專利名稱：：阿嗪霉素的生物合成基因簇的制作方法
技術領域：
：本發(fā)明屬于微生物基因資源和基因工程領域，具體涉及抗腫瘤抗生素阿嗪霉素(AzinomycinB)的生物合成基因簇的克隆、分析、功能研究及其應用。技術背景阿嗪霉素(Azinomycins)是一種能夠誘導DNA鏈間交聯(lián)的天然抗生物，由鏈霉菌S^e;tow少ceyM/zac/n'ra/NRRL2485所產(chǎn)生，結構十分獨特。它含有兩個活性很強的反應中心氮雜雙環(huán)(l-azabicyclo[3丄0]hexane)的脫水氨基酸殘基和環(huán)氧三元環(huán)。其中的氮環(huán)結構僅在天然化合物Ficellomycin中發(fā)現(xiàn)[J(1989)42,357-360]。作為一類小分子化合物，阿嗪霉素可以結合在DNA的大溝，選擇性地作用于序列5，-d(PuNpy)-3，[/爿附.C/ze附.Soc.(2002)124，13008-13017]，通過氮雜環(huán)上的C-10原子對DNA嘌呤氮的親電子攻擊形成烷基化的單聚絡合物，進而啟動環(huán)氧雜環(huán)系統(tǒng)的C-21原子對DNA互補鏈上鳥嘌呤G的N-7原子進行親電子攻擊，最終使DNA雙鏈發(fā)生鏈間交聯(lián)。阿嗪霉素具有良好的抗腫瘤活性，組分A和組分B在體外對于腫瘤細胞株L5178Y的1<35()抑制率分別為0.07和O.llug/ml[/JW^'oA(1986)39,1527-1532]。在體內(nèi)一系列實體瘤的生長抑制方面，B組分要優(yōu)于A組分，其治療效果與臨床上使用的ISC抗生物絲裂霉素C相當，但劑量更低(如能使腫瘤小鼠45天的存活率達到57%和生命期延長200%左右的阿嗪霉素B和絲裂霉素C的用量分別為32ug/kg/day和lmg/kg/day)[/爿"幼/W.(1987)40,60-65]。由于阿嗪菌素所誘導細胞DNA的ISC反應和烷基化反應受pH影響很大，在pH較低的反應環(huán)境中發(fā)生較快；而腫瘤細胞相對于正常細胞，通常由于產(chǎn)生過多的乳酸導致pH較低，這就使阿嗪霉素在腫瘤治療過程中具有一定的選擇性[J:歷oc/ew.(1977)55,630-635]。但是，阿嗪霉素化學結構的不穩(wěn)定嚴重限制了它們在臨床上的應用[j.Antibiot.(1986)39,1527-1532〗。鑒于阿嗪霉素良好的生物活性，近40種通過化學合成獲得的結構類似物被用于大量腫瘤細胞株的活性測試，包括部分藥物抗性細胞株[7^ra/ze^W7(2001)57,4467-4488;歷oorg.MM.C&附.(2005)15,653-656]。研究發(fā)現(xiàn)，只保留環(huán)氧系統(tǒng)的結構類似物仍然具有很高的細胞毒性(只發(fā)生DNA的烷基化，不引起ISC效應)，而只有氮雜雙環(huán)的結構類似物活性急劇下降，表明氮雜雙環(huán)的化學結構雖然對于DNA的烷基化十分重要，但并非阿嗪霉素生物活性所必須[5wwg.MedCte附.丄"A(2000)10，239-241;歷octe附/;y^y(2000)39,14968-14975;C/ew./脫￡d(2000)39,3467-3470]。同時，阿嗪霉素的抗腫瘤活性與環(huán)氧系統(tǒng)兩側的萘甲酸和氨基邊鏈取代基的微小變化都會引起活性的明顯變化，顯示兩者對活性中心環(huán)氧系統(tǒng)發(fā)生的效應有增強作用[Org.Lett.(2002)4,3545-3548]。構效關系研究顯示，誘導ISC的發(fā)生并非阿嗪霉素及其類似物產(chǎn)生細胞毒性的唯一途徑，可能還存在由環(huán)氧系統(tǒng)所介導的其它細胞效應方式。一系列具有較好生物活性的類似物的獲得，表明阿嗪霉素是一類很好的可用于新藥開發(fā)的先導化合物。阿嗪霉素的藥效基團和其它化學結構的重新組合，可能創(chuàng)造出更有臨床應用價值的新型藥物。但是，由于過于集中的化學官能團和結構的不穩(wěn)定，阿嗪菌素的獲得是合成化學的巨大挑戰(zhàn)。自1986年首次發(fā)現(xiàn)以來，2001年才首次完成了阿嗪霉素A的化學合成[7^ra/^Aow(2001)57，4467-4488;/^w.C&m.Soc.(2001)40,1736-1739]。通過化學合成所獲得的結構類似物即使在活性和結構穩(wěn)定性方面的性質(zhì)得到改善，也可能因為合成的復雜性而使實際生產(chǎn)成本過高。我們以微生物來源的阿嗪霉素為目標分子，從克隆生物合成基因簇出發(fā)，采用微生物學、分子生物學、生物化學及有機化學相結合的方法研究其生物合成，通過對其生物合成機制的研究揭示包括氮雜雙環(huán)在內(nèi)的獨特化學結構形成的酶學機理，在此基礎上運用代謝工程的原理，合理修飾阿嗪霉素的生物合成途徑，探索結構穩(wěn)定、活性更好、并能通過微生物發(fā)酵大量生產(chǎn)的新型藥物。
發(fā)明內(nèi)容本發(fā)明涉及一種由鏈霉菌S加ptowjcMsa/wc/z/ra/NRRL2485產(chǎn)生的具有抗腫瘤活性的抗生素一阿嗪霉素(Azinomycin)的生物合成基因簇的克隆、測序、分析、功能研究及其應用。本發(fā)明中整個基因簇共包含34個基因的核苷酸序列或互補序列(序列1),其中1個(azW)用于編碼重復使用的I型聚酮合成酶(PKS)，共包含5個功能域，負責萘甲酸基團的生物合成；2個基因(m^和mz'52)編碼的氧化酶和甲基轉移酶負責對萘環(huán)進行修飾；8個基因(az"7，flzM2，azMJ，az"4，azM5，flzM6，m"7，負責合成非核糖體聚肽骨架合成及修飾；11個基因(m/C7，"a'C2，az/C，az/CV，flz/C5，az/C<5，az/C7，az/CS，az/CP，az/C70，az/C7i)負責非天然氨基酸結構單元的合成酶；還包括1個抗性基因(az/五)、3個后修飾酶基因(azZD/，az/D2，以及8個功能不確定的基因(azz7/7，az///2，azZ/仏ok/F,az/G，az/f/7，az/L/2，az/^Z3)。本發(fā)明還提供了一個編碼包含AL，KR，PCP非核糖體聚肽合成酶結構域的的苷酸序列，由序列2中的氨基酸序列組成，命名為aziA3，其基因的核苷酸序列位于序列1中第2596-6387堿基處。本發(fā)明還提供了一個硫酯酶的核苷酸序列，由序列3中的氨基酸序列組成，命名為aziA6，其基因的核苷酸序列位于序列1中第6384-7205堿基處。本發(fā)明還提供了一個編碼包含C，A，PCP非核糖體肽合成酶結構域的核苷酸序列，由序列4中的氨基酸序列組成，命名為aziA4，其基因的核苷酸序列位于序列1中第7202-10513堿基處。本發(fā)明還提供了一個編碼硫酯酶的核苷酸序列，由序列5中的氨基酸序列組成，命名為aziA7，其基因的核苷酸序列位于序列1中第10796-11548堿基處。本發(fā)明還提供了一個編碼O-甲基轉移酶的核苷酸序列，由序列6中的氨基酸序列組成，命名為aziB2，其基因的核苷酸序列位于序列1中第11548-12585堿基處o本發(fā)明還提供了一個編碼細胞色素P450氧化酶的核苷酸序列，由序列7中的氨基酸序列組成，命名為aziBl，其基因的核苷酸序列位于序列1中第12587-13792堿基處。本發(fā)明還提供了一個編碼包含AL，PCP非核糖體肽合成酶結構域的核苷酸序列，由序列8中的氨基酸序列組成，命名為aziAl，其基因的核苷酸序列位于序列1中第13873-15756堿基處。本發(fā)明還提供了一個編碼轉運蛋白的核苷酸序列，由序列9中的氨基酸序列組成，命名為aziE，其基因的核苷酸序列位于序列1中第15839-17239堿基處。本發(fā)明還提供了一個編碼包含C，PCP，C非核糖體肽合成酶結構域的核苷酸序列，由序列10中的氨基酸序列組成，命名為aziA2，其基因的核苷酸序列位于序列1中第17541-20888堿基處。本發(fā)明還提供了一個編碼硫酸腺苷酰轉移酶亞單位1的核苷酸序列，由序列11中的氨基酸序列組成，命名為aziH2，其基因的核苷酸序列位于序列1中第20878-22185堿基處。本發(fā)明還提供了一個編碼硫酸腺苷酰轉移酶亞單位2的核苷酸序列，由序列12中的氨基酸序列組成，命名為aziHl，其基因的核苷酸序列位于序列1中第22185-22997堿基處。本發(fā)明還提供了一個編碼腺苷酰硫酸激酶的核苷酸序列，由序列13中的氨基酸序列組成，命名為aziH3，其基因的核苷酸序列位于序列1中第23101-23658堿基處。本發(fā)明還提供了一個編碼硫酯酶的核苷酸序列，由序列14中的氨基酸序列組成，命名為aziG，其基因的核苷酸序列位于序列1中第23726-24127堿基處。本發(fā)明還提供了一個編碼MbtH類似蛋白的核苷酸序列，由序列15中的氨基酸序列組成，命名為aziF，其基因的核苷酸序列位于序列1中第24328-24516堿基處。本發(fā)明還提供了一個編碼O-?；D移酶的核苷酸序列，由序列16中的氨基酸序列組成，命名為aziDl，其基因的核苷酸序列位于序列1中第24547-25758堿基處o本發(fā)明還提供了一個編碼PCP/ACP的核苷酸序列，由序列17中的氨基酸序列組成，命名為aziCll，其基因的核苷酸序列位于序列1中第25755-26018堿基處。本發(fā)明還提供了一個編碼酮基轉移酶C-端亞單位的核苷酸序列，由序列18中的氨基酸序列組成，命名為aziC6，其基因的核苷酸序列位于序列1中第26021-27043堿基處。本發(fā)明還提供了一個編碼酮基轉移酶N-端亞單位的核苷酸序列，由序列19中的氨基酸序列組成，命名為aziC5，其基因的核苷酸序列位于序列1中第27040-27975堿基處。本發(fā)明還提供了一個編碼氨基轉移酶的核苷酸序列，由序列20中的氨基酸序列組成，命名為aziC7，其基因的核苷酸序列位于序列1中第27972-29294堿基處。本發(fā)明還提供了一個編碼N-己酰-Y-谷氨酰磷酸還原酶的核苷酸序列，由序列21中的氨基酸序列組成，命名為aziC4，其基因的核苷酸序列位于序列1中第29735-30658堿基處。本發(fā)明還提供了一個編碼N-已酰谷氨酸激酶的核苷酸序列，由序列22中的氨基酸序列組成，命名為aziC3，其基因的核苷酸序列位于序列1中第30737-31615堿基處。本發(fā)明還提供了一個編碼N-已酰-鳥氨酸/N-已酰-賴氨酸脫?；傅暮塑账嵝蛄?，由序列23中的氨基酸序列組成，命名為aziC10，其基因的核苷酸序列位于序列1中第31608-32738堿基處。本發(fā)明還提供了一個編碼已酰-CoA脫氫酶/還原酶的核苷酸序列，由序列24中的氨基酸序列組成，命名為aziC8，其基因的核苷酸序列位于序列1中第32741-33559堿基處。本發(fā)明還提供了一個編碼包含C，A，PCP，RE非核糖體肽合成酶結構域的核苷酸序列，由序列25中的氨基酸序列組成，命名為aziA5，其基因的核苷酸序列位于序列1中第33638-38261堿基處。本發(fā)明還提供了一個編碼包含KS，AT，DH，KR，ACP結構域的重復使用的I型聚酮合成酶的核苷酸序列，由序列26中的氨基酸序列組成，命名為aziB，其基因的核苷酸序列位于序列1中第38515-43854堿基處。本發(fā)明還提供了一個編碼未知蛋白的核苷酸序列，由序列27中的氨基酸序列組成，命名為aziUl，其基因的核苷酸序列位于序列1中第44054-44827堿基處。本發(fā)明還提供了一個編碼未知蛋白的核苷酸序列，由序列28中的氨基酸序列組成，命名為aziU2，其基因的核苷酸序列位于序列1中第44838-45503堿基處。本發(fā)明還提供了一個編碼未知蛋白的核苷酸序列，由序列29中的氨基酸序列組成，命名為aziU3，其基因的核苷酸序列位于序列1中第45500-46513堿基處。本發(fā)明還提供了一個編碼賴氨酸生物合成酶的核苷酸序列，由序列30中的氨基酸序列組成，命名為aziC2，其基因的核苷酸序列位于序列1中第46593-47456堿基處。本發(fā)明還提供了一個編碼分枝氨基酸氨基轉移酶的核苷酸序列，由序列31中的氨基酸序列組成，命名為aziCl，其基因的核苷酸序列位于序列1中第47453-48184堿基處。本發(fā)明還提供了一個編碼細胞色素P450氧化酶的核苷酸序列，由序列32中的氨基酸序列組成，命名為aziC9，其基因的核苷酸序列位于序列1中第48254-49513堿基處。本發(fā)明還提供了一個編碼硫酯酶的核苷酸序列，由序列33中的氨基酸序列組成，命名為aziA8，其基因的核苷酸序列位于序列1中第49525-50274堿基處。本發(fā)明還提供了一個編碼已酰-CoA脫氫酶的核苷酸序列，由序列34中的氨基酸序列組成，命名為aziD3，其基因的核苷酸序列位于序列1中第50308-51456堿基處。本發(fā)明還提供了一個編碼分枝氨基酸氨基轉移酶的核苷酸序列，由序列35中的氨基酸序列組成，命名為aziC2，其基因的核苷酸序列位于序列1中第51462-52463堿基處。序列1的互補序列可根據(jù)DNA堿基互補原則隨時得到。序列1的核苷酸序列或部分核苷酸序列可以通過聚合酶鏈式反應(PCR)或用合適的限制性內(nèi)切酶酶切相應的DNA或使用其他合適的技術得到。本發(fā)明提供了得到至少包含部分序列1中DNA序列的重組DNA質(zhì)粒的途徑。本發(fā)明還提供了產(chǎn)生阿嗪霉素生物合成基因被中斷或加倍的微生物體的途徑，至少其中之一的基因包含有序列1中的核苷酸序列。本發(fā)明所提供的核苷酸序列或部分核苷酸序列，可利用聚合酶鏈式反應(PCR)的方法或包含本發(fā)明序列的DNA作為探針以Southern雜交等方法從其他生物體中得到與阿嗪霉素生物合成基因相似的基因。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的克隆DNA可用于從鏈霉菌5^p/ow_yc"ra/zacWra/NRRL2485基因組文庫中定位更多的文庫質(zhì)粒。這些文庫質(zhì)粒至少包含本發(fā)明中的部分序列，也包含有Sfre/^ow^c"M/wc/n'ra/NRRL2485基因組中以前鄰近區(qū)域未克隆的DNA。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列可以被修飾或突變。這些途徑包括插入、置換或缺失，聚合酶鏈式反應，錯誤介導聚合酶鏈式反應，位點特異性突變，不同序列的重新連接，序列的不同部分或與其他來源的同源序列進行定向進化(DNAshuffling)，或通過紫外線或化學試劑誘變等。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的克隆基因可以通過合適的表達體系在外源宿主中表達以得到相應的酶或其他更高的生物活性或廠里。込蘭Tiy爾怕土'巴:rti'低每困、iK平fbi困、人鵬rr困、才rarr困、酔可、植物和動物等。本發(fā)明所提供的氨基酸序列可以用來分離所需要的蛋白并可用于抗體的制備。包含本發(fā)明所提供的氨基酸序列或至少部分序列的多肽可能在去除或替代某些氨基酸之后仍有生物活性甚至有新的生物學活性，或者提高了產(chǎn)量或優(yōu)化了蛋白動力學特征或其他致力于得到的性質(zhì)。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的基因或基因簇可以在異源宿主中表達并通過DNA芯片技術了解它們在宿主代謝鏈中的功能。包含本發(fā)明所提供的核苷酸序列編碼的蛋白可以催化合成3-氧甲基-5-甲基萘甲酸及阿嗪霉素非天然氨基酸聚肽骨架，進一步催化合成抗生素。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的基因或基因簇可以通過遺傳重組來構建重組質(zhì)粒以獲得新型生物合成途徑，也可以通過插入、置換、缺失或失活進而獲得新型生物合成途徑。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的克隆基因或DNA片段可以通過中斷阿嗪霉素生物合成的一個或幾個步驟而得到新的阿嗪霉素結構類似物或前體。包含DNA片段或基因可以用來提高阿嗪霉素或其衍生物的產(chǎn)量，本發(fā)明提供了在基因工程微生物中提高產(chǎn)量的途徑。包含本發(fā)明所提供的非核糖體聚肽合成酶可以通過缺失、插入或失活來自于相同或不同的非核糖體聚肽合成酶系統(tǒng)的一個或多個非核糖體聚肽合成酶結構域、模塊或基因而產(chǎn)生新的聚肽化合物。包含本發(fā)明所提供的聚酮合成酶可以通過缺失、插入或失活來自于相同或不同的聚酮合成酶系統(tǒng)的一個或多個聚酮合成酶結構域、模塊或基因而產(chǎn)生新的聚酮化合物。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的片段或基因可以用來構建非核糖體聚肽合成酶庫或非核糖體聚肽合成酶衍生庫或組合庫。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的片段或基因可以用來構建聚酮合成酶庫或聚酮合成酶衍生庫或組合庫。本發(fā)明所提供的催化合成5-甲基萘甲酸和3-氧甲基-5-甲基-萘甲酸的基因可Pfl工人r4^r"ra廿龍msfiA工n,扭m甘cra甘龍ra3fi^/t]J口/J乂J-T巫眾T敗THJ-手、T巫-J-T"巫-統(tǒng)T1敗本發(fā)明所提供的阿嗪霉素骨架的后修飾基因提供了通過遺傳修飾得到類似物的途徑，所包含的氧化還原反應也可有其他應用?？傊景l(fā)明所提供的包含阿嗪霉素生物合成相關的所有基因和蛋白信息可以幫助人們理解阿嗪霉素類天然產(chǎn)物的生物合成機制，為進一步遺傳改造提供了材料和知識。本發(fā)明所提供的基因及其蛋白質(zhì)也可以用來尋找和發(fā)現(xiàn)可用于醫(yī)藥、工業(yè)或農(nóng)業(yè)的化合物或基因、蛋白。圖1:阿嗪霉素的化學結構圖2:阿嗪霉素生物合成基因簇的基因結構和限制性內(nèi)切酶譜。(A)5個交疊的粘粒代表了抗生鏈霉菌基因組80kb的DNA區(qū)域，B代表限制性內(nèi)切酶5awHI，實體表示已被DNA測序的部分，Probe-Pl和P2代表標記的探針部分；(B)阿嗪霉素生物合成基因簇的基因組成。圖3:提出的阿嗪霉素各組成單元的生物合成途徑。(A)3-甲氧基-5-甲基萘甲酸；(B)a-酮異戊酸；(C)氮丙啶[l，2a]吡啶氨基酸；(D)NRPS骨架合成和后修飾。圖4:基因置換與基因互補突變菌株發(fā)酵產(chǎn)物的生物活性檢測和高效液相色譜(HPLC)分析(A)生物活性檢測和(B)HPLC分析；(1)野生型發(fā)酵產(chǎn)物；(2)PKS基因置換的突變體(」az/5)發(fā)酵產(chǎn)物；(3)NRPS基因置換的突變體(Zlm"3)發(fā)酵產(chǎn)物；(4)az/C7基因置換的突變體(zlflZ!'C7)發(fā)酵產(chǎn)物；圖5:在S.a/6w中異源表達阿嗪霉素的生物合成基因(A)含有空載體pTGV2的S.a/6^發(fā)酵產(chǎn)物；(B)含有mW的突變株的發(fā)酵產(chǎn)物；(C)3-甲氧基-5_甲基-萘甲酸標準品；(D)含有^W，和m/萬2的突變株發(fā)酵產(chǎn)物。圖6:細菌中的重復使用的I型PKS(A)結構域組成和機制(I)AviM和CalO(II)ChlBl和MadB(III)、t__n/Tir、a—n/n、a——i八rm—1n1n>r_—m、t_.t\工naAAt丄NUSD、1V乂/\Z1D;VD乂/Wi丄Vl，LtUUJ，HllDl，，丄VlilLl13,，丄NCSJO>TUtJ、J同源性比較。符號說明圖1Azinomycin:阿嗪霉素。圖2Probe:探針；SkeletonAssembly:骨架生成；Naphthoate:萘甲酸；Tailoring:后修飾；BuildingBlocksfromaminoacid;FunctionalUnassigned:功能不確定；Resistance:抗性；UnknownFunction:未知功能；BeyondtheCluster:基因簇之外。圖3AL:酰基輔酶A連接酶；PCP:肽酰載體蛋白；C:縮合酶；KR:酮基還原酶A:腺苷化酶；RE:還原酶。圖6KS:酮基合成酶；AT:?；D移酶；DH:脫氫酶；KR:酮基還原酶；ACP:?；d體蛋白；2-hydroxyl-5-methyl-NPA:2-羥基-5-甲基一萘甲酸；5-methyl-NPA:5-甲基一萘甲酸。具體實施例方式以下結合圖1-圖6對本發(fā)明進一步詳細說明。1.克隆阿嗪霉素的生物合成基因片斷盡管在阿嗪霉素的化學合成和作用機制方面的研究很多，但有關其生物合成起源方面的認識卻非常少。近年來，采用13C-標記的乙酸對阿嗪霉素的生物合成進行同位素標記喂養(yǎng)實驗表明(Oe附.Owww".(2004)8，990-991;C&m.Comww".(2004)22,2600-2601)，其萘甲酸結構單元可能來源于聚酮的生物合成途徑，以已酰-CoA為起始，在聚酮合成酶(PKS)的催化下，于5個丙二酰-CoA之間的連續(xù)縮合反應形成線形的聚酮中間產(chǎn)物，經(jīng)過醛縮反應成環(huán)，脫水、發(fā)生甲基取代最終形成萘甲酸的結構單元。標記實驗同時顯示，烯醇殘基可能來源于蘇氨酸，含有氮雜雙環(huán)的脫水氨基酸殘基則可能來源于ci-酮戊二酸衍生物(如谷氨酸)。此外，推測環(huán)氧殘基可能來源于纈氨酸。由于氨基酸殘基的高度修飾，包含環(huán)氧系統(tǒng)和氮雜雙環(huán)的短肽結構單元可能是由非核糖體聚肽合成酶(NRPS)催化形成。阿嗪霉素的萘甲酸結構單元與新制癌菌素(neocarzinostatin)的萘甲酸結構單元非常相似。己有研究表明(C/2e附別o/(2005)12,293-302)，新制癌菌素的萘甲酸結構單元是由重復使用的I型PKS所合成。根據(jù)僅有的幾例重復使用的I型PKS的保守區(qū)域，設計了簡并性引物5，-GCGGACGGCTACGGSMGNGGNGARGG-3，禾卩5，-CGAGCCGTGGCCSGARAANACCCANAC-3，，采用快速PCR的方法(歷oc/ew5/op/^ie;yCoAwmm(2006)345:133-139)，從阿嗪霉素產(chǎn)生菌的總DNA中克隆編碼PKS基因部分序列，得到約0.8kb的PCR產(chǎn)物，克隆入pGEM-TEasy載體，經(jīng)限制性內(nèi)切酶分組，DNA順序分析表明與已知的重復使用的I型PKS基因有很高的同源性。2.阿嗪霉素生物合成基因簇的克隆，序列分析及功能分析將上述PKS基因片段用地高辛標記為探針Probe-l從5^印/ow少c^^!/zac/>o/NRRL2485的基因組文庫約6000個克隆中篩選，分離得到的粘粒涵蓋了染色體約50kb的區(qū)域。在此基礎上經(jīng)染色體步移得到了5個交疊的粘粒pAL1022，pAL1023,pAL10024，pAL1025,和pAL1026涵蓋了染色體約80kb的區(qū)域(圖2A)。DNA順序分析了63,549bp的染色體區(qū)域，GC含量71.48%。生物信息學分析包含了47個開放讀碼框(圖2B)。orf(-l)和詳細的分析結果列于表l。表l阿嗪霉素生物合成基因簇中各基因及編碼蛋白的功能分析基因氨基酸數(shù)目相似蛋白同一性/相似性％推測功能or飼143Spol正(CAK50935)75/62孢子化蛋白1263CesA(ABK00751)60/41NRPS(A-KR-PCP)273GrsT(YP—001106480)57/41硫酯酶1103EndB(ABD65957)50/38NRPS(C-A-PCP)250TeLB(AAT45287)65/52硫酯酶345Strop—0204(YP—001157067)64/50o-甲基轉移酶401Haur—3696(YP—001546460)48/31細胞色素P450羥化酶627i-v"、ai4i基a,vvy5W7WDPSCAT_DrD、466SpcT(AAF63340)49/31轉運蛋白1115NosD(AAF17281)45/30NRPS(C-PCP-C)435SCO6097(NP一630205)讓3硫酸腺苷酰轉移酶亞單位1270SCO6098(NP—630206)93/88硫酸腺苷酰轉移酶亞單位2185ORFQ(BAC79014)81/67腺苷酰硫酸激酶az/G133StropJ904(YP—001158744)66/55硫酯酶62SimX2(AAK06794)55/47MbtH類似蛋白403Srm6*(CAM96572)56/42O-己酰轉移酶87PPA1287(YP—055995)58/36PCP/ACP340TktC(NP—561213)61/41酮基轉移酶c-端亞單位311TM0954(NP一228762)61/44酮基轉移酶N-端亞單位az,C744001eN2(AAD55458)53/43氨基轉移酶307ArgC(YP—604156)55/45N-已酰-Y-谷氨酰磷酸還原醇292TTC1541(YP—005510)55/36N-已酰谷氨酸激酶376Caur—1204(YP—001634822)54/40N-已酰-鳥氨酸/N-已酰-賴氨酸脫已?；?72ORF39(ABD65959)63/47已酰-CoA脫氫酶1541SfmC(ABI22133)30/42NRPS(C-A-PCP-RE)1779NcsB(AAM77986)66/52重復使用I型PKS(KS-AT-DH-KR-ACP)az/W257Orf(-12)(AAN85502)50/32未知蛋白221Rxyl—2597(YP—645326)47/30未知蛋白337SMb20513(NP—437034)42/25未知蛋白287Rcas—3469(YP—001433537)61/44賴氨酸生物合成酶az/C7243At5g27410(Q9ASR4)43/28分枝氨基酸氨基轉移酶血'C9419CypLB(AAT45286)58/39細胞色素P450羥化酶249BorB(CAE45660)54/40硫酯酶382MAV一1338(YP一880582)53/33已酰-CoA脫氫酶333Acd一9(YP—326591)37/25已酰-CoA脫氫酶371Nfa26740(YP—118885)62/50未知蛋白175SACE—4220(YP—001106414)73/62未知蛋白128Franean1—6978(YP—001511217)63/52未知蛋白207Mmc1—2644(YP_866546)54/40轉座酶249ECA0747(YP—048859)71/59短鏈脫氫酶249SACE_4949(YP一OO1107140)91/84短鏈脫氫酶199SACE—4948(YP—001107139)88/83TetR-家族轉錄調(diào)節(jié)因子151StPAI005(AAW49299)60/47未知蛋白137Orf2(BAF46969)78/63轉錄調(diào)節(jié)因子286SAML0296(CAJ89283)62/45脫氫酶493SC02878(NP—627106)47/30未知蛋白198SAV一5747(NP一826924)畫l二元系統(tǒng)調(diào)節(jié)因子3.阿嗪霉素生物合成基因簇邊界的確定根據(jù)基因編碼蛋白的功能分析，阿嗪霉素的生物合成基因簇被確定為從基因az"3至Uaz/D2(圖2B),包含34個開放讀碼框。其中1個(azz'5)用于編碼重復使用的I型聚酮合成酶(PKS)，共包含5個功能域，負責萘甲酸基團的生物合成；2個基因(m/57和^W2)編碼的氧化酶和甲基轉移酶負責對萘環(huán)進行修飾；8個基因(flz/y4_/，azL42，azL43，az/",az"5，azL46，az^47，azL4S)負責合成非核糖體聚肽骨架合成及修飾；11個基因(m!'C7，az/C2，az/C3，az/OZ，az/C5，az/C6，az/C7，m/CS，az/C9，az/C7仏azZC77)負責非天然氨基酸結構單元的合成酶；還包括1個抗性基因(m/五)、3個后修飾酶基因("^X)/，azz'D2，az/Z)"以及8個功能不確定的基因(azz7/7，az///2，az///3，az/尸，az/G，az/W，dW，az/U"?；蛑袛嗉盎蛑脫Qflz/5,az/C7，和az"3完全抑制了阿嗪霉素的產(chǎn)生(圖4)，進一步證明了克隆到基因簇為阿嗪霉素的生物合成基因簇。4.萘甲酸結構單元的生物合成途徑萘甲酸結構單元的生物合成如圖3A所示。az^編碼的重復使用的聚酮合成酶(包括KS，AT，DH，KR，ACP結構域)催化1分子乙酰輔酶A和5分子丙二酰輔酶A縮合，脫除5分子水生成5-甲基萘甲酸。az^7編碼的P450羥化酶催化5-甲基萘甲酸在3-位的羥化反應，生成的羥基接著在m/52編碼的O-甲基轉移酶催化下進行甲基化修飾，從而完成阿嗪霉素中3-甲氧基-5-甲基萘甲酸單元的生物合成。5.來源于氨基酸的特殊結構單元的生物合成a-酮戊二酸推測由纈氨酸在m/C7所編碼分枝氨基酸氨基轉移酶的作用下發(fā)生脫氨基反應所產(chǎn)生(圖3B)，此過程與初級代謝中纈氨酸分解代謝的第一步反應一致。阿嗪霉素中含有一個非常特別的氮雜雙環(huán)[3丄0]己烷的螺環(huán)體系，該結構的生物合成過程如圖3C所示。az/C2編碼的N-乙?；D移酶催化谷氨酸a-氨基的乙?；?，接著在a力C3編碼的N-乙酰谷氨酸激酶和編碼的Y-谷氨酰磷酸還原酶的作用下生成N-乙酰谷氨酸Y-半醛。在m/C5與az/C6共同編碼的轉羥乙醛基酶的作用下，N-乙酰谷氨酸Y-半醛完成一次兩碳單元的延伸生成2-乙酰氨基-5，7-二羥基-6-酮庚酸。adC7編碼的轉氨酶對其進行轉氨反應后，在m/C§編碼的脫氫酶催化下形成a，P不飽和庚酸，此不飽和酸在6-氨基的親核進攻下發(fā)生分子內(nèi)Michael加成反應生成四氫吡咯環(huán)，隨后的進一步的修飾作用形成阿嗪霉素中的氮雜雙環(huán)結構，在此過程中還伴隨著a力C9編碼的P450羥化酶催化四氫吡咯環(huán)C4位的羥化反應。最后在flz/C/O編碼的脫乙?；傅拇呋饔孟旅撊グ被系囊阴；瑥亩砂⑧好顾刂械牡s雙環(huán)[3丄0]己烷的螺環(huán)結構單元。6.阿嗪霉素骨架合成及后修飾反應阿嗪霉素生物合成基因簇中有五個基因(az"7"5)，它們負責編碼阿嗪霉素骨架組裝的相關酶(AziAl-A5)。AziAl包括兩個功能域(AL-PCP)，其中AL域負責將起始萘甲酸單元的活化并將其裝載在PCP上。AziA3包含三個功能域(AL-KR-PCP)，其中的AL與一些a-酮酸腺苷化域同源，它負責將a-酮異戊酸活化并將其裝載在對應的PCP上，KR結構域?qū)νM行還原，生成第一個延伸單元a-羥基異戊酰-PCP。AziA2包含三個功能域(C-PCP-C)，它負責催化a-羥基異戊酰-PCP與起始單元的縮合。AziA4與AziA5則是兩個典型的NRPS，它們分別含有三個功能域(C-A-PCP)和四個功能域(C-A-PCP-Re)。AziA5中的A與很多負責蘇氨酸腺苷化的功能域同源，它負責蘇氨酸的活化與裝載，AziA4的A則負責特殊氮雜雙環(huán)氨基酸的活化與裝載。這兩個氨基酸延伸單元在各自對應的C功能域催化下與上游的?；l(fā)生縮合完成阿嗪霉素骨架的組裝，最后在AziA5的Re功能域的作用下將組裝好的?；?PCP還原，以醛基的形式從NRPS上釋放下來。釋放的化合物進一步在AziDl，AziD2禾口AziD3的催化作用下分別發(fā)生乙?；?、氧化和環(huán)氧化反應最終形成完整的阿嗪霉素分子。整個過程如圖3D所示。7.阿嗪霉素生物合成基因簇的應用一生物合成基因簇中的基因可以在異源宿主S.a/6ws中進行異源表達在克隆、分析了完整的阿嗪霉素生物合成基因簇，研究了各基因編碼蛋白可能的功能的基礎上，本發(fā)明對阿嗪霉素萘甲酸結構單元的生物合成機制進行了初步探討。將推測的萘甲酸結構單元的合成基因一重復使用的I型PKSaziB基因置于ErmE"^啟動子下于S.albus中進行異源表達，得到的突變株經(jīng)發(fā)酵、HPLC和LC-MS分析，顯示aziB導入的突變株可產(chǎn)生5-甲基萘甲酸(圖4H)。將我們推測的兩個萘甲酸修飾酶AziBl和AziB2—起導入S.albus,產(chǎn)生了預期的3-甲氧基-5-甲基萘甲酸(圖4IV)，證實了我們對阿嗪霉素萘甲酸結構單元生物合成途徑的推測。同時也證實阿嗪霉素生物合成基因簇中的基因可以在異源宿主S.albus中進行異源表達。以下進一步提供實施實例，這些實施實例有助于理解本發(fā)明，僅用作說明而不限制本發(fā)明的應用范圍。實施例l阿嗪霉素產(chǎn)生菌鏈霉菌S"加;tow;w5M/ac/2/ra/NRRL2485總DNA的提取將100pLlxl0851./mw由/ae314孢子懸液接種到3mLTSB液體培養(yǎng)基中，30°C，230rpm培養(yǎng)約24hr后達到對數(shù)生長期后期，取2mL接種到50mLTSB(含lOmM氯化鎂，0.1%甘氨酸)，3(TC，250rpm培養(yǎng)約23hr后達到穩(wěn)定生長期前期，呈乳黃色渾濁，將菌液4。C，3500rpm，離心15min收集菌絲，用裂解液洗滌，收集淡乳黃色菌絲0.5mL。向1mL菌絲中加入10mL裂解液(含溶菌酶5mg/mL)共四管，渦旋至均一，37。C水浴15mim。加入0.1mL蛋白酶K(10mg/mL，用裂解液新鮮配制)，1mL10%SDS，混勻后迅速放入70'C水浴15mim，呈澄清。置冰上冷卻，加入2.5mL5MKAc，冰上冷卻15min。加入10mL飽和酚，混勻，10mL氯仿，混勻，12000rpm，4。C離心20min。用破口的槍頭將水相吸出置于新的離心管，加等量的CHCl3-異戊醇(24:1)抽提，12000rpm，4。C離心10min。用破口的槍頭將水相吸出置于新的離心管，加2倍的無水乙醇，混勻，有大團的DNA出現(xiàn)。將其鉤出置于新的離心管，加5mL70。/。乙醇洗滌，將液體傾出，用槍吸凈，加5mLTE溶解，加RNaseA使終濃度為50pg/mL，37°C溫育0.5小時。依次用等體積的飽和酚抽提兩次，CHCl3-異戊醇抽提兩次，向水相中加入0.1體積的3MNaAc，2體積的無水乙醇，輕輕的混合充分，有絮狀DNA出現(xiàn)。將四管DNA合并到兩管(每管中有l(wèi)mL70Q/。乙醇用于洗滌)，將液體吸出，再加lmL無水乙醇洗滌，吸出乙醇，超凈臺中吹干，溶于適當體積的TE(pH8.0)中。實施例2阿嗪霉素產(chǎn)生菌鏈霉菌S的;/ow;/cewa/2acWra/NRRL2485遺傳轉移系統(tǒng)的建立培養(yǎng)含有適當質(zhì)粒的￡.co//S17-l至OD6oo0.3-0.4，20mLLB培養(yǎng)液中的細菌細胞離心收集，用等體積的LB洗兩次，重懸于2mLLB中，作為大腸桿菌供體細胞。取適量凍存于一8(TC的S加/^wy/c"ra/zac/n'ra/NRRL2485的20%甘油孢子懸液500tiL，用等體積的TES緩沖液(50mMTESNa,pH8.0)洗兩次，重懸于等體積的TES緩沖液，50'C熱激10min使孢子萌發(fā)。再加等體積的TSB培養(yǎng)基，37。C溫育2—5hr。離心重懸于0.5—lmLLB中作為鏈霉菌受體細胞。將不同濃度的受體細胞100pL與等體積的供體細胞混合直接涂布在含有10mMMgCl2的平板上，3(TC溫浴20hr后，采用無菌水輕輕洗滌平板表面以洗去大部分大腸桿菌，在每一平板的表面覆蓋3mL含萘啶酮酸(終濃度為50ng/nL)和相應抗生素的LB軟瓊脂或lmL無菌水。3CTC培養(yǎng)5天以上挑取接合子。由于S加;tow^c^w/zac/n'ro/NRRL2485對硫鏈絲菌肽，阿泊拉霉素和紅霉素都敏感，最后確定遺傳轉移所用抗生素的濃度硫鏈絲菌肽25pg/mL，阿泊拉霉素50pg/mL，紅霉素50pg/mL。IWL-4(可溶性淀粉1.0%，K2HPO40.1%，MgSO40.1%，NaC10.1%，(NH4)2SO40.2%，CaCO30.2%,FeS040扁1%，MnCl2.6H200.0001%，ZnS040.0001%，酵母提取物0.05%，胰化蛋白胨0.1%，瓊脂2.0%,pH7.2)培養(yǎng)基最好，接合轉移效率最高。在IWL-4培養(yǎng)基上，無論是在鏈霉菌中可以自主復制的質(zhì)粒pKC1139，pHZ1358，還是位點特異性整合的質(zhì)粒pSET152都可以得到相應的接合子。實施例3阿嗪霉素產(chǎn)生菌鏈霉菌S&印fomycessahac/n'raz'NRRL2485基因組文庫的構建首先通過一系列的稀釋實驗來確定Sau3AI的用量，在此基礎上大量酶切得到的DNA片段略大于40kb，脫磷。pOJ446先用HpaI從兩個cos序列中間切開并脫磷，然后再從多克隆位點處用BamHI切開，獲得兩個臂，與制備的40kb的DNA片段連接過夜。于冰上融化于一80。C取出地PromegaPackageneexract，立即加入10ul連接產(chǎn)物，輕彈混勻，于室溫(約22°C)放置3hr。加入445ulPhagebuffer,倒轉混合；加入25ul氯仿終止反應，離心使氯仿沉于底部，4'C保存。將凍存于-80'C的菌株E.coliLE392涂布在LB培養(yǎng)基上復蘇。調(diào)取單菌落接種于3MLLB培養(yǎng)基中(0.2%maltose10和mMMgS04)，37°C，220rpm振蕩培養(yǎng)過夜，轉接1%到50MLLB培養(yǎng)基中(0.2%maltose10和mMMgS04)，37°C，220rpm振蕩培養(yǎng)至OD600=0.6—0.8。取5ul包裝液，加入95ulphagebuffer,稀釋，再加入100ulE.ColiLE392(OD600=0.67)，混勻，37°C，30min，涂于LB(含100ug/mlApramycin)平板上。37'C倒置培養(yǎng)過夜，測定噬菌斑形成單位(pfu)以估算文庫的效價。取100ul包裝液加入3.9mlphagebuffer混勻，再加入4mlE.ColiLE392(OD600=0.72)菌液，室溫，30min，加入4mlLB，37°C，75min;2500rpm，10min，去上清，剩余1—3ml涂于LB(含100ug/mlApramycin)平板上。37。C倒置培養(yǎng)過夜。平板長有超過20，000個克隆，用LB刮下，加入甘油(終濃度18%)和Apramycin(終濃度50ug/ml)，分裝，于一8(TC保存。隨機從平板中調(diào)取10個克隆，接種于LB培養(yǎng)基中培養(yǎng)，按大腸桿菌的質(zhì)粒的堿法小量制備的方法抽提重組黏粒。用BamHI鑒定,于0.8%的瓊脂糖凝膠上電泳，根據(jù)限制酶切的電泳分析圖譜，我們判斷出黏粒被酶切產(chǎn)生的片斷大小，加和這些片斷，從而推算出整個黏粒的大小，實驗表明每個黏粒的插入片斷約為40Kb左右。對于鏈霉菌而言，其染色體DNA的大小約為8Mb，如果插入片斷為20kb的文庫效價是2000—5000cfu，就足以代表它的整個基因組。根據(jù)以上實驗，我們建立的文庫效價超過為10000pfu/ygDNA，插入片斷約為40kb左右，這表明我們建立的文庫具有良好的質(zhì)量，能夠滿足文庫篩選的需要。實施例4阿嗪霉素產(chǎn)生菌鏈霉菌5^eptow少cesM/wc/z/ra/NRRL2485的發(fā)酵、產(chǎn)物分離純化與鑒定取100ulS^e/to附少c^saAac/z/ra/NRRL2485孢子涂布在GYM(葡萄糖0.36%，酵母提取物0.4%，麥芽提取物1%，碳酸鈣0.3%，瓊脂2%，自來水，pH6.8)平板上，3(TC培養(yǎng)七天后，用接種一塊(約1cm2)長滿孢子的瓊脂塊接種到75%PS5+培養(yǎng)基(Pharmamedia0.5%，可溶性淀粉0.5%，葡萄糖0.2%，Caseinacidhydrolysate0.5%,硫酸銨0.2%,賴氨酸0.05%,鳥氨酸0.05%,甘氨酸0.05%，pH7.0)中，24小時后，轉接5ml至Ul00mL(500mL錐形瓶)75%PS5+培養(yǎng)基。。轉接15ml到100mL(500mL錐形瓶)25%PS5+培養(yǎng)基，30°C,250rpm培養(yǎng)72小時。離心收集上清，將二氯甲烷萃取兩次，有機相用無水硫酸鎂干燥，真空旋干后于-80'C保存。100ml發(fā)酵產(chǎn)物溶于100ul甲醇，取20pL經(jīng)HPLC檢測。HPLC分析UV=218nm;柱子AglientRp18column，4.6x250mm;流動相條件V=lmL/min;A=H20，B=CH3CN<table>tableseeoriginaldocumentpage22</column></row><table>實施例5PCR克隆阿嗪霉素的生物合成基因PCR體系包含DMSO(8%,v/v)，MgCl2(25mM)，dNTP(2.5mM)，兼并性引物(40mM)，TaqDNA聚合酶(2.5u)及適量模板SRe;rwwj;c^ra/wc/>o/NRRL2485總DNA。首先95°C，3min,1輪；然后94°C，lmin，68°C，lmin，72°C，2min,5輪;94°C，lmin，65。C,lmin,72°C,2min,30輪;最后72。C，10min,1輪。PCR結束后，1%瓊脂糖電泳檢査結果。低熔點膠回收預期大小的DNA片段，與pGEMTEasyvector連接，轉化大腸桿菌DH5ct感受態(tài)細胞，涂布在含有氨芐青霉素，IPTG(異丙基硫代—(3—D-半乳糖苷)和X-gal(5—溴—4—氯一3—吲哚—P—D-半乳糖苷)的LB平板上進行藍白斑篩選。挑取白色菌落過夜培養(yǎng)，抽提質(zhì)粒，五coRl酶切鑒定是否含有預期大小的DNA插入片段。插入有預期大小DNA片段的質(zhì)粒測序。實施例6核酸分子雜交1)DIGDNA標記將待標記的DNA用無菌水稀釋至總體積15^L，沸水浴中加熱變性10分鐘，立即置于冰鹽浴中冷卻。接著加入2pL引物混合物，2jiLdNTP混合物，lpL酶，混合均勻后，37。C水浴約16小時。加入0.8pL0.8MEDTA(pH8.0)以終止反應，加入2.5pL4MLiCl混合均勻，再加入75pL預冷的無水乙醇沉淀標記后的DNA，置于—8(TC沉降40分鐘。4°C，12000rpm離心20分鐘收集DNA，用預冷的70。/。乙醇洗滌DNA沉淀，真空干燥后重新溶于50pLTE((pH8.0)中。2)DIGDNA探針標記后的質(zhì)量檢測稀釋標記的DNA探針，至以下六個梯度，1、10"、l(T2、10'3、10'4、l(T5。稀釋標記的對照DNA至濃度分別為以下濃度lpg/mL，100ng/mL，10ng/mL，lng/mL，0.1ng/mL，0.01ng/mL。分別取lpL上述梯度的DNA樣品點在雜交用的尼龍膜上，根據(jù)7)所述步驟進行顯色反應，對比標記的DNA探針和DIG標記的對照DNA的顯色強度以決定標記的DNA探針濃度。3)菌落雜交(文庫篩選)的膜轉移將保存于—8(TC的基因文庫稍融，取50nL，用450^LLB稀釋得到10"的稀釋倍數(shù)，倍比稀釋得到l(T2，l(T3，104，l(T5，10—6。300^L涂平板(15cmxl5cm，平板為LB/50嗎/mL卡那霉素)。選取合適的比例，使每塊平板約1200—1500個克隆。照選定的比例均勻涂布四塊平板，37'C培養(yǎng)過夜。根據(jù)平板的大小剪取尼龍膜，小心地覆蓋于平板表面不要產(chǎn)生氣泡，做好位置標記，1分鐘后取下尼龍膜置于干燥濾紙上，干燥10分鐘直至菌落結合在尼龍膜上。原始的平板置于培養(yǎng)箱中4一5hr，使克隆重新生長作為原平板。將尼龍膜置于變性液(0.25MNaOH，L5MNaCl)飽和的濾紙上15分鐘(不要浸過膜)，轉移至中和液(1.0MTris.HCl，1.5MNaCl，pH7.5)飽和的濾紙上5分鐘。轉移至2xSSC(20xSSC儲備液(L"):NaCl，175.3g，擰檬酸鈉，88.2g，pH=7.0)飽和的濾紙上自然風干。取下尼龍膜置于烘箱中，120。C固定45分鐘。常溫下于3xSSC/0.1%SDS溶液中振蕩洗滌3小時，以除去細胞碎片。4)Southern雜交的膜轉移:DNA樣品在適當濃度的瓊脂糖凝膠上電泳至適當距離，做好標記。浸泡于400mL0.25MHCl中脫嘌呤20分鐘，使溴酚藍變黃，用去離子水洗數(shù)次。室溫下浸入堿性緩沖液(0.5MNaOH，1MNaCl)15分鐘并輕輕振蕩。換液一次繼續(xù)浸泡凝膠20分鐘，并輕輕振蕩，去離子水洗三次。取一張每邊都比凝膠大lmm的尼龍膜，用去離子水完全浸濕，做好標記。采用向上毛細管轉移方法，用10xSSC轉移緩沖液轉移8—24hr。用2xSSC略微洗膜，12(TC烘烤30分鐘。5)預雜交和雜交預熱雜交液(20mL/100cm2)至雜交溫度68'C，放入雜交尼龍膜，輕輕振蕩并保溫30分鐘。將DIG標記的DNA探針在沸水浴中變性5分鐘，立即置于冰鹽浴中冷卻。冷卻后，將DNA探針與合適體積的DIG雜交液(2.5mL/100cm"混合均勻。去除預雜交液并立即把DNA探針/DIG雜交液加入，輕輕振蕩保持雜交溫度64'C或68'C約16小時。6)雜交后嚴緊洗脫室溫下用2xSSC/0.1%SDS漂洗兩次，每次5分鐘。68。C，用0.1xSSC/0.1%SDS振蕩漂洗兩次，每次15分鐘。7)顯色反應和檢測嚴緊洗脫后的尼龍膜在洗滌緩沖液(0.1M馬來酸，0.15MNaCl，pH=7.5，0.3%(v/v)Tween20)中平衡1-5分鐘，接著在封閉緩沖液(封閉試劑以10%的濃度溶于0.1M馬來酸，0.15MNaCl，pH=7.5)中封閉30分鐘，然后在抗體中浸泡30分鐘。用洗滌緩沖液漂洗尼龍膜兩次后，用檢測緩沖液(0.1MTris-HCl，0.1MNaCl,pH-9.5)中平衡2-5分鐘，最后將尼龍膜置于10mL新配制的顯色溶液[NBT(nitrobluetetrazoliumchloride)溶于70%DMF，濃度為70mg/mL，BCIP(5-bromo-4-chloro-3-indolyl-phosphate)溶于水，濃度為50mg/mL。用時10mL顯色溶液中加45nLNBT，35pLBCIP]中，置于黑暗中顯色。顯色合適后用去離子水漂洗以終止反應。實施例7基因中斷突變菌株的獲得將獲得的轉化子接種到TSB液體培養(yǎng)基(Am25嗎/ml)中，3(TC振蕩約28hr。取出200nl涂布在ISP-4(Am50嗎/ml)平板，30。C培養(yǎng)6-8天，收孢子，保存于-80°C;取出10pl在ISP-24(Am50嗎/ml)平板畫線，37。C培養(yǎng)，放置2—3天。挑37。C整合生長的單菌落，接種至液體培養(yǎng)基ISP-4(Am25pg/ml),37X:，振蕩2—3天。取出涂布在ISP-4平板(Am5CHig/ml)，37。C整合2-3天，收孢子，保存于-8(TC。基因中斷或基因置換所用載體為pOJ260或pKC1139，基因置換用紅霉素抗性基因替代目標基因中間的DNA片段。構建好的質(zhì)粒通過實施例2所述屬間接合轉移的方式導入S&印/ow少CMw^adn7w'NRRL2485中得到雙交換突變體，所得突變體通過Southern雜交在基因型上加以證明。實施例8基因在S.albus中表達及發(fā)酵產(chǎn)物分析將目標基因和紅霉素啟動子克隆到pTGV2載體。正確的質(zhì)粒通過ET12567和S.AlusJ1074的屬間接合轉移得到各突變體的異源表達菌株將培養(yǎng)至對數(shù)生長期的細菌(約48hr)轉接0.5%的菌液于R5A(蔗糖100g/l，硫酸鉀0.25g/l，六水氯化鎂10.12g/l，葡萄糖10g/l，Hy-caseamino0.1g/l，酵母提取物5g/l，Mops21g/1，2mlR5traceelementssolution,調(diào)PH二6.85，高壓滅菌)的液體培養(yǎng)基中繼續(xù)培養(yǎng)120hr，把所有的發(fā)酵物(包括菌體和菌液)的PH調(diào)至2—3，超聲15min(10s/50s);用濾紙濾去大部分菌體，用等體積的乙酸乙酯萃取兩次，旋干，重旋于甲醇中。HPLC分析UV=218nm;柱子AglientRpl8column，4.6x250mm;流動相條件:V=lmL/min;A=H20(1%0TFA)，B=CH3CN(1%0TFA)<table>tableseeoriginaldocumentpage25</column></row><table>以下根據(jù)本
發(fā)明內(nèi)容提供的基因和蛋白序列氨基酸/核苷酸序列表:SEQUENCELISTING<110>中國科學院上海有機化學研究所<120>阿嗪霉素(Azinomycin)生物合成基因簇<130>說明書、權利要求書<160>1<170>Patentlnversion3.3<210>1<211>63549<212>DNA<213>鏈霉菌S,申o，eyra/wC'NRRL2485<400>1ggatcctggccgacctggacatggagaccgatctgccgatcctgatccgtgacagccgctgtcccstgggsgccgscctgggcctgtccggtgaccgactgctgctgtacacggacggcaagttcggacgggaccgtttcgtcgacttcatgcacgagaccctgcgccggctgatggccgacgacgacgccacggtcctgctcaccgaatcctgaagcgccgcgcacgcggaaggasacgcgagaacaagagcgcgccccgcacgtcgstgtcacgttccgaggggacctcgatctgcacccactgctggccaccgagggtcscagcgtggactccsccggcctgacgtgcctgatcgcgcgcctggcgctgstcgcgcccggtgaacgcgsccsggtcctgc3C3gct3cccgscccccgcaggctgacgtgggtcaaccgcggcgscc60ggatcaccgacctgtcctgtcccccggccg120tcaccgtggccaccgagcagctggagcccg180tcgtcgaagcccgcgacagacacggaacgg240tccgccgccatc3ctccggccggcacaccc3Q0ccgtcatggaacaccacgacggcaagctcg360ggcgcggcggccaccagcgcgagctgaccc420gagatgagcaccgatcagccgcccgccccc480gtgcsccgcactcgtgccggccgccatctg540accgctcctctcctggccgacaccctgcsg600ctcctggacctgtccggcgtcaccttcctg660gcctsccgcetccgccsg53ccaccggtgcc720gtgcgccacatgctggctctgaccggcgcg780g3tgcggc3cccggctgsccgccccgcccc840ggcgatcacatcagcgggccccgagggcctggatcagctccctggacacgggcgccggcgactgcgttcctcgcggtgctcgtcgtgtcggccgctcaccttccggcggtccgcacaacttgtgccgccaccacctcgagagccggcctgctcattctgaaacgctcagttggcgggggccggcgtcgagtgcggcgagcscggcgtcgc3cccgstcscgcsgccgtcccggscsgcs3tcctgccgtacttcgagtgggcgatgcgacccgtgtacggggcsggcagttcaggtcasctgtgcagcgatcgstgtcggccagagacsagagagccggagagcccaggcgacagcggggcatgtcgcgcggcgaaacggggcatccgtgccgcgatggtgtccgcagagtgtggggcaggtgtcagcasacttcgggaatgtcgatcacgttgcggtccggcaactcgtcgtatggggctatgctagtggscccatggaccctttgtgacacaaagatgsctacatttggatcatgcgaagtactcgtgtgtgacc;cgccaagggcgcgtsacggccgcatsgaagacgstsgcaggtcgtscggsggcgctgscgsgg33ccggcg3gcggaccgccacgg3t3gtcgssctggtggccagaggagcactcgacatcgacttcgagactgtgccctcctccctctccgagagcggatcccctgcagccgaagtacagaatcagcgcctcagagaagtcttcctcagactcaggagtgctcgttacaccgcacaggcatgggtggacscgaaatcgagcaaccccagactgcgcggcagcgggacgacgcctggacctgtgcgctgssaggcacgccgtttcctcsgccggcgatcctcgggccgggatgaggtctgcgggaggcgga900gtcgcgggascggccgcgcgttcggtcgcc960tcgtcgccgtggcgcgccgacggcagttgt1020gccgtgtcccggacggggttccgagcgcac1080cggcctcacctgcctccgccgtccgcggac1140ctgagtcgctaggggtaaccggctcgggac1200cgttcgccctcgccccctcggtcsgcgctc1260gctcagcggcagttcggtcgtgtagcggca1320gatcttccagagggtgatgcgacgatggtt1380tcttcggcgtgttgccgggtctgtgtcgac1440gtccgcaccgcaatcatgggtggatccgag1500atcgcagctctgccgtggggtccgctcggg1560gctcgatagcgggggtgtccggtcgaagac1620ggaagctgacagtgtgtgcagtctgtcgtg1680cgcaggtcgtggctctgttgtggggagacg1740acggacccgcgtgtctccgaggagagcgcg1800gccgtttactaaatcaacatagctttcgag1860atcactctcgcatgacccgggggggggatg1920tcgcgtcctcggccgctacgtctcatacgg1980atagtatgaaatggactattacggtcgaaa2040cgatcactgcagtgatgcsgagcgttcccg2100tcagagcctcggagcagggcgcgatccccc2160tgctggctcaggcggtgagcggggtcgctg2220cggagagtcaccggcctgcaagcgtgtccc2280cttaccgcaagatgcccgacccggacgagg2340tggggaaggtggcgcagcactacgscgtsc2400ggctgcgtcgtatgggacatctcgaagaag2460aaggacgtcggtaggaagtgtcaccggtgc2520cgtgccttccgggccgtgccscggcccgga2580ggccgccaggg3gcgg3ccgtcggggactc2640gtagagcgagcccaccgagaccggactgtccagctgcccgctcagcgcctccgcasgctg2700cacagccctgatcgaatcgcsccccaggctgsagaacgagtcgtccctgcccacgcgcgc2760gactccgagcacctcggccgcggcctcggccaacagtgcttcgagccgtccttcgggcgg2820cagataggaggcgccgcccgcgccgacggccgcgagtacagcggccgggtcgggggtgcc2880actgcggtcccgctctatccgtgaggtcaccacgacccgggcgaagatcccacggtcggc2940cagttgagccgacaccgcggcccgcacctgctgagggtccgtgtccgacgcgggtacgac3000cgccacggcgacggtgccgccgcccaattggtccgccgccaggagcggcttgatctgctc3060gttcgccggatccgcgccgatgagcaggtgtaccggcccggcgtgcsgggcgcccagcag3120ggccagcgtgccccgggtcgggtcgatcagggtcagtccccggtgcctgcccgccgcgac3180cagcggactgccctggttcatccccgggccgtcccacatgctccaggccaggctctgggc3240ccgtcgccccgccgcggcccagcgctgggcccagccgtccsgtaccgcgttggccgccgc3300gtacgcaccgaascccgtcccgccgaacagaccgttcacggacgagtacagcaccacggc3360ggtgtccggccgtgtggccagcsccccatcgagggccgcacagccaccggccttcggatg3420cagcatcgtcgcgagccactgcgcggactcggcccgcagttcgtggtcggtcaaggactc3480ccactgcggggcgaccgcggcaccggcgagatgcagcaccaggtcgaggccgcaccccca3540ggacgcctccgcttcggccaccgccccggccagcgcgtccgggtccgcggcatcggccgt3600gcggtagcggscctcgccgatggcgtgcaggacctccagcgcctcccgggccccggtgtc3660caggcgctcctgcggggtccgcccgacgatcagcagccgtgccccgtgcgtgaccaccag3720gtgctccgccscggctcgtcccaggccgccgagsccaccggtgatgaggcaggtcccgcc3780ccggggcagcacatcagcgggtacgtcgaagacctccggcaacggcacggcgcgcagccg3840ctgttgcagcggcctgccgtcacgcacggcgatcacgtcgccgctgtgccgcaccgtggc3900csgggccgcgagctcggccgcgccggcgtccgccggggcgtcgatcagtgcggtscgggc3960gsgaagccgttccgagcgcgcggtacgcsccagcgcggtcagtccggtccggcccggaac4020aaccgtgtcgcccggggcggccggcacggctccccgggtcagcacggtgsgctcggcatc4080ggggcggatccgcgccacggccgacgccacggcgaggaactccgacagcaccccggcgtc4140gccgtcggccggcgccgtcgcggggtccgaggcggcgacgaacagcacctgtggtacggc4200cccttcgtccgtgggcccctcgccggccgtcgcgaaggcccgctccagggccgaggccag4260ctccacggcgtccgggccgaccacgatctgagccgtggtcgacacgggcsgccagtcggg4320gacgtcsccgctgscacaggccscgsggggaccgtccggccgttgcctcsgccgtgcgtc4380ggccggttcccscacgaccgccagcgcggggacggsggcctccgagtccggcgcctcgtc4440cggtgcgacgccgtccgtcccaccgccgtagccttcgagcagccgctcgcgctgcaccttcgtcaccggcaccacgtaggtc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0>1ValThrValAlaGluValArgGlyAla65AspPro50AspAsp35GlyAlalie20ArgThr5AlaAlaValProAlaProGlyArgGinGlyLeu130CysAla145GlyGlyThr115ArgArg100TyrThr85GlyAspSerArgSerAlaAlaValProGinLeuSerLeuGluThrAlaArgLeuSerGlyGlyArgProAlaLeu70LeuGlyThrAla25ArgValThr40GlyGluAla55ValArg10GlyValArgAspAlaThrArgGlyArgGlyProProGinAlaThrThrTyrAlaGluLeuArgHisGlyPheLeuAspAsnAlaArgProPro195Ala180ValThr150ProThr165ValArgLeu135PheTyr120AlaLeu105GluLieu90LeuGlu75AlaGly60ProArg45ThrAla30AlaArgAlaAlaProAspGlyAspAlaArgProGlyAspValThrAlaProLeuAla!LysLeulieAlaAspPro200Ala185GlyTrp155ProAla170AlaAlaPro140TrpArg125ValSer110MetProGluTyrTrpAspLeuCysGluGly205Leu190Val15AlaAlaAlaSerAlaValProValLeuProArg95ThrPro80ValAsp!LeuGlyLeuLeuGinAlaCysValAla175Leu!Leu160ThrGlyArgSerLeuAlaGly210AlaAsp225AspLeuAlaLeuLysCysValArgLeuProGly215ProAspProGlu230lieValAsnLeu245GinHisArgGlyGluAlaProProAlaAspArgValLeu220ThrValProAl3SerTrp235LeuThrSerGlySerThrGlyHisArg250lieValAlaAlalieMetVal305ArgPro290PheAla275Leu一—Thr260265AlaAsnGlyPheThrGluHisAspVal280AspHisValGlyGlyMetlieMetTyr295300ArgSer270SerLeu285AsnLeuAspThr255TyrPro240ProAlaAsnTrpArgAspLeuAlaCysGluHisValAsnValArgThrGluSerVal310315ArgProLeuLeuTrpLeuAspCysLeuAspArgPheArg325330AsnThrTrpAlaProAsnPheAlaPheSerLeuVal340Ala335Arglie320ThrAlaGluGluCysAsn370GluLeu385TrpGlylie355AlaAlaAlaCysGin345350GlySerTrpAspLeuSerArgLeuThrAsnlie360GlyGluAlaValValAlaArgThrLeuAlaProMetArgAspAspAspGlyGlu435SerGlu405AlaAla420LeuArgHis390Thr375Ala!LeuProAlaAla380Ala365LeuArgPheValMetValProAsp395SerSerGlyValThrTyrThrArg410ValGlyThrValSerLeuVal425GluLeuProAlaGlyThrPro440ProAla430GlyAla445Met415SerCys400HisLeuValValValThrGluValGly450AspGinGluGlyGlu465ValSerGlyValProGlyValAlaLeuArglieVal460ValGlyArgSerPro455ValLeuProGluGly470ValLeuSerAspThr485AsnArgGluSerPheThrAlaAsp500ArgAspGlyArgGlyTyr490GlyTrpPheAspArg475AlaTyrAsnGluLeuArg495AspHis權AlaGlyPheLeu515MetVallieValAsnGly530ValGluGinValVal545GlyValProAla535GlyValValProThrGly505510PheLeuThrGlyArgArgLysAsn525GlulieGluAlaLeu520AsnPheProAlaPro550AspThrGlyThrAspGlu565ValProGlyThrAspAspValPro580ArgAspLeuAlaAlaLeuAla595ThrSerArgGluPhePro610EeuLeuGluGlyAsp570AlaLeuValAsp585ArgProThrTyrHis540AlaCysSerAlaVal555AlaLeuValValPhe575ArgAla560PheSerArg625GlyGlyGlyLeu600ArgHisAsnGlyGly615ArgGluGly!LeuAla工le590ValValProVal605ValGinArgGluTrp630GlyValAlaProLys620AspAspAi:gThrAsp645AlaSerValProAlaLeuAlaVal660ArgProAspGlyLeuArgGin675Pro680Val665LeuAsp650TrpPhe635GluAlaProAspGluCysSer655AlaTyr640GluArgProAlaAsp670ValAlaCysValSerGlyAsp685ValProAspTrpLeu690ProAspAlaValGlu705AlaGlyGluGlyProVal695LeuAlaSerAla710ThrAspGluGlySerThrThrAlaGinlieValValGly700ArgAlaPhePro725ValAlaAlaSerAspProAlaThr740ValLeuSerGluPheLeuAlaVal755760AlaGluLeuThrValLeuLeuGlu715AlaValProGinVal730ProAlaAspGlyAla745AlaSerAlaAlaThr720LeuPhe735AlaGlyAsp750ArglieArgProAsp770ProGlyAspThrVal785ArgThrAlaArgVal775ProGlyArgThrVal790GluArgLeuLeuValAla765ThrArgGlyAlaValProAlaAla780LeuThrAlaLeuSer805AlaProAlaAspAlaGlyAlaAla820ArgHisSerGlyAspVallie835LeuArgAlaValGinArg850AspValLeuProArg865Gly!LeuGlyArgGlu825AlaValArgAsp840LeuProGluValGlyLeuThrAlaLeuVal795800AlaArgThrAlaLeulieAsp810815LeuAlaAlaLeuAlaThrValPro855GlyGlyThrCysLeu870ValAlaGluHisAla830GlyArgProLeuGin845PheAspValProAla860ThrGlyGlyLeuAla885ArgLeuLeulieValGlyArgThr900AlaArgGluAlaLeuGluValLeu915920lie875ValValThi:His!Leu890GinGluArgLeuPro905HisAlalieGly895ThrGly880AlaGlyGlyGlu925Asp910ValArgTyrArgThrAlaAspAlaAlaAspProAspAlaLeuAlaGlyAlaValAla930935940GluAlaGluAlaSerTrpGlyCysGlyLeuAspLeuValLeuHisLeu945950955960AlaGlyAlaAlaValAlaProGinTrpGluSerLeuThrAspHisGlu965970975LeuArgAlaGluSerAlaGinTrpLeuAlaThrMetLeuHisProLys980985990AlaGlyGlyCysAlaAlaLeuAspGlyValLeuAlaThrArgProAsp99510001005ThrAlaValValLeuTyrSerSerValAsnGlyLeuPheGlyGlyThr101010151020GlyPheGlyAlaTyrAlaAlaAlaAsnAlaValLeuAspGlyTrpAla1025103010351040GinArgTrpAlaAlaAlaGlyArgArgAlaGinSerLeuAlaTrpSer104510501055MetTrpAspGlyProGlyMetAsnGinGlySerProLeuValAlaAla106010651070GlyArgHisArgGlyLeuThrLeulieAspProThrArgGlyThrLeu107510801085AlaLeuLeuGlyAlaLeuHisAlaGlyProValHisLeuLeulieGly109010951100AlaAspProAlaAsnGluGinlieLysProLeuLeuAlaAlaAspGin1105111011151120LeuGlyGlyGlyThrValAlaValAlaValValProAlaSerAspThr112511301135AspProGinGinValArgAlaAlaValSerAlaGinLeuAlaAspArg114011451150GlyliePheAlaArgValValValThrSerArglieGluArgAspArg1155.11601165SerGlyThrProAspProAlaAlaValLeuAlaAlaValGlyAlaGly117011751180GlyAlaSerTyrLeuProProGluGlyArgLeuGluAlaLeuLeuAla118511901195GluAlaAlaAlaGluValLeuGlyValAlaArgVal12051210SerPhePheSerLeuGlyCysAspSerlieArgAla12201225GluAlaLeuSerGlyGinLeuAspSerProValSer12351240TyrGluSerProThrValArgSerLeu125012551200GlyArgAspAsp1215ValGinLeuAla1230ValGlySerLeuAlaAlaArg12601245lieAlaGly<210>3<211〉273<212〉PRT<213>浙e/^om戸515^/wc/n,ra/NRRL2485<400>1LeuSerThrSerPheThrAlaArgValTyr50ThrVal65AlaArgVal35GinAspThrHisAlaGlyAla5Met20ArgAspSerProTrpLeulieCysArgThrAlaGinLeuProAla55ArgLeu40LeuVal25ProMetGluAspGly70lieGluThrLeuValThrAlaSerProArg10ArgArgValPheAlaGlyProSerTrp60ArgSerArg75CysSerlieSerGlyProGly45ValArg30GlyProVal15GluGinAlaSerGluThrLeuGluGluAlaLeuProPro80ArgPro85TyrThrThrMetPr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lAla505560ThrThrLeuGluAlaLeuGlyValProThrTyrAsnArgProArgVal65707580LeuAspValCysGlyAspLysLeuArgThrAlaLeuAlaPheHisSer859095AlaGlyLeuArgMetProGinAlaAlaAlaThrTrpGlyThrGlyAla<formula>formulaseeoriginaldocumentpage128</formula><formula>formulaseeoriginaldocumentpage129</formula>ArglieSer<210>32<211>419<212>PRT<213>Sf邵tom戸51ratoc/n'ra/NRRL2485<40'0>1MetlielieAspLeu!LeuSerLysProProPheCysProValGinPheGin5SerAsn_^2025TrpAlaAspLeuArgGluAsnGinPro10GlyPheHisAlaAlaThrAspMetLeuTyrArgGlyGly50ArgVal65LeuCysGin3540ArgGluProPheTrpValValThrArgLeuArgSerValLeuMetAlaAspAsnLysArgGlu130TyrAsp145ThrLeuThr100AlaPhe115LeuSerAsp85AspHis70Leu55GlyGluPheSerAsnThrAlaProProArgAlaProSerGinAsplieAlaHisMetGlyLys150LeuProLeu135ThrVal120lieHis105ValAsp90ArgSer75lieHis60ArgAla45GluHis30AspArgGlyAlaSerAspGluPheArgAlaThrGinGinSerValMetMetPheGlu110GluArg125LeuGluAla140MetAlaValPro155ProGluSerTrpGluArgLeuAlaAspSer15AlaLeuProGlyAspValSerThrlie80AsnMet9.5ProLeu!LeuLeuGlyValThrGly160GluLeu165170ValMetMetThrlieAlaTyrAspAspProAspTyrSer180185AlaGinAlaThrValArgGinAlaArgHisGlu195GluTyrAlaGinGinLys210lieGlyAlaMetVal225GluGinVal!LeuArg215MetAspLeuSerLeuPhe205ValAsp220GlyPro175AlaGlySer190GluTyrPheAla200SerArgAspAspValAspProAspValGlyMetAspLeuSerGlu230235AsnAlaPheValLeuLeulieGlyLeu245AlaLeuCysThrThrThrProHisAlaLeuCysThrLeu260265ProGluGinTyrArgAlaValGinGluAsnPro275280GinGluLeu!LeuAi:qTrpLeu250MetSerValMetSerSerProCysLeuGinGluLeu!LeuArg290295ArgTyrAlaValLysAspValGluMetHis305310GlyGluProValThrAlaTrplieGlyMetThrGin240AlaAsnVal255AlaGluHis270GluLeuArgAlaSer285ValThrAlaLeuMet300ArgThrGlyArgThrlieArgAla315320ThrAlaTrplieGlyAlaAlaAsnArgAspGluArg325330335ProTyrThrPheAspValAlaArgArgProAsnLys350AlaAsnLeuValPheProAspProTyrThrPheAsp340345HisLeuAlaPheGlyLeuGlyProHis355360ValGlyLeuAspliePheLeuAlaLys370GluSerlieGlulie385ValProGlyTyrLysLeuAsplie375GlyGluValAla390SerMetProValTyrCyslieGly365AspGluLeuLeuGluArgVal380LysHisValAlaSerHisPhe395400ArgPheThrProArgGluAlaHisValArg415<210>33<211>249<212>PRT<213>S加/fo，cessa/wc/n'raz'NRRL2485<400>1ValHisArgAlaThrAlaSerAspAlaArgLeuTrp5AlaThrLeuSer65AlaVal50AlaTyr35ProThr20TyrAlaGluProMetLeuAlaValArgProLeu145lie130ArgPhe100HisLeu115AlsProHisSer!LeuTyrAlaAspTrpValGinLeuLysAspLeuThr85GluVal70ArgPro55GinAla40GlyCys25ProThrLeuLeuValSerArgPheValSerValSerAspTyrGlyAla150His135AlaGly'120LeuArg105ArgPhe10LeuHisArgGlyLeuAlaGluTyr90!LeuProProArgAspGinThrAspGlyArgPheArgSer15ProPheAlaGlyGlySer30CysAlaSerProValGlu45GlyArgMetThrGluSer60GlulieAlaAspThrlie7580GlyHisSerMetGlyAla95GinThr!LeuAsnArgPro110AlaProThrlieValArg125AlaGluPhelieGinMet140ValPheGluHisAspGlu155160LeuLeuAspLeuLeuMetProMetlieArgAlaAspPheAlaMetlie165170175GluAsnTyrArgTyrGinGluAlaProArgLeuSerCysProlieSer180185190AlaTrpCysGlyAspAlaAspProGluValProProThrAlaMetArg195200205GlyTrpGlyAspGinThrSerGlyGluPheThrLeuSerValLeuArg210215220GlyGlyHisPhePheLeuThrGluHisArgAlaGlulieMetArgAla225230235240ValLeuAlaAlaValArgArgAlaArg245<210>34<211>382<212>PRT<213>Slf邵tow戸sra/zflfc/z/ra/NRRL2485<400>1ValProProGinSerValAspPheAlaAsnGluValGluGlulieLeu1.51015ThrAlaProGluThrGluArgLeuLeuArgGluLeuAlaThrArgGin202530AspGlyMetAspGlyAspProArgAlaLeuTyrArgHisLeuGlyAsp354045AlaGlylieLeuAlaProSerTrpProValGluTyrGlyGlyArgGly505560AlaAspPheThrAlaThrValMaLeuLeuGluLysLeuValMaHis65707580ArglieProGinAsnLeuTyrCyslieSerValGinAsnValGlySer8590LeulieLeuAlaAlaGlySerGluGinGinArgArg100105AlaLeuAlaSerThrGluLeuThrSerCyslieLeu115120GlyAsnGlySerAspLeuAlaGlylieThrAsn130ThrGlyTrpVal95ThrLeuLeuPro110PheThrGluPro125AlaValArgAspGly145AlaTyrAlaAlaGlylieThrThrThr135140LeuAsnGlyArgLysThrTyrAsnLeuLys150155AlaLeulieAlaValArgThrAspProAsplieAlaLeulieAlaVal165170SerGinTyrGluGlyLeuThrLeuPheLeuValPro180185lieArgProlieProSerLeuAlaAspGin175AlaSer160SerProGlyValVallieArgProliePro195200AsplieTrpPheThrAspValArgValAspGlu210215ValGlyGluGlyTrpSerLeulieThrGin225ArgThrGlyLeuAsp190GluGinPheHis205SerAlaLeuPheGly220MetPheAlaAlaArgThrLeuSer290GluLeuAsp245ArgVal260GluLeuAla275CysGinValTrpSerLeulieThrGin230235TyrTyrAlaArgGlyArgHisTrpLeuGly250ValAlaGinArgValAspArgAsnGlyAlaArgGinGly265ArgHisGluAlaArglieThrAsp255AlaGlu240LeuValGlu280MetGinAsnLeuGinAspGin295SerSerPheAlaLysTrpHisCysAlaGluSerSerPheAla305310lieAlaTrpTrpAlaLeuAspThrLeuSerAsp270AlaSerLysLeu285GlySerProAsplie300SerGluSerAlaGinArg315320GlyGinAspLeu!LeuGluProGlyProAspProGlyMet355LeuSerSer370325330335ProGlyAspArgThrLeuGluAlaAlaPheArgGlu340345350ThrlieSerGlyGlyAlaSerGluValMetLeu360365AlaArgValPheThrHisAlaProArgGluAsp375380AlaAsplie<210>35<211>333<212>PRT<213>浙e/to，cessa/zac/z/ra/NRRL2485<400>1MetAspLeuArgProAspProPheGinGinArgLeuArgSerAlaVal151015AspThrValLeuThrArgThrGlyAspLeuProAlaGinLeuAlaAsp202530lieGlyValProSerLeuGlyAlaProGluArgLeuGlyGlyPheAla354045LeuGlyLeuSerAlaAsplieValValAsnGluArgLeuGlyHisGly505560LeuGluProLeuAspSerTyrArgAlaThrAlaPheAlaLeuAspLeu65707580LeuAspCysASpGlyAlaProThrGluLeuLeuAlaGlulieLeuAla859095AspLeuTyrLysGlyThrArgHisAlaValValValGlyAlaArgGly100105110GlyGlyAlaValGinValArgAspGlyGlyArgLeuTrpGlyGluSer115120125Gly145CysSer130GluValAla150AlaGluSerLeuProAlaGlyAsplieAlaLeuCyslieValArgThrAla135140AspGlyAlaAlaTrpArgLeuAlalieProGluProGly155lieProAlaGluHisThrGluPro165ArgPheHisGlyAlaProSerGin180TrpGinArgAlaLeuAlaAlaAla195200lieAlaAspTrplieThrAspValLeuGly170SerLeuProValThr185ArglieSerArg175AlaThr160LeuHisThr190AlaLeuLeuLeuGly210ThrArgThrGinSer225HisArgLeuAlaVal245Alalie215GlyLysProLeuVal230!LeulieGlyGluArgGinAla205AlaArgAlaHisValAsn220GluArgGinThrValAla235240GluGlyTrpLysLeuLeuLeuAsnLysAla260AlaAlaMetLeuAlaValAla275LeuGinGlyGluGlyTrpLysLeu250255TrpGluPheAspArgGlyGlyArgProAsplieAla305ThrAla290AlaLeuAsp265GlyGluHisAlaGin280ValArgAlaGlyHisGly295SerValTyrArgMetValAlaVal310ProAlaAlaLeuTrpArgGluAlaGly325330Pro270LeuAlaSerArg285AlaHisSerThrMetLeu300GluSerAlaArgLeu315SerAlaAlaGly320權利要求1.一種抗腫瘤活性的抗生素-阿嗪霉素Azinomycin的生物合成基因簇，其特征在于編碼阿嗪霉素生物合成所涉及的34個基因，具體為1)1個重復使用的I型聚酮合成酶基因，即aziBaziB位于基因簇核苷酸序列第38515-43854個堿基處，長度為5340個堿基對，編碼I型聚酮合成酶，1779個氨基酸；2)萘環(huán)修飾酶基因，即aziB1，aziB2共個基因aziB1位于基因簇核苷酸序列第12587-13792個堿基處，長度為1206個堿基對，編碼細胞色素P450氧化酶，401個氨基酸；aziB2位于基因簇核苷酸序列第11548-12585個堿基處，長度為1038個堿基對，編碼O-甲基轉移酶，345個氨基酸；3)非核糖體聚肽骨架合成及修飾酶基因，即aziA1，aziA2，aziA3，aziA4，aziA5，aziA6，aziA7，aziA8共8個基因aziA1位于基因簇核苷酸序列第13873-15756個堿基處，長度為1884個堿基對，編碼非核糖體聚肽合成酶，627個氨基酸；aziA2位于基因簇核苷酸序列第17541-20888個堿基處，長度為3348個堿基對，編碼非核糖體聚肽合成酶，1115個氨基酸；aziA3位于基因簇核苷酸序列第2596-6387個堿基處，長度為3792個堿基對，編碼非核糖體聚肽合成酶，1263個氨基酸；aziA4位于基因簇核苷酸序列第7202-10513個堿基處，長度為3312個堿基對，編碼非核糖體聚肽合成酶，1103個氨基酸；aziA5位于基因簇核苷酸序列第33636-38261個堿基處，長度為4626個堿基對，編碼非核糖體聚肽合成酶，1541個氨基酸；aziA6位于基因簇核苷酸序列第6384-7205個堿基處，長度為822個堿基對，編碼硫酯酶，273個氨基酸；aziA7位于基因簇核苷酸序列第10796-11548個堿基處，長度為753個堿基對，編碼硫酯酶，250個氨基酸；aziA8位于基因簇核苷酸序列第49525-50274個堿基處，長度為750個堿基對，編碼硫酯酶，249個氨基酸；4)非天然氨基酸結構單元合成酶基因，即aziC1，aziC2，aziC3，aziC4，aziC5，aziC6，aziC7，aziC8，aziC9，aziC10，aziC11共11個基因aziC1位于基因簇核苷酸序列第個47453-48484堿基處，長度為732個堿基對，編碼分枝氨基酸氨基轉移酶，243個氨基酸；aziC2位于基因簇核苷酸序列第46593-47456個堿基處，長度為864個堿基對，編碼賴氨酸生物合成酶，287個氨基酸；aziC3位于基因簇核苷酸序列第30737-31615個堿基處，長度為879個堿基對，編碼N-己酰谷氨酸激酶，292個氨基酸；aziC4位于基因簇核苷酸序列第29735-30658個堿基處，長度為924個堿基對，編碼N-己酰-γ-谷氨酰磷酸還原酶，307個氨基酸；aziC5位于基因簇核苷酸序列第27040-27975個堿基處，長度為936個堿基對，編碼酮基轉移酶N-端亞單位，311個氨基酸；aziC6位于基因簇核苷酸序列第26021-27043個堿基處，長度為1023個堿基對，編碼酮基轉移酶C-端亞單位，340個氨基酸；aziC7位于基因簇核苷酸序列第27972-29294個堿基處，長度為1323個堿基對，編碼氨基轉移酶，440個氨基酸；aziC8位于基因簇核苷酸序列第32741-33559個堿基處，長度為819個堿基對，編碼己酰-CoA脫氫酶，272個氨基酸；aziC9位于基因簇核苷酸序列第48254-49513個堿基處，長度為1260個堿基對，編碼細胞色素P450氧化酶，419個氨基酸；aziC10位于基因簇核苷酸序列第31608-32738個堿基處，長度為1131個堿基對，編碼N-己酰鳥氨酸/N-己酰賴氨酸脫?；福?76個氨基酸；aziC11位于基因簇核苷酸序列第25755-26018個堿基處，長度為264個堿基對，編碼肽酰載體蛋白，87個氨基酸；5)抗性基因，即aziEaziE位于基因簇核苷酸序列第15839-17239個堿基處，長度為1401個堿基對，編碼轉運蛋白，466個氨基酸；6)后修飾酶基因，即aziD1，aziD2，aziD3共3個基因aziD1位于基因簇核苷酸序列第24547-25758個堿基處，長度為1212個堿基對，編碼O-?；D移酶，403個氨基酸；aziD2位于基因簇核苷酸序列第51462-52463個堿基處，長度為1002個堿基對，編碼己酰-CoA脫氫酶，333個氨基酸；aziD3位于基因簇核苷酸序列第50308-51456個堿基處，長度為1149個堿基對，編碼己酰-CoA脫氫酶，382個氨基酸；7)還包括8個功能不明確的基因，即aziH1，aziH2，aziH3，aziF，aziG，aziU1，aziU2，aziU3aziH1位于基因簇核苷酸序列第22185-22997個堿基處，長度為813個堿基對，編碼硫酸腺苷酰轉移酶亞單位2，270個氨基酸；aziH2位于基因簇核苷酸序列第20878-22185個堿基處，長度為1308個堿基對，編碼硫酸腺苷酰轉移酶亞單位1，435個氨基酸；aziH3位于基因簇核苷酸序列第23101-23658個堿基處，長度為558個堿基對，編碼腺苷酰硫酸激酶，185個氨基酸；aziF位于基因簇核苷酸序列第24328-24516個堿基處，長度為189個堿基對，編碼MbtH類似蛋白，62個氨基酸；aziG位于基因簇核苷酸序列第23726-24127個堿基處，長度為402個堿基對，編碼硫酯酶，133個氨基酸；aziU1位于基因簇核苷酸序列第44054-44827個堿基處，長度為774個堿基對，編碼未知功能蛋白，257個氨基酸；aziU2位于基因簇核苷酸序列第44838-45503個堿基處，長度為666個堿基對，編碼未知功能蛋白，221個氨基酸；aziU3位于基因簇核苷酸序列第45500-46513個堿基處，長度為1014個堿基對，編碼未知功能蛋白，337個氨基酸。2.根據(jù)權利要求1所述的阿嗪霉素的生物合成基因簇，其特征在于編碼的非核糖體聚肽合成酶包含下述模塊或結構域?；o酶A連接酶結構域AL、肽?？s合酶結構域C、腺苷化酶結構域A、肽酰基載體蛋白PCP、端基還原酶結構域RE。3.根據(jù)權利要求1所述的阿嗪霉素的生物合成基因簇，其特征在于編碼的聚酮合成酶包含下述模塊或結構域酮基合成酶結構域KS、酰基轉移酶結構域AT、酮基還原酶結構域KR、脫水酶結構域DH、?；d體蛋白ACP。4.根據(jù)權利要求1所述的阿嗪霉素的生物合成基因簇的編碼蛋白用于催化合成抗生素阿嗪菌素及其類似物。5.根據(jù)權利要求4所述的阿嗪霉素的生物合成基因簇的編碼蛋白用于催化合成3-甲氧基-5-甲基萘甲酸。6.根據(jù)權利要求4所述的阿嗪霉素的生物合成基因簇的編碼蛋白用于催化合成非天然氨基酸三肽骨架。7.根據(jù)權利要求4所述的阿嗪霉素的生物合成基因簇的用途，對其中的基因進行遺傳改造獲得的突變體經(jīng)生物發(fā)酵產(chǎn)生3-甲氧基-5-甲基萘甲酸。8.根據(jù)權利要求4所述的阿嗪霉素的生物合成基因簇的用途，其中的基因在S/wptowycMfl/ZnJ1074中進行異源表達產(chǎn)生5-甲基萘甲酸。9.根據(jù)權利要求4所述的阿嗪霉素的生物合成基因簇的用途，其中的基因在S加;tow;;c^a/6^J1074中進行異源表達產(chǎn)生3_甲氧基-5-甲基萘甲酸。全文摘要本發(fā)明是一種由鏈霉菌產(chǎn)生的具有抗腫瘤活性的抗生素——阿嗪霉素(AzinomycinB)的生物合成基因簇的克隆、測序、分析、功能研究及其應用。整個基因簇共包含34個基因1個重復使用的I型聚酮合成酶基因；2個萘環(huán)修飾酶基因；8個非核糖體聚肽骨架合成及修飾酶基因；11個非天然氨基酸結構單元合成酶基因；1個抗性基因；3個后修飾酶基因以及8個功能不確定的基因。通過對上述生物合成基因的遺傳操作可阻斷阿嗪霉素的合成；對萘環(huán)合成和修飾基因的異源表達可產(chǎn)生其前體化合物。本發(fā)明所提供的基因及其蛋白也可以用來尋找和發(fā)現(xiàn)可用于醫(yī)藥、工業(yè)或農(nóng)業(yè)的化合物或基因、蛋白。文檔編號C12N15/52GK101275141SQ200810034378公開日2008年10月1日申請日期2008年3月7日優(yōu)先權日2008年3月7日發(fā)明者文劉,趙群飛申請人:中國科學院上海有機化學研究所