雜氮源如玉米漿、大豆粉、大豆蛋白、酵母提取物、肉膏和其他。這些氮源可單獨地或者作為混合物使用。
培養基中可包含的無機鹽化合物包括鈣、鎂、鈉、鈷、鉬、鉀、錳、鋅、銅和鐵的鹽酸鹽、磷酸鹽或硫酸鹽。
含硫無機化合物，例如，硫酸鹽、亞硫酸鹽、連二亞硫酸鹽、連四硫酸鹽、硫代硫酸鹽、硫化物或其他有機硫化合物如硫醇和巰基類化合物可用作硫源以生產含硫精細化學品，尤其是甲硫氨酸。
磷酸、磷酸二氫鉀或磷酸氫二鉀或相應的含鈉鹽可用作磷源。
可向培養基中加入螯合劑以將金屬離子保留在溶液中。尤其適宜的螯合劑包括二羥基酚類，如兒茶酚或原兒茶酚和有機酸如檸檬酸。
根據本發明使用的發酵培養基通常也含有其他生長因子如維生素或生長促進物質，其包括，例如，生物素、核黃素、硫胺素、葉酸、煙酸、泛素和吡哆醇。生長因子和鹽經常來自複雜培養基組分如酵母提取物、糖蜜、玉米漿等等。此外可以向培養基加入適宜的前體。培養基的確切組成很大程度依賴於具體實驗並且應針對每個各例單獨決定。關於優化培養基的信息可在教科書「Applied Microbiol.Physiology，A Practical Approach」(編者P.M.Rhodes，P.F.Stanbury，IRL Press(1997)pp.53-73，ISBN 0 199635773)中發現。生長培養基也可從供應商得到，例如Standardl(Merck)或BHI(腦心浸液，DIFCO)等等。
所有培養基組分都通過加熱(1.5巴及121℃20分鐘)或者通過無菌過濾除菌。各組分可一起或者，如果需要，分開滅菌。所有培養基組分可以在培養開始時加入或者按需要連續地或者分批加入。
培養溫度通常為15℃到45℃，優選25℃到40℃，並且在實驗期間可保持恆定或者可以變化。培養基的pH應該為5到8.5，優選約7.0。可以在培養過程中通過加入鹼性化合物如氫氧化鈉、氫氧化鉀、氨和氨水或酸性化合物如磷酸或硫酸控制培養的pH。可通過使用消泡劑，例如，脂肪酸聚乙二醇酯控制起泡。為了保持質粒穩定，可以向培養基中加入具有選擇作用的適宜物質，例如抗生素。通過導入氧氣或含有氧氣的氣體混合物，例如，空氣到培養物中保持有氧條件。培養的溫度通常為20到45℃。持續培養直到期望產物的形成最大。該目標通常在10到160小時內實現。
以這種方法得到的發酵液，尤其是含有L-甲硫氨酸的發酵液通常含有按重量計7.5到25％的幹生物量。
此外，有利的是至少在發酵最後，但是尤其是在發酵期的至少30％後在糖限制下進行發酵。這意味著在該時間內發酵培養基中的可利用糖的濃度保持在或者減少到≥0到3g/l。
然後進一步處理髮酵液。根據需要，生物質可以通過分離方法，例如，離心、過濾、傾析或這些方法的組合從發酵液完全或部分除去或者完全留在所述發酵液中。
然後，使用公知的方法，例如，通過旋轉蒸發器、薄膜蒸發器、降膜蒸發器、反滲透或者通過納過濾將發酵液變稠或濃縮。
然而，也可以進一步純化含硫精細化學品，尤其是L-甲硫氨酸。為此，含有產物的發酵液在除去生物質後使用適宜樹脂進行層析，期望產物或汙染物被完全或部分保留在層析樹脂上。如果需要，可以使用相同或不同的層析樹脂重複這些層析步驟。技術人員熟悉適宜的層析樹脂的選擇和它們最有效的應用方式。純化的產物可通過過濾或超濾濃縮並保存在產物的穩定性最大的溫度。
分離的化合物的身份和純度可通過本領域技術確定。這些技術包括高效液相層析(HPLC)、光譜方法、染色方法、薄層層析、NIRS、酶測定法或微生物學測定法。這些分析方法概述於Patek等(1994)Appl.Environ.Microbiol.60133-140；Malakhova等(1996)Biotekhnologiya 11 27-32；和Schmidt等(1998)Bioprocess Engineer.1967-70；Ulmann’s Encyclopediaof Industrial Chemistry(1996)Bd.A27，VCHWeinheim，pp.89-90.pp.521-540，pp.540-547，pp.559-566，575-581和pp.581-587；Michal，G.，(1999)生物化學途徑生物化學和分子生物學手冊(Biochemical PathwaysAnAtlas of Biochemistry and Molecular Biology)，John Wiley and Sons；Fallon，A.等(1987)HPLC在生物化學中的應用，《生物化學和分子生物學實驗技術》(Laboratory Techniques in Biochemistry and MolecularBiology)，17卷。
下面的非限制性實施例更詳細描述本發明實施例1pCLiK5MCS的構建首先，通過聚合酶鏈式反應(PCR)使用寡核苷酸p1.3(SEQ ID NO53)和p2.3(SEQ ID NO54)擴增載體pBR322的氨苄青黴素抗性和複製起點。
p1.3(SEQ ID NO53)5』-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACAG-3』p2.3(SEQ ID NO54)5』-TCTAGACTCGAGCGGCCGCGGCCGGCCTTTAAATTGAAGACGAAAGGGCCTCG-3』除了與pBR322互補的序列，寡核苷酸p1.3(SEQ ID NO53)還以5』-3』方向含有限制性核酸酶SmaI、BamHI、NheI和AscI的切割位點，寡核苷酸p2.3(SEQ ID NO54)以5』-3』方向含有限制性內切核酸酶XbaI、XhoI、NotI和DraI的切割位點。根據標準方法如Innis等的方法(PCR Protocols.A Guide to Methods and Applications，Academic Press(1990))使用PfuTurbo聚合酶(Stratagen，La Jolla，USA)進行PCR反應。使用GFXTMPCR、DNA和凝膠條帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書純化所得大小約為2.1kb的DNA片段。使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據生產商的使用說明書將DNA片段的鈍端相互連接，並根據標準方法，如在Sambrook等(分子克隆實驗室手冊，冷泉港，(1989))中描述的方法將連接混合物轉化到感受態大腸桿菌XL-1Blue(Stragagen，La Jolla，USA)中。通過塗含有氨苄青黴素(50μg/ml)的LB瓊脂選擇攜帶質粒的細胞(Lennox，1955，Virology，1190)。
各單克隆的質粒DNA使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據生產商的使用說明書進行分離並通過限制性消化檢查。以這種方法得到的質粒被稱為pCLik1。
從用作PCR反應的模板的質粒pWLT1(Liebl等，1992)開始，使用寡核苷酸neo1(SEQ ID NO55)和neo2(SEQ ID NO56)擴增卡那黴素抗性盒。
neo1(SEQ ID NO55)
5』-GAGATCTAGACCCGGGGATCCGCTAGCGGGCTGCTAAAGGAAGCGGA-3』neo2(SEQ ID NO56)5』-GAGAGGCGCGCCGCTAGCGTGGGCGAAGAACTCCAGCA-3』，除了與pWLT1互補的序列，寡核苷酸neo1還以5』-3』方向含有限制性內切核酸酶XbaI、SmaI、BamHI、NheI的切割位點，寡核苷酸neo2(SEQID NO56)以5』-3』方向含有限制性內切核酸酶AscI和NheI的切割位點。根據標準方法如Innis等的方法(PCR Protocols.A Guide to Methods andApplications，Academic Press(1990))使用PfuTurbo聚合酶(Stratagen，LaJolla，USA)進行PCR反應。使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書純化所得大小約為1.3kb的DNA片段。DNA片段用限制性內切核酸酶XbaI和AscI(NewEngland Biolabs，Beverly，USA)切割，之後再次使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書純化。載體pCLiK1同樣用限制性核酸內切酶XbaI和AscI切割並使用鹼性磷酸酶(Roche Diagnostics，Mannheim)根據生產商的使用說明書去磷酸化。在濃度0.8％的瓊脂糖凝膠中電泳後，使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書分離線性化載體(約2.1kb)。使用快速DNA連接試劑盒(RocheDiagnostics，Mannheim)根據生產商的使用說明書將切割的PCR片段連接到載體片段上，並根據標準方法，如在Sambrook等(分子克隆實驗室手冊，冷泉港，(1989))中描述的方法將連接混合物轉化到感受態大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中，通過塗含有氨苄青黴素(50μg/ml)和卡那黴素(20μg/ml)的LB瓊脂選擇攜帶質粒的細胞(Lennox，1955，Virology，1190)。
各單克隆的質粒DNA使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據生產商的使用說明書進行分離並通過限制性消化檢查。以這種方法得到的質粒被稱為pCLik2。
載體pCLiK2用限制性內切核酸酶DraI(New England Biolabs，Beverly，USA)切割。在濃度0.8％的瓊脂糖凝膠中電泳後，使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書分離到約2.3kb載體片段。該載體片段使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據生產商的使用說明書重新連接，並根據標準方法，如在Sambrook等(分子克隆實驗室手冊，冷泉港，(1989))中描述的方法將連接混合物轉化到感受態大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中，通過塗含有卡那黴素(20μg/ml)的LB瓊脂選擇攜帶質粒的細胞(Lennox，1955，Virology，1190)。
各單克隆的質粒DNA使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據生產商的使用說明書進行分離並通過限制性消化檢查。以這種方法得到的質粒被稱為pC Lik3。
從用作PCR反應的模板的質粒pWLQ2(Liebl等，1992)開始，使用寡核苷酸cg1(SEQ ID NO57)和cg2(SEQ ID NO58)擴增pHM1519的複製起點。
cg1(SEQ ID NO57)5』-GAGAGGGCGGCCGCGCAAAGTCCCGCTTCGTGAA-3』cg2(SEQ ID NO58)5』-GAGAGGGCGGCCGCTCAAGTCGGTCAAGCCACGC-3』除了與pWLQ2互補的序列，寡核苷酸cg1(SEQ ID NO57)和cg2(SEQID NO58)還含有限制性核酸酶NotI的切割位點。根據標準方法如Innis等的方法(PCR Protocols.A Guide to Methods and Applications，AcademicPress(1990))使用PfuTurbo聚合酶(Stratagene，La Jolla，USA)進行PCR反應。使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書純化所得大小約為2.7kb的DNA片段。DNA片段用限制性內切核酸酶NotI(New England Biolabs，Beverly，USA)切割，之後再次使用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，Freiburg)根據生產商的使用說明書純化。載體pCLiK3同樣用限制性核酸內切酶NotI切割並使用鹼性磷酸酶(Roche Diagnostics，Mannheim)根據生產商的使用說明書進行去磷酸化。在濃度0.8％的瓊脂糖凝膠中電泳後，使用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，Freiburg)根據生產商的使用說明書分離線性化載體(約2.3kb)。使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據生產商的使用說明書將切割的PCR片段連接到此載體片段上，並根據標準方法，如在Sambrook等(分子克隆實驗室手冊，冷泉港，(1989))中描述的方法將連接混合物轉化到感受態大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中，通過塗含有卡那黴素(20μg/ml)的LB瓊脂選擇攜帶質粒的細胞(Lennox，1955，Virology，1190)。
單克隆的質粒DNA使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據生產商的使用說明書進行分離並通過限制性消化檢查。以這種方法得到的質粒被稱為pCLik5。
通過混合兩條合成的基本互補的含有限制性內切核酸酶SwaI、XhoI、AatI、ApaI、Asp718、MluI、NdeI、SpeI、EcoRV、SalI、ClaI、BamHI、XbaI和SmaI的切割位點的HS445((SEQ ID NO59)和HS446(SEQ IDNO60))寡核苷酸，通過將它們一起加熱到95℃然後緩慢冷卻得到雙鏈DNA片段，從而用多克隆位點(MCS)延伸pCLik5。
HS445(SEQ ID NO59)5′-TCGAATTTAAATCTCGAGAGGCCTGACGTCGGGCCCGGTACCACGCGTCATATGACTAGTTCGGACCTAGGGATATCGTCGACATCGATGCTCTTCTGCGTTAATTAACAATTGGGATCCTCTAGACCCGGGATTTAAAT-3『HS446(SEQ ID NO60)5′-GATCATTTAAATCCCGGGTCTAGAGGATCCCAATTGTTAATTAACGCAGAAGAGCATCGATGTCGACGATATCCCTAGGTCCGAACTAGTCATATGACGCGTGGTACCGGGCCCGACGTCAGGCCTCTCGAGATTTAAAT-3『載體pCLiK5用限制性核酸內切酶XhoI和BamHI(New EnglandBiolabs，Beverly，USA)切割並使用鹼性磷酸酶(Roche Diagnostics，Mannheim)根據生產商的使用說明書進行去磷酸化。在濃度0.8％的瓊脂糖凝膠中電泳後，使用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，Freiburg)根據生產商的使用說明書分離線性化載體(約5.0kb)。使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據生產商的使用說明書將合成的雙鏈DNA片段連接到此載體片段上，並根據標準方法，如在Sambrook等(分子克隆實驗室手冊，冷泉港，(1989))中描述的方法將連接混合物轉化到感受態大腸桿菌XL-1Blue(Stratagene，LaJolla，USA)中，通過塗含有卡那黴素(20μg/ml)的LB瓊脂選擇攜帶質粒的細胞(Lennox，1955，Virology，1190)。
單克隆的質粒DNA使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據生產商的使用說明書進行分離並通過限制性消化檢查。以這種方法得到的質粒被稱為pCLik5MCS。
根據Sanger等(1977)Proceedings of the National Acedemy of ScienceUSA 745463-5467進行測序反應。將測序反應物並通過ABI Prism 377(PEApplied Biosystems，Weiterstadt)分級分離並分析。
所得質粒pCLiK5MCS被列為SEQ ID NO63。
實施例2pCLik5MCS integrative sacB的構建從作為PCR反應模板的質粒pK 19mob(Sch_fer等，Gene 145，69-73(1994))開始，使用寡核苷酸BK1732和BK1733擴增枯草芽孢桿菌sacB基因(編碼果聚糖蔗糖酶)。
BK1732(SEQ ID NO61)5』-GAGAGCGGCCGCCGATCCTTTTTAACCCATCAC-3』BK1733(SEQ ID NO62)5』-AGGAGCGGCCGCCATCGGCATTTTCTTTTGCG-3』除了與pEK19mobsac互補的序列，寡核苷酸BK1732和BK1733還含有限制性核酸內切酶NotI的切割位點。根據標準方法如Innis等的方法(PCR Protocols. A Guide to Methods and Applications，AcademicPress(1990))使用PfuTurbo聚合酶(Stratagen，La Jolla，USA)進行PCR反應。使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書純化所得大小約為1.9kb的DNA片段。DNA片段用限制性內切核酸酶NotI(New England Biolabs，Beverly，USA)切割，之後再次使用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，Freiburg)根據生產商的使用說明書純化。
載體pCLiK5MCS(根據實施例1製備)同樣用限制性核酸內切酶NotI切割並使用鹼性磷酸酶(Roche Diagnostics，Mannheim)根據生產商的使用說明書進行去磷酸化。在濃度0.8％的瓊脂糖凝膠中電泳後，使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書分離大小約2.4kb的載體片段。使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據生產商的使用說明書將切割的PCR片段連接到此載體片段上，並根據標準方法，如在Sambrook等(分子克隆實驗室手冊，冷泉港，(1989))中描述的方法將連接混合物轉化到感受態大腸桿菌XL-1Blue(Stratgagene，La Jolla，USA)中，通過塗含有卡那黴素(20μg/ml)的LB瓊脂選擇攜帶質粒的細胞(Lennox，1955，Virology，1190)。
單克隆的質粒DNA使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據生產商的使用說明書進行分離並通過限制性消化檢查。以這種方法得到的質粒被稱為pCLik5MCS integrative sacB。
根據Sanger等(1977)Proceedings of the National Acedemy of ScienceUSA 745463-5467進行測序反應。將測序反應物並通過ABI Prism 377(PEApplied Biosystems，Weiterstadt)分級分離並分析。
所得質粒pCLik5MCS integrativ sacB被列為SEQ ID NO64。
可以以類似方法製備適於metH基因的本發明表達或過量生產的其它載體。
下面的實施例3到8描述了被稱為LU 1479 lysC 311ile ET-16 pCPhsdh metH Sc的改進的甲硫氨酸生產菌株的逐步構建。
實施例3從穀氨酸棒狀桿菌菌株LU1479分離lysC基因將在主幹構建的第一步進行下面稱為LU1479的穀氨酸棒狀桿菌ATCC13032中編碼天冬氨酸激酶的lysC野生型基因的等位基因交換。將在lysC基因中進行核苷酸交換從而使311位的胺基酸Thr在所得蛋白中被交換成胺基酸Ile。
從作為PCR反應的模板的LU1479染色體DNA開始，用寡核苷酸引物SEQ ID NO65和SEQ ID NO66 lysC通過Pfu-Turbo PCRSystem(Stratagene USA)，按照生產商的使用說明書進行擴增。按照Tauch等(1995)Plasmid 33168-179或Eikmanns等(1994)Microbiology 1401817-1828的方法製備穀氨酸棒狀桿菌ATCC 13032染色體DNA。擴增的片段在5』端側翼有一個SalI限制性切割位點，在3』端側翼有一個MluI限制性切割位點。克隆前，將擴增的片段通過這兩種限制酶消化並使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)純化。
SEQ ID NO655『-GAGAGAGAGACGCGTCCCAGTGGCTGAGACGCATC-3『SEQ ID NO665『-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG-3『將所得多核苷酸通過SalI和MluI反應切割，克隆到pCLiK5MCSintegrative SacB(此後稱為pCIS；實施例2的SEQ ID NO64)中並轉化大腸桿菌XL-1blue。通過塗含有卡那黴素(20μg/ml)的LB瓊脂選擇攜帶質粒的細胞(Lennox，1955，Virology，1190)。分離質粒並通過測序驗證預期的核苷酸序列。使用Qiagen的材料和方法進行質粒DNA的製備。
根據Sanger等(1977)Proceedings of the National Acedemy of ScienceUSA 745463-5467進行測序反應。將測序反應物通過ABI Prism 377(PEApplied Biosystems，Weiterstadt)分離並評價。所得質粒pCIS lysC被列為SEQ ID NO77。
序列SEQ ID NO77含有下面的基本部分-區域

1)編碼序列2)在互補菌株上實施例4穀氨酸棒狀桿菌lysC基因的誘變使用QuickChange試劑盒(Stratagene/USA)按照生產商的使用說明書進行穀氨酸棒狀桿菌lysC基因(實施例3)的定向誘變。誘變在質粒pCISlysC，SEQ ID NO77中進行。為了將thr311交換成311ile，通過Quickchange方法(Stratagene)合成下面的寡核苷酸引物SEQ ID NO675『-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG-3『SEQ ID NO685『-CGGAACGAGGGCAGGTGAAGATGATGTCGGTGGTGCCG-3『在Quickchange反應中這些寡核苷酸的使用引起lysC基因中932位核苷酸的交換(C被T代替)(參見SEQ ID NO75)和相應酶中311位的胺基酸置換(Thr-Ile)(參見，SEQ ID NO76)。在轉化大腸桿菌XL 1-blue和質粒製備後，LysC基因中所得胺基酸交換Thr311ile通過測序證實。
質粒被命名為pCIS lysC thr311ile並被列為SEQ ID NO78。
序列ID NO78含有下面的基本部分/區

1)編碼序列2)在互補菌株上通過如Liebl等(1989)FEMS Microbiology Letters 53299-303所描述的方法將質粒pCIS lysC thr311ile轉化到穀氨酸棒狀桿菌LU 1470中。該方案的修改之處在DE-A-10046870中描述。各個轉化體的lysC基因座的染色體安排使用Southern印跡和雜交按如Sambrook等(1989)，分子克隆實驗室指南，冷泉港的標準方法驗證。這樣確保轉化株是通過同源重組在lysC基因座整合了轉化的質粒的轉化株。這些菌落在沒有抗生素的培養基中生長過夜然後將細胞塗在蔗糖CM瓊脂培養基上(10％蔗糖)並在30℃孵育24小時。
由於sacB基因(其存在於載體pCIS lysC thr311ile中)將蔗糖轉化成毒性產物，因此只有通過野生型lysC基因和突變基因lysC thr311ile之間的第二同源重組步驟缺失掉sacB基因的那些菌落才能夠建立生長。在此同源重組步驟過程，野生型基因或突變基因可以與sacB基因一起缺失。如果sacB基因與野生型基因一起被除去，那麼導致突變的轉化體。
挑取具有已確立的生長的菌落並研究其卡那黴素敏感表型。缺失Sac基因的菌落一定同時顯示出卡那黴素敏感生長行為。在搖瓶中研究這種卡那黴素敏感克隆的賴氨酸生產力(見實施例6)。為了比較，生長未處理的菌株LU1479。選擇賴氨酸生產超過對照的克隆，得到染色體DNA，並將lysC基因的匹配區通過PCR反應擴增並測序。這種具有增加的賴氨酸合成和在lysC的932位具有確認的突變的克隆被稱為LU1479lysC 311ile。
實施例5乙硫氨酸抗性穀氨酸棒狀桿菌菌株的產生在主幹構建的第二步中，處理所得菌株LU1479lysC311ile(實施例4)以誘導乙硫氨酸抗性(Kase，H.Nakayama K.Agr.Biol.Chem.39，153-106，1975，通過穀氨酸棒狀桿菌的甲硫氨酸類似物抗性突變株生產L-甲硫氨酸)將BHI培養基(Difco)中的過夜培養物用檸檬酸緩衝液(50mM pH5.5)洗滌並在30℃下用N-甲基亞硝基胍(10mg/ml，於50mM檸檬酸鹽中，pH5.5)處理20分鐘。用化學誘變劑N-甲基亞硝基胍處理後，洗滌細胞(檸檬酸緩衝液50mM pH5.5)並塗布在由下面成分組成的培養基上，所述組成基於500ml為10g(NH4)2SO4，0.5g KH2PO4，0.5g K2HPO4，0.125gMgSO4.7H2O，21g MOPS，50mg CaCl2，15mg原兒茶酸，0.5mg生物素，1mg硫胺素，5g/l D，L-乙基硫氨酸(Sigma Chemicals德國)，pH7.0。此外，該培養基含有10g/l FeSO4.7H2O，1g/l MnSO4·H2O，0.1g/l ZnSO4·7H2O，0.02g/l CuSO4，0.002g/l NiCl2·6H2O的0.5ml微量營養鹽溶液。所有鹽溶於0.1M HCl中。將完成的培養基過濾除菌，加入40ml無菌50％葡萄糖溶液，加入液態無菌瓊脂至終濃度1.5％並將混合物導入培養皿中。
將已經經歷誘變處理的細胞塗布於含有上述培養基的平板中並在30℃下孵育3-7天。分離所得克隆，在選擇培養基上分離至少一次然後在搖瓶中培養基II中檢驗它們的甲硫氨酸生產力(見實施例6)。
實施例6使用菌株LU1479lysC311ile ET-16生產甲硫氨酸在實施例5中產生的菌株於30℃在含有CM培養基的瓊脂板上生長2天。
CM瓊脂10.0g/l D-葡萄糖，2.5g/l NaCl，2.0g/l尿素，10.0g/l細菌用-腖(Difco)，5.0g/l酵母提取物(Difco)，5.0g/l牛肉膏(Difco)，22.0g/l瓊脂(Difco)，高壓滅菌(20min，121℃)隨後從平板上刮下細胞並重懸於鹽水中。對於主要培養，在100ml錐形瓶的10ml培養基II和0.5g高壓滅菌的CaCO3(Riedel de Haen)中接種細胞懸浮物到OD600nm為1.5並在定軌搖床上以200rpm在30℃下孵育72小時。
培養基II40g/l蔗糖60g/l糖蜜(基於100％糖含量)10g/l(NH4)2SO40.4g/l MgSO4·7H2O0.6g/l KH2PO40.3mg/l 硫胺素·HCl1mg/l生物素(來自1mg/ml過濾除菌的母液，其已經用NH4OH調節到pH8.0)2mg/lFeSO42mg/lMnSO4用NH4OH調節到pH7.8並高壓滅菌(121℃，20min)。此外，將來自母液(200μg/ml，過濾除菌)的維生素B12(羥鈷胺素，Sigma Chemicals)加至終濃度100μg/l。
在Agilent 1100 Series LC System HPLC上通過來自Agilent的胺基酸確定方法確定培養液中形成的甲硫氨酸和其他胺基酸。柱分離前用鄰苯二醛衍生以便可以定量形成的胺基酸。在Hypersil AA柱(Agilent)上分離胺基酸混合物。
分離甲硫氨酸生產力是最初的菌株LU1479 lysC311ile的生產力的至少2倍的克隆。這種克隆的一株被用於隨後的實驗中並被命名為LU1479lysC 311ile ET-16。
實施例7從天藍色鏈黴菌克隆metH並克隆到質粒pCPhsdh metH Sc中a)從天藍色鏈黴菌菌株ATCC BAA-471(來自美國典型培養物保藏中心，(ATCC)Atlanta，USA，可通過目錄號BAA-471D得到)分離染色體DNA。穀氨酸棒狀桿菌ATCC 13032染色體DNA通過Tauch等(1995)Plasmid 33168-179或Eikmanns等(1994)Microbiology 1401817-1828的方法製備。
使用聚合酶鏈式反應(PCR)通過標準方法(如Innis等(1990)PCRProtocols，A Guide to Methods and Applications，Academic Press)，以穀氨酸棒狀桿菌DNA作為模板，利用Pfu Turbo聚合酶(Stratagene)，使用寡核苷酸引物SEQ ID NO69和SEQ ID NO70從高絲氨酸脫氫酶(HsDH)的非編碼5』區(啟動子區)擴增長度為約180鹼基對的DNA片段。擴增的片段5』端側翼為XhoI限制性切割位點，其3』端側翼為通過oligo導入的同源區，該同源區與天藍色鏈黴菌metH同源。
SEQ ID NO695』-GAGACTCGAGGGAAGGTGAATCGAATTTCGG-3』和SEQ ID NO705』-GTCCCGGGGAGAACGCACGATTCTCCAAAAATAATCGC-3』使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書純化所得DNA片段。
b)從作為PCR反應模板的天藍色鏈黴菌染色體DNA開始，通過GC-RICH PCR系統(Rocbe Diagnostics，Mannheim)按照生產商的使用說明書用寡核苷酸引物SEQ ID NO71和SEQ ID NO72擴增metH片段。該擴增的片段在其5』端側翼為通過oligo導入並且與穀氨酸棒狀桿菌HsDH啟動子區同源的區域。
SEQ ID NO715』-GAATCGTGCGTTCTCCCCGGGAC-3』和SEQ ID NO725』-GTAGTTGACCGAGTTGATCACC-3』
使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書純化所得約1.4kb的DNA片段。
c)在另一PCR反應中，上面所得的兩條片段一起用作模板。由於用寡核苷酸引物SEQ ID NO71和SEQ ID NO72(它們與相應的另一片段同源)導入的區域，兩條片段在PCR反應中相互退火，並且，由於使用聚合酶，這兩條片段延伸以形成連續DNA鏈。該標準方法被修改使得使用的寡核苷酸引物SEQ ID NO69和SEQ ID NO72僅僅在第二次循環中被加入反應混合物。
使用GFXTMPCR、DNA和凝膠帶純化試劑盒根據生產商的使用說明書純化擴增的約1.6kb的DNA片段。此後，將其用限制酶XhoI和NotI(Roche Diagnostics，Mannheim)切割並通過凝膠電泳分離。隨後使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)從瓊脂糖分離該約1.6kb DNA片段。
d)通過GC-RICH PCR系統(Roche Diagnostics，Mannheim)按照生產商的使用說明書用寡核苷酸引物SEQ ID NO73和SEQ ID NO74，從作為模板的天藍色鏈黴菌染色體DNA開始擴增metH3』區(仍然缺少該區域)。該擴增的片段在其3』端側翼為通過oligo導入的EcoRV限制性切割位點。
SEQ ID NO735』-CCGGCCTGGAGAAGCTCG-3』和SEQ ID NO745』-GAGAGATATCCCTCAGCGGGCGTTGAAG-3』使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，Freiburg)根據生產商的使用說明書純化所得約2.2kb的DNA片段。此後，將其用限制酶NotI和EcoRV(Roche Diagnostics，Mannheim)切割並通過凝膠電泳分離。隨後使用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，Freiburg)從瓊脂糖分離該約2.2kb DNA片段。
e)用限制酶NotI和EcoRV(Roche Diagnostics，Mannheim)切割載體pClik5MCS SEQ ID NO63(實施例1)並使用GFXTMPCR、DNA和凝膠帶純化試劑盒通過電泳分離後純化5kb片段。
使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據生產商的使用說明書連接載體片段以及兩條被切割並純化的PCR片段，並如在Sambrook等(分子克隆實驗室手冊，冷泉港，(1989))中描述的將連接反應物轉化到感受態大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中。通過塗含有卡那黴素(20μg/ml)的LB瓊脂選擇攜帶質粒的細胞(Lennox，1955，Virology，1190)。
使用來自Qiagen的材料和方法製備質粒DNA。根據如Sanger等(1977)Proceedings of the National Acedemy of Science USA 745463-5467描述的方法進行測序反應。將測序反應物分離並通過ABI Prism 377(PEApplied Biosystems，Weiterstadt)評價。
所得質粒pC Phsdh metH Sc(天藍色鏈黴菌)被列為SEQ ID NO79。
序列SEQ ID NO79含有下面的基本部分/區

1)編碼序列2)在互補菌株上實施例8用質粒pC Phsdh metH Sc轉化菌株LU1479 lysC 311 ileET-16通過如所描述的方法(Liebl等(1989)FEMS Microbiology Letters53299-303)用質粒pC Phsdh metH Sc(實施例7)轉化菌株LU1479 lysC 311ile ET-16(實施例5)。將轉化混合物塗布在補加20mg/l卡那黴素的CM板上以便選擇含有質粒的細胞。挑選並分離所得卡那黴素抗性克隆。在搖瓶實驗中研究克隆的甲硫氨酸生產力(見實施例6)。與LU1479 lysC 311 ileET-16相比，菌株LU1479 lysC 311 ile ET-16 pC Phsdh metH Sc產生明顯更多的甲硫氨酸。
序列表110巴斯福股份公司(BASF Aktiengesellschaft)120使用編碼metH的棒桿菌細菌發酵生產含硫精細化學品的方法130M/43120140
141
1607921012113597212DNA213天藍色鏈黴菌(Streptomyces coelicolor)220
221CDS222(1)..(3594)223RSX142544001gtg cgt tct ccc cgg gac gtc cca cga cgg gcg gca ccg ggc aga ggc 48Val Arg Ser Pro Arg Asp Val Pro Arg Arg Ala Ala Pro Gly Arg Gly1 5 10 15aaa gcc gac agc cgt cgc atc cta ggg agc cct ttc atg gcc tcg tcg 96Lys Ala Asp Ser Arg Arg Ile Leu Gly Ser Pro Phe Met Ala Ser Ser20 25 30cca tcc acc ccg ccc gcc gac acc cgc acc cgc gtg tcc gcc ctc cga144Pro Ser Thr Pro Pro Ala Asp Thr Arg Thr Arg Val Ser Ala Leu Arg35 40 45gag gcc ctc gcc acc cgc gtg gtg gtc gcc gac ggc gcc atg ggc acc192Glu Ala Leu Ala Thr Arg Val Val Val Ala Asp Gly Ala Met Gly Thr50 55 60atg ctc cag gcc cag aac ccc acg ctg gac gac ttc cag cag ctc gaa240Met Leu Gln Ala Gln Asn Pro Thr Leu Asp Asp Phe Gln Gln Leu Glu65 70 75 80ggg tgc aac gag gtc ctg aac ctc acc cgg ccc gac atc gtc cgc tcg288Gly Cys Asn Glu Val Leu Asn Leu Thr Arg Pro Asp Ile Val Arg Ser85 90 95gtg cac gag gag tac ttc gcg gcc ggc gtc gac tgc gtc gag acc aac336Val His Glu Glu Tyr Phe Ala Ala Gly Val Asp Cys Val Glu Thr Asn100 105 110acc ttc ggc gcc aac cac tcc gcc ctg ggc gag tac gac atc ccc gag384Thr Phe Gly Ala Asn His Ser Ala Leu Gly Glu Tyr Asp Ile Pro Glu115 120 125cgc gtc cac gaa ctg tcc gag gcc ggc gcc cgc gtc gcc cgc gag gtc432Arg Val His Glu Leu Ser Glu Ala Gly Ala Arg Val Ala Arg Glu Val130 135 140
gcc gac gag ttc ggc gcc cgc gac ggc cgg cag cgc tgg gtg ctg ggc 480Ala Asp Glu Phe Gly Ala Arg Asp Gly Arg Gln Arg Trp Val Leu Gly145 150 155 160tcc atg ggc ccc ggc acc aag ctc ccc acc ctc ggc cac gcc ccg tac 528Ser Met Gly Pro Gly Thr Lys Leu Pro Thr Leu Gly His Ala Pro Tyr165 170 175acc gtc ctg cgc gac gcc tac cag cgc aac gcc gag gga ctg gtc gcg 576Thr Val Leu Arg Asp Ala Tyr Gln Arg Asn Ala Glu Gly Leu Val Ala180 185 190ggc ggc gcg gac gca ctg ctg gtg gag acc acg cag gac ctg ctc cag 624Gly Gly Ala Asp Ala Leu Leu Val Glu Thr Thr Gln Asp Leu Leu Gln195 200 205acc aag gcc tcg gtg ctc ggc gcc cgg cgc gcc ctg gac gtc ctc ggc 672Thr Lys Ala Ser Val Leu Gly Ala Arg Arg Ala Leu Asp Val Leu Gly210 215 220ctc gac ctg ccg ctc atc gtg tcc gtc acc gtc gag acc acc ggc acc 720Leu Asp Leu Pro Leu Ile Val Ser Val Thr Val Glu Thr Thr Gly Thr225 230 235 240atg ctg ctc ggc tcg gag atc ggc gcc gcg ctc acc gcg ctg gaa ccg 768Met Leu Leu Gly Ser Glu Ile Gly Ala Ala Leu Thr Ala Leu Glu Pro245 250 255ctc ggc atc gac atg atc ggc ctg aac tgc gcc acc ggc ccc gcc gag 816Leu Gly Ile Asp Met Ile Gly Leu Asn Cys Ala Thr Gly Pro Ala Glu260 265 270atg agc gag cac ctg cgc tac ctc gcc cgg cac tcc cgc atc ccg ctg 864Met Ser Glu His Leu Arg Tyr Leu Ala Arg His Ser Arg Ile Pro Leu275 280 285acc tgc atg ccc aac gcc ggt ctg ccc gtc ctc ggc aag gac ggc gcc 912Thr Cys Met Pro Asn Ala Gly Leu Pro Val Leu Gly Lys Asp Gly Ala290 295 300cac tac ccg ctg acc gcg ccc gag ctg gcc gac gca cac gag acc ttc 960His Tyr Pro Leu Thr Ala Pro Glu Leu Ala Asp Ala His Glu Thr Phe305 310 315 320gtg cgc gag tac ggc ctg tcc ctg gtc ggc ggc tgc tgc ggc acc acg1008Val Arg Glu Tyr Gly Leu Ser Leu Val Gly Gly Cys Cys Gly Thr Thr325 330 335ccc gag cac ctg cgc cag gtc gtc gag cgg gtc cgg gac acc gcc ccc1056Pro Glu His Leu Arg Gln Val Val Glu Arg Val Arg Asp Thr Ala Pro340 345 350acc gca cgc gac ccg cgc ccc gag ccc ggc gcc gcc tcg ctc tac cag1104Thr Ala Arg Asp Pro Arg Pro Glu Pro Gly Ala Ala Ser Leu Tyr Gln355 360 365acc gtg ccc ttc cgc cag gac acc tcc tac ctg gcc atc ggc gag cgc1152Thr Val Pro Phe Arg Gln Asp Thr Ser Tyr Leu Ala Ile Gly Glu Arg370 375 380acc aac gcc aac ggg tcc aag aag ttc cgc gag gcc atg ctg gac ggc1200Thr Asn Ala Asn Gly Ser Lys Lys Phe Arg Glu Ala Met Leu Asp Gly
385 390 395 400cgc tgg gac gac tgc gtc gag atg gcc cgc gac cag atc cgc gaa ggc1248Arg Trp Asp Asp Cys Val Glu Met Ala Arg Asp Gln Ile Arg Glu Gly405 410 415gcg cac atg ctc gac ctc tgc gtc gac tac gtc ggc cgg gac ggc gtc1296Ala His Met Leu Asp Leu Cys Val Asp Tyr Val Gly Arg Asp Gly Val420 425 430gcc gac atg gag gaa ctg gcc ggc cgg ttc gcc acc gcc tcc acg ctg1344Ala Asp Met Glu Glu Leu Ala Gly Arg Phe Ala Thr Ala Ser Thr Leu435 440 445ccg atc gtc ctc gac tcc acc gag gtc gac gtc atc cgg gcc ggc ctg1392Pro Ile Val Leu Asp Ser Thr Glu Val Asp Val Ile Arg Ala Gly Leu450 455 460gag aag ctc ggc ggc cgc gcg gtg atc aac tcg gtc aac tac gag gac1440Glu Lys Leu Gly Gly Arg Ala Val Ile Asn Ser Val Asn Tyr Glu Asp465 470 475 480ggc gcc ggc ccc gag tcc cgg ttc gcc cgc gtc acg aag ctc gcc cgg1488Gly Ala Gly Pro Glu Ser Arg Phe Ala Arg Val Thr Lys Leu Ala Arg485 490 495gag cac ggc gcc gcg ctg atc gcg ctg acc atc gac gag gtg gga cag1536Glu His Gly Ala Ala Leu Ile Ala Leu Thr Ile Asp Glu Val Gly Gln500 505 510gcc cgc acc gcc gag aag aag gtc gag atc gcc gaa cgg ctc atc gac1584Ala Arg Thr Ala Glu Lys Lys Val Glu Ile Ala Glu Arg Leu Ile Asp515 520 525gac ctc acc ggc aac tgg ggc atc cac gag tcc gac atc ctc gtc gac1632Asp Leu Thr Gly Asn Trp Gly Ile His Glu Ser Asp Ile Leu Val Asp530 535 540tgc ctg acc ttc acc atc tgc acc ggc cag gag gag tcc cgc aag gac1680Cys Leu Thr Phe Thr Ile Cys Thr Gly Gln Glu Glu Ser Arg Lys Asp545 550 555 560ggc ctg gcc acc atc gag ggc atc cgg gaa ctc aag cgg cgc cac ccg1728Gly Leu Ala Thr Ile Glu Gly Ile Arg Glu Leu Lys Arg Arg His Pro565 570 575gac gtg cag acc acg ctc ggc ctg tcg aac atc tcc ttc ggc ctc aac1776Asp Val Gln Thr Thr Leu Gly Leu Ser Asn Ile Ser Phe Gly Leu Asn580 585 590ccg gcc gcc cgc atc ctg ctc aac tcc gtc ttc ctc gac gaa tgc gtc1824Pro Ala Ala Arg Ile Leu Leu Asn Ser Val Phe Leu Asp Glu Cys Val595 600 605aag gcc ggc ctg gac tcg gcc atc gtg cac gcg agc aag atc ctg ccg1872Lys Ala Gly Leu Asp Ser Ala Ile Val His Ala Ser Lys Ile Leu Pro610 615 620atc gcc cgc ttc gac gag gag cag gtc acc acc gcc ctc gac ttg atc1920Ile Ala Arg Phe Asp Glu Glu Gln Val Thr Thr Ala Leu Asp Leu Ile625 630 635 640
tac gac cgc cgc cgc gag ggc tac gac ccc ctg caa aag ctc atg cag1968Tyr Asp Arg Arg Arg Glu Gly Tyr Asp Pro Leu Gln Lys Leu Met Gln645 650 655ctc ttc gag ggc gcc acc gcc aag tcg ctg aag gcc tcc aag gcc gag2016Leu Phe Glu Gly Ala Thr Ala Lys Ser Leu Lys Ala Ser Lys Ala Glu660 665 670gaa ctg gcc gcc ctc ccg ctg gag gag cgc ctc aag cgc cgc atc atc2064Glu Leu Ala Ala Leu Pro Leu Glu Glu Arg Leu Lys Arg Arg Ile Ile675 680 685gac ggc gag aag aac ggc ctc gaa cag gac ctc gac gag gcc ctc cgg2112Asp Gly Glu Lys Asn Gly Leu Glu Gln Asp Leu Asp Glu Ala Leu Arg690 695 700gag cgc ccg gcc ctc gag atc gtc aac gac acc ctg ctc gac ggt atg2160Glu Arg Pro Ala Leu Glu Ile Val Asn Asp Thr Leu Leu Asp Gly Met705 710 715 720aag gtc gtc ggc gag ctg ttc ggc tcc ggc cag atg cag ctg ccg ttc2208Lys Val Val Gly Glu Leu Phe Gly Ser Gly Gln Met Gln Leu Pro Phe725 730 735gtg ctc cag tcc gcc gag gtc atg aag acc gcg gtg gcc cac ctg gag2256Val Leu Gln Ser Ala Glu Val Met Lys Thr Ala Val Ala His Leu Glu740 745 750ccg cac atg gag aag acc gac gac gac ggc aag ggc acg atc gtg ctg2304Pro His Met Glu Lys Thr Asp Asp Asp Gly Lys Gly Thr Ile Val Leu755 760 765gcc acc gtc cgc ggc gac gtc cac gac atc ggc aag aac ctc gtc gac2352Ala Thr Val Arg Gly Asp Val His Asp Ile Gly Lys Asn Leu Val Asp770 775 780atc atc ctg tcc aac aac ggc tac aac gtc gtc aac ctc ggc atc aag2400Ile Ile Leu Ser Asn Asn Gly Tyr Asn Val Val Asn Leu Gly Ile Lys785 790 795 800cag ccc gtc tcc gcg atc ctg gaa gcg gcc gac gag cac cgg gcc gac2448Gln Pro Val Ser Ala Ile Leu Glu Ala Ala Asp Glu His Arg Ala Asp805 810 815gtc atc ggc atg tcc ggc ctc ctc gtc aag tcc acg gtg atc atg aag2496Val Ile Gly Met Ser Gly Leu Leu Val Lys Ser Thr Val Ile Met Lys820 825 830gag aac ctg gag gag ctg aac cag cgc aag ctg gcc gcc gac tac ccg2544Glu Asn Leu Glu Glu Leu Asn Gln Arg Lys Leu Ala Ala Asp Tyr Pro835 840 845gtc atc ctc ggc ggc gcc gcc ctc acc agg gcc tac gtc gaa cag gac2592Val Ile Leu Gly Gly Ala Ala Leu Thr Arg Ala Tyr Val Glu Gln Asp850 855 860ctg cac gag atc tac gac ggc gag gtc cgc tac gcc cgc gac gcc ttc2640Leu His Glu Ile Tyr Asp Gly Glu Val Arg Tyr Ala Arg Asp Ala Phe865 870 875 880gag ggc ctg cgc ctc atg gac gcc ctc atc ggc atc aag cgc ggc gtg2688Glu Gly Leu Arg Leu Met Asp Ala Leu Ile Gly Ile Lys Arg Gly Val
885 890 895ccc ggc gcc aag ctg ccg gag ctg aag cag cgc cgg gtg cgg gcc gcc2736Pro Gly Ala Lys Leu Pro Glu Leu Lys Gln Arg Arg Val Arg Ala Ala900 905 910acc gtc gag atc gac gag cgc ccc gag gaa ggc cac gtc cgc tcc gac2784Thr Val Glu Ile Asp Glu Arg Pro Glu Glu Gly His Val Arg Ser Asp915 920 925gtc gcc acc gac aac ccg gtc ccg acc ccg ccc ttc cgc ggc acc cgc2832Val Ala Thr Asp Asn Pro Val Pro Thr Pro Pro Phe Arg Gly Thr Arg930 935 940gtc gtc aag ggc atc cag ctc aag gag tac gcc tcc tgg ctc gac gag2880Val Val Lys Gly Ile Gln Leu Lys Glu Tyr Ala Ser Trp Leu Asp Glu945 950 955 960ggc gcc ctc ttc aag ggc cag tgg ggc ctc aag cag gcc cgc acc ggc2928Gly Ala Leu Phe Lys Gly Gln Trp Gly Leu Lys Gln Ala Arg Thr Gly965 970 975gag gga ccc tcc tac gag gaa ctg gtc gag tcc gag ggc cgg ccg cgg2976Glu Gly Pro Ser Tyr Glu Glu Leu Val Glu Ser Glu Gly Arg Pro Arg980 985 990ctg cgc ggc ctg ctc gac cgg ctc cag acg gac aac ctt ttg gag gcg3024Leu Arg Gly Leu Leu Asp Arg Leu Gln Thr Asp Asn Leu Leu Glu Ala99510001005gcc gtg gtc tac ggc tac ttc ccc tgc gtc tcc aag gac gac gac ctg3072Ala Val Val Tyr Gly Tyr Phe Pro Cys Val Ser Lys Asp Asp Asp Leu101010151020atc gtc ctc gac gac gac ggc aac gaa cgc acc cgc ttc acc ttc ccc3120Ile Val Leu Asp Asp Asp Gly Asn Glu Arg Thr Arg Phe Thr Phe Pro1025 103010351040cgc cag cgc cgc ggc cgg cgc ctg tgc ctg gcc gac ttc ttc cgc ccg3168Arg Gln Arg Arg Gly Arg Arg Leu Cys Leu Ala Asp Phe Phe Arg Pro104510501055gag gag tcc ggc gag acc gac gtg gtc ggc ttc cag gtc gtc acc gtc3216Glu Glu Ser Gly Glu Thr Asp Val Val Gly Phe Gln Val Val Thr Val106010651070ggc tcc cgc atc ggc gag gag acg gcc cgc atg ttc gag gcc aac gcc3264Gly Ser Arg Ile Gly Glu Glu Thr Ala Arg Met Phe Glu Ala Asn Ala107510801085tac cgc gac tat ctc gag ctg cac ggc ctg tcc gtg cag ctc gcc gag3312Tyr Arg Asp Tyr Leu Glu Leu His Gly Leu Ser Val Gln Leu Ala Glu109010951100gcc ctc gcc gag tac tgg cac gcg cgc gtg cgc tcg gaa ctc ggc ttc3360Ala Leu Ala Glu Tyr Trp His Ala Arg Val Arg Ser Glu Leu Gly Phe1105 111011151120gcc ggg gag gac ccg gcc gag atg gag gac atg ttc gcc ctg aag tac3408Ala Gly Glu Asp Pro Ala Glu Met Glu Asp Met Phe Ala Leu Lys Tyr112511301135
cgg ggt gcc cgc ttc tcc ctc ggc tac ggc gcc tgc ccc gac ctg gag3456Arg Gly Ala Arg Phe Ser Leu Gly Tyr Gly Ala Cys Pro Asp Leu Glu114011451150gac cgc gcc aag atc gcc gcc ctg ctg gag ccc gag cgc atc ggc gtc3504Asp Arg Ala Lys Ile Ala Ala Leu Leu Glu Pro Glu Arg Ile Gly Val115511601165cac cta tcc gag gag ttc cag ctc cac ccc gag cag tcc acc gac gcc3552His Leu Ser Glu Glu Phe Gln Leu His Pro Glu Gln Ser Thr Asp Ala117011751180atc gtc atc cac cac ccg gag gcc aag tac ttc aac gcc cgc3594Ile Val Ile His His Pro Glu Ala Lys Tyr Phe Asn Ala Arg1185 11901195tga359721022111198212PRT 213天藍色鏈黴菌4002Val Arg Ser Pro Arg Asp Val Pro Arg Arg Ala Ala Pro Gly Arg Gly1 5 10 15Lys Ala Asp Ser Arg Arg Ile Leu Gly Ser Pro Phe Met Ala Ser Ser20 25 30Pro Ser Thr Pro Pro Ala Asp Thr Arg Thr Arg Val Ser Ala Leu Arg35 40 45Glu Ala Leu Ala Thr Arg Val Val Val Ala Asp Gly Ala Met Gly Thr50 55 60Met Leu Gln Ala Gln Asn Pro Thr Leu Asp Asp Phe Gln Gln Leu Glu65 70 75 80Gly Cys Asn Glu Val Leu Asn Leu Thr Arg Pro Asp Ile Val Arg Ser85 90 95Val His Glu Glu Tyr Phe Ala Ala Gly Val Asp Cys Val Glu Thr Asn100 105 110Thr Phe Gly Ala Asn His Ser Ala Leu Gly Glu Tyr Asp Ile Pro Glu115 120 125Arg Val His Glu Leu Ser Glu Ala Gly Ala Arg Val Ala Arg Glu Val130 135 140Ala Asp Glu Phe Gly Ala Arg Asp Gly Arg Gln Arg Trp Val Leu Gly145 150 155 160Ser Met Gly Pro Gly Thr Lys Leu Pro Thr Leu Gly His Ala Pro Tyr165 170 175Thr Val Leu Arg Asp Ala Tyr Gln Arg Asn Ala Glu Gly Leu Val Ala180 185 190Gly Gly Ala Asp Ala Leu Leu Val Glu Thr Thr Gln Asp Leu Leu Gln
195 200 205Thr Lys Ala Ser Val Leu Gly Ala Arg Arg Ala Leu Asp Val Leu Gly210 215 220Leu Asp Leu Pro Leu Ile Val Ser Val Thr Val Glu Thr Thr Gly Thr225 230 235 240Met Leu Leu Gly Ser Glu Ile Gly Ala Ala Leu Thr Ala Leu Glu Pro245 250 255Leu Gly Ile Asp Met Ile Gly Leu Asn Cys Ala Thr Gly Pro Ala Glu260 265 270Met Ser Glu His Leu Arg Tyr Leu Ala Arg His Ser Arg Ile Pro Leu275 280 285Thr Cys Met Pro Asn Ala Gly Leu Pro Val Leu Gly Lys Asp Gly Ala290 295 300His Tyr Pro Leu Thr Ala Pro Glu Leu Ala Asp Ala His Glu Thr Phe305 310 315 320Val Arg Glu Tyr Gly Leu Ser Leu Val Gly Gly Cys Cys Gly Thr Thr325 330 335Pro Glu His Leu Arg Gln Val Val Glu Arg Val Arg Asp Thr Ala Pro340 345 350Thr Ala Arg Asp Pro Arg Pro Glu Pro Gly Ala Ala Ser Leu Tyr Gln355 360 365Thr Val Pro Phe Arg Gln Asp Thr Ser Tyr Leu Ala Ile Gly Glu Arg370 375 380Thr Asn Ala Asn Gly Ser Lys Lys Phe Arg Glu Ala Met Leu Asp Gly385 390 395 400Arg Trp Asp Asp Cys Val Glu Met Ala Arg Asp Gln Ile Arg Glu Gly405 410 415Ala His Met Leu Asp Leu Cys Val Asp Tyr Val Gly Arg Asp Gly Val420 425 430Ala Asp Met Glu Glu Leu Ala Gly Arg Phe Ala Thr Ala Ser Thr Leu435 440 445Pro Ile Val Leu Asp Ser Thr Glu Val Asp Val Ile Arg Ala Gly Leu450 455 460Glu Lys Leu Gly Gly Arg Ala Val Ile Asn Ser Val Asn Tyr Glu Asp465 470 475 480Gly Ala Gly Pro Glu Ser Arg Phe Ala Arg Val Thr Lys Leu Ala Arg485 490 495Glu His Gly Ala Ala Leu Ile Ala Leu Thr Ile Asp Glu Val Gly Gln500 505 510Ala Arg Thr Ala Glu Lys Lys Val Glu Ile Ala Glu Arg Leu Ile Asp515 520 525
Asp Leu Thr Gly Asn Trp Gly Ile His Glu Ser Asp Ile Leu Val Asp530 535 540Cys Leu Thr Phe Thr Ile Cys Thr Gly Gln Glu Glu Ser Arg Lys Asp545 550 555 560Gly Leu Ala Thr Ile Glu Gly Ile Arg Glu Leu Lys Arg Arg His Pro565 570 575Asp Val Gln Thr Thr Leu Gly Leu Ser Asn Ile Ser Phe Gly Leu Asn580 585 590Pro Ala Ala Arg Ile Leu Leu Asn Ser Val Phe Leu Asp Glu Cys Val595 600 605Lys Ala Gly Leu Asp Ser Ala Ile Val His Ala Ser Lys Ile Leu Pro610 615 620Ile Ala Arg Phe Asp Glu Glu Gln Val Thr Thr Ala Leu Asp Leu Ile625 630 635 640Tyr Asp Arg Arg Arg Glu Gly Tyr Asp Pro Leu Gln Lys Leu Met Gln645 650 655Leu Phe Glu Gly Ala Thr Ala Lys Ser Leu Lys Ala Ser Lys Ala Glu660 665 670Glu Leu Ala Ala Leu Pro Leu Glu Glu Arg Leu Lys Arg Arg Ile Ile675 680 685Asp Gly Glu Lys Asn Gly Leu Glu Gln Asp Leu Asp Glu Ala Leu Arg690 695 700Glu Arg Pro Ala Leu Glu Ile Val Asn Asp Thr Leu Leu Asp Gly Met705 710 715 720Lys Val Val Gly Glu Leu Phe Gly Ser Gly Gln Met Gln Leu Pro Phe725 730 735Val Leu Gln Ser Ala Glu Val Met Lys Thr Ala Val Ala His Leu Glu740 745 750Pro His Met Glu Lys Thr Asp Asp Asp Gly Lys Gly Thr Ile Val Leu755 760 765Ala Thr Val Arg Gly Asp Val His Asp Ile Gly Lys Asn Leu Val Asp770 775 780Ile Ile Leu Ser Asn Asn Gly Tyr Asn Val Val Asn Leu Gly Ile Lys785 790 795 800Gln Pro Val Ser Ala Ile Leu Glu Ala Ala Asp Glu His Arg Ala Asp805 810 815Val Ile Gly Met Ser Gly Leu Leu Val Lys Ser Thr Val Ile Met Lys820 825 830Glu Asn Leu Glu Glu Leu Asn Gln Arg Lys Leu Ala Ala Asp Tyr Pro835 840 845Val Ile Leu Gly Gly Ala Ala Leu Thr Arg Ala Tyr Val Glu Gln Asp850 855 860
Leu His Glu Ile Tyr Asp Gly Glu Val Arg Tyr Ala Arg Asp Ala Phe865 870 875 880Glu Gly Leu Arg Leu Met Asp Ala Leu Ile Gly Ile Lys Arg Gly Val885 890 895Pro Gly Ala Lys Leu Pro Glu Leu Lys Gln Arg Arg Val Arg Ala Ala900 905 910Thr Val Glu Ile Asp Glu Arg Pro Glu Glu Gly His Val Arg Ser Asp915 920 925Val Ala Thr Asp Asn Pro Val Pro Thr Pro Pro Phe Arg Gly Thr Arg930 935 940Val Val Lys Gly Ile Gln Leu Lys Glu Tyr Ala Ser Trp Leu Asp Glu945 950 955 960Gly Ala Leu Phe Lys Gly Gln Trp Gly Leu Lys Gln Ala Arg Thr Gly965 970 975Glu Gly Pro Ser Tyr Glu Glu Leu Val Glu Ser Glu Gly Arg Pro Arg980 985 990Leu Arg Gly Leu Leu Asp Arg Leu Gln Thr Asp Asn Leu Leu Glu Ala99510001005Ala Val Val Tyr Gly Tyr Phe Pro Cys Val Ser Lys Asp Asp Asp Leu101010151020Ile Val Leu Asp Asp Asp Gly Asn Glu Arg Thr Arg Phe Thr Phe Pro1025 103010351040Arg Gln Arg Arg Gly Arg Arg Leu Cys Leu Ala Asp Phe Phe Arg Pro104510501055Glu Glu Ser Gly Glu Thr Asp Val Val Gly Phe Gln Val Val Thr Val106010651070Gly Ser Arg Ile Gly Glu Glu Thr Ala Arg Met Phe Glu Ala Asn Ala107510801085Tyr Arg Asp Tyr Leu Glu Leu His Gly Leu Ser Val Gln Leu Ala Glu109010951100Ala Leu Ala Glu Tyr Trp His Ala Arg Val Arg Ser Glu Leu Gly Phe1105 111011151120Ala Gly Glu Asp Pro Ala Glu Met Glu Asp Met Phe Ala Leu Lys Tyr112511301135Arg Gly Ala Arg Phe Ser Leu Gly Tyr Gly Ala Cys Pro Asp Leu Glu114011451150Asp Arg Ala Lys Ile Ala Ala Leu Leu Glu Pro Glu Arg Ile Gly Val115511601165His Leu Ser Glu Glu Phe Gln Leu His Pro Glu Gln Ser Thr Asp Ala117011751180Ile Val Ile His His Pro Glu Ala Lys Tyr Phe Asn Ala Arg
11851190119521032113537212DNA213魚腥藻屬種(Anabaena sp.)220
221CDS222(1)..(3534)223RAN037904003atg act cat cct ttc ctg aaa cgc ctg cac agt ccg gaa ctt ccg gtt48Met Thr His Pro Phe Leu Lys Arg Leu His Ser Pro Glu Leu Pro Val1 5 10 15atc gtc ttc gac ggt gca atg gga act aac cta caa acc caa aac ctc96Ile Val Phe Asp Gly Ala Met Gly Thr Asn Leu Gln Thr Gln Asn Leu20 25 30acg gct gag gat ttc ggc ggt gtg cag tat gaa ggt tgt aac gaa tac144Thr Ala Glu Asp Phe Gly Gly Val Gln Tyr Glu Gly Cys Asn Glu Tyr35 40 45cta gtc cac acc aaa ccc gaa gct gtc gcc aag gt tcac cgc gac ttt192Leu Val His Thr Lys Pro Glu Ala Val Ala Lys Val His Arg Asp Phe50 55 60ctc gct gtg ggt gca gat gtc atc gaa acc gac act ttc ggt gcg aca240Leu Ala Val Gly Ala Asp Val Ile Glu Thr Asp Thr Phe Gly Ala Thr65 70 75 80tcc att gtt ttg gcg gaa tat gac tta gca gac caa aca tat tac ctg288Ser Ile Val Leu Ala Glu Tyr Asp Leu Ala Asp Gln Thr Tyr Tyr Leu85 90 95aac aag aaa gcc gcc gaa ctg gcg aaa agt gtc gct gct gaa ttt tcc336Asn Lys Lys Ala Ala Glu Leu Ala Lys Ser Val Ala Ala Glu Phe Ser100 105 110aca cca gat aaa ccc cgg ttt gtt gct ggt tcc atc ggc ccc aca acc384Thr Pro Asp Lys Pro Arg Phe Val Ala Gly Ser Ile Gly Pro Thr Thr115 120 125aaa ctt ccc acc ttg gga cat atc gac ttt gac act ctc aaa act tgc432Lys Leu Pro Thr Leu Gly His Ile Asp Phe Asp Thr Leu Lys Thr Cys130 135 140ttt gct gaa caa gca gaa gcg ctg tta gat ggt ggc gtg gat tta ctt480Phe Ala Glu Gln Ala Glu Ala Leu Leu Asp Gly Gly Val Asp Leu Leu145 150 155 160ttg gtg gag act tgt caa gat gtg ctg caa atc aaa gcg gcg ctg aat528Leu Val Glu Thr Cys Gln Asp Val Leu Gln Ile Lys Ala Ala Leu Asn165 170 175ggg ata gaa gaa gtc ttt ggc aag aga ggg gaa cgc ata ccc ttg atg576Gly Ile Glu Glu Val Phe Gly Lys Arg Gly Glu Arg Ile Pro Leu Met180 185 190
gtg tcc gtg aca atg gaa agc atg ggg aca atg ttg gtc ggt tcc gaa624Val Ser Val Thr Met Glu Ser Met Gly Thr Met Leu Val Gly Ser Glu195 200 205atc aac gcc gtc ctg aca att tta gaa cct ttc cca att gac att ctc672Ile Asn Ala Val Leu Thr Ile Leu Glu Pro Phe Pro Ile Asp Ile Leu210 215 220ggt ctg aac tgt gcc aca ggc cca gac ttg atg aaa cca cat att aaa720Gly Leu Asn Cys Ala Thr Gly Pro Asp Leu Met Lys Pro His Ile Lys225 230 235 240tat ttg gct gaa cat tcg ccg ttt gtg gtt tct tgt att cct aac gcg768Tyr Leu Ala Glu His Ser Pro Phe Val Val Ser Cys Ile Pro Asn Ala245 250 255ggt tta cca gaa aac gtt ggt ggt caa gca cat tat cgc tta aca cca816Gly Leu Pro Glu Asn Val Gly Gly Gln Ala His Tyr Arg Leu Thr Pro260 265 270atg gaa tta cgc atg gcg ttg atg cac ttt gtt gaa gat ttg ggt gtc864Met Glu Leu Arg Met Ala Leu Met His Phe Val Glu Asp Leu Gly Val275 280 285caa gtg atc ggg ggt tgc tgt ggg aca cgt cca gaa cac att caa caa912Gln Val Ile Gly Gly Cys Cys Gly Thr Arg Pro Glu His Ile Gln Gln290 295 300tta gca gaa att gcc aag gat tta aag cca aag gtg aga cag cca agt960Leu Ala Glu Ile Ala Lys Asp Leu Lys Pro Lys Val Arg Gln Pro Ser305 310 315 320tta gaa cct gcg gct gca tca ata tat agt act caa ccc tac gaa caa1008Leu Glu Pro Ala Ala Ala Ser Ile Tyr Ser Thr Gln Pro Tyr Glu Gln325 330 335gat aat tct ttc ttg att gtg ggt gaa cgc ctc aac gcc agt ggt tcc1056Asp Asn Ser Phe Leu Ile Val Gly Glu Arg Leu Asn Ala Ser Gly Ser340 345 350aag aaa tgc cgt gat ttg ctg aat gcg gaa gat tgg gac gga ttg gta1104Lys Lys Cys Arg Asp Leu Leu Asn Ala Glu Asp Trp Asp Gly Leu Val355 360 365tca atg gcg cga tcg caa gtc aag gaa ggc gca cat atc ctt gat gtc1152Ser Met Ala Arg Ser Gln Val Lys Glu Gly Ala His Ile Lau Asp Val370 375 380aac gtt gat tat gtg gga cgg gac ggt gtg cgg gat atg cac gaa cta1200Asn Val Asp Tyr Val Gly Arg Asp Gly Val Arg Asp Met His Glu Leu385 390 395 400gtt tcc cgc att gtg aat aat gtt aca ctc ccc tta atg ctc gac tcc1248Val Ser Arg Ile Val Asn Asn Val Thr Leu Pro Leu Met Leu Asp Ser405 410 415acc gaa tgg gaa aag atg gag gcg ggt tta aag gtg gct ggt ggt aag1296Thr Glu Trp Glu Lys Met Glu Ala Gly Leu Lys Val Ala Gly Gly Lys420 425 430tgt ttg ctg aac tcc acc aac tac gaa gat ggg gaa cca cgt ttc tta1344Cys Leu Leu Asn Ser Thr Asn Tyr Glu Asp Gly Glu Pro Arg Phe Leu
435 440 445aaa gtg ttg gag ttg gcg aag aaa tat ggc gcg ggt gtt gtt att ggc1392Lys Val Leu Glu Leu Ala Lys Lys Tyr Gly Ala Gly Val Val Ile Gly450 455 460aca att gac gaa gaa ggg atg gcg cgg aca gcc gag aaa aag ttt caa1440Thr Ile Asp Glu Glu Gly Met Ala Arg Thr Ala Glu Lys Lys Phe Gln465 470 475 480att gcc cag cgt gcc tat cgt caa tcg gta gaa tat ggg att ccc ccc1488Ile Ala Gln Arg Ala Tyr Arg Gln Ser Val Glu Tyr Gly Ile Pro Pro485 490 495aca gaa ata ttc ttt gat acc tta gct tta cea att tct acc ggg att1536Thr Glu Ile Phe Phe Asp Thr Leu Ala Leu Pro Ile Ser Thr Gly Ile500 505 510gaa gaa gac cgg gaa aat ggc aag gcg aca att gaa tca att agc cgt1584Glu Glu Asp Arg Glu Asn Gly Lys Ala Thr Ile Glu Ser Ile Ser Arg515 520 525atc cgt aaa gaa ttg cca ggg tgt cat gtt att tta ggc gtg tca aat1632Ile Arg Lys Glu Leu Pro Gly Cys His Val Ile Leu Gly Val Ser Asn530 535 540ata tcc ttt ggc tta aat tca gcc tcg cgg atg gtc tta aac tcc gtg1680Ile Ser Phe Gly Leu Asn Ser Ala Ser Arg Met Val Leu Asn Ser Val545 550 555 560ttt ctc cat gaa gca atg act gct ggc atg gat gcg gcg atc gtc agt1728Phe Leu His Glu Ala Met Thr Ala Gly Met Asp Ala Ala Ile Val Ser565 570 575gct agc aag att cta cca ctg tcg aag att gaa gag cgt cat caa gaa1776Ala Ser Lys Ile Leu Pro Leu Ser Lys Ile Glu Glu Arg His Gln Glu580 585 590gtc tgc cgc cag tta att tat gac cag cgt aaa ttt gag ggt gat atc1824Val Cys Arg Gln Leu Ile Tyr Asp Gln Arg Lys Phe Glu Gly Asp Ile595 600 605tgc atc tat gac ccc tta aca gaa cta act aaa ttg ttt gag gga gtc1872Cys Ile Tyr Asp Pro Leu Thr Glu Leu Thr Lys Leu Phe Glu Gly Val610 615 620acc acc aaa cgt aac aaa ggc gtt gat gaa agc tta ccc atc gaa gaa1920Thr Thr Lys Arg Asn Lys Gly Val Asp Glu Ser Leu Pro Ile Glu Glu625 630 635 640cga ctc aag cgt cac att atc gac ggc gaa cgc att ggt tta gaa gcg1968Arg Leu Lys Arg His Ile Ile Asp Gly Glu Arg Ile Gly Leu Glu Ala645 650 655caa ctg aca aaa gcc tta gaa caa tat cca ccc cta gaa att atc aac2016Gln Leu Thr Lys Ala Leu Glu Gln Tyr Pro Pro Leu Glu Ile Ile Asn660 665 670act ttc cta cta gat ggg atg aaa gta gtc ggg gaa ttg ttc ggt tca2064Thr Phe Leu Leu Asp Gly Met Lys Val Val Gly Glu Leu Phe Gly Ser675 680 685
gga caa atg cag cta cct ttc gtt tta cag tca gcc gaa acc atg aaa2112Gly Gln Met Gln Leu Pro Phe Val Leu Gln Ser Ala Glu Thr Met Lys690 695 700gcg gcg gta gcc tac cta gaa ccg ttc atg gaa aaa tcg gaa agt ggc2160Ala Ala Val Ala Tyr Leu Glu Pro Phe Met Glu Lys Ser Glu Ser Gly705 710 715 720aac aat gcc aaa ggt aaa gta att att gcc acc gtg aaa ggc gat gtt2208Asn Asn Ala Lys Gly Lys Val Ile Ile Ala Thr Val Lys Gly Asp Val725 730 735cac gac att ggt aaa aac cta gta gac att atc ttg tcc aac aac ggc2256His Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Ser Asn Asn Gly740 745 750tac aag gta att aac ctg gga att aaa cag ccg gtg gaa aat atc atc2304Tyr Lys Val Ile Asn Leu Gly Ile Lys Gln Pro Val Glu Asn Ile Ile755 760 765gag gct tac aac caa cac aaa gct gat tgt att gcc atg agt ggc ttg2352Glu Ala Tyr Asn Gln His Lys Ala Asp Cys Ile Ala Met Ser Gly Leu770 775 780ctg gta aaa tcc acc gca ttc atg aaa gaa aat ttg gag gtc ttc aac2400Leu Val Lys Ser Thr Ala Phe Met Lys Glu Asn Leu Glu Val Phe Asn785 790 795 800gaa aaa ggc att aat gtt cct gta att tta ggt ggt gcg gca tta acc2448Glu Lys Gly Ile Asn Val Pro Val Ile Leu Gly Gly Ala Ala Leu Thr805 810 815ccg aaa ttc gtg cat aaa gat tgc caa aat acc tac aaa ggt aaa gtc2496Pro Lys Phe Val His Lys Asp Cys Gln Asn Thr Tyr Lys Gly Lys Val820 825 830att tat ggc aaa gat gct ttc tca gac ctg cat ttc atg gat aaa tta2544Ile Tyr Gly Lys Asp Ala Phe Ser Asp Leu His Phe Met Asp Lys Leu835 840 845atg cca gcc aaa gcc act ggc aaa tgg gac aat tcc tta gga ttc ttg2592Met Pro Ala Lys Ala Thr Gly Lys Trp Asp Asn Ser Leu Gly Phe Leu850 855 860gat gaa gta gaa acc gag gaa aca gaa cct acc aat cac aaa tcc cca2640Asp Glu Val Glu Thr Glu Glu Thr Glu Pro Thr Asn His Lys Ser Pro865 870 875 880atc ccc agt ccc caa tcc cca gtc ccc agt ccc cag tcc cca gtc cct2688Ile Pro Ser Pro Gln Ser Pro Val Pro Ser Pro Gln Ser Pro Val Pro885 890 895ata gac acc cga cgt tcc gaa gct gta gcc ata gac att ccc cgt ccc2736Ile Asp Thr Arg Arg Ser Glu Ala Val Ala Ile Asp Ile Pro Arg Pro900 905 910aca cca cca ttc tgg gga acg caa tta tta cag cct agc gat att tcc2784Thr Pro Pro Phe Trp Gly Thr Gln Leu Leu Gln Pro Ser Asp Ile Ser915 920 925tta gag gaa ata ttc tgg cac atg gat ttg caa gcc ttg att gcg gga2832Leu Glu Glu Ile Phe Trp His Met Asp Leu Gln Ala Leu Ile Ala Gly930 935 940
caa tgg caa ttc cgc aaa ccc aaa gaa caa tca aag gaa gaa tat caa2880Gln Trp Gln Phe Arg Lys Pro Lys Glu Gln Ser Lys Glu Glu Tyr Gln945 950 955 960gct ttc ttg aat gag aaa gtg tat cca gtt cta gaa act tgg aaa cag2928Ala Phe Leu Asn Glu Lys Val Tyr Pro Val Leu Glu Thr Trp Lys Gln965 970 975cgc atc att gca gaa aac ttg tta cat ccc cag gta att tat ggg tat2976Arg Ile Ile Ala Glu Asn Leu Leu His Pro Gln Val Ile Tyr Gly Tyr980 985 990ttt cct tgt caa tct gag ggt aat act tta tat gtt tac gaa aca aac3024Phe Pro Cys Gln Ser Glu Gly Asn Thr Leu Tyr Val Tyr Glu Thr Asn99510001005agc cca aat gcc aca gaa atc act cag ttt gaa ttc ccc cga caa aag3072Ser Pro Asn Ala Thr Glu Ile Thr Gln Phe Glu Phe Pro Arg Gln Lys101010151020tca tca aaa cga tta tgt att gcc gat ttc ttt gca ccg aaa gat tca3120Ser Ser Lys Arg Leu Cys Ile Ala Asp Phe Phe Ala Pro Lys Asp Ser1025 103010351040gga atc att gat gtc ttc ccc atg cag gcg gtg act gta ggc gaa att3168Gly Ile Ile Asp Val Phe Pro Met Gln Ala Val Thr Val Gly Glu Ile104510501055gct aca gag ttc gcg caa aaa ttg ttt gca aac aat caa tac act gat3216Ala Thr Glu Phe Ala Gln Lys Leu Phe Ala Asn Asn Gln Tyr Thr Asp106010651070tat ctg tat ttt cac ggt ttg gcg gtg caa gta gca gaa gcc ttg gcc3264Tyr Leu Tyr Phe His Gly Leu Ala Val Gln Val Ala Glu Ala Leu Ala107510801085gag tgg aca cac gcc aga atc cgc cgt gag tta ggg ttc ggt gct gaa3312Glu Trp Thr His Ala Arg Ile Arg Arg Glu Leu Gly Phe Gly Ala Glu109010951100gaa ccg gat aat atc cgg gat att ttg gca caa cgc tat cag ggt tcc3360Glu Pro Asp Asn Ile Arg Asp Ile Leu Ala Gln Arg Tyr Gln Gly Ser1105 111011151120cgg tat agt ttt ggc tac cca gct tgt ccc aat att caa gac cag ttt3408Arg Tyr Ser Phe Gly Tyr Pro Ala Cys Pro Asn Ile Gln Asp Gln Phe112511301135aag cag ctg gat ttg ttg gag act agc aga att aac tta tac atg gat3456Lys Gln Leu Asp Leu Leu Glu Thr Ser Arg Ile Asn Leu Tyr Met Asp114011451150gaa agt gag caa ctt tat cca gaa cag tct acg acg gcg att att act3504Glu Ser Glu Gln Leu Tyr Pro Glu Gln Ser Thr Thr Ala Ile Ile Thr115511601165tat cac cca gta gct aag tac ttc acc gcg taa3537Tyr His Pro Val Ala Lys Tyr Phe Thr Ala11701175
21042111178212PRT213魚腥藻屬種4004Met Thr His Pro Phe Leu Lys Arg Leu His Ser Pro Glu Leu Pro Val1 5 10 15Ile Val Phe Asp Gly Ala Met Gly Thr Asn Leu Gln Thr Gln Asn Leu20 25 30Thr Ala Glu Asp Phe Gly Gly Val Gln Tyr Glu Gly Cys Asn Glu Tyr35 40 45Leu Val His Thr Lys Pro Glu Ala Val Ala Lys Val His Arg Asp Phe50 55 60Leu Ala Val Gly Ala Asp Val Ile Glu Thr Asp Thr Phe Gly Ala Thr65 70 75 80Ser Ile Val Leu Ala Glu Tyr Asp Leu Ala Asp Gln Thr Tyr Tyr Leu85 90 95Asn Lys Lys Ala Ala Glu Leu Ala Lys Ser Val Ala Ala Glu Phe Ser100 105 110Thr Pro Asp Lys Pro Arg Phe Val Ala Gly Ser Ile Gly Pro Thr Thr115 120 125Lys Leu Pro Thr Leu Gly His Ile Asp Phe Asp Thr Leu Lys Thr Cys130 135 140Phe Ala Glu Gln Ala Glu Ala Leu Leu Asp Gly Gly Val Asp Leu Leu145 150 155 160Leu Val Glu Thr Cys Gln Asp Val Leu Gln Ile Lys Ala Ala Leu Asn165 170 175Gly Ile Glu Glu Val Phe Gly Lys Arg Gly Glu Arg Ile Pro Leu Met180 185 190Val Ser Val Thr Met Glu Ser Met Gly Thr Met Leu Val Gly Ser Glu195 200 205Ile Asn Ala Val Leu Thr Ile Leu Glu Pro Phe Pro Ile Asp Ile Leu210 215 220Gly Leu Asn Cys Ala Thr Gly Pro Asp Leu Met Lys Pro His Ile Lys225 230 235 240Tyr Leu Ala Glu His Ser Pro Phe Val Val Ser Cys Ile Pro Asn Ala245 250 255Gly Leu Pro Glu Asn Val Gly Gly Gln Ala His Tyr Arg Leu Thr Pro260 265 270Met Glu Leu Arg Met Ala Leu Met His Phe Val Glu Asp Leu Gly Val275 280 285Gln Val Ile Gly Gly Cys Cys Gly Thr Arg Pro Glu His Ile Gln Gln290 295 300
Leu Ala Glu Ile Ala Lys Asp Leu Lys Pro Lys Val Arg Gln Pro Ser305 310 315 320Leu Glu Pro Ala Ala Ala Ser Ile Tyr Ser Thr Gln Pro Tyr Glu Gln325 330 335Asp Asn Ser Phe Leu Ile Val Gly Glu Arg Leu Asn Ala Ser Gly Ser340 345 350Lys Lys Cys Arg Asp Leu Leu Asn Ala Glu Asp Trp Asp Gly Leu Val355 360 365Ser Met Ala Arg Ser Gln Val Lys Glu Gly Ala His Ile Leu Asp Val370 375 380Asn Val Asp Tyr Val Gly Arg Asp Gly Val Arg Asp Met His Glu Leu385 390 395 400Val Ser Arg Ile Val Asn Asn Val Thr Leu Pro Leu Met Leu Asp Ser405 410 415Thr Glu Trp Glu Lys Met Glu Ala Gly Leu Lys Val Ala Gly Gly Lys420 425 430Cys Leu Leu Asn Ser Thr Asn Tyr Glu Asp Gly Glu Pro Arg Phe Leu435 440 445Lys Val Leu Glu Leu Ala Lys Lys Tyr Gly Ala Gly Val Val Ile Gly450 455 460Thr Ile Asp Glu Glu Gly Met Ala Arg Thr Ala Glu Lys Lys Phe Gln465 470 475 480Ile Ala Gln Arg Ala Tyr Arg Gln Ser Val Glu Tyr Gly Ile Pro Pro485 490 495Thr Glu Ile Phe Phe Asp Thr Leu Ala Leu Pro Ile Ser Thr Gly Ile500 505 510Glu Glu Asp Arg Glu Asn Gly Lys Ala Thr Ile Glu Ser Ile Ser Arg515 520 525Ile Arg Lys Glu Leu Pro Gly Cys His Val Ile Leu Gly Val Ser Asn530 535 540Ile Ser Phe Gly Leu Asn Ser Ala Ser Arg Met Val Leu Asn Ser Val545 550 555 560Phe Leu His Glu Ala Met Thr Ala Gly Met Asp Ala Ala Ile Val Ser565 570 575Ala Ser Lys Ile Leu Pro Leu Ser Lys Ile Glu Glu Arg His Gln Glu580 585 590Val Cys Arg Gln Leu Ile Tyr Asp Gln Arg Lys Phe Glu Gly Asp Ile595 600 605Cys Ile Tyr Asp Pro Leu Thr Glu Leu Thr Lys Leu Phe Glu Gly Val610 615 620Thr Thr Lys Arg Asn Lys Gly Val Asp Glu Ser Leu Pro Ile Glu Glu625 630 635 640
Arg Leu Lys Arg His Ile Ile Asp Gly Glu Arg Ile Gly Leu Glu Ala645 650 655Gln Leu Thr Lys Ala Leu Glu Gln Tyr Pro Pro Leu Glu Ile Ile Asn660 665 670Thr Phe Leu Leu Asp Gly Met Lys Val Val Gly Glu Leu Phe Gly Ser675 680 685Gly Gln Met Gln Leu Pro Phe Val Leu Gln Ser Ala Glu Thr Met Lys690 695 700Ala Ala Val Ala Tyr Leu Glu Pro Phe Met Glu Lys Ser Glu Ser Gly705 710 715 720Asn Asn Ala Lys Gly Lys Val Ile Ile Ala Thr Val Lys Gly Asp Val725 730 735His Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Ser Asn Asn Gly740 745 750Tyr Lys Val Ile Asn Leu Gly Ile Lys Gln Pro Val Glu Asn Ile Ile755 760 765Glu Ala Tyr Asn Gln His Lys Ala Asp Cys Ile Ala Met Ser Gly Leu770 775 780Leu Val Lys Ser Thr Ala Phe Met Lys Glu Asn Leu Glu Val Phe Asn785 790 795 800Glu Lys Gly Ile Asn Val Pro Val Ile Leu Gly Gly Ala Ala Leu Thr805 810 815Pro Lys Phe Val His Lys Asp Cys Gln Asn Thr Tyr Lys Gly Lys Val820 825 830Ile Tyr Gly Lys Asp Ala Phe Ser Asp Leu His Phe Met Asp Lys Leu835 840 845Met Pro Ala Lys Ala Thr Gly Lys Trp Asp Asn Ser Leu Gly Phe Leu850 855 860Asp Glu Val Glu Thr Glu Glu Thr Glu Pro Thr Asn His Lys Ser Pro865 870 875 880Ile Pro Ser Pro Gln Ser Pro Val Pro Ser Pro Gln Ser Pro Val Pro885 890 895Ile Asp Thr Arg Arg Ser Glu Ala Val Ala Ile Asp Ile Pro Arg Pro900 905 910Thr Pro Pro Phe Trp Gly Thr Gln Leu Leu Gln Pro Ser Asp Ile Ser915 920 925Leu Glu Glu Ile Phe Trp His Met Asp Leu Gln Ala Leu Ile Ala Gly930 935 940Gln Trp Gln Phe Arg Lys Pro Lys Glu Gln Ser Lys Glu Glu Tyr Gln945 950 955 960Ala Phe Leu Asn Glu Lys Val Tyr Pro Val Leu Glu Thr Trp Lys Gln
965 970 975Arg Ile Ile Ala Glu Asn Leu Leu His Pro Gln Val Ile Tyr Gly Tyr980 985 990Phe Pro Cys Gln Ser Glu Gly Asn Thr Leu Tyr Val Tyr Glu Thr Asn99510001005Ser Pro Asn Ala Thr Glu Ile Thr Gln Phe Glu Phe Pro Arg Gln Lys101010151020Ser Ser Lys Arg Leu Cys Ile Ala Asp Phe Phe Ala Pro Lys Asp Ser1025 103010351040Gly Ile Ile Asp Val Phe Pro Met Gln Ala Val Thr Val Gly Glu Ile104510501055Ala Thr Glu Phe Ala Gln Lys Leu Phe Ala Asn Asn Gln Tyr Thr Asp106010651070Tyr Leu Tyr Phe His Gly Leu Ala Val Gln Val Ala Glu Ala Leu Ala107510801085Glu Trp Thr His Ala Arg Ile Arg Arg Glu Leu Gly Phe Gly Ala Glu109010951100Glu Pro Asp Asn Ile Arg Asp Ile Leu Ala Gln Arg Tyr Gln Gly Ser1105 111011151120Arg Tyr Ser Phe Gly Tyr Pro Ala Cys Pro Asn Ile Gln Asp Gln Phe112511301135Lys Gln Leu Asp Leu Leu Glu Thr Ser Arg Ile Asn Leu Tyr Met Asp114011451150Glu Ser Glu Gln Leu Tyr Pro Glu Gln Ser Thr Thr Ala Ile Ile Thr115511601165Tyr His Pro Val Ala Lys Tyr Phe Thr Ala1170117521052113588212DNA213集胞藻屬種(Synechocystis sp.)220
221CDS222(1)..(3585)223RCY359654005atg aaa agt gct ttt tta gac cgt atc cac agt ccc gat cgc ccg gta48Met Lys Ser Ala Phe Leu Asp Arg Ile His Ser Pro Asp Arg Pro Val1 5 10 15tta gtc ttt gac ggg gct atg ggt aca aac ctg cag gta cag aac cta96Leu Val Phe Asp Gly Ala Met Gly Thr Asn Leu Gln Val Gln Asn Leu20 25 30acg gcg gcg gat ttt ggt ggg gcg gaa tac gaa ggt tgc aat gaa tat144
Thr Ala Ala Asp Phe Gly Gly Ala Glu Tyr Glu Gly Cys Asn Glu Tyr35 40 45tta gtc cat acc aag cca gag gcc gtg gct acg gtg cat cgt gct ttt192Leu Val His Thr Lys Pro Glu Ala Val Ala Thr Val His Arg Ala Phe50 55 60tac gaa gcg ggg gcc gat gtc gtg gaa acg gat act ttt ggg gga acg240Tyr Glu Ala Gly Ala Asp Val Val Glu Thr Asp Thr Phe Gly Gly Thr65 70 75 80ccc ctg gtg ctg gcg gag tac gat tta gca gac caa agt tat tac tta288Pro Leu Val Leu Ala Glu Tyr Asp Leu Ala Asp Gln Ser Tyr Tyr Leu85 90 95aat aaa gca gcg gcg gag ttg gcc aag gcg gta gca gcg gaa ttt tct336Asn Lys Ala Ala Ala Glu Leu Ala Lys Ala Val Ala Ala Glu Phe Ser100 105 110acc cca gaa aag cct cga ttc gtg gcc ggc tcc atg gga cca ggc acc384Thr Pro Glu Lys Pro Arg Phe Val Ala Gly Ser Met Gly Pro Gly Thr115 120 125aag cta ccc acc cta ggt cat gtg gac tac gat agt ctc aag gat gcc432Lys Leu Pro Thr Leu Gly His Val Asp Tyr Asp Ser Leu Lys Asp Ala130 135 140tat gtg gtt cag gtg cgg ggt tta tac gat ggc gga gtg gat tta ttg480Tyr Val Val Gln Val Arg Gly Leu Tyr Asp Gly Gly Val Asp Leu Leu145 150 155 160cta gtg gaa acc tgc cag gat gtg ctg caa att aaa gcg gcc ttg aac528Leu Val Glu Thr Cys Gln Asp Val Leu Gln Ile Lys Ala Ala Leu Asn165 170 175gcc att gaa cag gtc ttt gcc gaa aaa ggc gat cgc cta ccg ttg atg576Ala Ile Glu Gln Val Phe Ala Glu Lys Gly Asp Arg Leu Pro Leu Met180 185 190gtg tca gta acc atg gaa acc atg ggg acc atg ctg gtg ggt acg gag624Val Ser Val Thr Met Glu Thr Met Gly Thr Met Leu Val Gly Thr Glu195 200 205atg gcg gcg gcc ctg gcc att ttg gag ccc tat ccc atc gat att ttg672Met Ala Ala Ala Leu Ala Ile Leu Glu Pro Tyr Pro Ile Asp Ile Leu210 215 220ggg cta aac tgc gcc acc ggg cca gat ttg atg aag gaa cac gtt aaa720Gly Leu Asn Cys Ala Thr Gly Pro Asp Leu Met Lys Glu His Val Lys225 230 235 240tat ctt tcc gaa cat tcc ccc ttt gtg gtg tcc tgt att ccc aat gct768Tyr Leu Ser Glu His Ser Pro Phe Val Val Ser Cys Ile Pro Asn Ala245 250 255ggt ttg cca gaa aac gtt ggc ggt caa gct ttt tat cgc ctc acc ccg816Gly Leu Pro Glu Asn Val Gly Gly Gln Ala Phe Tyr Arg Leu Thr Pro260 265 270atg gaa ctg caa atg tcc ctg atg cac ttc atc gaa gac ctg gga gta864Met Glu Leu Gln Met Ser Leu Met His Phe Ile Glu Asp Leu Gly Val275 280 285
cag gta att ggt ggt tgt tgt ggc act aga ccc gat cac atc aag gcc912Gln Val Ile Gly Gly Cys Cys Gly Thr Arg Pro Asp His Ile Lys Ala290 295 300ctg gcg gat att gcc aag gat ctc cag ccc aaa caa cgc caa cct cac960Leu Ala Asp Ile Ala Lys Asp Leu Gln Pro Lys Gln Arg Gln Pro His305 310 315 320tac gaa ccc agc gcc gct tcc att tat tcc acc caa acc tac gcc caa1008Tyr Glu Pro Ser Ala Ala Ser Ile Tyr Ser Thr Gln Thr Tyr Ala Gln325 330 335gaa aat tct ttt tta atc att ggc gaa cgg ctc aat gcc agt ggc tcg1056Glu Asn Ser Phe Leu Ile Ile Gly Glu Arg Leu Asn Ala Ser Gly Ser340 345 350aaa aaa tgt cga gat ctg ctc aat gct gaa gat tgg gac agc cta gtt1104Lys Lys Cys Arg Asp Leu Leu Asn Ala Glu Asp Trp Asp Ser Leu Val355 360 365tcc ctg gct aaa tcc caa gtc aag gaa gga gcc caa atc ctt gac gtc1152Ser Leu Ala Lys Ser Gln Val Lys Glu Gly Ala Gln Ile Leu Asp Val370 375 380aac gtg gat tac gtt ggt cga gat ggg gta agg gac atg aaa gaa tta1200Asn Val Asp Tyr Val Gly Arg Asp Gly Val Arg Asp Met Lys Glu Leu385 390 395 400gct tcc cga cta gtc aat aat gtc acc ctg ccg ttg atg ttg gac tcc1248Ala Ser Arg Leu Val Asn Asn Val Thr Leu Pro Leu Met Leu Asp Ser405 410 415acc gaa tgg caa aaa atg gag gcg ggt tta aaa gtt gca ggg gga aaa1296Thr Glu Trp Gln Lys Met Glu Ala Gly Leu Lys Val Ala Gly Gly Lys420 425 430tgt att ctc aat tcc acc aac tac gaa gac ggg gaa gaa cgg ttt tat1344Cys Ile Leu Asn Ser Thr Asn Tyr Glu Asp Gly Glu Glu Arg Phe Tyr435 440 445aaa gtg tta gaa att gcc aaa gaa tat gga gct ggt att gtc att ggc1392Lys Val Leu Glu Ile Ala Lys Glu Tyr Gly Ala Gly Ile Val Ile Gly450 455460acc atc gat gaa gat ggc atg gga cgc act gca gat aaa aaa ttt gag1440Thr Ile Asp Glu Asp Gly Met Gly Arg Thr Ala Asp Lys Lys Phe Glu465 470 475 480att gcc aaa cgg gcc tac gaa gcg gcg atc gcc ttt ggc att ccg gcc1488Ile Ala Lys Arg Ala Tyr Glu Ala Ala Ile Ala Phe Gly Ile Pro Ala485 490 495aca gaa att ttc ttt gat cct tta gct ctg cct att tcc acc ggc att1536Thr Glu Ile Phe Phe Asp Pro Leu Ala Leu Pro Ile Ser Thr Gly Ile500 505 510gaa gaa gac agg gag aac ggt aaa gcc acc gtg gat gct atc cgc aga1584Glu Glu Asp Arg Glu Asn Gly Lys Ala Thr Val Asp Ala Ile Arg Arg515 520 525att cgc cag gaa ttg ccc gat tgt cat att ttg ttg ggg gtt tct aac1632
Ile Arg Gln Glu Leu Pro Asp Cys His Ile Leu Leu Gly Val Ser Asn530 535 540gtt tcc ttt ggc ttg aat ccc gcc gct cgc cag gta ctc aat tcc atc1680Val Ser Phe Gly Leu Asn Pro Ala Ala Arg Gln Val Leu Asn Ser Ile545 550 555 560ttt ctc cac gaa tgt atg cag gtg ggc atg gat gcg gcc att gtc agt1728Phe Leu His Glu Cys Met Gln Val Gly Met Asp Ala Ala Ile Val Ser565 570 575gcc aat aag att tta ccc ctg gca aaa att gac cca gaa caa caa caa1776Ala Asn Lys Ile Leu Pro Leu Ala Lys Ile Asp Pro Glu Gln Gln Gln580 585 590gtc tgt cta gat tta atc tat gac cgc cgg gaa ttt gaa gga gag cgc1824Val Cys Leu Asp Leu Ile Tyr Asp Arg Arg Glu Phe Glu Gly Glu Arg595 600 605tgt aca tat gac ccg tta acc aaa ctc acc act tta ttt gaa ggt aaa1872Cys Thr Tyr Asp Pro Leu Thr Lys Leu Thr Thr Leu Phe Glu Gly Lys610 615 620acc acc aaa cgg gat aaa tcc ggt gat gcc aat tta ccg gtg gaa gaa1920Thr Thr Lys Arg Asp Lys Ser Gly Asp Ala Asn Leu Pro Val Glu Glu625 630 635 640aga tta aaa cgc cac atc att gat ggg gaa aga ttg ggc tta gaa gag1968Arg Leu Lys Arg His Ile Ile Asp Gly Glu Arg Leu Gly Leu Glu Glu645 650 655gcc ctc aat gaa gct tta aaa ctt tac gct ccc tta gat atc att aac2016Ala Leu Asn Glu Ala Leu Lys Leu Tyr Ala Pro Leu Asp Ile Ile Asn660 665 670atc tat ttg ttg gat ggc atg aaa gtg gtg ggg gaa cta ttt ggt tcc2064Ile Tyr Leu Leu Asp Gly Met Lys Val Val Gly Glu Leu Phe Gly Ser675 680 685ggg caa atg cag ttg ccc ttt gtg ttg cag tcg gcc caa acc atg aaa2112Gly Gln Met Gln Leu Pro Phe Val Leu Gln Ser Ala Gln Thr Met Lys690 695 700gcg gcg gtg gct ttt tta gaa ccc cat atg gat aag gat gat tcc gcc2160Ala Ala Val Ala Phe Leu Glu Pro His Met Asp Lys Asp Asp Ser Ala705 710 715 720gac aat gct aag ggt act ttt tta att gcc act gtt aag ggg gat gtc2208Asp Asn Ala Lys Gly Thr Phe Leu Ile Ala Thr Val Lys Gly Asp Val725 730 735cat gat att ggc aaa aac tta gtg gat att atc ctt tcc aac aat ggc2256His Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Ser Asn Asn Gly740 745 750tat cga gtg gtc aac cta ggc att aaa cag cca gtg gaa aat att atc2304Tyr Arg Val Val Asn Leu Gly Ile Lys Gln Pro Val Glu Asn Ile Ile755 760 765gaa gcc tac aaa aaa cac agg ccc gat tgc att gcc atg agt ggt ttg2352Glu Ala Tyr Lys Lys His Arg Pro Asp Cys Ile Ala Met Ser Gly Leu770 775 780
ttg gtc aaa tca act gct ttt atg aag gaa aat tta gaa gtt ttc aac2400Leu Val Lys Ser Thr Ala Phe Met Lys Glu Asn Leu Glu Val Phe Asn785 790 795 800caa gag ggc att act gtt ccc gtc att ctt ggt ggt gct gct tta acg2448Gln Glu Gly Ile Thr Val Pro Val Ile Leu Gly Gly Ala Ala Leu Thr805 810 815cct aaa ttt gtt cac cag gac tgc caa aat acc tac aaa ggc caa gta2496Pro Lys Phe Val His Gln Asp Cys Gln Asn Thr Tyr Lys Gly Gln Val820 825 830att tac ggc aaa gat gcg ttc gcc gat tta cat ttc atg gat aag cta2544Ile Tyr Gly Lys Asp Ala Phe Ala Asp Leu His Phe Met Asp Lys Leu835 840 845atg ccc gct aaa aat agc cac aat tgg gat gat ttc cag ggc ttt tta2592Met Pro Ala Lys Asn Ser His Asn Trp Asp Asp Phe Gln Gly Phe Leu850 855 860ggg gaa tat gca acg gaa aat ggc cat aat gtg acc act gat gat ggt2640Gly Glu Tyr Ala Thr Glu Asn Gly His Asn Val Thr Thr Asp Asp Gly865 870 875 880gct aaa act aat ttt ggc att gaa gaa gaa aaa tta att gac gct agt2688Ala Lys Thr Asn Phe Gly Ile Glu Glu Glu Lys Leu Ile Asp Ala Ser885 890 895gag cag tct agg gag ccg gag gta att gat act gtt cgt tct gaa gcg2736Glu Gln Ser Arg Glu Pro Glu Val Ile Asp Thr Val Arg Ser Glu Ala900 905 910gtg gac cct gat cta gaa aga cct gtg cca cct ttt tgg ggc act aaa2784Val Asp Pro Asp Leu Glu Arg Pro Val Pro Pro Phe Trp Gly Thr Lys915 920 925att ttg caa tcc agt gat att tcc ctc gat gaa gtc ttc cct tta ctg2832Ile Leu Gln Ser Ser Asp Ile Ser Leu Asp Glu Val Phe Pro Leu Leu930 935 940gat tta caa gca tta ttt gtt ggt cag tgg cag ttt cgc aaa cct agg2880Asp Leu Gln Ala Leu Phe Val Gly Gln Trp Gln Phe Arg Lys Pro Arg945 950 955 960gag caa tcc agg gaa gaa tac gag caa ttc cta gcg gaa aaa gtt cat2928Glu Gln Ser Arg Glu Glu Tyr Glu Gln Phe Leu Ala Glu Lys Val His965 970 975ccc att ttg gct gag tgg aaa ggt aag gtc atg gca gaa aat tta ctc2976Pro Ile Leu Ala Glu Trp Lys Gly Lys Val Met Ala Glu Asn Leu Leu980 985 990cat cct acg gtg gtt tat ggt tat ttt ccc tgt caa tcc cag ggc aat3024His Pro Thr Val Val Tyr Gly Tyr Phe Pro Cys Gln Ser Gln Gly Asn99510001005acc ttg tta att tat gac cca gaa ttg gtc agc caa aat aat ggc caa3072Thr Leu Leu Ile Tyr Asp Pro Glu Leu Val Ser Gln Asn Asn Gly Gln101010151020att ccc cca gac gca acg gcg atc gcc aaa ttt gag ttt ccc cgg caa3120
Ile Pro Pro Asp Ala Thr Ala Ile Ala Lys Phe Glu Phe Pro Arg Gln1025 103010351040aaa tca ggg cgg cgg ctc tgt att gcg gac ttt ttt gct tca aaa gaa3168Lys Ser Gly Arg Arg Leu Cys Ile Ala Asp Phe Phe Ala Ser Lys Glu104510501055tcg ggg att act gat gtt ttt cct ttg caa gcg gtt aca gtg ggg gaa3216Ser Gly Ile Thr Asp Val Phe Pro Leu Gln Ala Val Thr Val Gly Glu106010651070atc gcg acg gaa tat gca agg aaa ctt ttt gct ggc gat aat tac acc3264Ile Ala Thr Glu Tyr Ala Arg Lys Leu Phe Ala Gly Asp Asn Tyr Thr107510801085gat tac ctc tac ttc cac ggc atg gcg gtg cag atg gcg gaa gct tta3312Asp Tyr Leu Tyr Phe His Gly Met Ala Val Gln Met Ala Glu Ala Leu109010951100gcg gag tgg act cac caa cgg ata cgt cag gaa ttg ggc ttt ggc cat3360Ala Glu Trp Thr His Gln Arg Ile Arg Gln Glu Leu Gly Phe Gly His1105 111011151120tta gat cca gat aac atc cgt gat ctt ctc cag caa cgt tac caa ggt3408Leu Asp Pro Asp Asn Ile Arg Asp Leu Leu Gln Gln Arg Tyr Gln Gly112511301135tcc cgc tac agt ttt ggt tat ccc gct tgt ccc aac atg cag gat caa3456Ser Arg Tyr Ser Phe Gly Tyr Pro Ala Cys Pro Asn Met Gln Asp Gln114011451150tac aca caa tta gaa ttg tta caa acc gaa cga att ggc ttg tat atg3504Tyr Thr Gln Leu Glu Leu Leu Gln Thr Glu Arg Ile Gly Leu Tyr Met115511601165gat gaa agt gaa cag gtt tat cca gaa caa tcc acc acg gcg att att3552Asp Glu Ser Glu Gln Val Tyr Pro Glu Gln Ser Thr Thr Ala Ile Ile117011751180tcc tat cat cct gcg gct aaa tat ttc agc gct taa3588Ser Tyr His Pro Ala Ala Lys Tyr Phe Ser Ala1185 1190119521062111195212PRT213集胞藻屬種4006Met Lys Ser Ala Phe Leu Asp Arg Ile His Ser Pro Asp Arg Pro Val1 5 10 15Leu Val Phe Asp Gly Ala Met Gly Thr Asn Leu Gln Val Gln Asn Leu20 25 30Thr Ala Ala Asp Phe Gly Gly Ala Glu Tyr Glu Gly Cys Asn Glu Tyr35 40 45Leu Val His Thr Lys Pro Glu Ala Val Ala Thr Val His Arg Ala Phe50 55 60Tyr Glu Ala Gly Ala Asp Val Val Glu Thr Asp Thr Phe Gly Gly Thr
65 70 75 80Pro Leu Val Leu Ala Glu Tyr Asp Leu Ala Asp Gln Ser Tyr Tyr Leu85 90 95Asn Lys Ala Ala Ala Glu Leu Ala Lys Ala Val Ala Ala Glu Phe Ser100 105 110Thr Pro Glu Lys Pro Arg Phe Val Ala Gly Ser Met Gly Pro Gly Thr115 120 125Lys Leu Pro Thr Leu Gly His Val Asp Tyr Asp Ser Leu Lys Asp Ala130 135 140Tyr Val Val Gln Val Arg Gly Leu Tyr Asp Gly Gly Val Asp Leu Leu145 150 155 160Leu Val Glu Thr Cys Gln Asp Val Leu Gln Ile Lys Ala Ala Leu Asn165 170 175Ala Ile Glu Gln Val Phe Ala Glu Lys Gly Asp Arg Leu Pro Leu Met180 185 190Val Ser Val Thr Met Glu Thr Met Gly Thr Met Leu Val Gly Thr Glu195 200 205Met Ala Ala Ala Leu Ala Ile Leu Glu Pro Tyr Pro Ile Asp Ile Leu210 215 220Gly Leu Asn Cys Ala Thr Gly Pro Asp Leu Met Lys Glu His Val Lys225 230 235 240Tyr Leu Ser Glu His Ser Pro Phe Val Val Ser Cys Ile Pro Asn Ala245 250 255Gly Leu Pro Glu Asn Val Gly Gly Gln Ala Phe Tyr Arg Leu Thr Pro260 265 270Met Glu Leu Gln Met Ser Leu Met His Phe Ile Glu Asp Leu Gly Val275 280 285Gln Val Ile Gly Gly Cys Cys Gly Thr Arg Pro Asp His Ile Lys Ala290 295 300Leu Ala Asp Ile Ala Lys Asp Leu Gln Pro Lys Gln Arg Gln Pro His305 310 315 320Tyr Glu Pro Ser Ala Ala Ser Ile Tyr Ser Thr Gln Thr Tyr Ala Gln325 330 335Glu Asn Ser Phe Leu Ile Ile Gly Glu Arg Leu Asn Ala Ser Gly Ser340 345 350Lys Lys Cys Arg Asp Leu Leu Asn Ala Glu Asp Trp Asp Ser Leu Val355 360 365Ser Leu Ala Lys Ser Gln Val Lys Glu Gly Ala Gln Ile Leu Asp Val370 375 380Asn Val Asp Tyr Val Gly Arg Asp Gly Val Arg Asp Met Lys Glu Leu385 390 395 400
Ala Ser Arg Leu Val Asn Asn Val Thr Leu Pro Leu Met Leu Asp Ser405 410 415Thr Glu Trp Gln Lys Met Glu Ala Gly Leu Lys Val Ala Gly Gly Lys420 425 430Cys Ile Leu Asn Ser Thr Asn Tyr Glu Asp Gly Glu Glu Arg Phe Tyr435 440 445Lys Val Leu Glu Ile Ala Lys Glu Tyr Gly Ala Gly Ile Val Ile Gly450 455 460Thr Ile Asp Glu Asp Gly Met Gly Arg Thr Ala Asp Lys Lys Phe Glu465 470 475 480Ile Ala Lys Arg Ala Tyr Glu Ala Ala Ile Ala Phe Gly Ile Pro Ala485 490 495Thr Glu Ile Phe Phe Asp Pro Leu Ala Leu Pro Ile Ser Thr Gly Ile500 505 510Glu Glu Asp Arg Glu Asn Gly Lys Ala Thr Val Asp Ala Ile Arg Arg515 520 525Ile Arg Gln Glu Leu Pro Asp Cys His Ile Leu Leu Gly Val Ser Asn530 535 540Val Ser Phe Gly Leu Asn Pro Ala Ala Arg Gln Val Leu Asn Ser Ile545 550 555 560Phe Leu His Glu Cys Met Gln Val Gly Met Asp Ala Ala Ile Val Ser565 570 575Ala Asn Lys Ile Leu Pro Leu Ala Lys Ile Asp Pro Glu Gln Gln Gln580 585 590Val Cys Leu Asp Leu Ile Tyr Asp Arg Arg Glu Phe Glu Gly Glu Arg595 600 605Cys Thr Tyr Asp Pro Leu Thr Lys Leu Thr Thr Leu Phe Glu Gly Lys610 615 620Thr Thr Lys Arg Asp Lys Ser Gly Asp Ala Asn Leu Pro Val Glu Glu625 630 635 640Arg Leu Lys Arg His Ile Ile Asp Gly Glu Arg Leu Gly Leu Glu Glu645 650 655Ala Leu Asn Glu Ala Leu Lys Leu Tyr Ala Pro Leu Asp Ile Ile Asn660 665 670Ile Tyr Leu Leu Asp Gly Met Lys Val Val Gly Glu Leu Phe Gly Ser675 680 685Gly Gln Met Gln Leu Pro Phe Val Leu Gln Ser Ala Gln Thr Met Lys690 695 700Ala Ala Val Ala Phe Leu Glu Pro His Met Asp Lys Asp Asp Ser Ala705 710 715 720Asp Asn Ala Lys Gly Thr Phe Leu Ile Ala Thr Val Lys Gly Asp Val725 730 735
His Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Ser Asn Asn Gly740 745 750Tyr Arg Val Val Asn Leu Gly Ile Lys Gln Pro Val Glu Asn Ile Ile755 760 765Glu Ala Tyr Lys Lys His Arg Pro Asp Cys Ile Ala Met Ser Gly Leu770 775 780Leu Val Lys Ser Thr Ala Phe Met Lys Glu Asn Leu Glu Val Phe Asn785 790 795 800Gln Glu Gly Ile Thr Val Pro Val Ile Leu Gly Gly Ala Ala Leu Thr805 810 815Pro Lys Phe Val His Gln Asp Cys Gln Asn Thr Tyr Lys Gly Gln Val820 825 830Ile Tyr Gly Lys Asp Ala Phe Ala Asp Leu His Phe Met Asp Lys Leu835 840 845Met Pro Ala Lys Asn Ser His Asn Trp Asp Asp Phe Gln Gly Phe Leu850 855 860Gly Glu Tyr Ala Thr Glu Asn Gly His Asn Val Thr Thr Asp Asp Gly865 870 875 880Ala Lys Thr Asn Phe Gly Ile Glu Glu Glu Lys Leu Ile Asp Ala Ser885 890 895Glu Gln Ser Arg Glu Pro Glu Val Ile Asp Thr Val Arg Ser Glu Ala900 905 910Val Asp Pro Asp Leu Glu Arg Pro Val Pro Pro Phe Trp Gly Thr Lys915 920 925Ile Leu Gln Ser Ser Asp Ile Ser Leu Asp Glu Val Phe Pro Leu Leu930 935 940Asp Leu Gln Ala Leu Phe Val Gly Gln Trp Gln Phe Arg Lys Pro Arg945 950 955 960Glu Gln Ser Arg Glu Glu Tyr Glu Gln Phe Leu Ala Glu Lys Val His965 970 975Pro Ile Leu Ala Glu Trp Lys Gly Lys Val Met Ala Glu Asn Leu Leu980 985 990His Pro Thr Val Val Tyr Gly Tyr Phe Pro Cys Gln Ser Gln Gly Asn99510001005Thr Leu Leu Ile Tyr Asp Pro Glu Leu Val Ser Gln Asn Asn Gly Gln101010151020Ile Pro Pro Asp Ala Thr Ala Ile Ala Lys Phe Glu Phe Pro Arg Gln1025 103010351040Lys Ser Gly Arg Arg Leu Cys Ile Ala Asp Phe Phe Ala Ser Lys Glu104510501055Ser Gly Ile Thr Asp Val Phe Pro Leu Gln Ala Val Thr Val Gly Glu
106010651070Ile Ala Thr Glu Tyr Ala Arg Lys Leu Phe Ala Gly Asp Asn Tyr Thr107510801085Asp Tyr Leu Tyr Phe His Gly Met Ala Val Gln Met Ala Glu Ala Leu109010951100Ala Glu Trp Thr His Gln Arg Ile Arg Gln Glu Leu Gly Phe Gly His1105 111011151120Leu Asp Pro Asp Asn Ile Arg Asp Leu Leu Gln Gln Arg Tyr Gln Gly112511301135Ser Arg Tyr Ser Phe Gly Tyr Pro Ala Cys Pro Asn Met Gln Asp Gln114011451150Tyr Thr Gln Leu Glu Leu Leu Gln Thr Glu Arg Ile Gly Leu Tyr Met115511601165Asp Glu Ser Glu Gln Val Tyr Pro Glu Gln Ser Thr Thr Ala Ile Ile117011751180Ser Tyr His Pro Ala Ala Lys Tyr Phe Ser Ala1185 1190119521072113561212DNA213海洋原綠球藻(Prochlorococcus marinus)220
221CDS222(1)..(3558)223RCK008304007atg gtt tca ttt aga aat tat tta aat aga gat gat aaa cca att att48Met Val Ser Phe Arg Asn Tyr Leu Asn Arg Asp Asp Lys Pro Ile Ile1 5 10 15att ttc gat ggt ggg aca ggt act tct ttt caa aat tta aat tta tca96Ile Phe Asp Gly Gly Thr Gly Thr Ser Phe Gln Asn Leu Asn Leu Ser20 25 30tca cat gat ttt ggt gga gat gat tta gag ggt tgc aat gaa aac tta144Ser His Asp Phe Gly Gly Asp Asp Leu Glu Gly Cys Asn Glu Asn Leu35 40 45gtt cta tcc tct cct aat act gtt gaa caa gta cat aat tca ttt ctt192Val Leu Ser Ser Pro Asn Thr Val Glu Gln Val His Asn Ser Phe Leu50 55 60gaa gca ggt tgt cat gta att gaa acc aat aca ttt ggt gct tca tct240Glu Ala Gly Cys His Val Ile Glu Thr Asn Thr Phe Gly Ala Ser Ser65 70 75 80att gtt tta gac gaa tat agt att tct aat aaa gct tat gaa atc aat288Ile Val Leu Asp Glu Tyr Ser Ile Ser Asn Lys Ala Tyr Glu Ile Asn85 90 95
aaa aaa gca gct cag ata gct aaa aaa tgt gca aat tta ttt tca tct336Lys Lys Ala Ala Gln Ile Ala Lys Lys Cys Ala Asn Leu Phe Ser Ser100 105 110att aat act cct aga ttt gtc gct gga tca att ggg cca act aca aaa384Ile Asn Thr Pro Arg Phe Val Ala Gly Ser Ile Gly Pro Thr Thr Lys115 120 125tta cca aca tta ggt cat att agt ttt gat aag ctt aaa gat tca tat432Leu Pro Thr Leu Gly His Ile Ser Phe Asp Lys Leu Lys Asp Ser Tyr130 135 140gaa gaa caa ata aat ggt cta att gac gga ggt att gac ctt cta ttg480Glu Glu Gln Ile Asn Gly Leu Ile Asp Gly Gly Ile Asp Leu Leu Leu145 150 155 160att gaa aca tgc caa gat gtt tta caa ata aaa tca gca tta tct gct528Ile Glu Thr Cys Gln Asp Val Leu Gln Ile Lys Ser Ala Leu Ser Ala165 170 175tct caa gaa gtt att aaa aac agg aat att gaa tta cca ata atg ata576Ser Gln Glu Val Ile Lys Asn Arg Asn Ile Glu Leu Pro Ile Met Ile180 185 190tcc ata act atg gaa acc aca gga acg atg ctt gtc ggg tca gat ata624Ser Ile Thr Met Glu Thr Thr Gly Thr Met Leu Val Gly Ser Asp Ile195 200 205gct tct gca tta aca ata tta gag cca tac aat att gat att ctg gga672Ala Ser Ala Leu Thr Ile Leu Glu Pro Tyr Asn Ile Asp Ile Leu Gly210 215 220ctg aat tgt gca act ggt cca gtt caa atg aaa gaa cat att aag tat720Leu Asn Cys Ala Thr Gly Pro Val Gln Met Lys Glu His Ile Lys Tyr225 230 235 240tta gct gaa aat tca cct ttt gca att agt tgt ata cct aat gca gga768Leu Ala Glu Asn Ser Pro Phe Ala Ile Ser Cys Ile Pro Asn Ala Gly245 250 255tta cct gaa aat ata gga ggt gtt gct cac tat aaa tta act cca ttg816Leu Pro Glu Asn Ile Gly Gly Val Ala His Tyr Lys Leu Thr Pro Leu260 265 270gag ttg aaa atg cag tta atg aac ttt att tat gat ttt aac gta caa864Glu Leu Lys Met Gln Leu Met Asn Phe Ile Tyr Asp Phe Asn Val Gln275 280 285ctt att ggc gga tgt tgt ggt act act cct gaa cat atc aag cat tta912Leu Ile Gly Gly Cys Cys Gly Thr Thr Pro Glu His Ile Lys His Leu290 295 300tca tca atc att gag gaa ata gtt gat aaa aaa ata aat aaa aga ctt960Ser Ser Ile Ile Glu Glu Ile Val Asp Lys Lys Ile Asn Lys Arg Leu305 310 315 320cct act gta aaa aca aat ttt gtt cct tca gca gct tct ata tat aac1008Pro Thr Val Lys Thr Asn Phe Val Pro Ser Ala Ala Ser Ile Tyr Asn325 330 335gca gtt cca tat aaa caa gat aac tca ata tta ata gtt gga gaa cgt1056Ala Val Pro Tyr Lys Gln Asp Asn Ser Ile Leu Ile Val Gly Glu Arg
340 345 350tta aat gct agt gga tca aaa aaa gta agg gaa tta cta aat gaa gat1104Leu Asn Ala Ser Gly Ser Lys Lys Val Arg Glu Leu Leu Asn Glu Asp355 360 365gat tgg gac ggc ctg cta tca att gct aaa caa cag caa aaa gaa aat1152Asp Trp Asp Gly Leu Leu Ser Ile Ala Lys Gln Gln Gln Lys Glu Asn370 375 380gct cac ata cta gat gtc aat gtt gat tat gta gga aga gat gga gtt1200Ala His Ile Leu Asp Val Asn Val Asp Tyr Val Gly Arg Asp Gly Val385 390 395 400aaa gat atg aaa gaa att acc tca aga tta gtt aca aat ata aat ctt1248Lys Asp Met Lys Glu Ile Thr Ser Arg Leu Val Thr Asn Ile Asn Leu405 410 415cca tta atg ata gat tca aca gaa gca gat aaa atg gaa agt gga tta1296Pro Leu Met Ile Asp Ser Thr Glu Ala Asp Lys Met Glu Ser Gly Leu420 425 430aag act gta gga gga aaa tgc att ata aat tca aca aac tac gaa gat1344Lys Thr Val Gly Gly Lys Cys Ile Ile Asn Ser Thr Asn Tyr Glu Asp435 440 445gga gat gac aga ttt aat cag gtc tta aga ctt gca tta gat tat ggt1392Gly Asp Asp Arg phe Asn Gln Val Leu Arg Leu Ala Leu Asp Tyr Gly450 455 460gct gga ata gta att gga act att gat gaa gat gga atg gca aga aca1440Ala Gly Ile Val Ile Gly Thr Ile Asp Glu Asp Gly Met Ala Arg Thr465 470 475 480tca cag aaa aaa tat gac att gca aaa aga gca tta att aaa act aga1488Ser Gln Lys Lys Tyr Asp Ile Ala Lys Arg Ala Leu Ile Lys Thr Arg485 490 495tca agt ggc ctc gct gat tat gag ata ttt ttt gat cct cta gca ttg1536Ser Ser Gly Leu Ala Asp Tyr Glu Ile Phe Phe Asp Pro Leu Ala Leu500 505 510cca ata tct act gga att gaa gaa gat aga tta aat gct aaa gca act1584Pro Ile Ser Thr Gly Ile Glu Glu Asp Arg Leu Asn Ala Lys Ala Thr515 520 525att gaa gct ata tca aaa ata aga aaa agc ttt cca gat att cat att1632Ile Glu Ala Ile Ser Lys Ile Arg Lys Ser Phe Pro Asp Ile His Ile530 535 540att tta ggg ata tct aat att agt ttc ggg ctt tca cca tta tca aga1680Ile Leu Gly Ile Ser Asn Ile Ser Phe Gly Leu Ser Pro Leu Ser Arg545 550 555 560att aat cta aat tca ata ttt ctc gat gaa tgt ata aag gca gga tta1728Ile Asn Leu Asn Ser Ile Phe Leu Asp Glu Cys Ile Lys Ala Gly Leu565 570 575gat tca gcg att att gca cca aat aaa ata ttg cct ctt tca aaa ata1776Asp Ser Ala Ile Ile Ala Pro Asn Lys Ile Leu Pro Leu Ser Lys Ile580 585 590
tct gcg gaa aca aaa aaa tta tgt tta gat tta att tat gac aga aga1824Ser Ala Glu Thr Lys Lys Leu Cys Leu Asp Leu Ile Tyr Asp Arg Arg595 600 605aat ttc gaa aat gaa ata tgt ata tat gat cca tta gtt gaa cta aca1872Asn Phe Glu Asn Glu Ile Cys Ile Tyr Asp Pro Leu Val Glu Leu Thr610 615 620aaa gca ttc caa gat ata aca atc agt gac ttt aaa aaa gga tct act1920Lys Ala Phe Gln Asp Ile Thr Ile Ser Asp Phe Lys Lys Gly Ser Thr625 630 635 640tca aac aaa aac ctc acc tta gaa gaa aaa ctt aaa aac cat att gta1968Ser Asn Lys Asn Leu Thr Leu Glu Glu Lys Leu Lys Asn His Ile Val645 650 655gat ggg gaa aaa ata ggt tta gaa gaa caa tta aat aat gcg ctt aaa2016Asp Gly Glu Lys Ile Gly Leu Glu Glu Gln Leu Asn Asn Ala Leu Lys660 665 670aag tac aaa cca ctt gaa ata att aat act tat tta tta gat gga atg2064Lys Tyr Lys Pro Leu Glu Ile Ile Asn Thr Tyr Leu Leu Asp Gly Met675 680 685aaa gta gtc ggt gaa cta ttt gga tcc ggc caa atg caa tta cct ttt2112Lys Val Val Gly Glu Leu Phe Gly Ser Gly Gln Met Gln Leu Pro Phe690 695 700gta ttg caa tca gcg gaa aca atg aaa ttt gct gtt tca gtg ctt gaa2160Val Leu Gln Ser Ala Glu Thr Met Lys Phe Ala Val Ser Val Leu Glu705 710 715 720cct cat atg gaa aca gta gat gaa aaa ata tct aac gga aaa tta cta2208Pro His Met Glu Thr Val Asp Glu Lys Ile Ser Asn Gly Lys Leu Leu725 730 735ata gca act gtt aaa gga gat gtt cat gat ata ggt aaa aat tta gtt2256Ile Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Leu Val740 745 750gat ata att ctc tca aat aat ggt ttt gat gta atc aac ctt gga att2304Asp Ile Ile Leu Ser Asn Asn Gly Phe Asp Val Ile Asn Leu Gly Ile755 760 765aag caa gat gtt tca gcg att att gat gca caa aaa aaa cat aaa gca2352Lys Gln Asp Val Ser Ala Ile Ile Asp Ala Gln Lys Lys His Lys Ala770 775 780gac tgt att gct atg agt ggt tta ctt gtt aaa tct aca gca ttt atg2400Asp Cys Ile Ala Met Ser Gly Leu Leu Val Lys Ser Thr Ala Phe Met785 790 795 800aag gat aat tta gaa gca ttt aac aat gct gaa att aat gtt cca gtt2448Lys Asp Asn Leu Glu Ala Phe Asn Asn Ala Glu Ile Asn Val Pro Val805 810 815att ctt gga ggt gca gca tta act cca aaa ttt gtg aat gaa gat tgt2496Ile Leu Gly Gly Ala Ala Leu Thr Pro Lys Phe Val Asn Glu Asp Cys820 825 830agt cag ata tat aaa ggt aaa att ttg tat ggg aaa gat gct ttt aca2544Ser Gln Ile Tyr Lys Gly Lys Ile Leu Tyr Gly Lys Asp Ala Phe Thr
835 840 845gat tta caa ttt atg aat gac tat atg gat agt aaa aag aag ggc aat2592Asp Leu Gln phe Met Asn Asp Tyr Met Asp Ser Lys Lys Lys Gly Asn850 855 860tgg tct aat gaa aat ggt ttt act aat act gat gat att caa att aaa2640Trp Ser Asn Glu Asn Gly Phe Thr Asn Thr Asp Asp Ile Gln Ile Lys865 870 875 880tta gct tcc cca agg tct tcc gct aaa gat aaa aat tta aat aaa aat2688Leu Ala Ser Pro Arg Ser Ser Ala Lys Asp Lys Asn Leu Asn Lys Asn885 890 895ttt gaa aaa acc aaa agt att caa tta att gag aat ttt aat aga tct2736Phe Glu Lys Thr Lys Ser Ile Gln Leu Ile Glu Asn Phe Asn Arg Ser900 905 910aat ttt gta gag gaa gag gaa cct ata aag gct cca ttt ttg gga act2784Asn Phe Val Glu Glu Glu Glu Pro Ile Lys Ala Pro Phe Leu Gly Thr915 920 925aga gtt ctt caa gat att gaa ata gac ttt gac aaa cta att ttt tat2832Arg Val Leu Gln Asp Ile Glu Ile Asp Phe Asp Lys Leu Ile Phe Tyr930 935 940cta gat aaa aaa gca tta ttt agt ggt caa tgg caa att aaa aaa aat2880Leu Asp Lys Lys Ala Leu Phe Ser Gly Gln Trp Gln Ile Lys Lys Asn945 950 955 960aaa ggt caa tca gta gaa gaa tac aat aat tat tta gat tca tat gca2928Lys Gly Gln Ser Val Glu Glu Tyr Asn Asn Tyr Leu Asp Ser Tyr Ala965 970 975aat cca tta ctt gaa aaa tgg att aat att att tta gat aaa ggc tta2976Asn Pro Leu Leu Glu Lys Trp Ile Asn Ile Ile Leu Asp Lys Gly Leu980 985 990att tca cca aaa gta gtc tat ggc tac ttc cgt tgc ggg agg aat gat3024Ile Ser Pro Lys Val Val Tyr Gly Tyr Phe Arg Cys Gly Arg Asn Asp99510001005aat agt att tat ctc ttt gat aat gta tca aat aaa aga att tct gaa3072Asn Ser Ile Tyr Leu Phe Asp Asn Val Ser Asn Lys Arg Ile Ser Glu101010151020ttt aac ttt cct aga caa aaa tcg gga aat aat ctt tgt att gca gat3120Phe Asn Phe Pro Arg Gln Lys Ser Gly Asn Asn Leu Cys Ile Ala Asp1025 103010351040ttt tac tgt gat ctt aaa aat aat gat cca gta gat ata ttt cca atg3168Phe Tyr Cys Asp Leu Lys Asn Asn Asp Pro Val Asp Ile Phe Pro Met104510501055caa gca gta aca atg ggg gaa ata gct agc gaa tat tcc caa gaa tta3216Gln Ala Val Thr Met Gly Glu Ile Ala Ser Glu Tyr Ser Gln Glu Leu106010651070ttt aaa gct gat aaa tat agt gat tat tta ata ttt cat ggt tta acc3264Phe Lys Ala Asp Lys Tyr Ser Asp Tyr Leu Ile Phe His Gly Leu Thr107510801085
gtt caa tta gca gaa gct ctt gca gaa tat gtt cat tca ata gta aga3312Val Gln Leu Ala Glu Ala Leu Ala Glu Tyr Val His Ser Ile Val Arg109010951100att gaa tgc gga ttt aaa tca tat gag cca aac aat aac cgt gat ata3360Tle Glu Cys Gly Phe Lys Ser Tyr Glu Pro Asn Asn Asn Arg Asp Ile1105 111011151120tta gct caa aaa tat aga gga gct aga tac tca ttt ggt tat cca gct3408Leu Ala Gln Lys Tyr Arg Gly Ala Arg Tyr Ser Phe Gly Tyr Pro Ala112511301135tgt cct aaa gtt tct gat tca aat ata cag tta tca tta ttg gat aca3456Cys Pro Lys Val Ser Asp Ser Asn Ile Gln Leu Ser Leu Leu Asp Thr114011451150aaa agg att aat tta aca atg gat gaa tca gag caa tta cat cct gaa3504Lys Arg Ile Asn Leu Thr Met Asp Glu Ser Glu Gln Leu His Pro Glu115511601165caa agt act act gct ata att tca ctt cat tca aaa gca aaa tat ttt3552Gln Ser Thr Thr Ala Ile Ile Ser Leu His Ser Lys Ala Lys Tyr Phe117011751180agt gcc taa3561Ser Ala118521082111186212PRT213海洋原綠球藻4008Met Val Ser Phe Arg Asn Tyr Leu Asn Arg Asp Asp Lys Pro Ile Ile1 5 10 15Ile Phe Asp Gly Gly Thr Gly Thr Ser Phe Gln Asn Leu Asn Leu Ser20 25 30Ser His Asp Phe Gly Gly Asp Asp Leu Glu Gly Cys Asn Glu Asn Leu35 40 45Val Leu Ser Ser Pro Asn Thr Val Glu Gln Val His Asn Ser Phe Leu50 55 60Glu Ala Gly Cys His Val Ile Glu Thr Asn Thr Phe Gly Ala Ser Ser65 70 75 80Ile Val Leu Asp Glu Tyr Ser Ile Ser Asn Lys Ala Tyr Glu Ile Asn85 90 95Lys Lys Ala Ala Gln Ile Ala Lys Lys Cys Ala Asn Leu Phe Ser Ser100 105 110Ile Asn Thr Pro Arg Phe Val Ala Gly Ser Ile Gly Pro Thr Thr Lys115 120 125Leu Pro Thr Leu Gly His Ile Ser Phe Asp Lys Leu Lys Asp Ser Tyr130 135 140
Glu Glu Gln Ile Asn Gly Leu Ile Asp Gly Gly Ile Asp Leu Leu Leu145 150 155 160Ile Glu Thr Cys Gln Asp Val Leu Gln Ile Lys Ser Ala Leu Ser Ala165 170 175Ser Gln Glu Val Ile Lys Asn Arg Asn Ile Glu Leu Pro Ile Met Ile180 185 190Ser Ile Thr Met Glu Thr Thr Gly Thr Met Leu Val Gly Ser Asp Ile195 200 205Ala Ser Ala Leu Thr Ile Leu Glu Pro Tyr Asn Ile Asp Ile Leu Gly210 215 220Leu Asn Cys Ala Thr Gly Pro Val Gln Met Lys Glu His Ile Lys Tyr225 230 235 240Leu Ala Glu Asn Ser Pro Phe Ala Ile Ser Cys Ile Pro Asn Ala Gly245 250 255Leu Pro Glu Asn Ile Gly Gly Val Ala His Tyr Lys Leu Thr Pro Leu260 265 270Glu Leu Lys Met Gln Leu Met Asn Phe Ile Tyr Asp Phe Asn Val Gln275 280 285Leu Ile Gly Gly Cys Cys Gly Thr Thr Pro Glu His Ile Lys His Leu290 295 300Ser Ser Ile Ile Glu Glu Ile Val Asp Lys Lys Ile Asn Lys Arg Leu305 310 315 320Pro Thr Val Lys Thr Asn Phe Val Pro Ser Ala Ala Ser Ile Tyr Asn325 330 335Ala Val Pro Tyr Lys Gln Asp Asn Ser Ile Leu Ile Val Gly Glu Arg340 345 350Leu Asn Ala Ser Gly Ser Lys Lys Val Arg Glu Leu Leu Asn Glu Asp355 360 365Asp Trp Asp Gly Leu Leu Ser Ile Ala Lys Gln Gln Gln Lys Glu Asn370 375 380Ala His Ile Leu Asp Val Asn Val Asp Tyr Val Gly Arg Asp Gly Val385 390 395 400Lys Asp Met Lys Glu Ile Thr Ser Arg Leu Val Thr Asn Ile Asn Leu405 410 415Pro Leu Met Ile Asp Ser Thr Glu Ala Asp Lys Met Glu Ser Gly Leu420 425 430Lys Thr Val Gly Gly Lys Cys Ile Ile Asn Ser Thr Asn Tyr Glu Asp435 440 445Gly Asp Asp Arg Phe Asn Gln Val Leu Arg Leu Ala Leu Asp Tyr Gly450 455 460Ala Gly Ile Val Ile Gly Thr Ile Asp Glu Asp Gly Met Ala Arg Thr465 470 475 480
Ser Gln Lys Lys Tyr Asp Ile Ala Lys Arg Ala Leu Ile Lys Thr Arg485 490 495Ser Ser Gly Leu Ala Asp Tyr Glu Ile Phe Phe Asp Pro Leu Ala Leu500 505 510Pro Ile Ser Thr Gly Ile Glu Glu Asp Arg Leu Asn Ala Lys Ala Thr515 520 525Ile Glu Ala Ile Ser Lys Ile Arg Lys Ser Phe Pro Asp Ile His Ile530 535 540Ile Leu Gly Ile Ser Asn Ile Ser Phe Gly Leu Ser Pro Leu Ser Arg545 550 555 560Ile Asn Leu Asn Ser Ile Phe Leu Asp Glu Cys Ile Lys Ala Gly Leu565 570 575Asp Ser Ala Ile Ile Ala Pro Asn Lys Ile Leu Pro Leu Ser Lys Ile580 585 590Ser Ala Glu Thr Lys Lys Leu Cys Leu Asp Leu Ile Tyr Asp Arg Arg595 600 605Asn Phe Glu Asn Glu Ile Cys Ile Tyr Asp Pro Leu Val Glu Leu Thr610 615 620Lys Ala Phe Gln Asp Ile Thr Ile Ser Asp Phe Lys Lys Gly Ser Thr625 630 635 640Ser Asn Lys Asn Leu Thr Leu Glu Glu Lys Leu Lys Asn His Ile Val645 650 655Asp Gly Glu Lys Ile Gly Leu Glu Glu Gln Leu Asn Asn Ala Leu Lys660 665 670Lys Tyr Lys Pro Leu Glu Ile Ile Asn Thr Tyr Leu Leu Asp Gly Met675 680 685Lys Val Val Gly Glu Leu Phe Gly Ser Gly Gln Met Gln Leu Pro Phe690 695 700Val Leu Gln Ser Ala Glu Thr Met Lys Phe Ala Val Ser Val Leu Glu705 710 715 720Pro His Met Glu Thr Val Asp Glu Lys Ile Ser Asn Gly Lys Leu Leu725 730 735Ile Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Leu Val740 745 750Asp Ile Ile Leu Ser Asn Asn Gly Phe Asp Val Ile Asn Leu Gly Ile755 760 765Lys Gln Asp Val Ser Ala Ile Ile Asp Ala Gln Lys Lys His Lys Ala770 775 780Asp Cys Ile Ala Met Ser Gly Leu Leu Val Lys Ser Thr Ala Phe Met785 790 795 800Lys Asp Asn Leu Glu Ala Phe Asn Asn Ala Glu Ile Asn Val Pro Val
805 810 815Ile Leu Gly Gly Ala Ala Leu Thr Pro Lys Phe Val Asn Glu Asp Cys820 825 830Ser Gln Ile Tyr Lys Gly Lys Ile Leu Tyr Gly Lys Asp Ala Phe Thr835 840 845Asp Leu Gln Phe Met Asn Asp Tyr Met Asp Ser Lys Lys Lys Gly Asn850 855 860Trp Ser Asn Glu Asn Gly Phe Thr Asn Thr Asp Asp Ile Gln Ile Lys865 870 875 880Leu Ala Ser Pro Arg Ser Ser Ala Lys Asp Lys Asn Leu Asn Lys Asn885 890 895Phe Glu Lys Thr Lys Ser Ile Gln Leu Ile Glu Asn Phe Asn Arg Ser900 905 910Asn Phe Val Glu Glu Glu Glu Pro Ile Lys Ala Pro Phe Leu Gly Thr915 920 925Arg Val Leu Gln Asp Ile Glu Ile Asp Phe Asp Lys Leu Ile Phe Tyr930 935 940Leu Asp Lys Lys Ala Leu Phe Ser Gly Gln Trp Gln Ile Lys Lys Asn945 950 955 960Lys Gly Gln Ser Val Glu Glu Tyr Asn Asn Tyr Leu Asp Ser Tyr Ala965 970 975Asn Pro Leu Leu Glu Lys Trp Ile Asn Ile Ile Leu Asp Lys Gly Leu980 985 990Ile Ser Pro Lys Val Val Tyr Gly Tyr Phe Arg Cys Gly Arg Asn Asp99510001005Asn Ser Ile Tyr Leu Phe Asp Asn Val Ser Asn Lys Arg Ile Ser Glu101010151020Phe Asn Phe Pro Arg Gln Lys Ser Gly Asn Asn Leu Cys Ile Ala Asp1025 103010351040Phe Tyr Cys Asp Leu Lys Asn Asn Asp Pro Val Asp Ile Phe Pro Met104510501055Gln Ala Val Thr Met Gly Glu Ile Ala Ser Glu Tyr Ser Gln Glu Leu106010651070Phe Lys Ala Asp Lys Tyr Ser Asp Tyr Leu Ile Phe His Gly Leu Thr107510801085Val Gln Leu Ala Glu Ala Leu Ala Glu Tyr Val His Ser Ile Val Arg109010951100Ile Glu Cys Gly Phe Lys Ser Tyr Glu Pro Asn Asn Asn Arg Asp Ile1105 111011151120Leu Ala Gln Lys Tyr Arg Gly Ala Arg Tyr Ser Phe Gly Tyr Pro Ala112511301135
Cys Pro Lys Val Ser Asp Ser Asn Ile Gln Leu Ser Leu Leu Asp Thr114011451150Lys Arg Ile Asn Leu Thr Met Asp Glu Ser Glu Gln Leu His Pro Glu115511601165Gln Ser Thr Thr Ala Ile Ile Ser Leu His Ser Lys Ala Lys Tyr Phe117011751180Ser Ala118521092113048212DNA213嗜熱棲熱菌(Thermus thermophilus)220
221CDS222(1)..(3045)223RTT002664009atg cgg gcc tac aag gag gcg gca cgg ggg ctt ctt aag ggc ggg gtg48Met Arg Ala Tyr Lys Glu Ala Ala Arg Gly Leu Leu Lys Gly Gly Val1 5 10 15gac ctc atc ctc ttg gag acc gcc cag gac atc ctc cag gtg cgc tgc96Asp Leu Ile Leu Leu Glu Thr Ala Gln Asp Ile Leu Gln Val Arg Cys20 25 30gcc gtc ttg gcg gtg cgg gag gcc atg gcc gag gtg ggc cgg gag gtg144Ala Val Leu Ala Val Arg Glu Ala Met Ala Glu Val Gly Arg Glu Val35 40 45ccc ctc cag gtc cag gtg acc ttt gag gcc acg ggg acg atg ctc gtg192Pro Leu Gln Val Gln Val Thr Phe Glu Ala Thr Gly Thr Met Leu Val50 55 60ggc acg gac gag cag gcg gcc ctg gcc gct ctg gag agc ctc ccc gtg240Gly Thr Asp Glu Gln Ala Ala Leu Ala Ala Leu Glu Ser Leu Pro Val65 70 75 80gac gtg gtg ggg atg aac tgc gcc acg ggc ccc gac ctc atg gac agc288Asp Val Val Gly Met Asn Cys Ala Thr Gly Pro Asp Leu Met Asp Ser85 90 95aag gtg cgc tac ttc gcc gag cac agc acc cgc ttc gtc tcc tgc ctc336Lys Val Arg Tyr Phe Ala Glu His Ser Thr Arg Phe Val Ser Cys Leu100 105 110ccg aec gcg ggc ctg ccc cgg aac gag ggg ggg agg gtg gtc tac gac384Pro Asn Ala Gly Leu Pro Arg Asn Glu Gly Gly Arg Val Val Tyr Asp115 120 125ctc acc ccc gag gag ctc gcc aag tgg cac ctc aag ttc gtg gcc gag432Leu Thr Pro Glu Glu Leu Ala Lys Trp His Leu Lys Phe Val Ala Glu130 135 140tac ggg gtg aac gcc gtg ggg gga tgc tgc ggc acg ggg ccc gag cac480Tyr Gly Val Asn Ala Val Gly Gly Cys Cys Gly Thr Gly Pro Glu His
145 150 155 160ata agg aag gtg gcc gag gcg gtg aag ggg ctc gcc ccg aag cca agg528Ile Arg Lys Val Ala Glu Ala Val Lys Gly Leu Ala Pro Lys Pro Arg165 170 175ccc gaa agc ttc cct ccc cag gtg gcc tcc ttg tac cag gcg gtg tcc576Pro Glu Ser Phe Pro Pro Gln Val Ala Ser Leu Tyr Gln Ala Val Ser180 185 190ctc aag cag gag gcg agc ctt ttc ctc gtg ggg gag agg ctc aac gcc624Leu Lys Gln Glu Ala Ser Leu Phe Leu Val Gly Glu Arg Leu Asn Ala195 200 205acg ggg agc aag cgc ttc cgg gag atg ctc ttc gcg aga gac ctc gag672Thr Gly Ser Lys Arg Phe Arg Glu Met Leu Phe Ala Arg Asp Leu Glu210 215 220ggc atc ctc gcc ctc gcc cgg gag cag gtg gag gag ggg gcc cac gcc720Gly Ile Leu Ala Leu Ala Arg Glu Gln Val Glu Glu Gly Ala His Ala225 230 235 240ctg gac ctc tcc gtg gcc tgg acg ggg cgg gac gag ctt gag gac ctc768Leu Asp Leu Ser Val Ala Trp Thr Gly Arg Asp Glu Leu Glu Asp Leu245 250 255cgg tgg ctc ctt ccc cat ctc gcc acc gcc ctt acc gtc ccc gtc atg816Arg Trp Leu Leu Pro His Leu Ala Thr Ala Leu Thr Val Pro Val Met260 265 270gtg gac tcc acc tcc cct gag gcc atg gag ctc gcc ctc aaa tac ctc864Val Asp Ser Thr Ser Pro Glu Ala Met Glu Leu Ala Leu Lys Tyr Leu275 280 285ccg ggc cgg gtc ctc ctg aac tcc gcc aac ctc gag gat ggc tta gag912Pro Gly Arg Val Leu Leu Asn Ser Ala Asn Leu Glu Asp Gly Leu Glu290 295 300cgc ttt gac cgg gtg gcc tcc ctg gcc aag gcc cac ggg gcg gcc ctc960Arg Phe Asp Arg Val Ala Ser Leu Ala Lys Ala His Gly Ala Ala Leu305 310 315 320gtg gtc ctc gcc att gac gag aag ggg atg gcc aag acc cgg gag gag1008Val Val Leu Ala Ile Asp Glu Lys Gly Met Ala Lys Thr Arg Glu Glu325 330 335aag gtg cgg gtg gcc ctg agg atg tac gag cgc ctc acg gag cac cac1056Lys Val Arg Val Ala Leu Arg Met Tyr Glu Arg Leu Thr Glu His His340 345 350ggc ctc cgc ccc gag gac ctc ctc ttt gac ctc ctt acc ttc ccc atc1104Gly Leu Arg Pro Glu Asp Leu Leu Phe Asp Leu Leu Thr Phe Pro Ile355 360 365acc caa ggg gac gag gag agc cgc cct ctg gcc aag gag acc ctc ctc1152Thr Gln Gly Asp Glu Glu Ser Arg Pro Leu Ala Lys Glu Thr Leu Leu370 375 380gcc ata gag gag cta cgg gag agg ctt ccc ggg gtg ggc ttc gtc ctt1200Ala Ile Glu Glu Leu Arg Glu Arg Leu Pro Gly Val Gly Phe Val Leu385 390 395 400
cgg gtc tcc aac gtc tcc ttc ggg ctc aag ccc cgg gcg agg cgc gtc1248Arg Val Ser Asn Val Ser Phe Gly Leu Lys Pro Arg Ala Arg Arg Val405 410 415ctg aac tcc gtc ttc ctg gac gag gcg agg aaa cgg ggc ctc acc gcg1296Leu Asn Ser Val Phe Leu Asp Glu Ala Arg Lys Arg Gly Leu Thr Ala420 425 430gcc atc gtg gac gcg ggg aag atc ctc ccc ata agc cag atc ccc gag1344Ala Ile Val Asp Ala Gly Lys Ile Leu Pro Ile Ser Gln Ile Pro Glu435 440 445gag gcc tac gcc ctc gcc tta gac ctc atc tac gac cgc cgc aag gag1392Glu Ala Tyr Ala Leu Ala Leu Asp Leu Ile Tyr Asp Arg Arg Lys Glu450 455 460ggc ttt gac ccc ctc ctc gcc ttc atg gcc tac ttt gag gcc cac aag1440Gly Phe Asp Pro Leu Leu Ala Phe Met Ala Tyr Phe Glu Ala His Lys465 470 475 480gag gac ccg ggg aag agg gag gac gcc ttc ctg gcc ctt ccc ctt ctg1488Glu Asp Pro Gly Lys Arg Glu Asp Ala Phe Leu Ala Leu Pro Leu Leu485 490 495gag agg ctc aag cgc cgc gtg gtg gag ggg agg aag cag ggc ctc gag1536Glu Arg Leu Lys Arg Arg Val Val Glu Gly Arg Lys Gln Gly Leu Glu500 505 510gcc gac ctg gag gag gcc ctg aag gcg ggg cac aag ccc ttg gac ctc1584Ala Asp Leu Glu Glu Ala Leu Lys Ala Gly His Lys Pro Leu Asp Leu515 520 525atc aac ggc ccc ctc ctc gcg ggg atg aag gag gtg ggg gac ctc ttc1632Ile Asn Gly Pro Leu Leu Ala Gly Met Lys Glu Val Gly Asp Leu Phe530 535 540ggg gcg ggg aag atg cag ctc ccc ttc gtc ctc cag gcc gcc gag gtg1680Gly Ala Gly Lys Met Gln Leu Pro Phe Val Leu Gln Ala Ala Glu Val545 550 555 560atg aag cgg gcg gtg gcc tac ctc gag ccc cac atg gag aag aag ggg1728Met Lys Arg Ala Val Ala Tyr Leu Glu Pro His Met Glu Lys Lys Gly565 570 575gag ggc aag ggt acc ctg gtc ctc gcc acc gtc aag ggg gac gtg cac1776Glu Gly Lys Gly Thr Leu Val Leu Ala Thr Val Lys Gly Asp Val His580 585 590gac atc ggc aag aac ctg gtg gac atc atc ctc agc aac aac ggc tac1824Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Ser Asn Asn Gly Tyr595 600 605cgg gtg gtg aac ctg ggg atc aag gtg ccc att gag gag atc ctg aag1872Arg Val Val Asn Leu Gly Ile Lys Val Pro Ile Glu Glu Ile Leu Lys610 615 620gcc gtg gag gcg cac aag ccc cac gcc gtg ggc atg tcg ggc ctc ctg1920Ala Val Glu Ala His Lys Pro His Ala Val Gly Met Ser Gly Leu Lau625 630 635 640gtg aag agc acc ctg gtg atg aag gag aac ctg gag tac atg cgg gat1968Val Lys Ser Thr Leu Val Met Lys Glu Asn Leu Glu Tyr Met Arg Asp
645 650 655agg ggc tac acc ctc ccc gtg atc ctg ggc ggg gcc gcc ctc acc cgg2016Arg Gly Tyr Thr Leu Pro Val Ile Leu Gly Gly Ala Ala Leu Thr Arg660 665 670agc tac gtg gag gag ctt aag gcc atc tac ccc aac gtc tac tac gcc2064Ser Tyr Val Glu Glu Leu Lys Ala Ile Tyr Pro Asn Val Tyr Tyr Ala675 680 685gag gac gcc ttt gag ggc tta agg ctc atg gag gag ctc acg ggc cac2112Glu Asp Ala Phe Glu Gly Leu Arg Leu Met Glu Glu Leu Thr Gly His690 695 700gcc cct ccc gag ctc acc cgg aag gcc cca gct agg ccc aag cgg gag2160Ala Pro Pro Glu Leu Thr Arg Lys Ala Pro Ala Arg Pro Lys Arg Glu705 710 715 720gcc ccc aag gtg gcg ccc cgc gct cgg ccc gtg ggg gag gcc ccc gcc2208Ala Pro Lys Val Ala Pro Arg Ala Arg Pro Val Gly Glu Ala Pro Ala725 730 735gtc ccc cgg ccc ccc ttc ttc ggc gtg cgg gtg gag gaa ggc ttg gac2256Val Pro Arg Pro Pro Phe Phe Gly Val Arg Val Glu Glu Gly Leu Asp740 745 750ctc gcc acc atc gcc cac tac gtc aac aag ctc gcc ctc tac cgg ggc2304Leu Ala Thr Ile Ala His Tyr Val Asn Lys Leu Ala Leu Tyr Arg Gly755 760 765cag tgg ggc tac agc cgc aag ggc ttt ccc ggg agg cgt ggc agg ccc2352Gln Trp Gly Tyr Ser Arg Lys Gly Phe Pro Gly Arg Arg Gly Arg Pro770 775 780tgg tgg agc ggg agg cgg agc ctg tct tcc aga ggc tcc tca agg agg2400Trp Trp Ser Gly Arg Arg Ser Leu Ser Ser Arg Gly Ser Ser Arg Arg785 790 795 800cga tgg cgg aag ggt ggc ttg aac cca agg tcc tct acg gct tct tcc2448Arg Trp Arg Lys Gly Gly Leu Asn Pro Arg Ser Ser Thr Ala Ser Ser805 810 815ccg tgg ccc ggg agg gga gga gct tct cgt ctt ctc ccc aga gac ggg2496Pro Trp Pro Gly Arg Gly Gly Ala Ser Arg Leu Leu Pro Arg Asp Gly820 825 830gga ggt gct gga gcg ctt ccg ctt ccc ccg gca aag ggg cgg ggg cct2544Gly Gly Ala Gly Ala Leu Pro Leu Pro Pro Ala Lys Gly Arg Gly Pro835 840 845gag cct cgt gga cta ctt ccg ccc ccg gtt tgc cgc gcc ttt ggg gga2592Glu Pro Arg Gly Leu Leu Pro Pro Pro Val Cys Arg Ala Phe Gly Gly850 855 860cga ggc gga ctg gat gcc caa gga ggc ctt ccg ggc ggg ggc cgg gac2640Arg Gly Gly Leu Asp Ala Gln Gly Gly Leu Pro Gly Gly Gly Arg Asp865 870 875 880gtc ctc ggg gtc cag ctc gtc acc atg ggg gag gcc cct tcc cga aag2688Val Leu Gly Val Gln Leu Val Thr Met Gly Glu Ala Pro Ser Arg Lys885 890 895
gcc cag gcc ctc ttt gcg tcc ggg gcc tac cag gac tac ctc ttc gtc2736Ala Gln Ala Leu Phe Ala Ser Gly Ala Tyr Gln Asp Tyr Leu Phe Val900 905 910cac ggc ttc agc gtg gag atg acc gag gcc ttg gcg gag tac tgg cac2784His Gly Phe Ser Val Glu Met Thr Glu Ala Leu Ala Glu Tyr Trp His915 920 925aag agg atg cgg cag atg tgg ggc atc gcc cac aag gac gcc acc gag2832Lys Arg Met Arg Gln Met Trp Gly Ile Ala His Lys Asp Ala Thr Glu930 935 940atc cag aag ctc ttc cag cag ggc tac cag ggg gcc cgc tac tcc ttc2880Ile Gln Lys Leu Phe Gln Gln Gly Tyr Gln Gly Ala Arg Tyr Ser Phe945 950 955 960ggc tac ccc gcc tgc ccg gac ctc gcc gac cag gcc aag ctg gac cgg2928Gly Tyr Pro Ala Cys Pro Asp Leu Ala Asp Gln Ala Lys Leu Asp Arg965 970 975ctc atg ggc ttc cac cgg gtg ggg gtg cac ctc acg gag aac ttc cag2976Leu Met Gly Phe His Arg Val Gly Val His Leu Thr Glu Asn Phe Gln980 985 990ctg gag ccg gag cac gcc acc agc gcc ctc gtg gtc cac cac ccc gag3024Leu Glu Pro Glu His Ala Thr Ser Ala Leu Val Val His His Pro Glu99510001005gcc cgc tac ttc agc gtg gac tag3048Ala Arg Tyr Phe Ser Val Asp10101015210102111015212PRT213嗜熱棲熱菌40010Met Arg Ala Tyr Lys Glu Ala Ala Arg Gly Leu Leu Lys Gly Gly Val1 5 10 15Asp Leu Ile Leu Leu Glu Thr Ala Gln Asp Ile Leu Gln Val Arg Cys20 25 30Ala Val Leu Ala Val Arg Glu Ala Met Ala Glu Val Gly Arg Glu Val35 40 45Pro Leu Gln Val Gln Val Thr Phe Glu Ala Thr Gly Thr Met Leu Val50 55 60Gly Thr Asp Glu Gln Ala Ala Leu Ala Ala Leu Glu Ser Leu Pro Val65 70 75 80Asp Val Val Gly Met Asn Cys Ala Thr Gly Pro Asp Leu Met Asp Ser85 90 95Lys Val Arg Tyr Phe Ala Glu His Ser Thr Arg Phe Val Ser Cys Leu100 105 110Pro Asn Ala Gly Leu Pr0 Arg Asn Glu Gly Gly Arg Val Val Tyr Asp115 120 125
Leu Thr Pro Glu Glu Leu Ala Lys Trp His Leu Lys Phe Val Ala Glu130 135 140Tyr Gly Val Asn Ala Val Gly Gly Cys Cys Gly Thr Gly Pro Glu His145 150 155 160Ile Arg Lys Val Ala Glu Ala Val Lys Gly Leu Ala Pro Lys Pro Arg165 170 175Pro Glu Ser Phe Pro Pro Gln Val Ala Ser Leu Tyr Gln Ala Val Ser180 185 190Leu Lys Gln Glu Ala Ser Leu Phe Leu Val Gly Glu Arg Leu Asn Ala195 200 205Thr Gly Ser Lys Arg Phe Arg Glu Met Leu Phe Ala Arg Asp Leu Glu210 215 220Gly Ile Leu Ala Leu Ala Arg Glu Gln Val Glu Glu Gly Ala His Ala225 230 235 240Leu Asp Leu Ser Val Ala Trp Thr Gly Arg Asp Glu Leu Glu Asp Leu245 250 255Arg Trp Leu Leu Pro His Leu Ala Thr Ala Leu Thr Val Pro Val Met260 265 270Val Asp Ser Thr Ser Pro Glu Ala Met Glu Leu Ala Leu Lys Tyr Leu275 280 285Pro Gly Arg Val Leu Leu Asn Ser Ala Asn Leu Glu Asp Gly Leu Glu290 295 300Arg Phe Asp Arg Val Ala Ser Leu Ala Lys Ala His Gly Ala Ala Leu305 310 315 320Val Val Leu Ala Ile Asp Glu Lys Gly Met Ala Lys Thr Arg Glu Glu325 330 335Lys Val Arg Val Ala Leu Arg Met Tyr Glu Arg Leu Thr Glu His His340 345 350Gly Leu Arg Pro Glu Asp Leu Leu Phe Asp Leu Leu Thr Phe Pro Ile355 360 365Thr Gln Gly Asp Glu Glu Ser Arg Pro Leu Ala Lys Glu Thr Leu Leu370 375 380Ala Ile Glu Glu Leu Arg Glu Arg Leu Pro Gly Val Gly Phe Val Leu385 390 395 400Arg Val Ser Asn Val Ser Phe Gly Leu Lys Pro Arg Ala Arg Arg Val405 410 415Leu Asn Ser Val Phe Leu Asp Glu Ala Arg Lys Arg Gly Leu Thr Ala420 425 430Ala Ile Val Asp Ala Gly Lys Ile Leu Pro Ile Ser Gln Ile Pro Glu435 440 445Glu Ala Tyr Ala Leu Ala Leu Asp Leu Ile Tyr Asp Arg Arg Lys Glu
450 455 460Gly Phe Asp Pro Leu Leu Ala Phe Met Ala Tyr Phe Glu Ala His Lys465 470 475 480Glu Asp Pro Gly Lys Arg Glu Asp Ala Phe Leu Ala Leu Pro Leu Leu485 490 495Glu Arg Leu Lys Arg Arg Val Val Glu Gly Arg Lys Gln Gly Leu Glu500 505 510Ala Asp Leu Glu Glu Ala Leu Lys Ala Gly His Lys Pro Leu Asp Leu515 520 525Ile Asn Gly Pro Leu Leu Ala Gly Met Lys Glu Val Gly Asp Leu Phe530 535 540Gly Ala Gly Lys Met Gln Leu Pro Phe Val Leu Gln Ala Ala Glu Val545 550 555 560Met Lys Arg Ala Val Ala Tyr Leu Glu Pro His Met Glu Lys Lys Gly565 570 575Glu Gly Lys Gly Thr Leu Val Leu Ala Thr Val Lys Gly Asp Val His580 585 590Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Ser Asn Asn Gly Tyr595 600 605Arg Val Val Asn Leu Gly Ile Lys Val Pro Ile Glu Glu Ile Leu Lys610 615 620Ala Val Glu Ala His Lys Pro His Ala Val Gly Met Ser Gly Leu Leu625 630 635 640Val Lys Ser Thr Leu Val Met Lys Glu Asn Leu Glu Tyr Met Arg Asp645 650 655Arg Gly Tyr Thr Leu Pro Val Ile Leu Gly Gly Ala Ala Leu Thr Arg660 665 670Ser Tyr Val Glu Glu Leu Lys Ala Ile Tyr Pro Asn Val Tyr Tyr Ala675 680 685Glu Asp Ala Phe Glu Gly Leu Arg Leu Met Glu Glu Leu Thr Gly His690 695 700Ala Pro Pro Glu Leu Thr Arg Lys Ala Pro Ala Arg Pro Lys Arg Glu705 710 715 720Ala Pro Lys Val Ala Pro Arg Ala Arg Pro Val Gly Glu Ala Pro Ala725 730 735Val Pro Arg Pro Pro Phe Phe Gly Val Arg Val Glu Glu Gly Leu Asp740 745 750Leu Ala Thr Ile Ala His Tyr Val Asn Lys Leu Ala Leu Tyr Arg Gly755 760 765Gln Trp Gly Tyr Ser Arg Lys Gly Phe Pro Gly Arg Arg Gly Arg Pro770 775 780
Trp Trp Ser Gly Arg Arg Ser Leu Ser Ser Arg Gly Ser Ser Arg Arg785 790 795 800Arg Trp Arg Lys Gly Gly Leu Asn Pro Arg Ser Ser Thr Ala Ser Ser805 810 815Pro Trp Pro Gly Arg Gly Gly Ala Ser Arg Leu Leu Pro Arg Asp Gly820 825 830Gly Gly Ala Gly Ala Leu Pro Leu Pro Pro Ala Lys Gly Arg Gly Pro835 840 845Glu Pro Arg Gly Leu Leu Pro Pro Pro Val Cys Arg Ala Phe Gly Gly850 855 860Arg Gly Gly Leu Asp Ala Gln Gly Gly Leu Pro Gly Gly Gly Arg Asp865 870 875 880Val Leu Gly Val Gln Leu Val Thr Met Gly Glu Ala Pro Ser Arg Lys885 890 895Ala Gln Ala Leu Phe Ala Ser Gly Ala Tyr Gln Asp Tyr Leu Phe Val900 905 910His Gly Phe Ser Val Glu Met Thr Glu Ala Leu Ala Glu Tyr Trp His915 920 925Lys Arg Met Arg Gln Met Trp Gly Ile Ala His Lys Asp Ala Thr Glu930 935 940Ile Gln Lys Leu Phe Gln Gln Gly Tyr Gln Gly Ala Arg Tyr Ser Phe945 950 955 960Gly Tyr Pro Ala Cys Pro Asp Leu Ala Asp Gln Ala Lys Leu Asp Arg965 970 975Leu Met Gly Phe His Arg Val Gly Val His Leu Thr Glu Asn Phe Gln980 985 990Leu Glu Pro Glu His Ala Thr Ser Ala Leu Val Val His His Pro Glu99510001005Ala Arg Tyr Phe Ser Val Asp10101015210112113441212DNA213Bacillus halodurans220
221CDS222(1)..(3438)223RHD0 555040011atg act aaa tcg ttg ttt gaa caa cag tta gag cga aaa atc gtc atc48Met Thr Lys Ser Leu Phe Glu Gln Gln Leu Glu Arg Lys Ile Val Ile1 5 10 15ctt gat ggg gcg atg ggg acc atg tta caa gcc gcg aat cta acc gct96
Leu Asp Gly Ala Met Gly Thr Met Leu Gln Ala Ala Asn Leu Thr Ala20 25 30gat gac ttt ggc gga gaa gag tat gaa ggg tgt aat gaa tat tta aat144Asp Asp Phe Gly Gly Glu Glu Tyr Glu Gly Cys Asn Glu Tyr Leu Asn35 40 45gag acg gcc ccc cat gtc gtt gag gac att cat cgc gca tac tta gag192Glu Thr Ala Pro His Val Val Glu Asp Ile His Arg Ala Tyr Leu Glu50 55 60gca gga gca gac gtc att gcg acg aac acg ttc ggg gca aca gat atc240Ala Gly Ala Asp Val Ile Ala Thr Asn Thr Phe Gly Ala Thr Asp Ile65 70 75 80gtt ctt gac gat tat gat ctc gga tac aaa gca gag gag tta aac ata288Val Leu Asp Asp Tyr Asp Leu Gly Tyr Lys Ala Glu Glu Leu Asn Ile85 90 95tgc gcg gtg aaa atc gct aaa cgt gta gct gaa gag ttt tcc act cca336Cys Ala Val Lys Ile Ala Lys Arg Val Ala Glu Glu Phe Ser Thr Pro100 105 110gat tgg cct cga ttc gtt gca ggg gcg atg ggg ccg acg acg aaa tct384Asp Trp Pro Arg Phe Val Ala Gly Ala Met Gly Pro Thr Thr Lys Ser115 120 125ctt tcc gtc aca ggg ggc gcg aca ttc gaa caa ctt atc gag tct tat432Leu Ser Val Thr Gly Gly Ala Thr Phe Glu Gln Leu Ile Glu Ser Tyr130 135 140cgc cag caa gct aca ggt cta att aaa ggc ggg gcg gat att tta tta480Arg Gln Gln Ala Thr Gly Leu Ile Lys Gly Gly Ala Asp Ile Leu Leu145 150 155 160ctc gaa acg agc cag gat atg cga aac gtg aag gcg gct tat tta gga528Leu Glu Thr Ser Gln Asp Met Arg Asn Val Lys Ala Ala Tyr Leu Gly165 170 175ctg agc caa gcg caa aaa gag cta gag gtg aaa ctg cct ctc att att576Leu Ser Gln Ala Gln Lys Glu Leu Glu Val Lys Leu Pro Leu Ile Ile180 185 190tct gga acg att gaa ccg atg gga aca acg ctc gcc ggc caa aac atc624Ser Gly Thr Ile Glu Pro Met Gly Thr Thr Leu Ala Gly Gln Asn Ile195 200 205gag gcg ttc tat ttg tca tta gag cat atg aat ccc gtc gtt gtc ggt672Glu Ala Phe Tyr Leu Ser Leu Glu His Met Asn Pro Val Val Val Gly210 215 220ctc aac tgc gct aca gga cca gaa ttt atg cgc gat cac ctc cgt tct720Leu Asn Cys Ala Thr Gly Pro Glu Phe Met Arg Asp His Leu Arg Ser225 230 235 240ctt tca gac ctt gcg acc tgc tct gta agc tgt tat ccg aat gct ggg768Leu Ser Asp Leu Ala Thr Cys Ser Val Ser Cys Tyr Pro Asn Ala Gly245 250 255tta cct gat gaa gag ggg aac tat cac gaa tcc cca gaa tca tta gca816Leu Pro Asp Glu Glu Gly Asn Tyr His Glu Ser Pro Glu Ser Leu Ala260 265 270
gcc aag ctc gca ggt ttt gcg gaa aag ggc tgg ttg aat atg gtt ggt864Ala Lys Leu Ala Gly Phe Ala Glu Lys Gly Trp Leu Asn Met Val Gly275 280 285ggc tgt tgc ggg acg act cca gac cac att cgt gct ctt ttg gac gtt912Gly Cys Cys Gly Thr Thr Pro Asp His Ile Arg Ala Leu Leu Asp Val290 295 300atg aag caa ttt gag ccg aga caa cca aaa ggg gat cac ccc cac tcg960Met Lys Gln Phe Glu Pro Arg Gln Pro Lys Gly Asp His Pro His Ser305 310 315 320gtc tca gga att gag cca ctg tta tac gat gac agc atg cgt cca cta1008Val Ser Gly Ile Glu Pro Leu Leu Tyr Asp Asp Ser Met Arg Pro Leu325 330 335ttt gtc ggt gaa cgg aca aac gtc atc ggg tct cgt aaa ttt aaa cgg1056Phe Val Gly Glu Arg Thr Asn Val Ile Gly Ser Arg Lys Phe Lys Arg340 345 350ttg atc gaa gaa gaa aaa tat gaa gaa gcc tca gaa att gca aga tcc1104Leu Ile Glu Glu Glu Lys Tyr Glu Glu Ala Ser Glu Ile Ala Arg Ser355 360 365caa gtg aag aaa ggg gcc cac gtt atc gat gtt tgt ctt gct gat ccg1152Gln Val Lys Lys Gly Ala His Val Ile Asp Val Cys Leu Ala Asp Pro370 375 380gat cgc gat gaa atg gag gac atg gag gaa ttt tta aaa ttc gtg atc1200Asp Arg Asp Glu Met Glu Asp Met Glu Glu Phe Leu Lys Phe Val Ile385 390 395 400aac aaa gtg aag gta ccg ctc atg att gac tcc acc gac gaa aag gta1248Asn Lys Val Lys Val Pro Leu Met Ile Asp Ser Thr Asp Glu Lys Val405 410 415att gaa caa gcg ctt acg tat tca caa ggg aaa gcg atc att aat tcg1296Ile Glu Gln Ala Leu Thr Tyr Ser Gln Gly Lys Ala Ile Ile Asn Ser420 425 430atc aac tta gag gac ggc gaa gaa cgt ttt gaa aaa gtg gtc ccg ctc1344Ile Asn Leu Glu Asp Gly Glu Glu Arg Phe Glu Lys Val Val Pro Leu435 440 445gtc cat aag tat gga gcc gcg gtt gtc gtt ggt acg atc gac gaa gaa1392Val His Lys Tyr Gly Ala Ala Val Val Val Gly Thr Ile Asp Glu Glu450 455 460gga atg gcg att acg gca gaa aaa aaa tta gcg gtt gcg aaa cga tca1440Gly Met Ala Ile Thr Ala Glu Lys Lys Leu Ala Val Ala Lys Arg Ser465 470 475 480tac gac ctg ctc gta aac aaa tac aac att cgt ccg agc gat att att1488Tyr Asp Leu Leu Val Asn Lys Tyr Asn Ile Arg Pro Ser Asp Ile Ile485 490 495ttt gat ccg ctc gtg ttc cca gta gga aca ggc gat gag caa tac att1536Phe Asp Pro Leu Val Phe Pro Val Gly Thr Gly Asp Glu Gln Tyr Ile500 505 510ggc tcg gcg aat gag acg gtg gaa gga att agg agg atc aaa gaa gag1584
Gly Ser Ala Asn Glu Thr Val Glu Gly Ile Arg Arg Ile Lys Glu Glu515 520 525ctc cct gaa tgt tta acg att ctt gga gtt agt aac gtg tcg ttc ggt1632Leu Pro Glu Cys Leu Thr Ile Leu Gly Val Ser Asn Val Ser Phe Gly530 535 540ctt ccg cct gtc gga aga gag gtg ctg aac gcg gcg tac tta tac cat1680Leu Pro Pro Val Gly Arg Glu Val Leu Asn Ala Ala Tyr Leu Tyr His545 550 555 560tgt aca caa gct ggc ctt gat tac gct atc gtg aac aca gaa aag ctt1728Cys Thr Gln Ala Gly Leu Asp Tyr Ala Ile Val Asn Thr Glu Lys Leu565 570 575gag cgt tat gcc tcg att tct gat gaa gaa aaa gaa ttg tca agg aag1776Glu Arg Tyr Ala Ser Ile Ser Asp Glu Glu Lys Glu Leu Ser Arg Lys580 585 590ctc tta ttt gaa acg aca gat gaa acg ctc gct gag ttc acc gcc ttt1824Leu Leu Phe Glu Thr Thr Asp Glu Thr Leu Ala Glu Phe Thr Ala Phe595 600 605tat cga ggg aaa aaa gca gag aaa aaa gtg gag act tct aat tta act1872Tyr Arg Gly Lys Lys Ala Glu Lys Lys Val Glu Thr Ser Asn Leu Thr610 615 620ttg gaa gag cgg ttg gca aac tac att gtt gaa ggg tca aag gac gga1920Leu Glu Glu Arg Leu Ala Asn Tyr Ile Val Glu Gly Ser Lys Asp Gly625 630 635 640ctg aca gaa gat tta gat aaa gcg ctc gcg aaa tat gat gat ccg ctt1968Leu Thr Glu Asp Leu Asp Lys Ala Leu Ala Lys Tyr Asp Asp Pro Leu645 650 655gat atc att aac ggc ccg ctc atg aat gga atg gac gaa gtc ggt cgt2016Asp Ile Ile Asn Gly Pro Leu Met Asn Gly Met Asp Glu Val Gly Arg660 665 670ttg ttt aac aat aac gag ctt att gtc gct gaa gta ttg caa agc gct2064Leu Phe Asn Asn Asn Glu Leu Ile Val Ala Glu Val Leu Gln Ser Ala675 680 685gag gtt atg aag gct tcc gtc gcc cac ctt gag cca cat atg gaa aag2112Glu Val Met Lys Ala Ser Val Ala His Leu Glu Pro His Met Glu Lys690 695 700aaa gca gac gat cat gga aaa gga aaa atc att ctt gcc acg gtc aag2160Lys Ala Asp Asp His Gly Lys Gly Lys Ile Ile Leu Ala Thr Val Lys705 710 715 720ggc gat gtt cac gat atc ggg aaa aat cta gtg gaa att att ttg agc2208Gly Asp Val His Asp Ile Gly Lys Asn Leu Val Glu Ile Ile Leu Ser725 730 735aat aat ggt ttc cgc atc gtg aac cta gga att aaa gtt acc tct aat2256Asn Asn Gly Phe Arg Ile Val Asn Leu Gly Ile Lys Val Thr Ser Asn740 745 750gag ctg att gaa gcg gtg gcg aga gaa aat cca gat gcg att ggc ttg2304Glu Leu Ile Glu Ala Val Ala Arg Glu Asn Pro Asp Ala Ile Gly Leu755 760 765
tca ggg ttg ctc gtc aaa tca gca caa caa atg gta ctt acc gcc caa2352Ser Gly Leu Leu Val Lys Ser Ala Gln Gln Met Val Leu Thr Ala Gln770 775 780gat ttg aag caa caa caa att tcc att ccg att tta gtc gga ggc gca2400Asp Leu Lys Gln Gln Gln Ile Ser Ile Pro Ile Leu Val Gly Gly Ala785 790 795 800gcc ctt acg cgg aaa ttt acg aat aca aaa atc gct cca gag tat gat2448Ala Leu Thr Arg Lys Phe Thr Asn Thr Lys Ile Ala Pro Glu Tyr Asp805 810 815ggt ctc gtc gtc tac gcg aag gat gcg atg aac ggg tta gag ctt gcc2496Gly Leu Val Val Tyr Ala Lys Asp Ala Met Asn Gly Leu Glu Leu Ala820 825 830aat aaa tta atg aaa cct gat gaa cga gaa aag cta gcg gtc tcc ctc2544Asn Lys Leu Met Lys Pro Asp Glu Arg Glu Lys Leu Ala Val Ser Leu835 840 845cat gaa gcg aag gag cag gcg aac tcg agg aca caa atg gga gga ggc2592His Glu Ala Lys Glu Gln Ala Asn Ser Arg Thr Gln Met Gly Gly Gly850 855 860gga act gca gtt gcg gta aag ccg act cga tcc cat gtt tcg aca acg2640Gly Thr Ala Val Ala Val Lys Pro Thr Arg Ser His Val Ser Thr Thr865 870 875 880gtg cct gta gcg gtc cca cct gat gtg aag ccg cac att ttg cgc cac2688Val Pro Val Ala Val Pro Pro Asp Val Lys Pro His Ile Leu Arg His885 890 895cat agc att gcc cat tta gag ccg tat att aac atg cag atg ttg tta2736His Ser Ile Ala His Leu Glu Pro Tyr Ile Asn Met Gln Met Leu Leu900 905 910gga cgt cac tta ggc tta caa ggg aaa gtg agc cgc ctg ctt gca gaa2784Gly Arg His Leu Gly Leu Gln Gly Lys Val Ser Arg Leu Leu Ala Glu915 920 925aaa gac gag aag gct ctt gaa tta aaa gaa aaa gtt gat gcg cta ctc2832Lys Asp Glu Lys Ala Leu Glu Leu Lys Glu Lys Val Asp Ala Leu Leu930 935 940acc agg gtg aaa gag gag cag ctc atg gaa gcc cat ggc atg tat cag2880Thr Arg Val Lys Glu Glu Gln Leu Met Glu Ala His Gly Met Tyr Gln945 950 955 960ttt ttt cct gcc cag tcg gat ggg gac gat att gtc att tat gat caa2928Phe Phe Pro Ala Gln Ser Asp Gly Asp Asp Ile Val Ile Tyr Asp Gln965 970 975acg gga aca aat gaa atc gag cga ttc cat ttt ccg cgt cag aat aag2976Thr Gly Thr Asn Glu Ile Glu Arg Phe His Phe Pro Arg Gln Asn Lys980 985 990gag cct tat ctg tgt ctt gcc gat ttc ctt cgc cca gtt tcc agt ggg3024Glu Pro Tyr Leu Cys Leu Ala Asp Phe Leu Arg Pro Val Ser Ser Gly995 10001005gaa atg gac tat gtt ggc ttc ctt gct gta acc gca gga aaa ggc att3072
Glu Met Asp Tyr Val Gly Phe Leu Ala Val Thr Ala Gly Lys Gly Ile101010151020cgt gaa tta ggg gag cag gcg aaa gag gct gga gac tat tta ttc agt3120Arg Glu Leu Gly Glu Gln Ala Lys Glu Ala Gly Asp Tyr Leu Phe Ser1025103010351040cac tta atc caa gca aca gcc tta gag atg gcg gaa ggg ttt gcc gag3168His Leu Ile Gln Ala Thr Ala Leu Glu Met Ala Glu Gly Phe Ala Glu104510501055cgt gtc cat cag ctc atg cgt gat aag tgg ggg ttt cct gat tcg gct3216Arg Val His Gln Leu Met Arg Asp Lys Trp Gly Phe Pro Asp Ser Ala106010651070gac ttt aca atg gaa gag cgt ttc gct gca aaa tac cgt ggc atc cgt3264Asp Phe Thr Met Glu Glu Arg Phe Ala Ala Lys Tyr Arg Gly Ile Arg107510801085gta tcg ttt ggc tac cct gca tgc cct gac ttg gat gac caa gca aag3312Val Ser Phe Gly Tyr Pro Ala Cys Pro Asp Leu Asp Asp Gln Ala Lys109010951100ttg ttt aag ctg ttg aag cct gga aag atc gga att gag ttg acg gaa3360Leu Phe Lys Leu Leu Lys Pro Gly Lys Ile Gly Ile Glu Leu Thr Glu1105111011151120ggg ttt atg atg gag cca gaa gcc tcc gtc acc gcg atg gtg ttt gcc3408Gly Phe Met Met Glu Pro Glu Ala Ser Val Thr Ala Met Val Phe Ala112511301135cat cct gag gct cgc tat ttt aat gtt tta tag3441His Pro Glu Ala Arg Tyr Phe Asn Val Leu11401145210122111146212PRT213Bacillus halodurans40012Met Thr Lys Ser Leu Phe Glu Gln Gln Leu Glu Arg Lys Ile Val Ile1 5 10 15Leu Asp Gly Ala Met Gly Thr Met Leu Gln Ala Ala Asn Leu Thr Ala20 25 30Asp Asp Phe Gly Gly Glu Glu Tyr Glu Gly Cys Asn Glu Tyr Leu Asn35 40 45Glu Thr Ala Pro His Val Val Glu Asp Ile His Arg Ala Tyr Leu Glu50 55 60Ala Gly Ala Asp Val Ile Ala Thr Asn Thr Phe Gly Ala Thr Asp Ile65 70 75 80Val Leu Asp Asp Tyr Asp Leu Gly Tyr Lys Ala Glu Glu Leu Asn Ile85 90 95Cys Ala Val Lys Ile Ala Lys Arg Val Ala Glu Glu Phe Ser Thr Pro100 105 110
Asp Trp Pro Arg Phe Val Ala Gly Ala Met Gly Pro Thr Thr Lys Ser115 120 125Leu Ser Val Thr Gly Gly Ala Thr Phe Glu Gln Leu Ile Glu Ser Tyr130 135 140Arg Gln Gln Ala Thr Gly Leu Ile Lys Gly Gly Ala Asp Ile Leu Leu145 150 155 160Leu Glu Thr Ser Gln Asp Met Arg Asn Val Lys Ala Ala Tyr Leu Gly165 170 175Leu Ser Gln Ala Gln Lys Glu Leu Glu Val Lys Leu Pro Leu Ile Ile180 185 190Ser Gly Thr Ile Glu Pro Met Gly Thr Thr Leu Ala Gly Gln Asn Ile195 200 205Glu Ala Phe Tyr Leu Ser Leu Glu His Met Asn Pro Val Val Val Gly210 215 220Leu Asn Cys Ala Thr Gly Pro Glu Phe Met Arg Asp His Leu Arg Ser225 230 235 240Leu Ser Asp Leu Ala Thr Cys Ser Val Ser Cys Tyr Pro Asn Ala Gly245 250 255Leu Pro Asp Glu Glu Gly Asn Tyr His Glu Ser Pro Glu Ser Leu Ala260 265 270Ala Lys Leu Ala Gly Phe Ala Glu Lys Gly Trp Leu Asn Met Val Gly275 280 285Gly Cys Cys Gly Thr Thr Pro Asp His Ile Arg Ala Leu Leu Asp Val290 295 300Met Lys Gln Phe Glu Pro Arg Gln Pro Lys Gly Asp His Pro His Ser305 310 315 320Val Ser Gly Ile Glu Pro Leu Leu Tyr Asp Asp Ser Met Arg Pro Leu325 330 335Phe Val Gly Glu Arg Thr Asn Val Ile Gly Ser Arg Lys Phe Lys Arg340 345 350Leu Ile Glu Glu Glu Lys Tyr Glu Glu Ala Ser Glu Ile Ala Arg Ser355 360 365Gln Val Lys Lys Gly Ala His Val Ile Asp Val Cys Leu Ala Asp Pro370 375 380Asp Arg Asp Glu Met Glu Asp Met Glu Glu Phe Leu Lys Phe Val Ile385 390 395 400Asn Lys Val Lys Val Pro Leu Met Ile Asp Ser Thr Asp Glu Lys Val405 410 415Ile Glu Gln Ala Leu Thr Tyr Ser Gln Gly Lys Ala Ile Ile Asn Ser420 425 430Ile Asn Leu Glu Asp Gly Glu Glu Arg Phe Glu Lys Val Val Pro Leu
435 440 445Val His Lys Tyr Gly Ala Ala Val Val Val Gly Thr Ile Asp Glu Glu450 455 460Gly Met Ala Ile Thr Ala Glu Lys Lys Leu Ala Val Ala Lys Arg Ser465 470 475480Tyr Asp Leu Leu Val Asn Lys Tyr Asn Ile Arg Pro Ser Asp Ile Ile485 490 495Phe Asp Pro Leu Val Phe Pro Val Gly Thr Gly Asp Glu Gln Tyr Ile500 505 510Gly Ser Ala Asn Glu Thr Val Glu Gly Ile Arg Arg Ile Lys Glu Glu515 520 525Leu Pro Glu Cys Leu Thr Ile Leu Gly Val Ser Asn Val Ser Phe Gly530 535 540Leu Pro Pro Val Gly Arg Glu Val Leu Asn Ala Ala Tyr Leu Tyr His545 550 555 560Cys Thr Gln Ala Gly Leu Asp Tyr Ala Ile Val Asn Thr Glu Lys Leu565 570 575Glu Arg Tyr Ala Ser Ile Ser Asp Glu Glu Lys Glu Leu Ser Arg Lys580 585 590Leu Leu Phe Glu Thr Thr Asp Glu Thr Leu Ala Glu Phe Thr Ala Phe595 600 605Tyr Arg Gly Lys Lys Ala Glu Lys Lys Val Glu Thr Ser Asn Leu Thr610 615 620Leu Glu Glu Arg Leu Ala Asn Tyr Ile Val Glu Gly Ser Lys Asp Gly625 630 635 640Lau Thr Glu Asp Leu Asp Lys Ala Leu Ala Lys Tyr Asp Asp Pro Leu645 650 655Asp Ile Ile Asn Gly Pro Leu Met Asn Gly Met Asp Glu Val Gly Arg660 665 670Leu Phe Asn Asn Asn Glu Leu Ile Val Ala Glu Val Leu Gln Ser Ala675 680 685Glu Val Met Lys Ala Ser Val Ala His Leu Glu Pro His Met Glu Lys690 695 700Lys Ala Asp Asp His Gly Lys Gly Lys Ile Ile Leu Ala Thr Val Lys705 710 715 720Gly Asp Val His Asp Ile Gly Lys Asn Leu Val Glu Ile Ile Leu Ser725 730 735Asn Asn Gly Phe Arg Ile Val Asn Leu Gly Ile Lys Val Thr Ser Asn740 745 750Glu Leu Ile Glu Ala Val Ala Arg Glu Asn Pro Asp Ala Ile Gly Leu755 760 765
Ser Gly Leu Leu Val Lys Ser Ala Gln Gln Met Val Leu Thr Ala Gln770 775 780Asp Leu Lys Gln Gln Gln Ile Ser Ile Pro Ile Leu Val Gly Gly Ala785 790 795 800Ala Leu Thr Arg Lys Phe Thr Asn Thr Lys Ile Ala Pro Glu Tyr Asp805 810 815Gly Leu Val Val Tyr Ala Lys Asp Ala Met Asn Gly Leu Glu Leu Ala820 825 830Asn Lys Leu Met Lys Pro Asp Glu Arg Glu Lys Leu Ala Val Ser Leu835 840 845His Glu Ala Lys Glu Gln Ala Asn Ser Arg Thr Gln Met Gly Gly Gly850 855 860Gly Thr Ala Val Ala Val Lys Pro Thr Arg Ser His Val Ser Thr Thr865 870 875 880Val Pro Val Ala Val Pro Pro Asp Val Lys Pro His Ile Leu Arg His885 890 895His Ser Ile Ala His Leu Glu Pro Tyr Ile Asn Met Gln Met Leu Leu900 905 910Gly Arg His Leu Gly Leu Gln Gly Lys Val Ser Arg Leu Leu Ala Glu915 920 925Lys Asp Glu Lys Ala Leu Glu Leu Lys Glu Lys Val Asp Ala Leu Leu930 935 940Thr Arg Val Lys Glu Glu Gln Leu Met Glu Ala His Gly Met Tyr Gln945 950 955 960Phe Phe Pro Ala Gln Ser Asp Gly Asp Asp Ile Val Ile Tyr Asp Gln965 970 975Thr Gly Thr Asn Glu Ile Glu Arg Phe His Phe Pro Arg Gln Asn Lys980 985 990Glu Pro Tyr Leu Cys Leu Ala Asp Phe Leu Arg Pro Val Ser Ser Gly99510001005Glu Met Asp Tyr Val Gly Phe Leu Ala Val Thr Ala Gly Lys Gly Ile101010151020Arg Glu Leu Gly Glu Gln Ala Lys Glu Ala Gly Asp Tyr Leu Phe Ser1025 103010351040His Leu Ile Gln Ala Thr Ala Leu Glu Met Ala Glu Gly Phe Ala Glu104510501055Arg Val His Gln Leu Met Arg Asp Lys Trp Gly Phe Pro Asp Ser Ala106010651070Asp Phe Thr Met Glu Glu Arg Phe Ala Ala Lys Tyr Arg Gly Ile Arg107510801085Val Ser Phe Gly Tyr Pro Ala Cys Pro Asp Leu Asp Asp Gln Ala Lys109010951100
Leu Phe Lys Leu Leu Lys Pro Gly Lys Ile Gly Ile Glu Leu Thr Glu1105 111011151120Gly Phe Met Met Glu Pro Glu Ala Ser Val Thr Ala Met Val Phe Ala112511301135His Pro Glu Ala Arg Tyr Phe Asn Val Leu11401145210132113411212DNA213嗜熱脂肪芽孢桿菌(Bacillus stearothermophilus)220
221CDS222(1)..(3408)223RBE0204440013atg gct aac gtc acc tta gaa cag caa ctg caa aga aaa att ctt gtc48Met Ala Asn Val Thr Leu Glu Gln Gln Leu Gln Arg Lys Ile Leu Val1 5 10 15atc gat ggc gcc atg ggc acg atg atc caa agc gcc aac cta tcg gcc96Ile Asp Gly Ala Met Gly Thr Met Ile Gln Ser Ala Asn Leu Ser Ala20 25 30gcc gac ttt ggc ggc gag gcg tat gaa ggg tgc aac gaa tat ttg acc144Ala Asp Phe Gly Gly Glu Ala Tyr Glu Gly Cys Asn Glu Tyr Leu Thr35 40 45ctc acc gcc ccg cat gtc atc cgc cgc att cat gaa gcg tac cta gaa192Leu Thr Ala Pro His Val Ile Arg Arg Ile His Glu Ala Tyr Leu Glu50 55 60gcc ggt gct gat atc att gaa acg aac acg ttc gga gcg aca cgc atc240Ala Gly Ala Asp Ile Ile Glu Thr Asn Thr Phe Gly Ala Thr Arg Ile65 70 75 80gtg ctt gac gaa tat ggc ctc ggt cat ttg gcg ctt gag ctg aac atc288Val Leu Asp Glu Tyr Gly Leu Gly His Leu Ala Leu Glu Leu Asn Ile85 90 95gaa gcg gcc aaa ctc gcc aaa caa acg gct gag tcg ttc tcc acc ccg336Glu Ala Ala Lys Leu Ala Lys Gln Thr Ala Glu Ser Phe Ser Thr Pro100 105 110gac tgg ccg cgc ttt gtc gcc ggt tcg atg ggg ccg acg acg aaa acg384Asp Trp Pro Arg Phe Val Ala Gly Ser Met Gly Pro Thr Thr Lys Thr115 120 125ttg tcg gtg aca ggc ggc gca acg ttt gaa gaa ctc gtc gcc gcc tac432Leu Ser Val Thr Gly Gly Ala Thr Phe Glu Glu Leu Val Ala Ala Tyr130 135 140gaa gaa caa gcg cgc gga ctg ctc tta gga ggc gtc gac ctt ctc cta480Glu Glu Gln Ala Arg Gly Leu Leu Leu Gly Gly Val Asp Leu Leu Leu145 150 155 160
ctc gag acg tgc caa gat acg ctg aat gtc aaa gcc ggt ttt ctc ggc528Leu Glu Thr Cys Gln Asp Thr Leu Asn Val Lys Ala Gly Phe Leu Gly165 170 175att tcg aag gcg ttt gaa gcg gtc ggc cgc cgc gtg ccg ctc atg att576Ile Ser Lys Ala Phe Glu Ala Val Gly Arg Arg Val Pro Leu Met Ile180 185 190tcc ggc acg atc gaa ccg atg ggc acg acg ctc gcc ggg cag gcg atc624Ser Gly Thr Ile Glu Pro Met Gly Thr Thr Leu Ala Gly Gln Ala Ile195 200 205gat gcg ttt ttc atc tcg gtg cgc cat atg aag ccg atc gcc gtc ggc672Asp Ala Phe Phe Ile Ser Val Arg His Met Lys Pro Ile Ala Val Gly210 215 220tta aac tgc gca acc ggt cog gag ttt atg acc gac cat ttg cgc acg720Leu Asn Cys Ala Thr Gly Pro Glu Phe Met Thr Asp His Leu Arg Thr225 230 235 240ctc gcc tcg ctc gct gac acg gcg gtc agc tgc tac ccg aac gcc ggt768Leu Ala Ser Leu Ala Asp Thr Ala Val Ser Cys Tyr Pro Asn Ala Gly245 250 255ctg ccg gat gag gaa ggc cac tat cat gaa acg ccg aat atg ctg gca816Leu Pro Asp Glu Glu Gly His Tyr His Glu Thr Pro Asn Met Leu Ala260 265 270gag aaa atc cgc cgc ttt gcc gaa aag gga tgg atc aac atc gtc ggc864Glu Lys Ile Arg Arg Phe Ala Glu Lys Gly Trp Ile Asn Ile Val Gly275 280 285ggg tgt tgc ggc acg acg ccg gat cat atc cgc gcc att gct gaa gcg912Gly Cys Cys Gly Thr Thr Pro Asp His Ile Arg Ala Ile Ala Glu Ala290 295 300gtg cgt gat ctc ccg ccg cgg gcg att ccg tct tcg ttt gat gtc cac960Val Arg Asp Leu Pro Pro Arg Ala Ile Pro Ser Ser Phe Asp Val His305 310 315 320gcc gtt tcc ggc atc gag gcg ctc atc tat gat gaa acg atg cgc ccg1008Ala Val Ser Gly Ile Glu Ala Leu Ile Tyr Asp Glu Thr Met Arg Pro325 330 335ctc ttt gtc ggc gag cgg aca aac gtg atc ggc tcg cgc aaa ttc aag1056Leu Phe Val Gly Glu Arg Thr Asn Val Ile Gly Ser Arg Lys Phe Lys340 345 350cgc ctc atc gcc gaa ggg aaa tac gaa gaa gcg gcg gaa atc gcc cgc1104Arg Leu Ile Ala Glu Gly Lys Tyr Glu Glu Ala Ala Glu Ile Ala Arg355 360 365gcc caa gtg aaa aac ggc gcc cat gtc atc gac att tgc ctc gcc gac1152Ala Gln Val Lys Asn Gly Ala His Val Ile Asp Ile Cys Leu Ala Asp370 375 380cca gac cgc gac gaa ctc cat gac atg gag cag ttc gtc cgc gaa gtc1200Pro Asp Arg Asp Glu Leu His Asp Met Glu Gln Phe Val Arg Glu Val385 390 395 400gtg aaa aaa gtg aaa gtg ccg ctt gtc atc gat tcg acc gac gag cgc1248Val Lys Lys Val Lys Val Pro Leu Val Ile Asp Ser Thr Asp Glu Arg
405 4l0 415gtc atc gaa cgc gcc ctt acg tat tcg caa ggg aag gcg atc atc aac1296Val Ile Glu Arg Ala Leu Thr Tyr Ser Gln Gly Lys Ala Ile Ile Asn420 425 430tcg atc aac ctc gaa gat ggc gaa gag cgg ttt gcg aag gtc gtt cct1344Ser Ile Asn Leu Glu Asp Gly Glu Glu Arg Phe Ala Lys Val Val Pro435 440 445ctc ctg cat caa tac ggc gcc gcc gtt gtc gtc ggc acg atc gat gag1392Leu Leu His Gln Tyr Gly Ala Ala Val Val Val Gly Thr Ile Asp Glu450 455 460caa gga atg gcg gtt aca gcc gaa cgg aaa ttg gaa atc gcc ttg cgt1440Gln Gly Met Ala Val Thr Ala Glu Arg Lys Leu Glu Ile Ala Leu Arg465 470 475 480tcg tat gac ttg ctg gtg aac cgc tac ggc gtc ccc gag cgc gac atc1488Ser Tyr Asp Leu Leu Val Asn Arg Tyr Gly Val Pro Glu Arg Asp Ile485 490 495att ttc gac ccg ctc gtc ttc ccg gtc ggc acc ggc gat gag caa tac1536Ile Phe Asp Pro Leu Val Phe Pro Val Gly Thr Gly Asp Glu Gln Tyr500 505 510atc ggc gcg gcg aaa gaa acc att gag ggc atc cgc ctc att aaa gag1584Ile Gly Ala Ala Lys Glu Thr Ile Glu Gly Ile Arg Leu Ile Lys Glu515 520 525cgg ctg cct cat tgc ttg acg atg ctt ggc atc agc aac gtc tcg ttc1632Arg Leu Pro His Cys Leu Thr Met Leu Gly Ile Ser Asn Val Ser Phe530 535 540ggc ttg ccg ccg gcc gga cgc gag gtg ctc aac tcc gtc ttt ttg tac1680Gly Leu Pro Pro Ala Gly Arg Glu Val Leu Asn Ser Val Phe Leu Tyr545 550 555 560cat tgc acg caa gcc ggg ctc gat tac gcc atc gtc aac acc gag aaa1728His Cys Thr Gln Ala Gly Leu Asp Tyr Ala Ile Val Asn Thr Glu Lys565 570 575ttg gag cgg ttc gcc tcg att ccg gaa gag gaa gtg cga atg gct gag1776Leu Glu Arg Phe Ala Ser Ile Pro Glu Glu Glu Val Arg Met Ala Glu580 585 590gca ctt ctt ttt gac aca aac gac gaa aca tta aac gcc ttt atc gaa1824Ala Leu Leu Phe Asp Thr Asn Asp Glu Thr Leu Asn Ala Phe Ile Glu595 600 605ttt tac cga agc aaa atc acc gcc gcc aaa ccg gcg cag acg aac ttg1872Phe Tyr Arg Ser Lys Ile Thr Ala Ala Lys Pro Ala Gln Thr Asn Leu610 615 620agc ttg gaa gag cgg ctc gcc cgc tac gtt att gaa ggg tcg aaa gac1920Ser Leu Glu Glu Arg Leu Ala Arg Tyr Val Ile Glu Gly Ser Lys Asp625 630 635 640ggg ctc att ctc gat ttg gaa aag gcg ctt gag acc tac tcc gat ccg1968Gly Leu Ile Leu Asp Leu Glu Lys Ala Leu Glu Thr Tyr Ser Asp Pro645 650 655
ctg tcc atc atc aac ggt ccg ctc atg gcc ggc atg gat gaa gtc ggg2016Leu Ser Ile Ile Asn Gly Pro Leu Met Ala Gly Met Asp Glu Val Gly660 665 670cgg ctg ttc aac aac aac cag ctc atc gtc gct gaa gta ttg caa agc2064Arg Leu Phe Asn Asn Asn Gln Leu Ile Val Ala Glu Val Leu Gln Ser675 680 685gcg gaa gtg atg aaa gca gcg gtc gcc ttt tta gag ctg tat atg gaa2112Ala Glu Val Met Lys Ala Ala Val Ala Phe Leu Glu Leu Tyr Met Glu690 695 700aag aaa gaa gga agc aca aaa gga aaa gtc att ctc gcc acc gtc aaa2160Lys Lys Glu Gly Ser Thr Lys Gly Lys Val Ile Leu Ala Thr Val Lys705 710 715 720ggc gat gtg cat gac atc ggc aaa aac ttg gtc gac atc att tta agc2208Gly Asp Val His Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Ser725 730 735aac aac ggc tac gag gtg atc gac ctc ggc att aaa gtc gct ccg cag2256Asn Asn Gly Tyr Glu Val Ile Asp Leu Gly Ile Lys Val Ala Pro Gln740 745 750caa ctc att gaa gcg gtg cgc gaa cat cag ccg gac atc atc ggg ttg2304Gln Leu Ile Glu Ala Val Arg Glu His Gln Pro Asp Ile Ile Gly Leu755 760 765tcg ggc ttg ctt gtg aaa tcg gct caa cag atg gtc gtc acc gcc caa2352Ser Gly Leu Leu Val Lys Ser Ala Gln Gln Met Val Val Thr Ala Gln770 775 780gac ttg cgc caa gcg ggc atc tcg acc ccg att tta gtc ggc ggc gcc2400Asp Leu Arg Gln Ala Gly Ile Ser Thr Pro Ile Leu Val Gly Gly Ala785 790 795 800gcc ttg acg cgc aaa ttt acg gaa aac aaa atc gcg ccc gag tac gac2448Ala Leu Thr Arg Lys Phe Thr Glu Asn Lys Ile Ala Pro Glu Tyr Asp805 810 815ggc gtt gtc ttg tac gcg aaa gac gcc atg gac ggg ctc gcc ctt gcc2496Gly Val Val Leu Tyr Ala Lys Asp Ala Met Asp Gly Leu Ala Leu Ala820 825 830aac caa atc cag cag ggc gag att gac tac aag aaa aaa gaa acg gcc2544Asn Gln Ile Gln Gln Gly Glu Ile Asp Tyr Lys Lys Lys Glu Thr Ala835 840 845gaa agc gag cca acg cgg caa acg acg gtg gtc aca gcg gtc aaa tcg2592Glu Ser Glu Pro Thr Arg Gln Thr Thr Val Val Thr Ala Val Lys Ser850 855 860acc gtc tcg acc gac gtt ccc gtc tac atc ccg gcc gat ctc gag cgc2640Thr Val Ser Thr Asp Val Pro Val Tyr Ile Pro Ala Asp Leu Glu Arg865 870 875 880cac gcg ctg cga aat gtg ccg ctt gac cac att ttg ccg tac gtc aac2688His Ala Leu Arg Asn Val Pro Leu Asp His Ile Leu Pro Tyr Val Asn885 890895tgg caa atg gtg ctc ggc cac cac ctc ggc ttg aaa gga aaa gtg aaa2736Trp Gln Met Val Leu Gly His His Leu Gly Leu Lys Gly Lys Val Lys
900 905 910cgg ctg ctt gaa gag aaa gac gaa aaa gcg ttg gcg tta aaa gcg gtc2784Arg Leu Leu Glu Glu Lys Asp Glu Lys Ala Leu Ala Leu Lys Ala Val915 920 925gtc gac gaa ctg ctc gcc gaa gcg aaa gag cgc cgc tgg att cag ccc2832Val Asp Glu Leu Leu Ala Glu Ala Lys Glu Arg Arg Trp Ile Gln Pro930 935 940gcc ggc gtc tac cgc ttc ttc ccg gcg caa agc gac ggc aac cgg gtt2880Ala Gly Val Tyr Arg Phe Phe Pro Ala Gln Ser Asp Gly Asn Arg Val945 950 955 960tac att tac gat ccg act gac ggc aaa aca gtg ctc gag atg ttc gac2928Tyr Ile Tyr Asp Pro Thr Asp Gly Lys Thr Val Leu Glu Met Phe Asp965 970 975ttt ccg cgc caa ccg cgg gcg ccg tat ctt tgc ctc gcc gat tat ttg2976Phe Pro Arg Gln Pro Arg Ala Pro Tyr Leu Cys Leu Ala Asp Tyr Leu980 985 990aaa tcg aaa gaa agc ggc gaa atg gat tac gtc ggt ttg ttc gcc gtc3024Lys Ser Lys Glu Ser Gly Glu Met Asp Tyr Val Gly Leu Phe Ala Val99510001005acc gct ggg cat ggc gtc cgc gaa ctc gcc cag cgc tgg aag gaa gaa3072Thr Ala Gly His Gly Val Arg Glu Leu Ala Gln Arg Trp Lys Glu Glu101010151020ggc gaa ttt ttg aaa agc cat gcc atc caa gcg ttg gcg ctc gag att3120Gly Glu Phe Leu Lys Ser His Ala Ile Gln Ala Leu Ala Leu Glu Ile1025 103010351040gcc gaa ggg ttc gcc gaa cga atc cat caa att atg cgc gac cgc tgg3168Ala Glu Gly Phe Ala Glu Arg Ile His Gln Ile Met Arg Asp Arg Trp104510501055ggc ttc ccg gac gac ccg gat ttc acg atg gaa gag cgc ttc gcc gcc3216Gly Phe Pro Asp Asp Pro Asp Phe Thr Met Glu Glu Arg Phe Ala Ala106010651070aaa tac cag ggc cag cgc tac tcg ttc ggc tac ccg gcc tgt ccg aac3264Lys Tyr Gln Gly Gln Arg Tyr Ser Phe Gly Tyr Pro Ala Cys Pro Asn1075 1080 1085ttg gaa gac cag gag aaa ctg ttc cgt ctg ctt cat cca gaa gac atc3312Leu Glu Asp Gln Glu Lys Leu Phe Arg Leu Leu His Pro Glu Asp Ile109010951100ggc atc cgt ctc acc gac ggc tat atg atg gaa ccc gaa gca tcg gtt3360Gly Ile Arg Leu Thr Asp Gly Tyr Met Met Glu Pro Glu Ala Ser Val1105 111011151120tcg gcg atc gtc ttc gcc cat ccg gaa gcg cgg tat ttc aat gtg tta3408Ser Ala Ile Val Phe Ala His Pro Glu Ala Arg Tyr Phe Asn Val Leu112511301135taa341121014
2111136212PRT213嗜熱脂肪芽孢桿菌40014Met Ala Asn Val Thr Leu Glu Gln Gln Leu Gln Arg Lys Ile Leu Val1 5 10 15Ile Asp Gly Ala Met Gly Thr Met Ile Gln Ser Ala Asn Leu Ser Ala20 25 30Ala Asp Phe Gly Gly Glu Ala Tyr Glu Gly Cys Asn Glu Tyr Leu Thr35 40 45Leu Thr Ala Pro His Val Ile Arg Arg Ile His Glu Ala Tyr Leu Glu50 55 60Ala Gly Ala Asp Ile Ile Glu Thr Asn Thr Phe Gly Ala Thr Arg Ile65 70 75 80Val Leu Asp Glu Tyr Gly Leu Gly His Leu Ala Leu Glu Leu Asn Ile85 90 95Glu Ala Ala Lys Leu Ala Lys Gln Thr Ala Glu Ser Phe Ser Thr Pro100 105 110Asp Trp Pro Arg Phe Val Ala Gly Ser Met Gly Pro Thr Thr Lys Thr115 120 125Leu Ser Val Thr Gly Gly Ala Thr Phe Glu Glu Leu Val Ala Ala Tyr130 135 140Glu Glu Gln Ala Arg Gly Leu Leu Leu Gly Gly Val Asp Leu Leu Leu145 150 155 160Leu Glu Thr Cys Gln Asp Thr Leu Asn Val Lys Ala Gly Phe Leu Gly165 170 175Ile Ser Lys Ala Phe Glu Ala Val Gly Arg Arg Val Pro Leu Met Ile180 185 190Ser Gly Thr Ile Glu Pro Met Gly Thr Thr Leu Ala Gly Gln Ala Ile195 200 205Asp Ala Phe Phe Ile Ser Val Arg His Met Lys Pro Ile Ala Val Gly210 215 220Leu Asn Cys Ala Thr Gly Pro Glu Phe Met Thr Asp His Leu Arg Thr225 230 235 240Leu Ala Ser Leu Ala Asp Thr Ala Val Ser Cys Tyr Pro Asn Ala Gly245 250 255Leu Pro Asp Glu Glu Gly His Tyr His Glu Thr Pro Asn Met Leu Ala260 265 270Glu Lys Ile Arg Arg Phe Ala Glu Lys Gly Trp Ile Asn Ile Val Gly275 280 285Gly Cys Cys Gly Thr Thr Pro Asp His Ile Arg Ala Ile Ala Glu Ala290 295 300
Val Arg Asp Leu Pro Pro Arg Ala Ile Pro Ser Ser Phe Asp Val His305 310 315 320Ala Val Ser Gly Ile Glu Ala Leu Ile Tyr Asp Glu Thr Met Arg Pro325 330 335Leu Phe Val Gly Glu Arg Thr Asn Val Ile Gly Ser Arg Lys Phe Lys340 345 350Arg Leu Ile Ala Glu Gly Lys Tyr Glu Glu Ala Ala Glu Ile Ala Arg355 360 365Ala Gln Val Lys Asn Gly Ala His Val Ile Asp Ile Cys Leu Ala Asp370 375 380Pro Asp Arg Asp Glu Leu His Asp Met Glu Gln Phe Val Arg Glu Val385 390 395 400Val Lys Lys Val Lys Val Pro Leu Val Ile Asp Ser Thr Asp Glu Arg405 410 415Val Ile Glu Arg Ala Leu Thr Tyr Ser Gln Gly Lys Ala Ile Ile Asn420 425 430Ser Ile Asn Leu Glu Asp Gly Glu Glu Arg Phe Ala Lys Val Val Pro435 440 445Leu Leu His Gln Tyr Gly Ala Ala Val Val Val Gly Thr Ile Asp Glu450 455 460Gln Gly Met Ala Val Thr Ala Glu Arg Lys Leu Glu Ile Ala Leu Arg465 470 475 480Ser Tyr Asp Leu Leu Val Asn Arg Tyr Gly Val Pro Glu Arg Asp Ile485 490 495Ile Phe Asp Pro Leu Val Phe Pro Val Gly Thr Gly Asp Glu Gln Tyr500 505 510Ile Gly Ala Ala Lys Glu Thr Ile Glu Gly Ile Arg Leu Ile Lys Glu515 520 525Arg Leu Pro His Cys Leu Thr Met Leu Gly Ile Ser Asn Val Ser Phe530 535 540Gly Leu Pro Pro Ala Gly Arg Glu Val Leu Asn Ser Val Phe Leu Tyr545 550 555 560His Cys Thr Gln Ala Gly Leu Asp Tyr Ala Ile Val Asn Thr Glu Lys565 570 575Leu Glu Arg Phe Ala Ser Ile Pro Glu Glu Glu Val Arg Met Ala Glu580 585 590Ala Leu Leu Phe Asp Thr Asn Asp Glu Thr Leu Asn Ala Phe Ile Glu595 600 605Phe Tyr Arg Ser Lys Ile Thr Ala Ala Lys Pro Ala Gln Thr Asn Leu610 615 620Ser Leu Glu Glu Arg Leu Ala Arg Tyr Val Ile Glu Gly Ser Lys Asp625 630 635 640
Gly Leu Ile Leu Asp Leu Glu Lys Ala Leu Glu Thr Tyr Ser Asp Pro645 650 655Leu Ser Ile Ile Asn Gly Pro Leu Met Ala Gly Met Asp Glu Val Gly660 665 670Arg Leu Phe Asn Asn Asn Gln Leu Ile Val Ala Glu Val Leu Gln Ser675 680 685Ala Glu Val Met Lys Ala Ala Val Ala Phe Leu Glu Leu Tyr Met Glu690 695 700Lys Lys Glu Gly Ser Thr Lys Gly Lys Val Ile Leu Ala Thr Val Lys705 710 715 720Gly Asp Val His Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Ser725 730 735Asn Asn Gly Tyr Glu Val Ile Asp Leu Gly Ile Lys Val Ala Pro Gln740 745 750Gln Leu Ile Glu Ala Val Arg Glu His Gln Pro Asp Ile Ile Gly Leu755 760 765Ser Gly Leu Leu Val Lys Ser Ala Gln Gln Met Val Val Thr Ala Gln770 775 780Asp Leu Arg Gln Ala Gly Ile Ser Thr Pro Ile Leu Val Gly Gly Ala785 790 795 800Ala Leu Thr Arg Lys Phe Thr Glu Asn Lys Ile Ala Pro Glu Tyr Asp805 810 815Gly Val Val Leu Tyr Ala Lys Asp Ala Met Asp Gly Leu Ala Leu Ala820 825 830Asn Gln Ile Gln Gln Gly Glu Ile Asp Tyr Lys Lys Lys Glu Thr Ala835 840 845Glu Ser Glu Pro Thr Arg Gln Thr Thr Val Val Thr Ala Val Lys Ser850 855 860Thr Val Ser Thr Asp Val Pro Val Tyr Ile Pro Ala Asp Leu Glu Arg865 870 875 880His Ala Leu Arg Asn Val Pro Leu Asp His Ile Leu Pro Tyr Val Asn885 890 895Trp Gln Met Val Leu Gly His His Leu Gly Leu Lys Gly Lys Val Lys900 905 910Arg Leu Leu Glu Glu Lys Asp Glu Lys Ala Leu Ala Leu Lys Ala Val915 920 925Val Asp Glu Leu Leu Ala Glu Ala Lys Glu Arg Arg Trp Ile Gln Pro930 935 940Ala Gly Val Tyr Arg Phe Phe Pro Ala Gln Ser Asp Gly Asn Arg Val945 950 955 960Tyr Ile Tyr Asp Pro Thr Asp Gly Lys Thr Val Leu Glu Met Phe Asp
965 970 975Phe Pro Arg Gln Pro Arg Ala Pro Tyr Leu Cys Leu Ala Asp Tyr Leu980 985 990Lys Ser Lys Glu Ser Gly Glu Met Asp Tyr Val Gly Leu Phe Ala Val99510001005Thr Ala Gly His Gly Val Arg Glu Leu Ala Gln Arg Trp Lys Glu Glu101010151020Gly Glu Phe Leu Lys Ser His Ala Ile Gln Ala Leu Ala Leu Glu Ile1025 103010351040Ala Glu Gly Phe Ala Glu Arg Ile His Gln Ile Met Arg Asp Arg Trp104510501055Gly Phe Pro Asp Asp Pro Asp Phe Thr Met Glu Glu Arg Phe Ala Ala106010651070Lys Tyr Gln Gly Gln Arg Tyr Ser Phe Gly Tyr Pro Ala Cys Pro Asn107510801085Leu Glu Asp Gln Glu Lys Leu Phe Arg Leu Leu His Pro Glu Asp Ile109010951100Gly Ile Arg Leu Thr Asp Gly Tyr Met Met Glu Pro Glu Ala Ser Val1105 111011151120Ser Ala Ile Val Phe Ala His Pro Glu Ala Arg Tyr Phe Asn Val Leu112511301135210152113681212DNA213霍亂弧菌(vibrio cholerae)220
221CDS222(1)..(3678)223RVC0426540015gtg gga aaa gaa gta aga caa caa ctc gaa cag caa ttg aaa caa cgt48Val Gly Lys Glu Val Arg Gln Gln Leu Glu Gln Gln Leu Lys Gln Arg1 5 10 15atc cta ctg att gat ggt ggt atg ggt acc atg att cag agt tat aag96Ile Leu Leu Ile Asp Gly Gly Met Gly Thr Met Ile Gln Ser Tyr Lys20 25 30tta caa gag gaa gac tat cgc ggt gca cga ttt gtc gat tgg cac tgt144Leu Gln Glu Glu Asp Tyr Arg Gly Ala Arg Phe Val Asp Trp His Cys35 40 45gat ttg aaa gga aat aac gac ctc tta gtg ctt act cag ccg caa att192Asp Leu Lys Gly Asn Asn Asp Leu Leu Val Leu Thr Gln Pro Gln Ile50 55 60
att aaa gag att cac tcc gct tac ctt gaa gcg ggg gcg gat att ctt240Ile Lys Glu Ile His Ser Ala Tyr Leu Glu Ala Gly Ala Asp Ile Leu65 70 75 80gag acc aac acc ttt aac tca acc acg att gcc atg gca gac tat gac288Glu Thr Asn Thr Phe Asn Ser Thr Thr Ile Ala Met Ala Asp Tyr Asp85 90 95atg caa tcg ctc agt gct gaa att aac ttt gcc gcg gct aag ctt gca336Met Gln Ser Leu Ser Ala Glu Ile Asn Phe Ala Ala Ala Lys Leu Ala100 105 110cgt gaa gtc gcg gat gag tgg acg gct aaa gat cca agt cgg cca cgc384Arg Glu Val Ala Asp Glu Trp Thr Ala Lys Asp Pro Ser Arg Pro Arg115 120 125tat gtg gct ggt gtg ctt ggg cca acc aac cgt act tgc tct att tcg432Tyr Val Ala Gly Val Leu Gly Pro Thr Asn Arg Thr Cys Ser Ile Ser130 135 140cca gat gtg aac gat cca gga ttt cgt aac gtc act ttt gat ggg ctt480Pro Asp Val Asn Asp Pro Gly Phe Arg Asn Val Thr Phe Asp Gly Leu145 150 155 160gtt gaa gcc tat tcc gaa tcg acg cgc gct ttg atc aaa ggt ggc agc528Val Glu Ala Tyr Ser Glu Ser Thr Arg Ala Leu Ile Lys Gly Gly Ser165 170 175gat ctg atc ctc att gaa acc atc ttc gat aca ctt aac gcc aaa gcc576Asp Leu Ile Leu Ile Glu Thr Ile Phe Asp Thr Leu Asn Ala Lys Ala180 185 190tgt gcg ttt gcg gtc gat agc gta ttt gaa gag ctg ggc atc agc tta624Cys Ala Phe Ala Val Asp Ser Val Phe Glu Glu Leu Gly Ile Ser Leu195 200 205cct gtg atg att tcc ggc acg att acc gat gcc tct ggg cga act ctg672Pro Val Met Ile Ser Gly Thr Ile Thr Asp Ala Ser Gly Arg Thr Leu210 215 220tca gga cag aca acg gaa gct ttc tac aac gcc ttg cgt cat gta cgg720Ser Gly Gln Thr Thr Glu Ala Phe Tyr Asn Ala Leu Arg His Val Arg22