用于epothilone生物合成的基因的制作方法

专利名称：用于epothilone生物合成的基因的制作方法
技术领域：
本发明主要涉及聚酮化合物(polyketide)和用于合成它们的基因。特别的，本发明涉及从纤维堆囊菌(Sorangium cellulosum)中分离和鉴定生物合成epothiloneA和B必需的新的聚酮化合物合酶和非核糖体肽合成酶基因。
背景技术：
聚酮化合物是由二碳结构单元合成的化合物，其β-碳总是携带酮基，由此命名为聚酮化合物。这些化合物包括许多重要的抗生素、免疫抑制剂、癌症化疗剂、和其它具有广泛生物学特性的化合物。巨大的结构多样性源于聚酮化合物链的不同长度、(作为二碳结构单元的一部分或在聚酮化合物主链形成之后)引入的不同侧链、和这些基团的立体化学。酮基还可以被还原成羟基、烯酰基、或完全除去。每一轮二碳的加入是由叫做聚酮化合物合酶(polyketide synthase,PKS)的酶复合物以与脂肪酸生物合成相似的方式进行的。
已经分离得到并测序的聚酮化合物的生物合成基因与日俱增。例如，见美国专利号5,639,949、5,693,774、和5,716,849描述了用于soraphen生物合成的基因，所有这些此处引用作为参考。还可见Schupp等人，FEMS微生物学通讯(FEMS Microbiology Letters)159:201-207(1998)和WO98/07868(其中描述了用于利福霉素生物合成的基因)，和美国专利号5,876,991(其中描述了用于tylactone生物合成的基因，所有这些此处引用作为参考。编码的蛋白质通常分为两类第一类和第二类。第一类蛋白质是多功能的，有多个进行不同酶步骤的催化结构域共价连接在一起(如用于红霉素、soraphen、利福霉素、和除虫菌素的PKS(MacNeil等人，工业微生物基础和应用分子遗传学(Industrial Microorganisms:Basic and AppliedMolecular Genetics)，(编辑Baltz等人)，美国微生物学协会，华盛顿特区，pp.245-256(1993))；而第二类蛋白质是单功能的(Hutchinson等人，工业微生物基础和应用分子遗传学，(编辑Baltz等人)，美国微生物学协会，华盛顿特区，pp.203-216(1993))。
对于较简单的聚酮化合物诸如放线菌紫素(由天蓝色链霉菌(Streptomyces coelicolor)产生)，几轮二碳的加入在由一组PKS基因编码的PKS酶上重复进行。相反的，更复杂的化合物诸如红霉素和soraphen的合成涉及组织成模块的PKS酶，由此每个模块进行一轮二碳的加入(为了回顾，见Hopwood等人，工业微生物基础和应用分子遗传学，(编辑Baltz等人)，美国微生物学协会，华盛顿特区，pp.267-275(1993))。
复杂的聚酮化合物和次级代谢物通常可能包含氨基酸、而非简单的羧酸衍生的亚结构。这些结构单元的掺入由非核糖体多肽合成酶(non-ribosomal polypeptide synthetase,NRPS)完成。NRPS是组织成模块的多酶。每个模块负责一个氨基酸结构单元的加入(和额外的处理，如果需要)。NRPS通过形成氨酰腺苷酸激活氨基酸，并将激活的氨基酸捕获于肽基载体蛋白结构域的磷酸泛酰巯基乙胺辅基的硫醇基上。然后，NRPS通过立体异构化、N-甲基化、或环化(如果需要)来修饰氨基酸，并催化被酶结合的氨基酸之间肽键的形成。NRPS负责肽次级代谢物如环孢菌素的生物合成，可以如在雷帕霉素中提供聚酮化合物链终止单位，或如在耶尔森菌素(yersiniabactin)生物合成中与PKS形成混合系统。
EpothiloneA和B是16元大环聚酮化合物，具有由细菌纤维堆囊菌菌株So ce90(Gerth等人，抗生素杂志(J.Antibiotics)49:560-563(1996)，此处引用作为参考)产生的酰基半胱氨酸衍生的起始单位。EpothiloneA和B的结构是(其中R在epothiloneA中表示氢，在epothiloneB中表示甲基) epothilone具有窄的抗真菌谱，且在动物细胞培养物中尤其显示高细胞毒性(见Hfle等人，德国专利4138042(1993)，此处引用作为参考)。极其重要的是，epothilone在体内和在培养细胞中都模仿紫杉醇的生物学效应(Bollag等人，癌症研究(Cancer Research)55:2325-2333(1995)，此处引用作为参考)。稳定细胞微管的紫杉醇和泰索帝(taxotere)是对各种人实体瘤具有显著活性的癌症化疗剂(Rowinsky等人，国家癌症学会会刊(J.Natl.Cancer Inst.)83:1778-1781(1991))。竞争研究已经揭示了epothilone作为紫杉醇与微管结合的竞争抑制剂，与它们共有相同的微管结合位点和拥有与紫杉醇相似的微管亲和力的解释一致。然而，epothilone具有比紫杉醇显著的优势，因为对多药抗性细胞系epothilone的效力降低比紫杉醇低许多(Bollag等人(1995))。而且，与紫杉醇相比，较少的epothilone有效通过P-糖蛋白排出细胞(Gerth等人(1996))。此外，多种epothilone类似物已经被合成，如它们增强的诱导微管聚合和稳定的能力所示，具有比epothiloneA或epothiloneB更高的细胞毒性活性(WO98/25929，此处引用作为参考)。
尽管epothilone有希望作为抗癌药，目前这些化合物的产量限制了它们的商业潜力。这些化合物对于工业规模的化学合成过于复杂，所以必须通过发酵生产。用于粘细菌诸如纤维堆囊菌基因操作的技术描述于美国专利号5,686,295，此处引用作为参考。然而，纤维堆囊菌难以发酵而且epothilone的产量因此低。在更适合发酵的异源宿主中的epothilone重组生产可以解决当前的产量问题。然而，编码负责epothilone生物合成的多肽的基因至今还未分离得到。而且，产生epothilone的菌株，即So ce90，还产生至少一种其它的聚酮化合物spirangien，可能使分离专门负责epothilone生物合成的基因更加复杂。
因此，综上所述，本发明的一个目的是分离涉及epothilone生物合成的基因，特别是在堆囊粘菌/-多囊粘菌属的粘细菌，即纤维堆囊菌菌株So ce90中涉及epothiloneA和B合成的基因。本发明的另一个目的是提供应用于抗癌制剂的epothilone的重组生产方法。
发明概述为了促进上述和其它目的，本发明出乎意料的克服了上面提出的困难，首次提供了包含编码至少一种涉及epothilone生物合成的多肽的核苷酸序列的核酸分子。在优选的实施方案中，该核苷酸序列是从属于粘细菌目的物种，最优选纤维堆囊菌中分离的。
在另一个优选的实施方案中，本发明提供了分离的核酸分子，其包含编码至少一种涉及epothilone生物合成的多肽的核苷酸序列，其中多肽包含与选自下组的氨基酸序列基本上相似的氨基酸序列:SEQID NO:2,SEQ ID NO:2的氨基酸11-437,SEQ ID NO:2的氨基酸543-864,SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:3,SEQ ID NO:3的氨基酸72-81,SEQID NO:3的氨基酸118-125,SEQ ID NO:3的氨基酸199-212,SEQID NO:3的氨基酸353-363,SEQ ID NO:3的氨基酸549-565,SEQID NO:3的氨基酸588-603,SEQ ID NO:3的氨基酸669-684,SEQID NO:3的氨基酸815-821,SEQ ID NO:3的氨基酸868-892,SEQID NO:3的氨基酸903-912,SEQ ID NO:3的氨基酸918-940,SEQID NO:3的氨基酸1268-1274,SEQ ID NO:3的氨基酸1285-1297,SEQ ID NO:3的氨基酸973-1256,SEQ ID NO:3的氨基酸1344-1351,SEQ ID NO:4,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5,SEQ ID NO:5的氨基酸39-457,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸3555-3876,SEQ ID NO:5的氨基酸3886-4048,SEQ IDNO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸4729-4974,SEQID NO:5的氨基酸5010-5082,SEQ ID NO:5的氨基酸5103-5525,SEQ ID NO:5的氨基酸5631-5951,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:5的氨基酸6542-6837,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:5的氨基酸7140-7211,SEQ ID NO:6,SEQID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸561-881,SEQ IDNO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸1430-1503,SEQID NO:6的氨基酸1522-1946,SEQ ID NO:6的氨基酸2053-2373,SEQ ID NO:6的氨基酸2383-2551,SEQ ID NO:6的氨基酸2671-3045,SEQ ID NO:6的氨基酸3392-3636,SEQ ID NO:6的氨基酸3673-3745,SEQ ID NO:7,SEQ ID NO:7的氨基酸32-450,SEQ IDNO:7的氨基酸556-877,SEQ ID NO:7的氨基酸887-1051,SEQ IDNO:7的氨基酸1478-1790,SEQ ID NO:7的氨基酸1810-2055,SEQID NO:7的氨基酸2093-2164,SEQ ID NO:7的氨基酸2165-2439,SEQ ID NO:8,SEQ ID NO:10,SEQ ID NO:11，和SEQ ID NO:22。
在更优选的实施方案中，本发明提供了一种分离的核酸分子，其包含编码至少一种涉及epothilone生物合成的多肽的核苷酸序列，其中该多肽包含选自下组的氨基酸序列:SEQ ID NO:2,SEQ ID NO:2的氨基酸11-437,SEQ ID NO:2的氨基酸543-864,SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:3,SEQ ID NO:3的氨基酸72-81,SEQ ID NO:3的氨基酸118-125,SEQ ID NO:3的氨基酸199-212,SEQ ID NO:3的氨基酸353-363,SEQ ID NO:3的氨基酸549-565,SEQ ID NO:3的氨基酸588-603,SEQ ID NO:3的氨基酸669-684,SEQ ID NO:3的氨基酸815-821,SEQ ID NO:3的氨基酸868-892,SEQ ID NO:3的氨基酸903-912,SEQ ID NO:3的氨基酸918-940,SEQ ID NO:3的氨基酸1268-1274,SEQ ID NO:3的氨基酸1285-1297,SEQ ID NO:3的氨基酸973-1256,SEQ ID NO:3的氨基酸1344-1351,SEQ ID NO:4,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5,SEQ ID NO:5的氨基酸39-457,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸3555-3876,SEQ IDNO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸4433-4719,SEQID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸5010-5082,SEQ ID NO:5的氨基酸5103-5525,SEQ ID NO:5的氨基酸5631-5951,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:5的氨基酸6542-6837,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:5的氨基酸7140-7211,SEQ ID NO:6,SEQ ID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸561-881,SEQ ID NO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸1522-1946,SEQ ID NO:6的氨基酸2053-2373,SEQ ID NO:6的氨基酸2383-2551,SEQ ID NO:6的氨基酸2671-3045,SEQ ID NO:6的氨基酸3392-3636,SEQ ID NO:6的氨基酸3673-3745,SEQ ID NO:7,SEQ ID NO:7的氨基酸32-450,SEQ ID NO:7的氨基酸556-877,SEQ ID NO:7的氨基酸887-1051,SEQ ID NO:7的氨基酸1478-1790,SEQ ID NO:7的氨基酸1810-2055,SEQ ID NO:7的氨基酸2093-2164,SEQ ID NO:7的氨基酸2165-2439,SEQ ID NO:8,SEQ ID NO:10,SEQ ID NO:11，和SEQ ID NO:22。
在另一个优选的实施方案中，本发明提供了一种分离的核酸分子，其包含编码至少一种涉及epothilone生物合成的多肽的核苷酸序列，其中所述核苷酸序列与选自下组的核苷酸序列基本上相似:SEQ ID NO:1的核苷酸1900-3171的互补序列，SEQ ID NO:1的核苷酸3415-5556,SEQ ID NO:1的核苷酸7610-11875,SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:1的核苷酸11549-11764,SEQ IDNO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:1的核苷酸14313-14334,SEQID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639,SEQ ID NO:1的核苷酸15901-15924,SEQ ID NO:1的核苷酸16251-21749,SEQ ID NO:1的核苷酸16269-17546,SEQID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸21746-43519,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸26045-26263,SEQID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸35930-36667,SEQID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸41369-42256,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸43524-54920,SEQ ID NO:1的核苷酸43626-44885,SEQID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸48087-49361,SEQ ID NO:1的核苷酸49680-50642,SEQ ID NO:1的核苷酸50670-51176,SEQ ID NO:1的核苷酸51534-52657,SEQ ID NO:1的核苷酸53697-54431,SEQ ID NO:1的核苷酸54540-54758,SEQID NO:1的核苷酸54935-62254,SEQ ID NO:1的核苷酸55028-56284,SEQ ID NO:1的核苷酸56600-57565,SEQ ID NO:1的核苷酸57593-58087,SEQ ID NO:1的核苷酸59366-60304,SEQ ID NO:1的核苷酸60362-61099,SEQ ID NO:1的核苷酸61211-61426,SEQ ID NO:1的核苷酸61427-62254,SEQ ID NO:1的核苷酸62369-63628,SEQID NO:1的核苷酸67334-68251，和SEQ ID NO:1的核苷酸1-68750。
在特别优选的实施方案中，本发明提供了包含编码至少一种涉及epothilone生物合成的多肽的核苷酸序列的核酸分子，其中所述核苷酸序列选自下组:SEQ ID NO:1的核苷酸1900-3171的互补序列，SEQ ID NO:1的核苷酸3415-5556,SEQ ID NO:1的核苷酸7610-11875,SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:1的核苷酸11549-11764,SEQ ID NO:1的核苷酸11872-16104,SEQID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:1的核苷酸14313-14334,SEQ ID NO:1的核苷酸14473-14547,SEQID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639,SEQ ID NO:1的核苷酸15901-15924,SEQ ID NO:1的核苷酸16251-21749,SEQ ID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸17865-18827,SEQID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸21746-43519,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸26045-26263,SEQ ID NO:1的核苷酸26318-27595,SEQID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸35930-36667,SEQ ID NO:1的核苷酸36773-36991,SEQID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸41369-42256,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸43524-54920,SEQ ID NO:1的核苷酸43626-44885,SEQ ID NO:1的核苷酸45204-46166,SEQID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸48087-49361,SEQ ID NO:1的核苷酸49680-50642,SEQ ID NO:1的核苷酸50670-51176,SEQ ID NO:1的核苷酸51534-52657,SEQ ID NO:1的核苷酸53697-54431,SEQ ID NO:1的核苷酸54540-54758,SEQ ID NO:1的核苷酸54935-62254,SEQID NO:1的核苷酸55028-56284,SEQ ID NO:1的核苷酸56600-57565,SEQ ID NO:1的核苷酸57593-58087,SEQ ID NO:1的核苷酸59366-60304,SEQ ID NO:1的核苷酸60362-61099,SEQ ID NO:1的核苷酸61211-61426,SEQ ID NO:1的核苷酸61427-62254,SEQ ID NO:1的核苷酸62369-63628,SEQ ID NO:1的核苷酸67334-68251，和SEQ ID NO:1的核苷酸1-68750。
在另一个优选的实施方案中，本发明提供了分离的核酸分子，其包含编码至少一种涉及epothilone生物合成的多肽的核苷酸序列，其中所述核苷酸序列包含与选自下组的核苷酸序列中相应的连续20、25、30、35、40、45或50(优选20)个碱基对的部分序列相同的连续20、25、30、35、40、45或50(优选20)个碱基对的核苷酸部分SEQ IDNO:1的核苷酸1900-3171的互补序列，SEQ ID NO:1的核苷酸3415-5556,SEQ ID NO:1的核苷酸7610-11875,SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:1的核苷酸11549-11764,SEQID NO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:1的核苷酸14313-14334,SEQID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639,SEQ ID NO:1的核苷酸15901-15924,SEQ ID NO:1的核苷酸16251-21749,SEQ ID NO:1的核苷酸16269-17546,SEQID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸21746-43519,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸26045-26263,SEQID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸35930-36667,SEQID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸37052-38320,SEQID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸41369-42256,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸43524-54920,SEQ ID NO:1的核苷酸43626-44885,SEQID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸48087-49361,SEQ ID NO:1的核苷酸49680-50642,SEQ ID NO:1的核苷酸50670-51176,SEQ ID NO:1的核苷酸51534-52657,SEQ ID NO:1的核苷酸53697-54431,SEQ ID NO:1的核苷酸54540-54758,SEQID NO:1的核苷酸54935-62254,SEQ ID NO:1的核苷酸55028-56284,SEQ ID NO:1的核苷酸56600-57565, SEQ ID NO:1的核苷酸57593-58087,SEQ ID NO:1的核苷酸59366-60304,SEQ ID NO:1的核苷酸60362-61099,SEQ ID NO:1的核苷酸61211-61426,SEQ ID NO:1的核苷酸61427-62254,SEQ ID NO:1的核苷酸62369-63628,SEQID NO:1的核苷酸67334-68251，和SEQ ID NO:1的核苷酸1-68750。
本发明还提供包含与本发明的核酸分子可操作连接的异源启动子序列的嵌合基因。其次，本发明提供了包含这种嵌合基因的重组载体，其中所述载体能够被稳定的转化到宿主细胞中。再次，本发明提供了包含这种嵌合基因的重组宿主细胞，其中宿主细胞能够表达编码至少一种epothilone生物合成必需的多肽的核苷酸序列。在优选的实施方案中，重组宿主细胞是属于放线菌目的细菌，而且在更优选的实施方案中重组宿主细胞是链霉菌菌株。在其它实施方案中，重组宿主细胞是任何其它适合发酵的细菌，诸如假单胞菌或大肠杆菌。再其次，本发明提供了包含本发明的核酸分子的Bac克隆，优选Bac克隆pEPO15。
在另一方面，本发明提供了分离的核酸分子，其包含编码epothilone合酶结构域的核苷酸序列。
根据一个实施方案，epothilone合酶结构域是β-酮脂酰合酶(KS)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQID NO:2的氨基酸11-437,SEQ ID NO:4的氨基酸7-432,SEQ IDNO:5的氨基酸39-457,SEQ ID NO:5的氨基酸1524-1950,SEQ IDNO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸5103-5525,SEQID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸1522-1946，和SEQ ID NO:7的氨基酸32-450。根据这个实施方案，优选所述KS结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸11-437,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:5的氨基酸39-457,SEQID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸5103-5525,SEQ ID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸1522-1946，和SEQ ID NO:7的氨基酸32-450。而且，根据这个实施方案，优选所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸21860-23116,SEQID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸43626-44885,SEQ ID NO:l的核苷酸48087-49361，和SEQ ID NO:1的核苷酸55028-56284。根据这个实施方案，更优选该核苷酸序列包含与选自下组的核苷酸序列中相应的连续20、25、30、35、40、45、或50(优选20)个碱基对部分序列相同的连续20、25、30、35、40、45、或50(优选20)个碱基对的核苷酸部分SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸26318-27595,SEQID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸43626-44885,SEQ ID NO:1的核苷酸48087-49361，和SEQ ID NO:1的核苷酸55028-56284。此外，根据这个实施方案，最优选该核苷酸序列选自下组SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸37052-38320,SEQID NO:1的核苷酸43626-44885,SEQ ID NO:1的核苷酸48087-49361，和SEQ ID NO:1的核苷酸55028-56284。
根据另一个实施方案，epothilone合酶结构域是酰基转移酶(AT)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQID NO:2的氨基酸543-864,SEQ ID NO:4的氨基酸539-859,SEQID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸3555-3876,SEQ ID NO:5的氨基酸5631-5951,SEQ ID NO:6的氨基酸561-881,SEQ ID NO:6的氨基酸2053-2373，和SEQ ID NO:7的氨基酸556-877。根据这个实施方案，优选所述AT结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸543-864,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸3555-3876,SEQ ID NO:5的氨基酸5631-5951,SEQ IDNO:6的氨基酸561-881,SEQ ID NO:6的氨基酸2053-2373，和SEQ ID NO:7的氨基酸556-877。而且，根据这个实施方案，优选所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸27911-28876,SEQID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸49680-50642，和SEQ ID NO:1的核苷酸56600-57565。根据这个实施方案，更优选所述核苷酸序列包含与选自下组的核苷酸序列中相应的连续20、25、30、35、40、45、或50(优选20)个碱基对的部分序列相同的连续20、25、30、35、40、45、或50(优选20)个碱基对的核苷酸部分SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸23431-24397,SEQ IDNO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸49680-50642，和SEQ ID NO:1的核苷酸56600-57565。此外，根据这个实施方案，最优选该核苷酸序列选自下组:SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸23431-24397,SEQID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸49680-50642，和SEQ ID NO:1的核苷酸56600-57565。
根据另一个实施方案，该epothilone合酶结构域是烯酰基还原酶(ER)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸6542-6837，和SEQ ID NO:7的氨基酸1478-1790。根据这个实施方案，优选所述ER结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸6542-6837，和SEQID NO:7的氨基酸1478-1790。而且，根据这个实施方案，优选所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸41369-42256，和SEQ ID NO:1的核苷酸59366-60304。根据这个实施方案，更优选该核苷酸序列包含与选自下组的核苷酸序列中相应的连续20、25、30、35、40、45、或50(优选20)个碱基对的部分序列相同的连续20、25、30、35、40、45、或50(优选20)个碱基对的核苷酸部分SEQ ID NO:1的核苷酸10529-11428,SEQ IDNO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸41369-42256,和SEQ ID NO:1的核苷酸59366-60304。此外，根据这个实施方案，最优选该核苷酸序列选自下组SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸41369-42256，和SEQ ID NO:1的核苷酸59366-60304。
根据另一个实施方案，所述epothilone合酶结构域是酰基载体蛋白(ACP)结构域，其中所述多肽包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸5010-5082,SEQ IDNO:5的氨基酸7140-7211,SEQ ID NO:6的氨基酸1430-1503,SEQID NO:6的氨基酸3673-3745，和SEQ ID NO:7的氨基酸2093-2164。根据这个实施方案，优选所述ACP结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸5010-5082,SEQ ID NO:5的氨基酸7140-7211,SEQ ID NO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸3673-3745，和SEQ ID NO:7的氨基酸2093-2164。而且，根据这个实施方案，优选所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸11549-11764,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸26045-26263,SEQID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸54540-54758，和SEQ ID NO:1的核苷酸61211-61426。根据这个实施方案，更优选所述核苷酸序列包含与选自下组的核苷酸序列中相应的连续20、25、30、35、40、45、或50(优选20)个碱基对的部分序列相同的连续20、25、30、35、40、45、或50(优选20)个碱基对的核苷酸部分SEQ ID NO:1的核苷酸11549-11764,SEQ ID NO:1的核苷酸21414-21626,SEQ IDNO:1的核苷酸26045-26263,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸54540-54758，和SEQ ID NO:1的核苷酸61211-61426。此外，根据这个实施方案，最优选该核苷酸序列选自下组SEQ ID NO:1的核苷酸11549-11764,SEQ ID NO:1的核苷酸21414-21626,SEQ IDNO:1的核苷酸26045-26263,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸54540-54758，和SEQ ID NO:1的核苷酸61211-61426。
根据另一个实施方案，所述epothilone合酶结构域是脱水酶(DH)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQID NO:4的氨基酸869-1037,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:6的氨基酸2383-2551，和SEQ ID NO:7的氨基酸887-1051。根据这个实施方案，优选所述DH结构域包含选自下组的氨基酸序列SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:6的氨基酸2383-2551，和SEQ IDNO:7的氨基酸887-1051。而且，根据这个实施方案，优选所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸50670-51176，和SEQ ID NO:1的核苷酸57593-58087。根据这个实施方案，更优选所述核苷酸序列包含与选自下组的核苷酸序列中相应的连续20、25、30、35、40、45、或50(优选20)个碱基对的部分序列相同的连续20、25、30、35、40、45、或50(优选20)个碱基对的核苷酸部分SEQ IDNO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸50670-51176，和SEQ ID NO:1的核苷酸57593-58087。此外，根据这个实施方案，最优选该核苷酸序列选自下组SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸50670-51176，和SEQ IDNO:1的核苷酸57593-58087。
根据另一个实施方案，所述epothilone合酶结构域是β-酮还原酶(KR)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸3392-3636，和SEQID NO:7的氨基酸1810-2055。根据这个实施方案，优选所述KR结构域包含选自下组的氨基酸序列SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸3392-3636,和SEQ ID NO:7的氨基酸1810-2055。而且，根据这个实施方案，优选所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸35930-36667,SEQ ID NO:1的核苷酸42314-43048,SEQID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸53697-54431,和SEQ ID NO:1的核苷酸60362-61099。根据这个实施方案，更优选该核苷酸序列包含与选自下组的核苷酸序列中相应的连续20、25、30、35、40、45、或50(优选20)个碱基对的部分序列相同的连续20、25、30、35、40、45、或50(优选20)个碱基对的核苷酸部分SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸35930-36667,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸53697-54431，和SEQ ID NO:1的核苷酸60362-61099。此外，根据这个实施方案，最优选该核苷酸序列选自下组SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸35930-36667,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸53697-54431，和SEQ ID NO:1的核苷酸60362-61099。
根据另一个实施方案，所述epothilone合酶结构域是甲基转移酶(MT)结构域，其包含与SEQ ID NO:6的氨基酸2671-3045基本相似的氨基酸序列。根据这个实施方案，优选所述MT结构域包含SEQ IDNO:6的氨基酸2671-3045。而且，根据这个实施方案，优选所述核苷酸序列与SEQ ID NO:1的核苷酸51534-52657基本相似。根据这个实施方案，更优选该核苷酸序列包含与SEQ ID NO:1的核苷酸51534-52657中相应的连续20、25、30、35、40、45或50(优选20)个碱基对的部分序列相同的连续20、25、30、35、40、45或50(优选20)个碱基对的核苷酸部分。此外，根据这个实施方案，最优选该核苷酸序列是SEQ ID NO:1的核苷酸51534-52657。
根据另一个实施方案，所述epothilone合酶结构域是硫酯酶(TE)结构域，其包含与SEQ ID NO:7的氨基酸2165-2439基本相似的氨基酸序列。根据这个实施方案，优选所述TE结构域包含SEQ ID NO:7的氨基酸2165-2439。而且，根据这个实施方案，优选所述核苷酸序列与SEQ ID NO:1的核苷酸61427-62254基本相似。根据这个实施方案，更优选该核苷酸序列包含与SEQ ID NO:1的核苷酸61427-62254中相应的连续20、25、30、35、40、45或50(优选20)个碱基对部分序列相同的连续20、25、30、35、40、45或50(优选20)个碱基对的核苷酸部分。此外，根据这个实施方案，最优选该核苷酸序列是SEQ ID NO:1的核苷酸61427-62254。
在另一方面，本发明提供了分离的核酸分子，其包含编码非核糖体肽合成酶的核苷酸序列，其中所述非核糖体肽合成酶包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:3,SEQ ID NO:3的氨基酸72-81,SEQ ID NO:3的氨基酸118-125,SEQ ID NO:3的氨基酸199-212,SEQ ID NO:3的氨基酸353-363,SEQ ID NO:3的氨基酸549-565,SEQ ID NO:3的氨基酸588-603,SEQ ID NO:3的氨基酸669-684,SEQ ID NO:3的氨基酸815-821,SEQ ID NO:3的氨基酸868-892,SEQ ID NO:3的氨基酸903-912,SEQ ID NO:3的氨基酸918-940,SEQ ID NO:3的氨基酸1268-1274,SEQ ID NO:3的氨基酸1285-1297,SEQ ID NO:3的氨基酸973-1256，和SEQ IDNO:3的氨基酸1344-1351。根据这个实施方案，优选所述非核糖体肽合成酶包含选自下组的氨基酸序列SEQ ID NO:3,SEQ ID NO:3的氨基酸72-81,SEQ ID NO:3的氨基酸118-125,SEQ ID NO:3的氨基酸199-212,SEQ ID NO:3的氨基酸353-363,SEQ ID NO:3的氨基酸549-565,SEQ ID NO:3的氨基酸588-603,SEQ ID NO:3的氨基酸669-684,SEQ ID NO:3的氨基酸815-821,SEQ ID NO:3的氨基酸868-892,SEQ ID NO:3的氨基酸903-912,SEQ ID NO:3的氨基酸918-940,SEQ ID NO:3的氨基酸1268-1274,SEQ ID NO:3的氨基酸1285-1297,SEQ ID NO:3的氨基酸973-1256，和SEQ IDNO:3的氨基酸1344-1351。而且，根据这个实施方案，优选所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:l的核苷酸14313-14334,SEQ ID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639,和SEQ ID NO:1的核苷酸15901-15924。根据这个实施方案，更优选该核苷酸序列包含与选自下组的核苷酸序列中相应的连续20、25、30、35、40、45或50(优选20)个碱基对的部分序列相同的连续20、25、30、35、40、45或50(优选20)个碱基对的核苷酸部分SEQ IDNO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:1的核苷酸14313-14334,SEQID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639，和SEQ ID NO:1的核苷酸15901-15924。此外，根据这个实施方案，最优选该核苷酸序列选自下组SEQ ID NO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ IDNO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:1的核苷酸14313-14334,SEQ ID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639，和SEQ ID NO:1的核苷酸15901-15924。
本发明还提供了一种分离的核酸分子，其包含编码包括选自SEQID NO:2-23的氨基酸序列的多肽的核苷酸序列。
根据另一方面，本发明还提供了用于重组生产聚酮化合物诸如epothilone的方法，其生产产量足够用于它们的纯化和药用制剂诸如用于治疗癌症。这些生产方法的特殊优势是产生的分子有手性；在转基因有机体中进行生产，避免了产生大量的外消旋混合物，其中一些对映体可能具有降低的活性。特别的，本发明提供了在重组宿主中异源表达epothilone的方法，包括(a)将包含与本发明的核酸分子(其包括编码至少一种涉及epothilone生物合成的多肽的核苷酸序列)可操作连接的异源启动子序列的嵌合基因导入宿主；和(b)在适合宿主中生物合成epothilone的条件下培养宿主。本发明还提供了生产epothilone的方法，包括(a)用上述方法在重组宿主中表达epothilone；和(b)从重组宿主中提取epothilone。
根据另一方面，本发明提供了一种分离的多肽，其包括由epothilone合酶结构域组成的氨基酸序列。
根据一个实施方案，所述epothilone合酶结构域是β-酮脂酰合酶(KS)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸11-437,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:5的氨基酸39-457,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸5103-5525,SEQ ID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸1522-1946，和SEQ ID NO:7的氨基酸32-450。根据这个实施方案，优选所述KS结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸11-437,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:5的氨基酸39-457,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸5103-5525,SEQ IDNO:6的氨基酸35-454,SEQ ID NO:6的氨基酸1522-1946，和SEQID NO:7的氨基酸32-450。
根据另一个实施方案，所述epothilone合酶结构域是酰基转移酶(AT)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸543-864,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸3555-3876,SEQ ID NO:5的氨基酸5631-5951,SEQ ID NO:6的氨基酸561-881,SEQ ID NO:6的氨基酸2053-2373，和SEQ ID NO:7的氨基酸556-877。根据这个实施方案，优选所述AT结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸543-864,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸3555-3876,SEQ ID NO:5的氨基酸5631-5951,SEQ IDNO:6的氨基酸561-881,SEQ ID NO:6的氨基酸2053-2373，和SEQ ID NO:7的氨基酸556-877。
根据另一个实施方案，所述epothilone合酶结构域是烯酰基还原酶(ER)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸6542-6837，和SEQ ID NO:7的氨基酸1478-1790。根据这个实施方案，优选该ER结构域包含选自下组的氨基酸序列:SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸6542-6837，和SEQ IDNO:7的氨基酸1478-1790。
根据另一个实施方案，所述epothilone合酶结构域是酰基载体蛋白(ACP)结构域，其中所述多肽包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸5010-5082,SEQ IDNO:5的氨基酸7140-7211,SEQ ID NO:6的氨基酸1430-1503,SEQID NO:6的氨基酸3673-3745，和SEQ ID NO:7的氨基酸2093-2164。根据这个实施方案，优选所述ACP结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸5010-5082,SEQ ID NO:5的氨基酸7140-7211,SEQ ID NO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸3673-3745，和SEQ ID NO:7的氨基酸2093-2164。
根据另一个实施方案，所述epothilone合酶结构域是脱水酶(DH)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQID NO:4的氨基酸869-1037,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:6的氨基酸2383-2551，和SEQ ID NO:7的氨基酸887-1051。根据这个实施方案，优选所述DH结构域包含选自下组的氨基酸序列SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:6的氨基酸2383-2551，和SEQ IDNO:7的氨基酸887-1051。
根据另一个实施方案，所述epothilone合酶结构域是β-酮还原酶(KR)结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸3392-3636，和SEQID NO:7的氨基酸1810-2055。根据这个实施方案，优选所述KR结构域包含选自下组的氨基酸序列SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸3392-3636，和SEQ ID NO:7的氨基酸1810-2055。
根据另一个实施方案，所述epothilone合酶结构域是甲基转移酶(MT)结构域，其包含与SEQ ID NO:6的氨基酸2671-3045基本相似的氨基酸序列。根据这个实施方案，优选所述MT结构域包含SEQ IDNO:6的氨基酸2671-3045。
根据另一个实施方案，所述epothilone合成酶结构域是硫酯酶(TE)结构域，其包含与SEQ ID NO:7的氨基酸2165-2439基本相似的氨基酸序列。根据这个实施方案，优选所述TE结构域包含SEQ IDNO:7的氨基酸2165-2439。
本发明的其它方面和优点对于那些本领域的技术人员，在研究了本发明的下列描述和非限制性实施例后将是显而易见的。
定义在本发明的描述中，将使用下列术语，并做如下定义。
相关联/可操作连接指物理或功能相关的两种DNA序列。例如，如果将启动子或调控DNA序列与编码RNA或蛋白质的DNA可操作连接或放置，以至于调控DNA序列将影响编码或结构DNA序列的表达水平，则该启动子或调控序列被认为是与该DNA序列“相关联”。
嵌合基因重组DNA序列，其中启动子或调控DNA序列与编码mRNA或表达为蛋白质的DNA序列可操作连接，或与之相关联，以至于调控DNA序列能够调控该相关联的DNA序列的转录或表达。正如在自然界中发现的，嵌合基因的调控DNA序列通常不与该相关联的DNA序列可操作连接的。
编码DNA序列在有机体中翻译成蛋白质的DNA序列。
结构域聚酮化合物合酶中对于特定的独特活性必需的那部分。例子包括酰基载体蛋白(acyl carrier protein,ACP)、β-酮合酶(β-ketosynthase,KS)、酰基转移酶(acyltransferae,AT)、β-酮还原酶(β-ketoreductase,KR)、脱水酶(dehydratase,DH)、烯酰基还原酶(enoylreductase,ER)、和硫酯酶(thioesterase,TE)结构域。
Epothilone由细菌纤维堆囊菌菌株So ce90天然产生的16元大环聚酮化合物，模仿紫杉醇的生物学效应。在本申请中，“epothilone”指聚酮化合物类，包括epothiloneA和epothiloneB，以及它们的类似物诸如WO98/25929中描述的那些。
Epothilone合酶负责epothilone生物合成的聚酮化合物合酶。
基因位于基因组内，除了上述编码DNA序列，还包括主要是调控DNA序列(负责调控编码DNA序列的表达即转录和翻译的DNA序列)的确定区域。
异源DNA序列与导入的宿主细胞天然不相关联的DNA序列，包括天然DNA序列的非自然产生的多拷贝。
同源DNA序列与导入的宿主细胞天然相关联的DNA序列。
同源重组同源DNA分子之间互相交换DNA片段。
分离的在本发明的内容中，分离的核酸分子或分离的酶是由人工实现，脱离其自然环境存在并因而不是天然产物的核酸分子或酶。分离的核酸分子或酶可以以纯化形式存在，或者可以在非天然环境诸如重组宿主细胞中存在。
模块编码单轮聚酮化合物生物合成(即一个缩合步骤和所有与此有关的β-羰基处理步骤)需要的所有独特活性的基因元件。每个模块编码ACP、KS、和AT活性以实现生物合成的缩合部分，编码选定的缩合后活性以进行β-羰基处理。
NRPS非核糖体多肽合成酶，是负责将氨基酸加入到次级代谢物中的酶活性，包括，例如，氨基酸腺苷酸化、异构化、N-甲基化、环化、肽酰基载体蛋白、和缩合结构域的复合物。有功能的NRPS催化将氨基酸加入到次级代谢物中。
NRPS基因编码在一个或多个相容的控制元件的指导下产生有功能的次级代谢物(例如epothiloneA和B)的NRPS的一个或多个基因。
核酸分子可以从任何来源分离的单链或双链DNA或RNA的线性片段。在本发明的内容中，核酸分子优选是DNA片段。
ORF开放阅读框架。
PKS聚酮化合物合酶，是负责聚酮化合物生物合成的酶活性(结构域)，包括，例如酮还原酶、脱水酶、酰基载体蛋白、烯酰基还原酶、酮脂酰ACP合酶、和酰基转移酶的复合物。有功能的PKS催化聚酮化合物的合成。
PKS基因当在一个或多个相容的控制元件的指导下，编码产生有功能的聚酮化合物(如epothiloneA和B)需要的各种多肽的一个或多个基因。
基本相似对于核酸，是指具有与参考核酸分子至少60％序列相同的核酸分子。在优选的实施方案中，基本相似的DNA序列与参考DNA序列至少80％相同；在更优选的实施方案中，基本相似的DNA序列与参考DNA序列至少90％相同；而在最优选的实施方案中，基本相似的DNA序列与参考DNA序列至少95％相同。基本相似的DNA序列优选编码具有与参考DNA序列编码的蛋白质或肽基本相同活性的蛋白质或肽。基本相似的核苷酸序列通常可与参考核酸分子或它们的片段在下列条件下杂交在7％十二烷基硫酸钠(SDS)、0.5M NaPO4pH7.0、1mM EDTA中于50℃杂交；用2×SSC、1％SDS于50℃漂洗。对于蛋白质或肽，基本相似的氨基酸序列是与参考蛋白质或肽的氨基酸序列至少90％相同而且具有与参考蛋白质或肽基本相同活性的氨基酸序列。
转化将异源核酸导入宿主细胞或有机体的过程。
经转化的/转基因的/重组的指已经导入了异源核酸分子的宿主有机体诸如细菌。核酸分子可以稳定的整合到宿主的基因组中，或者核酸分子也可以作为染色体外分子存在。这样的染色体外分子可以自主复制。经转化的细胞、组织、或植株应理解为不仅包含转化过程的终产物，而且还有它的转基因后代。“非转化的”、“非转基因的”、或“非重组的”宿主指野生型有机体，即细菌，其中不含有异源核酸分子。
核苷酸由它们的碱基依照下列标准缩写表示腺嘌呤(adenine,A)，胞嘧啶(cytosine,C)，胸腺嘧啶(thymine,T)，和鸟嘌呤(guanine,G)。氨基酸类似的依照下列标准缩写表示丙氨酸(alanine；Ala；A)，精氨酸(arginine；Arg；R)，天冬酰胺(asparagine；Asn；N)，天冬氨酸(aspartic acid；Asp；D)，半胱氨酸(cysteine；Cys；C)，谷氨酰胺(glutamine；Gln；Q)，谷氨酸(glutamicacid；Glu；E)，甘氨酸(glycine；Gly；G)，组氨酸(histidine；His；H)，异亮氨酸(isoleucine；Ile；I)，亮氨酸(leucine；Leu；L)，赖氨酸(lysine；Lys；K)，甲硫氨酸(methionine；Met；M)，苯丙氨酸(phenylalanine；Phe；F)，脯氨酸(proline；Pro；P)，丝氨酸(serine；Ser；S)，苏氨酸(threonine；Thr；T)，色氨酸(trytophan；Trp；W)，酪氨酸(tyrosine；Tyr；Y)，和缬氨酸(valine；Val；V)。此外，(Xaa；X)代表任何氨基酸。
序列表中序列的描述SEQ ID NO:1是含有22个开放阅读框架(ORF)的68750bp毗连序列群的核苷酸序列，包括epothilone生物合成基因。
SEQ ID NO:2是由epoA(SEQ ID NO:1的核苷酸7610-11875)编码的第一类聚酮化合物合酶(EPOS A)的蛋白质序列。
SEQ ID NO:3是由epoP(SEQ ID NO:1的核苷酸11872-16104)编码的非核糖体肽合成酶(EPOS P)的蛋白质序列。
SEQ ID NO:4是由epoB(SEQ ID NO:1的核苷酸16251-21749)编码的第一类聚酮化合物合酶(EPOS B)的蛋白质序列。
SEQ ID NO:5是由epoC(SEQ ID NO:1的核苷酸21746-43519)编码的第一类聚酮化合物合酶(EPOS C)的蛋白质序列。
SEQ ID NO:6是由epoD(SEQ ID NO:1的核苷酸43524-54920)编码的第一类聚酮化合物合酶(EPOS D)的蛋白质序列。
SEQ ID NO:7是由epoE(SEQ ID NO:1的核苷酸54935-62254)编码的第一类聚酮化合物合酶(EPOS E)的蛋白质序列。
SEQ ID NO:8是由epoF(SEQ ID NO:1的核苷酸62369-63628)编码的细胞色素P450加氧酶同源物(EPOS F)的蛋白质序列。
SEQ ID NO:9是由orf1(SEQ ID NO:1的核苷酸1-1826)编码的部分蛋白质序列(部分Orf1)。
SEQ ID NO:10是由orf2(SEQ ID NO:1反向互补链的核苷酸3171-1900)编码的蛋白质序列(Orf2)。
SEQ ID NO:11是由orf3(SEQ ID NO:1的核苷酸3415-5556)编码的蛋白质序列(Orf3)。
SEQ ID NO:12是由orf4(SEQ ID NO:1反向互补链的核苷酸5992-5612)编码的蛋白质序列(Orf4)。
SEQ ID NO:13是由orf5(SEQ ID NO:1的核苷酸6226-6675)编码的蛋白质序列(Orf5)。
SEQ ID NO:14是由orf6(SEQ ID NO:1的核苷酸63779-64333)编码的蛋白质序列(Orf6)。
SEQ ID NO:15是由orf7(SEQ ID NO:1反向互补链的核苷酸64290-63853)编码的蛋白质序列(Orf7)。
SEQ ID NO:16是由orf8(SEQ ID NO:1的核苷酸64363-64920)编码的蛋白质序列(Orf8)。
SEQ ID NO:17是由orf9(SEQ ID NO:1反向互补链的核苷酸64727-64287)编码的蛋白质序列(Orf9)。
SEQ ID NO:18是由orf10(SEQ ID NO:1的核苷酸65063-65767)编码的蛋白质序列(Orf10)。
SEQ ID NO:19是由orf11(SEQ ID NO:1反向互补链的核苷酸65874-65008)编码的蛋白质序列(Orf11)。
SEQ ID NO:20是由orf12(SEQ ID NO:1反向互补链的核苷酸66338-65871)编码的蛋白质序列(Orf12)。
SEQ ID NO:21是由orf13(SEQ ID NO:1的核苷酸66667-67137)编码的蛋白质序列(Orf13)。
SEQ ID NO:22是由orf14(SEQ ID NO:1的核苷酸67334-68251)编码的蛋白质序列(Orf14)。
SEQ ID NO:23是由orf15(SEQ ID NO:1的核苷酸68346-68750)编码的部分蛋白质序列(部分Orf15)。
SEQ ID NO:24是通用的反向PCR引物序列。
SEQ ID NO:25是通用的正向PCR引物序列。
SEQ ID NO:26是NH24末端“B”PCR引物序列。
SEQ ID NO:27是NH2末端“A”PCR引物序列。
SEQ ID NO:28是NH2末端“B”PCR引物序列。
SEQ ID NO:29是pEPO15-NH6“B”PCR引物序列。
SEQ ID NO:30是pEPO15-H2.7“A”PCR引物序列。
保藏信息下列物质已依照国际承认用于专利程序的微生物保藏布达佩斯条约，已经储存于农业研究服务中心(Agricultural Research Service)，专利培养物收藏中心(Patent Culture Collection(NRRL)),1815北方大学街，Peoria，伊利诺斯州61604。所有对获取保藏物的限制在取得专利的授权后将消除。
保藏物保藏号储存日期pEPO15NRRL B-300331998年6月11日pEPO32NRRL B-301191999年4月16日发明的详细描述可以使用本发明的技术分离得到涉及epothilone生物合成的基因。分离epothilone生物合成基因的优选步骤，需要从经鉴定产生epothiloneA和B的有机体中分离基因组DNA，并将分离的合适质粒或载体上的DNA转移到通常不产生聚酮化合物的宿主有机体中，随后鉴定具有epothilone生产能力的经转化的宿主菌落。使用诸如λ::Tn5转座子诱变(de Bruijn和Lupski，基因(Gene)27:131-149(1984))技术，转化的赋予epothilone的DNA的精确区域可以更准确确定。可替代地或额外地，可以将转化的赋予epothilone的DNA切割成较小的片段，而且可以进一步鉴定维持授予epothilone能力的最小片段。然而，缺乏epothilone生产能力的宿主有机体可能是与产生聚酮化合物的物种不同的物种，这种技术的变更涉及将宿主DNA转化到epothilone生产能力已经被诱变破坏的相同宿主中。在这种方法中，产生epothilone的有机体被突变了，并分离得到不产生epothilone的突变体。然后将这些突变体用从产生epothilone的亲本菌株中分离的基因组DNA弥补。
可以用来分离epothilone生物合成需要的基因的进一步的实例，是使用转座子诱变来产生产生epothilone的有机体的突变体，其被诱变后不能产生聚酮化合物。这样，宿主基因组负责产生epothilone的区域被转座子标以标签，而且可以回收并作为探针使用来从亲代菌株中分离天然基因。由于它们与序列已知的生物合成基因诸如利福霉素或soraphen生物合成的PKS基因的序列同源性，可以分离得到合成聚酮化合物需要的、且与已知的PKS基因相似的PKS基因。利用同源性的合适分离技术，包括由DNA杂交进行常规文库筛选。
可以从在已知聚酮化合物合成中起作用的基因或其它DNA序列中得到的DNA片段，作为优选的探针分子使用。一种优选的探针分子包括编码soraphen PKS第四个模块的酮合酶结构域的1.2kb SmaⅠDNA片段(美国专利号5,716,849)，更优选的探针分子包括利福霉素PKS第一和第二模块的β-酮脂酰合酶结构域(Schupp等人，FEMS微生物通讯159:201-207(1998))。这些探针可以用来探测产生epothilone的微生物的基因库，以分离负责epothilone生物合成的PKS基因。
尽管众所周知分离PKS基因通常很困难，尽管预期分离epothilone生物合成基因将特别困难，通过使用此说明书中描述的方法，可以令人惊讶的从产生epothiloneA和B的微生物中克隆得到该聚酮化合物的生物合成基因。使用此说明书中描述的基因操作的方法和重组产物，可以修饰和在转基因宿主有机体中表达被克隆的PKS基因。
可以在异源宿主中表达分离的epothilone生物合成基因，以比天然宿主更高效的生产聚酮化合物。用于这些基因操作的技术，对于不同的可获得的宿主是特异的，而且本领域的技术人员是知道的。例如，使用诸如那些描述于McDaniel等人，科学(Science)262:1546-1550(1993)和Kao等人，科学265:509-512(1994)(此处引用作为参考)的技术，可以在链霉菌和其它放线菌中表达异源基因。还可见，Rowe等人，基因(Gene)216:215-226(1998)；Holmes等人，欧洲分子生物学杂志(EMBO Journal)12(8):3183-3191(1993)和Bibb等人，基因38:215-226(1985)，此处引用作为参考。
或者，还可以在其它宿主有机体诸如假单胞菌和大肠杆菌中表达负责聚酮化合物生物合成的基因，即epothilone生物合成基因。用于这些基因操作的技术对于不同的可获得的宿主是特异的，而且本领域的技术人员是知道的。例如，使用pT7-7载体(使用T7启动子)，已经在大肠杆菌中成功的表达了PKS基因。见，Tabor等人，美国国家科学院进展(Proc.Natl.Acad.Sci.USA)82:1074-1078(1985)，此处引用作为参考。此外，可以用表达载体pKK223-3和pKK223-2在大肠杆菌中表达异源基因，或以转录或以翻译融合方式，在tac或trc启动子之后。对于编码多ORF的操纵子的表达，最简单的步骤是以转录融合方式将操纵子插入到载体诸如pKK223-3中，使得能够使用异源基因的同类核糖体结合位点。用于在革兰氏阳性物种诸如芽孢杆菌中过量表达的技术，本领域的技术人员也是知道的，而且可以用于本发明中(Quax等人，工业微生物学基础和应用分子遗传学，编辑Baltz等人，美国微生物学学会，华盛顿(1993))。
其它可以与本发明的epothilone生物合成基因使用的表达系统，包括酵母和杆状病毒表达系统。见，例如，“重组蛋白质在酵母中的表达”，P.E.Sudbery，生物技术的流行观点(Curr.Opin.Biotechnol.)7(5):517-524(1996)；“在酵母中表达重组蛋白质的方法”，Mackay等人，编辑Paul R.Carey，蛋白质工程评论(Protein Eng.Des.)105-153，出版商Academic，圣地亚哥，加利福尼亚(1996)；“异源基因产物在酵母中的表达”，Pichuantes等人，编辑J.L.Cleland,C.S.Craik，蛋白质工程(Protein Eng.)129-161，出版商Wiley-Liss，纽约，纽约(1996)；WO98/27203；Kealey等人，美国国家科学院进展95:505-509(1998)；“昆虫细胞培养蛋白质生产的最新进展，生物工程挑战和意义”，Palomares等人，编辑EnriqueGalindo；Octavio T.Ramirez，高级生物工程(Adv.Bioprocess Eng.)第2卷，Invited Pap.Int.Symp.，第二版(1998)25-52，出版商Kluwer,Dordrecht,Neth；“杆状病毒表达载体”，Donald L.Jarvis,编辑Lois K.Miller，杆状病毒(Baculoviruses)389-431，出版商Plenum，纽约，纽约(1997)；“使用杆状病毒/昆虫表达系统生产异源蛋白质”，Grittiths等人，分子生物学方法(Methods Mol.Biol.)(N.J.Totowa)75(基础细胞培养方案(第二版))427-440(1997)；和“昆虫细胞表达技术”，Verne A.Luckow，蛋白质工程183-218，出版商Wiley-Liss，纽约，纽约(1996)；所有文献均此处引用作为参考。
在异源宿主中表达PKS基因的另一个要考虑的问题，是PKS酶的翻译后修饰(在它们能够合成聚酮化合物之前要磷酸泛酰巯基乙胺基化)需要酶。然而，负责第一类PKS酶的这种修饰的酶，磷酸泛酰巯基乙胺基(P-pant)转移酶在许多宿主诸如大肠杆菌中通常不存在。这个问题可以通过P-pant转移酶与PKS基因在异源宿主中的共表达解决，如Kealey等人，美国国家科学院进展95:505-509(1998)描述的，此处引用作为参考。
因此，为了生产聚酮化合物，选择宿主有机体的重要标准是它的操作容易，生长(即发酵)迅速，拥有适当的加工诸如翻译后修饰的分子机制，和对过量表达聚酮化合物不敏感。最优选的宿主有机体是放线菌诸如链霉菌菌株。其它优选的宿主有机体是假单胞菌和大肠杆菌。生产聚酮化合物的上述方法比制备该类化合物使用的现行技术具有明显优势。这些优势包括生产成本更低，生产更大量的化合物的能力，和生产优选生物学对映体的化合物的能力，这与有机合成生产不可避免的外消旋混合物相反。由异源宿主产生的化合物可以用于医疗(例如用epothilone治疗癌症)以及农业应用。
实验本发明将由下列详细实施例作为参考进一步描述。提供这些实施例只是为了例证，而并不是为了限制(除非特殊说明)。此处使用的常规重组DNA和分子克隆技术是本领域的技术人员熟知的，而且由Ausubel(编辑)，分子生物学现行方案(Current Protocols inMolecular Biology),John Wiley and Sons Inc.(1994)；T.Maniatis,E.F.Fritsch和J.Sambrook，分子克隆实验室手册(MolecularCloning:A Laboratory Manual)，冷泉港实验室，冷泉港，纽约(1989)；和T.J.Silhavy,M.L.Berman，和L.W.Enquist，基因融合实验(Experiments with Gene Fusion)，冷泉港实验室，冷泉港，纽约(1994)描述。
实施例1生产epothilone的纤维堆囊菌菌株的培养将纤维堆囊菌菌株90(DSM 6773,Deutsche Sammlung vonMikroorganismen und Zellkulturen,Braunschweig)在SolE培养基(0.35％葡萄糖，0.05％胰蛋白胨，0.15％MgSO4·7H2O,0.05％硫酸铵，0.1％CaCl2,0.006％K2HPO4,0.01％连二亚硫酸钠，0.0008％Fe-EDTA,1.2％HEPES,3.5％[vol/vo/]经灭菌的稳定期纤维堆囊菌培养物的上清液)pH调到7.4的琼脂平板上划线，并于30℃培养。挑取来自1cm2的细胞，接种到5ml G51t液体培养基(0.2％葡萄糖，0.5％淀粉，0.2％胰蛋白胨，0.1％probionS,0.05％CaCl2·2H2O,0.05％MgSO4·7H2O,1.2％HEPES,pH调到7.4)中，并于30℃振摇225rpm培养。4天后，将培养物转移到50ml G51t中，并如上所述培养5天。用此培养物接种500ml G51t，并如上所述培养6天。将此培养物以4000rpm离心10分钟，并将细胞沉淀重悬于50ml G51t中。
实施例2细菌人工染色体文库的构建为了构建Bac文库，将如实施例1所述培养的纤维堆囊菌细胞包埋于琼脂糖块中，裂解，并将释放出来的基因组DNA用限制酶HindⅢ部分消化。在琼脂糖凝胶上用脉冲电泳分离经消化的DNA。从琼脂糖凝胶中分离大片段DNA(大约90-150kb)，并连接到载体pBelobacⅡ中。pBelobacⅡ包含编码氯霉素抗性的基因、位于lacZ基因中在适当培养基上提供蓝白斑选择的多克隆位点、以及复制和在每个细胞中维持一个或两个质粒拷贝需要的基因。使用常规电穿孔技术，将连接混合物转化大肠杆菌DH10B电感受态细胞。将氯霉素抗性重组体(白斑、lacZ突变)菌落转移到带正电的384 3×3栅格的尼龙滤膜上。裂解克隆并将DNA交联到滤膜上。将同一克隆以液体培养物的形式保存于-80℃。
实施例3在纤维堆囊菌90的Bac文库中筛选第一类聚酮化合物合酶的相关序列使用常规Southern杂交步骤探测Bac文库滤膜。使用的探针编码利福霉素聚酮化合物合酶第一和第二模块的β-酮脂酰合酶结构域(Schupp等人，FEMS微生物学通讯159:210-207(1998))。以质粒pNE95(pNE95即Schupp等人(1998)中描述的粘粒2)作为模板，使用每个酮合酶结构域两侧的引物通过PCR产生探针DNA。从0.5％琼脂糖凝胶分离得到25ng PCR扩增的DNA，并使用随机引物标记试剂盒(Gibco-BRL,Methesda,MD，美国)按照供应商的指示用32P-dCTP标记。杂交于65℃进行36小时，并在高度严谨条件下洗膜(0.1×SSC和0.5％SDS于65℃20分钟3次)。将经标记的斑点暴露于磷光屏上，并在Phospholmager445SI上检测信号(屏和445SI购自MolecularDynamics)。结果某些Bac克隆与探针强杂交。选择这些克隆，并在5ml Luria肉汤(LB)培养基中于37℃培养过夜。使用典型的微量制备步骤，从感兴趣的Bac克隆中分离Bac DNA。将细胞重悬于200μl溶菌酶溶液(50mM葡萄糖，10mM EDTA,25mM Tris-HCl,5mg/ml溶菌酶)，在400μl裂解液(0.2N NaOH和2％SDS)中裂解，沉淀蛋白质(3.0M醋酸钾，用乙酸调到pH5.2)，并用异丙醇沉淀Bac DNA。将DNA重悬于20μl无核酸酶的蒸馏水中，用BamHⅠ(New England Biolabs,Inc.)限制酶消化，并在0.7％琼脂糖凝胶上分离。如上所述通过Southern杂交将凝胶转膜，并在如上所述的条件下用编码soraphen聚酮化合物合酶第四模块的酮合酶结构域的1.2kb SmaⅠDNA片段作为探针(见，美国专利号5,716,849)探测。观察到五个不同的杂交样式。每种选择一个克隆作为代表，并分别命名为pEPO15、pEPO20、pEPO30、pEPO31、和pEPO33。
实施例4pEPO15、pEPO20、pEPO30、pEPO31、和pEPO33的BamHⅠ片段的亚克隆用BamHⅠ消化五个选择的Bac克隆的DNA，并将随机片段亚克隆到pBluescriptⅡSK+(Stragagene)的BamHⅠ位点。选择插入片段大小在2-10kb之间的亚克隆，以测定插入片段两侧末端的序列，并且还用如上所述的1.2SmaⅠ探针探测。将与已知聚酮化合物合酶具有高度序列同源性和/或与soraphen酮合酶结构域强杂交的亚克隆用于基因破坏实验。
实施例5制备纤维堆囊菌菌株So ce90的链霉素抗性自发突变体取0.1ml纤维堆囊菌菌株So ce90在液体培养基G52-H(0.2％酵母提取物，0.2％脱脂大豆粉，0.8％土豆淀粉，0.2％葡萄糖，0.1％MgSO4·7H2O,0.1％CaCl2·2H2O,0.008％Fe-EDTA，用KOH调到pH7.4)中培养3天的培养物，铺在添加100μg/ml链霉素的SolE培养基琼脂平板上。平板于30℃培养2星期。生长在这种培养基上的菌落是链霉素抗性的突变体，将其在含链霉素的相同琼脂培养基上划线并再一次培养以纯化。选择这些链霉素抗性突变体中的一个，并命名为BCE28/2。
实施例6纤维堆囊菌BCE28/2中的基因破坏(使用亚克隆的BamHⅠ片段)分离如上所述由五个选择的Bac克隆产生的亚克隆的BamHⅠ插入片段，并连接到质粒pCIB132(见美国专利号5,716,849)的单一BamHⅠ位点。将携带插入片段的pCIB132衍生物转化含辅助质粒pUZ8的大肠杆菌ED8767(Hedges和Matthew，质粒(Plasmid)2:269-278(1979))。使用转化体作为接合实验中的供体，而纤维堆囊菌BCE28/2作为受体。为了接合，将5-10×109个在液体培养基G51b(G51b相当于G51t，其中用蛋白胨替代胰蛋白胨)中于30℃培养的纤维堆囊菌BCE28/2稳定期早期培养物(达到大约5×108细胞/ml)，以1∶1的细胞比率，与含pCIB132衍生物(携带亚克隆的BamHⅠ片段)和辅助质粒pUZ8的大肠杆菌ED8767的指数晚期培养物(在LB液体培养基中)混合。然后将混合的细胞以4000rpm离心10分钟，并重悬于0.5ml G51b培养基中。然后将此细胞悬浮液滴一滴在含50mg/l卡那霉素的So1E琼脂平板的中央铺板。于30℃培养24小时后收获细胞，并重悬于0.8mlG51b培养基，并将0.1-0.3ml此悬浮液在含腐草霉素(30mg/l)、链霉素(300mg/l)、和卡那霉素(50mg/l)的选择性So1E固体培养基上铺板。使用链霉素进行供体大肠杆菌菌株的负选择。于30℃培养8-12天后，用塑料环分离生长在这种选择培养基上的菌落，并在相同琼脂培养基上划线和培养以进行第二轮选择和纯化。于30℃培养7天后生长在此选择琼脂培养基上的菌落衍生的培养物，是通过携带亚克隆BamHⅠ片段的pCIB132衍生物的接合转移获得了腐草霉素抗性的纤维堆囊菌BCE28/2的转接合体。
通过Southern杂交检验证实pCIB132衍生的质粒通过同源重组整合到了纤维堆囊菌BCE28/2的染色体中。为了这个实验，应用Pospiech和Neumann,Trends Genet.11:217(1995)描述的方法，每种转移BamHⅠ片段的转接合体取5-10个分离完整DNA(于培养基G52-H中生长了3天的10ml培养物)。为了Southern杂交转膜，将如上所述分离的DNA用限制酶BglⅡ、ClaⅠ、或NotⅠ切割，并使用相应的BamHⅠ插入片段或pCIB132作为32P标记的探针。
实施例7分析整合的BamHⅠ片段对纤维堆囊菌在基因破坏后对epothilone生产的影响用无菌塑料环将生长在第二轮选择的选择性So1E平板表面上大约1cm-的转接合细胞(见实施例6)转移到10ml G52-H培养基中(装在50ml Erlenmeyer烧瓶中)。于30℃和180rpm培养3天后，将培养物转移到50ml G52-H培养基中(装在200ml Erlenmeyer烧瓶中)。于30℃和180rpm培养4-5天后，将10ml此培养物转移到50ml 23B3培养基(0.2％葡萄糖，2％土豆淀粉，1.6％脱脂大豆粉，0.0008％Fe-EDTA钠盐，0.5％HEPES(4-(2-羟乙基)-哌嗪-1-乙烷磺酸)，2％vol/vol聚苯乙烯树脂XAD16(Rohm和Haas)，用NaOH调pH到7.8)中(装在200ml Erlenmeyer烧瓶中)。
培养物于30℃和180rpm培养7天后，进行产生的epothilone的定量实验。将全部培养液通过150μm尼龙滤膜抽滤过滤。然后将留在滤膜上的树脂重悬于10ml异丙醇，并通过将悬浮液以180rpm振摇1小时进行抽提。从此悬浮液中取出1ml，并在Eppendorff微量离心机中以12,000rpm离心。通过HPLC和用UV_DAD检测仪(用Waters-Symetry C18层析柱进行HPLC并用0.02％磷酸60％-0％和乙腈40％-100％的梯度)于250nm检测的方法测定其中的epothiloneA和B的量。
以上述方法测试含有从pEPO15亚克隆的三种不同的BamHⅠ整合片段的转接合体，即含有质粒pEPO15-21的BamHⅠ片段的转接合体、含有质粒pEPO15-4-5的BamHⅠ片段的转接合体、和含有质粒pEPO15-4-1的BamHⅠ片段的转接合体。HPLC分析揭示所有转接合体不再产生epothiloneA和B。相反的，在整合有pEPO20、pEPO30、pEPO31、pEPO33的BamHⅠ片段的转接合体和亲本菌株BCE28/2中检测到epothiloneA和B浓度为2-4mg/l。
实施例8测定克隆的片段的核苷酸序列并构建毗连序列群A．质粒pEPO15-21的BamHⅠ插入片段从大肠杆菌DH10B[pEPO15-21]菌株中分离质粒DNA，并测定插入pEPO15-21的2.3kb BamHⅠ片段的核苷酸序列。自动DNA测序是在双链DNA模板上通过双脱氧核苷酸链终止方法进行的，使用应用Applied Biosystems377型测序仪。使用的引物是通用反向引物(5’GGA AAC AGC TAT GAC CAT G3’(SEQ ID NO:24))和通用正向引物(5’GTA AAA CGA CGG CCA GT3’(SEQ ID NO:25))。在随后几轮测序反应中，使用为先前测定的序列的3’末端设计而定制合成的寡核苷酸来延伸并连接毗连序列群。两条链都完全测序，而且每个核苷酸至少测序两次。使用3.0版Sequencher程序(Gene Codes Corporation)编辑核苷酸序列，并使用威斯康星遗传学计算机组程序(WisconsinGenetics Computer Group programs)分析。2213-bp插入片段的核苷酸序列相应于SEQ ID NO:1的核苷酸20779-22991。
B．质粒pEPO15-4-1的BamHⅠ插入片段从大肠杆菌DH10B[pEPO15-4-1]菌株中分离质粒DNA，并如上(A)所述测定插入pEPO15-4-1的3.9kb BamHⅠ片段的核苷酸序列。3909-bp插入片段的核苷酸序列相应于SEQ ID NO:1的核苷酸16876-20784。
C．质粒pEPO15-4-5的BamHⅠ插入片段从大肠杆菌DH10B[pEPO15-4-5]菌株中分离质粒DNA，并如上(A)所述测定插入pEPO15-4-5的2.3kb BamHⅠ片段的核苷酸序列。2233-bp插入片段的核苷酸序列相应于SEQ ID NO:1的核苷酸42528-44760。
实施例9含有epothilone生物合成基因的pEPO15中的DNA片段的亚克隆和排序将pEPO15用限制酶HindⅢ完全消化，并将产生的片段亚克隆到已经用HindⅢ切割且用小牛肠碱性磷酸酶去磷酸化的pBluescriptⅡSK-或pNEB193(New England Biolabs)中。产生了六个不同的克隆，并命名为pEPO15-NH1、pEPO15-NH2、pEPO15-NH6、pEPO15-NH24(都由pNEB193衍生)、和pEPO15-H2.7和pEPO15-H3.0(都由pBluescriptⅡSK-衍生)。
分离并DIG标记(非放射性DNA标记和检测系统，BoehringerMannheim)pEPO15-21的BamHⅠ插入片段，并在DNA杂交实验中在高度严谨条件下作为针对pEPO15-NH1、pEPO15-NH2、pEPO15-NH6、pEPO15-NH24、pEPO15-H2.7和pEPO15-H3.0的探针使用。对pEPO15-NH24检测到强杂交信号，说明pEPO15-NH24包含pEPO15-21。
如上所述分离并DIG标记pEPO15-4-1的BamHⅠ插入片段，并在DNA杂交实验中在高度严谨条件下作为针对pEPO15-NH1、pEPO15-NH2、pEPO15-NH6、pEPO15-NH24、pEPO15-H2.7和pEPO15-H3.0的探针使用。对pEPO15-NH24和pEPO15-H2.7检测到强杂交信号。由pEPO15-NH24和pEPO15-H2.7的一个末端得到的核苷酸序列信息还与先前测定的pEPO15-4-1的BamHⅠ插入片段的序列完全一致。这些实验证明pEPO15-4-1(含有一个内部的HindⅢ位点)与pEPO15-H2.7和pEPO15-NH24重叠，而且pEPO15-H2.7和pEPO15-NH24是以这个顺序相邻的。
如上所述分离并DIG标记pEPO15-4-5的BamHⅠ插入片段，并在DNA杂交实验中在高度严谨条件下作为针对pEPO15-NH1、pEPO15-NH2、pEPO15-NH6、pEPO15-NH24、pEPO15-H2.7和pEPO15-H3.0的探针使用。对pEPO15-NH2检测到强杂交信号，说明pEPO15-NH2包含pEPO15-4-5。
由pEPO15-NH2的两个末端和pEPO15-H24不与pEPO15-4-1重叠的那个末端得到核苷酸序列信息。朝向HindⅢ位点的PCR引物NH24末端“B”:GTGACTGGCGCCTGGAATCTGCATGAGC(SEQ ID NO:26)、NH2末端“A”:AGCGGGAGCTTGCTAGACATTCTGTTTC(SEQ ID NO:27)、和NH2末端“B”:GACGCGCCTCGGGCAGCGCCCCAA(SEQ ID NO:28)是根据这些序列设计的，并用于以pEPO15和在个别实验中以纤维堆囊菌So ce90基因组DNA作为模板的扩增反应。以NH24末端“B”和NH2末端“A”作为引物对，在两种模板中都发现特异扩增。将扩增物克隆到pBluescriptⅡSK-中并完全测序。扩增物的序列相同，而且还与pEPO15-NH24和pEPO15-NH2的末端序列完全一致，并在HindⅢ位点融合，确定了pEPO15-NH2和pEPO15-NH24的HindⅢ片段是以此顺序相邻的。
如上所述分离并DIG标记pEPO2.7的HindⅢ插入片段，并在DNA杂交实验中在高度严谨条件下作为针对经NotⅠ消化的pEPO15的探针使用。大小大约为9kb的一个NotⅠ片段显示强杂交信号，将其进一步亚克隆到已经用NotⅠ消化且用小牛肠碱性磷酸酶去磷酸化的pBluescriptⅡSK-中，从而产生pEPO15-N9-16。如上所述分离并DIG标记pEPO15-N9-16的NotⅠ插入片段，并在DNA杂交实验中在高度严谨条件下作为针对pEPO15-NH1、pEPO15-NH2、pEPO15-NH6、pEPO15-NH24、pEPO15-H2.7和pEPO15-H3.0的探针使用。对pEPO15-NH6、以及预期的克隆pEPO15-H2.7和pEPO15-NH24检测到强杂交信号。由pEPO15-NH6的两个末端和pEPO15-H2.7不与pEPO15-4-1重叠的那个末端得到核苷酸序列信息。设计朝向HindⅢ位点的PCR引物并用于以pEPO15和在个别实验中以纤维堆囊菌So ce90基因组DNA作为模板的扩增反应。以pEPO15-NH6末端“B”:CACCGAAGCGTCGATCTGGTCCATC(SEQ ID NO:29)和pEPO15-H2.7末端“A”:CGGTCAGATCGACGACGGGCTTTCC(SEQ ID NO:30)作为引物对，在两种模板中都发现特异扩增。将扩增物克隆到pBluescriptⅡSK-中并完全测序。扩增物的序列是相同的，而且还与pEPO15-NH6和pEPO15-NH2.7的末端序列完全一致，在HindⅢ位点融合，确定了pEPO15-NH6和pEPO15-NH2.7的HindⅢ片段是以此顺序相邻的。
所有这些实验，综合起来，确定了覆盖大约55kb区域并由pEPO15-NH6、pEPO15-H2.7、pEPO15-NH24、和pEPO15-NH2的HindⅢ插入片段以此顺序组成的HindⅢ片段毗连序列群。剩余的两个HindⅢ亚克隆的插入片段(命名为pEPO15-NH1和pEPO15-H3.0)未发现是这个毗连序列群的部分。
实施例10覆盖epothilone生物合成基因的亚克隆毗连序列群的进一步延伸将pEPO15-NH2插入片段下游末端产生、并因此代表实施例9中描述的亚克隆毗连序列群的下游末端的大约2.2kb BamHⅠ-HindⅢ片段分离出来、DIG标记、并用于针对经多种酶消化的pEPO15和pEPO15-NH2DNA的Southern杂交实验。总是发现在两种目的DNA之间强杂交条带大小相同，说明克隆到pEPO15中的纤维堆囊菌So ce9O基因组DNA片段结束于位于pEPO15-NH2下游末端的HindⅢ位点。
使用已确定的步骤，在pScosTriplex-Ⅱ(Ji等人，基因组学(Genomics)31:185-192(1996))中构建纤维堆囊菌So ce90的粘粒DNA文库。简单的说，将纤维堆囊菌So ce90的高分子量基因组DNA用限制酶Sau3AⅠ部分消化以提供平均大小为大约40kb的片段，并连接到经BamHⅠ和XbaⅠ消化的pScosTriplex-Ⅱ中。连接混合物用GigapackⅢXL(Stratagene)包装，并用于转染大肠杆菌XL1 BlueMR细胞。
用所述大约2.2kb的BamHⅠ-HindⅢ片段(pEPO15-NH2插入片段的下游末端产生、作为菌落杂交中的探针使用)筛选粘粒文库，选择到一个强杂交克隆，命名为pEPO4E7。
将pEPO4E7 DNA分离、用多种限制性内切酶消化、并用2.2kb BamHⅠ-HindⅢ片段在Southern杂交中探测。选择到一个大小大约为9kb的强杂交的NotⅠ片段，并亚克隆到pBluescriptⅡSK-以产生pEPO4E7-N9-8。进一步的Southern杂交实验揭示了pEPO4E7-N9-8大约9kb的NotⅠ插入片段与pEPO15-NH2的NotⅠ-HindⅢ片段超过6kb重叠，而剩余的大约3kb HindⅢ-NotⅠ将延伸实施例9中描述的亚克隆毗连序列群。然而，末端测序揭示pEPO4E7-N9-8插入片段的下游末端包含pScosTriplex-Ⅱ的BamHⅠ-NotⅠ多克隆位点，由此说明pEPO4E7的基因组DNA插入片段终止于HindⅢ-NotⅠ延伸片段中的Sau3AⅠ位点，而NotⅠ位点来自pScosTriplex-Ⅱ。
将pEPO4E7-N9-8的大约3kb HindⅢ-NotⅠ延伸亚片段衍生的大约1.6kb PstⅠ-SalⅠ片段(不含载体，只包含纤维堆囊菌So ce90衍生的序列)，作为针对实施例2中描述的细菌人工染色体文库的探针使用。除了先前分离的EPO15，还发现命名为EPO32的Bac克隆与该探针强杂交。将pEPO32分离、用多种限制性内切酶消化、并与大约1.6kb的PstⅠ-SalⅠ探针杂交。发现大小大约为13kb的HindⅢ-EcoRV片段与该探针强杂交，将其亚克隆到经HindⅢ和HincⅡ消化的pBluescriptⅡSK-中以产生pEPO32-HEV15。
根据pEPO15-NH2的下游末端序列和pEPO32-HEV15的上游(HindⅢ)末端序列设计寡核苷酸引物，并用于以pEPO4E7-N9-8为模板的测序反应。序列揭示了在常规限制性分析中检测不到的一个24bp小HindⅢ片段(EPO4E7-H0.02)的存在，分隔pEPO15-NH2下游末端的HindⅢ位点和pEPO32-HEV15上游末端的HindⅢ位点。
由此，描述于实施例9中的亚克隆毗连序列群延伸到了包括EPO4E7-H0.02的HindⅢ片段和pEPO32-HEV15的插入片段，而且由pEPO15-NH6、pEPO15-H2.7、pEPO15-NH24、pEPO15-NH2、EPO4E7-H0.02和pEPO32-HEV15的插入片段以此顺序组成。
实施例11测定覆盖epothilone生物合成基因的亚克隆毗连序列群的核苷酸序列如下测定实施例10中描述的亚克隆毗连序列群的核苷酸序列。
pEPO15-H2.7。从大肠杆菌DH10B[pEPO15-H2.7]菌株中分离质粒DNA，并测定pEPO15-H2.7的2.7kb BamHⅠ插入片段的核苷酸序列。自动DNA测序在双链DNA模板上通过双脱氧核苷酸链终止方法进行，使用Applied Biosystems377型测序仪。使用的引物是通用反向引物(5’GGA AAC AGC TAT GAC CAT G3’(SEQ ID NO:24))和通用正向引物(5’GTA AAA CGA CGG CCA GT3’(SEQ ID NO:25))。在随后几轮测序反应中，使用为先前测定的序列的3’末端设计而定制合成的寡核苷酸来延伸并连接毗连序列群。
pEPO15-NH6、pEPO15-NH24和pEPO15-NH2。分离这些质粒的HindⅢ插入片段并用于随机片段化，使用Hydroshear apparatus(GenomicInstrumentation Services,Inc.)以产生平均大小为1-2kb的片段。使用T4 DNA聚合酶和Klenow DNA聚合酶在三磷酸脱氧核苷酸存在时对片段进行末端修复，并用T4 DNA激酶在ribo-ATP存在时磷酸化。从琼脂糖凝胶中分离在1.5-2.2kb大小范围内的片段，并连接到经EcoRV切割和去磷酸化的pBluescriptⅡSK-中。使用通用反向引物和通用正向引物对随机亚克隆测序。
pEPO32-HEV15。将pEPO32-HEV15用HindⅢ和SspⅠ消化，分离得到包含纤维堆囊菌So ce90的大约13kb HindⅢ-EcoRV插入片段和pBluescriptⅡSK-的0.3kb HincⅡ-SspⅠ片段的大约13.3kb片段，并用HaeⅢ部分消化以产生平均大小为1-2kb的片段。从琼脂糖凝胶中分离在1.5-2.2kb大小范围内的片段，并连接到经EcoRV切割和去磷酸化的pBluescriptⅡSK-中。使用通用反向引物和通用正向引物对随机亚克隆测序。
分析了色谱图并用Phred、Phrap和Consed程序(Ewing等人，基因组研究(Genome Res.)8(3):175-185(1998)；Ewing等人，基因组研究8(3):186-194(1998)；Gordon等人，基因组研究8(3):195-202(1998))装配成毗连序列群。填充毗连序列群缺口，分析序列差异，并对低准确度区域使用定制设计的用于原始亚克隆或从随机亚克隆文库中选定克隆测序的寡核苷酸引物重新测序。两条链都完全测序，而且每个碱基对的累计Phred得分至少40(置信度99.99％)。
该68750bp毗连序列群的核苷酸序列显示如SEQ ID NO:1。
实施例12分析epothilone生物合成基因的核苷酸序列发现SEQ ID NO:1包含22个ORF，详见下表1表1
*在反向互补链上。编号根据SEQ ID NO:1。
epoA(SEQ ID NO:1的核苷酸7610-11875)编码EPOS A(SEQ IDNO:2)，一种第一类聚酮化合物合酶，由单个模块组成，并包含下列结构域β-酮脂酰合酶(KS)(SEQ ID NO:1的核苷酸7643-8920,SEQID NO:2的氨基酸11-437)、酰基转移酶(acyltransferase,AT)(SEQID NO:1的核苷酸9236-10201,SEQ ID NO:2的氨基酸543-864)、烯酰基还原酶(enoyl reductase,ER)(SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:2的氨基酸974-1273)、和酰基载体蛋白同源结构域(ACP)(SEQ ID NO:1的核苷酸11549-11764,SEQ ID NO:2的氨基酸1314-1385)。序列比较和基元分析(Haydock等人,FEBS通讯(FEBS Lett.)374:246-248(1995)；Tang等人，基因216:255-265(1998))揭示了EPOS A编码的AT对丙二酸单酰CoA是特异的。EPOS A应当是通过将乙酸根单元上载到多酶复合物上最终形成2-甲基噻唑环的部分(C26和C20)，而涉及epothilone生物合成的起始。
epoP(SEQ ID NO:1的核苷酸11872-16104)编码EPOS P(SEQ IDNO:3)，一种非核糖体肽合成酶，含有单个模块。EPOS P包含下列结构域●肽键形成结构域，由基元K(SEQ ID NO:3的氨基酸72-81[FPLTDIQESY]，相应于SEQ ID NO:1的核苷酸位置12085-12114)、基元L(SEQ ID NO:3的氨基酸118-125[VVARHDML]，相应于SEQ ID NO:1的核苷酸位置12223-12246)、基元M(SEQ ID NO:3的氨基酸199-212[SIDLINVDLGSLSI]，相应于SEQ ID NO:1的核苷酸位置12466-12507)、和基元O(SEQ ID NO:3的氨基酸353-363[GDFTSMVLLDI]，相应于SEQ ID NO:1的核苷酸位置12928-12960)描绘；●氨酰基腺苷酸形成结构域，由基元A(SEQ ID NO:3的氨基酸549-565[LTYEELSRRSRRLGARL]，相应于SEQ ID NO:1的核苷酸位置13516-13566)、基元B(SEQ ID NO:3的氨基酸588-603[VAVLAVLESGAAYVPI]，相应于SEQ ID NO:1的核苷酸位置13633-13680)、基元C(SEQ ID NO:3的氨基酸669-684[AYVIYTSGSTGLPKGV]，相应于SEQ ID NO:1的核苷酸位置13876-13923)、基元D(SEQ ID NO:3的氨基酸815-821[SLGGATE]，相应于SEQ ID NO:1的核苷酸位置14313-14334)、基元E(SEQ ID NO:3的氨基酸868-892[GQLYIGGVGLALGYWRDEEKTRKSF]，相应于SEQ ID NO:1的核苷酸位置14473-14547)、基元F(SEQ ID NO:3的氨基酸903-912[YKTGDLGRYL]，相应于SEQ ID NO:1的核苷酸位置14578-14607)、基元G(SEQ ID NO:3的氨基酸918-940[EFMGREDNQIKLRGYRVELGEIE]，相应于SEQ ID NO:1的核苷酸位置14623-14692)、基元H(SEQ ID NO:3的氨基酸1268-1274[LPEYMVP]，相应于SEQ ID NO:1的核苷酸位置15673-15693)、和基元I(SEQ ID NO:3的氨基酸1285-1297[LTSNGKVDRKALR]，相应于SEQ ID NO:1的核苷酸位置15724-15762)描绘；●一个未知结构域，插入在氨酰基腺苷酸形成结构域的基元G和H之间(SEQ ID NO:3的氨基酸973-1256，相应于SEQ ID NO:1的核苷酸位置14788-15639)；和●肽基载体蛋白同源结构域(PCP)，由基元J(SEQ ID NO:3的氨基酸1344-1351[GATSIHIV]，相应于SEQ ID NO:1的核苷酸位置15901-15924)描绘。
有人提出EPOS P涉及半胱氨酸的腺苷酸化活化(结合经活化的半胱氨酸成为氨酰基-S-PCP，在酶结合的半胱氨酸和EPOS A提供的乙酰基-S-ACP之间形成肽键)和通过分子内杂环化形成最初的噻唑啉环。EPOS P的未知结构域显示与来自芽孢杆菌物种的NAD(P)H氧化酶和还原酶有非常弱的同源性。由此，这个未知结构域和/或EPOS A的ER结构域可能涉及最初的2-甲基噻唑啉环氧化成2-甲基噻唑。
epoB(SEQ ID NO:1的核苷酸16251-21749)编码EPOS B(SEQ IDNO:4)，一种第一类聚酮化合物合酶，由单个模块组成，并包含下列结构域KS(SEQ ID NO:1的核苷酸16269-17546,SEQ ID NO:4的氨基酸7-432)、AT(SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:4的氨基酸539-859)、脱水酶(DH)(SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:4的氨基酸869-1037)、β-酮还原酶(KR)(SEQ IDNO:1的核苷酸20565-21302,SEQ ID NO:4的氨基酸1439-1684)和ACP(SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:4的氨基酸1722-1792)。序列比较和基元分析揭示了EPOS B编码的AT对甲基丙二酸单酰CoA是特异的。EPOS A可能通过催化2-甲基-4-噻唑羧基-S-PCP起始基团与甲基丙二酸单酰-S-ACP的Claisen样缩合和伴随的C17的β-酮基还原成烯酰基，而参与第一种聚酮化合物链的延伸。
epoC(SEQ ID NO:1的核苷酸21746-43519)编码EPOS C(SEQ IDNO:5)，一种第一类聚酮化合物合酶，由4个模块组成。第一个模块包含KS结构域(SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:5的氨基酸39-457)、丙二酸单酰CoA特异的AT(SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:5的氨基酸563-884)、KR(SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:5的氨基酸1147-1399)、和ACP(SEQ ID NO:1的核苷酸26045-26263,SEQ ID NO:5的氨基酸1434-1506)。这个模块引入乙酸基延伸单元(C14-C13)，并将位于C15的β-酮基还原成参与epothilone大内酯环最终内酯化的羟基基团。EPOS C的第二个模块包含KS(SEQ ID NO:1的核苷酸26318-27595,SEQ ID NO:5的氨基酸1524-1950)、丙二酸单酰CoA特异的AT(SEQID NO:1的核苷酸27911-28876,SEQ ID NO:5的氨基酸2056-2377)、KR(SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:5的氨基酸2645-2895)、和ACP(SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:5的氨基酸2932-3005)。这个模块引入乙酸基延伸单元(C12-C11)，并将位于C13的β-酮基还原成羟基基团。由此，epothilone的初生聚酮化合物链相当于epothiloneA，而在epothiloneB中的C12引入甲基侧链将需要PKS后续C-甲基转移酶活性。位于C13-C12的环氧环的形成也将需要PKS后续氧化步骤。EPOS C的第三个模块包含KS(SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:5的氨基酸3024-3449)、丙二酸单酰CoA特异的AT(SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:5的氨基酸3555-3876)、DH(SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:5的氨基酸3886-4048)、ER(SEQ IDNO:1的核苷酸35042-35902,SEQ ID NO:5的氨基酸4433-4719)、KR(SEQ ID NO:1的核苷酸35930-36667,SEQ ID NO:5的氨基酸4729-4974)、和ACP(SEQ ID NO:1的核苷酸36773-36991,SEQ ID NO:5的氨基酸5010-5082)。这个模块引入乙酸基延伸单元((C10-C9)，并完全还原位于C11的β-酮基。EPOS C的第四个模块包含KS(SEQ IDNO:1的核苷酸37052-38320,SEQ ID NO:5的氨基酸5103-5525)；甲基丙二酸单酰CoA特异的AT(SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:5的氨基酸5631-5951)、DH(SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:5的氨基酸5964-6132)、ER(SEQ ID NO:1的核苷酸41369-42256,SEQ ID NO:5的氨基酸6542-6837)、KR(SEQID NO:1的核苷酸42314-43048,SEQ ID NO:5的氨基酸6857-7101)、和ACP(SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:5的氨基酸7140-7211)。这个模块引入丙酸基延伸单元(C24和C8-C7)，并完全还原位于C9的β-酮基。
epoD(SEQ ID NO:1的核苷酸43524-54920)编码EPOS D(SEQ IDNO:6)，一种第一类聚酮化合物合酶，由2个模块组成。第一个模块包含KS结构域(SEQ ID NO:1的核苷酸43626-44885,SEQ ID NO:6的氨基酸35-454)、甲基丙二酸单酰CoA特异的AT(SEQ ID NO:1的核苷酸45204-46166,SEQ ID NO:6的氨基酸561-881)、KR(SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:6的氨基酸1143-1393)、和ACP(SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:6的氨基酸1430-1503)。这个模块引入丙酸基延伸单元(C23和C6-C5)，并将位于C7的β-酮基还原成羟基基团。第二个模块包含KS(SEQ ID NO:1的核苷酸48087-49361,SEQ ID NO:6的氨基酸1522-1946)、甲基丙二酸单酰CoA特异的AT(SEQ ID NO:1的核苷酸49680-50642,SEQ IDNO:6的氨基酸2053-2373)、DH(SEQ ID NO:1的核苷酸50670-51176,SEQ ID NO:6的氨基酸2383-2551)、甲基转移酶(MT,SEQ ID NO:1的核苷酸51534-52657,SEQ ID NO:6的氨基酸2671-3045)、KR(SEQID NO:1的核苷酸53697-54431,SEQ ID NO:6的氨基酸3392-3636)、和ACP(SEQ ID NO:1的核苷酸54540-54758,SEQ ID NO:6的氨基酸3673-3745)。这个模块引入丙酸基延伸单元(C21或C22和C4-C3)，并将位于C5的β-酮基还原成羟基基团。这个还原有些出乎意料，因为epothilone在C5含有酮基。然而，PKS模块推断的还原活性和最终聚酮化合物产物相应位置的氧化还原状态之间的这种不一致，已经在文献(见，例如，Schweche等人，美国国家科学院进展92:7839-7843(1995)和Schupp等人，FEMS微生物学通讯159:201-207(1998))中有报导。epothilone的一个重要特征是位于C4的偕甲基侧链基团(C21和C22)的存在。预测EPOS D的第二个模块将丙酸基单元引入到增长的聚酮化合物链，在C4提供一个甲基侧链。这个模块还包含整合到PKS中DH和KR结构域之间的甲基转移酶结构域，与在HMWP1耶尔森菌素合酶(A.M.Gehring,E.DeMoll,J.D.Fetherston,I.Mori,G.F.Mayhew,F.R.Blattner,C.T.Walsh，和R.D.Perry，鼠疫中的铁获取鼠疫耶尔森菌(Yersinia pestis)产生的耶尔森菌素的酶生源论中的模块原理(Iron acquisition in plague:modularlogic in enzymatic biogenesis of yersiniabactin by Yersiniapestis)化学生物学(Chem.Biol.)5,573-586,1998)中看到的排列相似。有人提出EPOS D中的这个MT结构域负责在C4引入该第二个甲基侧链基团(C21或C22)。
epoE(SEQ ID NO:1的核苷酸54935-62254)编码EPOS E(SEQ IDNO:7)，一种第一类聚酮化合物合酶，由一个模块组成，包含KS(SEQID NO:1的核苷酸55028-56284,SEQ ID NO:7的氨基酸32-450)、丙二酸单酰CoA特异的AT(SEQ ID NO:1的核苷酸56600-57565,SEQ IDNO:7的氨基酸556-877)、DH(SEQ ID NO:1的核苷酸57593-58087,SEQ ID NO:7的氨基酸887-1051)、很可能无功能的ER(SEQ ID NO:1的核苷酸59366-60304,SEQ ID NO:7的氨基酸1478-1790)、KR(SEQID NO:1的核苷酸60362-61099,SEQ ID NO:7的氨基酸1810-2055)、ACP(SEQ ID NO:1的核苷酸61211-61426,SEQ ID NO:7的氨基酸2093-2164)、和硫酯酶(TE)(SEQ ID NO:1的核苷酸61427-62254,SEQID NO:7的氨基酸2165-2439)。这个模块中的ER结构域包含活性位点基元，其中具有一些高度不平常的氨基酸取代，可能使得这个结构域无活性。该模块引入乙酸基延伸单元(C2-C1)，并将位于C3的β-酮基还原成烯酰基基团。epothilone在C3含有羟基基团，所以这个还原也显得多余，如在EPOS D的第二个模块中讨论的。EPOS E的TE结构域参与已增长的聚酮化合物链经由C1羧基基团和C15羟基基团之间的内酯化的释放和环化。
在被测序区域中，epoA上游检测到五个ORF。部分测序的orf1在序列数据库中没有同源序列。orf2(SEQ ID NO:1反向互补链上的核苷酸3171-1900)推导的蛋白质产物(Orf2,SEQ ID NO:10)显示与分枝杆菌(Mycobacterium)和天蓝色链霉菌(Streptomycescoelicolor)的假设ORF有强相似性，以及与不同细菌的羧肽酶和DD-肽酶有更远的相似性。orf3(SEQ ID NO:1的核苷酸3415-5556)推导出来的蛋白质产物，Orf3(SEQ ID NO:11)显示与不同细菌的Na/H反向转运蛋白有同源性。Orf3可能参与从生产菌株中运出epothilone。orf4和orf5在序列数据库中没有同源序列。
在被测序区域中，epoE下游检测到十一个ORF。epoF(SEQ ID NO:1的核苷酸62369-63628)编码EPOS F(SEQ ID NO:8)，一种推导出来的与细胞色素P450加氧酶有强序列相似性的蛋白质。EPOS F可能参与调整碳C12、C5、和/或C3的氧化还原状态。orf14(SEQ ID NO:1的核苷酸67334-68251)推导出来的蛋白质，Orf14(SEQ ID NO:22)显示与GI:3293544(没有推测出功能的来自天蓝色链霉菌的假设蛋白质)及GI:2654559(人胚肺蛋白质)有强相似性。它还更远的与来自热自养甲烷杆菌(Methanobacterium thermoautotrophicum)阳离子流出系统蛋白质类GI:2623026相关，所以它可能还参与从生产细胞中运出epothilone。剩余的ORF(orf6-orf13和orf15)与序列数据库中的条目不显示同源性。
实施例13epothilone生物合成基因的重组表达为了达到比纤维堆囊菌发酵更高的产量，在异源有机体中表达本发明的epothilone合酶基因。用于异源表达的一种优选的宿主是链霉菌，例如天然产生聚酮化合物放线菌紫素的天蓝色链霉菌。用于在此宿主中表达重组PKS基因的技术描述于McDaniel等人，科学262:1546-1550(1993)和Kao等人，科学265:509-512(1994)。还可见，Holmes等人，欧洲分子生物学杂志12(8):3183-3191(1993)和Bibb等人，基因38:215-226(1985)，以及美国专利号5,521,077,5,672,491，和5,712,146，此处引用作为参考。
根据某种方法，处理异源宿主菌株以包含放线菌紫素(act)基因簇的染色体删除。通过将DNA从温度敏感的供体质粒转移到大肠杆菌中的受体穿梭载体(McDaniel等人(1993)和Kao等人(1994))，以至于合酶基因通过载体中的同源重组实现转移，由此构建了含有本发明的epothilone合酶基因的表达质粒。或者，将epothilone合酶基因簇通过限制性片段连接导入载体。按照例如Kao等人(1994)描述的选择后，根据Hopwood等人，链霉菌的基因操作实验室手册(Genetic Manipulation of Streptomyces:A Laboratory Manual)(John Innes Foundation,Norwich,United Kingdom,1985)提出的方案，将来自载体的DNA导入act-的天蓝色链霉菌菌株(此处引用作为参考)。在R2YE培养基(Hopwood等人(1985))上培养重组链霉菌菌株并生产epothilone。或者，在其它宿主有机体诸如假单胞菌、芽孢杆菌、酵母、昆虫细胞和/或大肠杆菌中表达本发明的epothilone合酶基因。PKS和NRPS基因优选在大肠杆菌中使用pT7-7载体(使用T7启动子)表达。见Tabor等人，美国国家科学院进展82:1074-1078(1985)。在另一个实施方案中，使用表达载体pKK223-3和pKK223-2在大肠杆菌中，以转录或翻译融合形式在tac或trc启动子后表达PKS和NRPS基因。在天然不含有PKS酶翻译后修饰需要的磷酸泛酰巯基乙胺基(phosphopantetheinyl,P-pant)转移酶的异源宿主中表达PKS和NRPS基因，需要在宿主中共表达P-pant转移酶，如Kealey等人，美国国家科学院进展95:505-509(1998)描述的。
实施例14从生产菌株中分离epothiloneWO93/10121(此处引用作为参考)，在美国专利号5,639,949的实施例57中，在Gerth等人，抗生素杂志49:560-563(1996)，和在瑞士专利申请号396/98(申请日期1998年2月19)，和美国专利申请号09/248,910(还公开了优选的纤维堆囊菌突变菌株)中(此处引用作为参考)给出了培养、发酵、和用来分离聚酮化合物的提取步骤的实施例，对从天然和本发明的重组宿主中提取epothilone有用。下列步骤可以用来从人工培养的纤维堆囊菌菌株诸如So ce90中分离epothilone，而且还可以用来从重组宿主中分离epothilone。A:epothilone生产菌株的培养菌株纤维堆囊菌Soce-90或本发明的重组宿主菌株菌株保存液氮培养基预培养和中间培养G52主要培养1B12G52培养基酵母提取物，低盐(BioSpringer,Maison Alfort，法国) 2g/lMgSO4(7H2O) 1g/lCaCl2(2H2O) 1g/l脱脂大豆粉Soyamine 50T(Lucas Meyer，汉堡，德国) 2g/1土豆淀粉Noredux A-150(Blattmann,Waedenswil，瑞士) 8g/l葡萄糖，无水 2g/lEDTA-Fe(Ⅲ)-Na盐(8g/l)1ml/l用KOH调到pH 7.4灭菌20mins,120℃
1B12培养基土豆淀粉Noredux A-150(Blattmann,Waedenswil，瑞士) 20g/l脱脂大豆粉Soyamine 50T(Lucas Meyer，汉堡，德国) 11g/lEDTA-Fe(Ⅲ)-Na盐 8mg/l用KOH调到pH 7.8灭菌20mins,120℃添加环糊精和环糊精衍生物不同浓度的环糊精(Fluka,Buchs，瑞士，或Wacker Chemie,Munich,德国)单独灭菌并在接种前加到1B12培养基中。培养从液氮安瓿瓶中取1ml纤维堆囊菌Soce-90悬浮液，转移到10m1 G52培养基中(装在50mlErlenmeyer烧瓶中)并在摇床中于180rpm和30℃，25mm振幅培养3天。取5ml此培养物，加到45ml G52培养基中(装在200mlErlenmeyer烧瓶中)并在摇床中于180rpm和30℃，25mm振幅培养3天。然后取50ml此培养物，加到450ml G52培养基中(装在2升的Erlenmeyer烧瓶中)并在摇床中于180rpm和30℃，50mm振幅培养3天。维持培养每3-4天通过将50ml培养物加入到450ml G52培养基中(装在2升的Erlenmeyer烧瓶中)将培养物过量接种。所有实验和发酵都是用这种维持培养物开始进行的。烧瓶中的测试(ⅰ)在摇瓶中的预培养由500ml维持培养物开始，用50ml维持培养物接种1×450ml G52培养基并在摇床中180rpm和30℃，50mm振幅培养4天。(ⅱ)在摇瓶中的主要培养将添加了5g/14-吗啉代-丙烷磺酸(=MOPS)粉的40ml 1B12培养基(装在200ml Erlenmeyer烧瓶中)与5ml 10×浓缩环糊精溶液混和，接种10ml预培养物并在摇床中以180rpm和30℃，50mm振幅培养5天。发酵以10升、100升和500升的量进行发酵。20升和100升发酵作为中间培养步骤。预培养和中间培养接种10％(v/v)维持培养物，而主要培养接种20％(v/v)中间培养物。重要与振摇培养相反，发酵培养基的成分是根据最终培养液体积(包括接种液)计算的。如果，例如，18升培养基+2升接种液混和，那么称量用于20升的物质但是只配制成18升。摇瓶中的预培养物由500ml维持培养物开始，4×450ml G52培养基(装在2升Erlenmeyer烧瓶中)每个用50ml维持培养物接种并在摇床中以180rpm和30℃，50mm振幅培养4天。中间培养物，20升或100升20升装在总容量30升的发酵罐中的18升G52培养基接种2升预培养物。培养持续3-4天，条件是30℃,250rpm,0.5升空气每升液体每分钟，0.5巴超压，无pH控制。100升装在总容量150升的发酵罐中的90升G52培养基接种20升中间培养物中的10升。培养持续3-4天，条件是30℃,150rpm,0.5升空气每升液体每分钟，0.5巴超压，无pH控制。主要培养物，10升、100升或500升10升用于10升1B12的培养基物质在7升水中灭菌，然后加入1升无菌的10％2-(羟丙基)-β-环糊精溶液，并接种2升的20升中间培养物。主要培养的持续时间是6-7天，条件是30℃,250rpm,0.5升空气每升液体每分钟，0.5巴超压，pH用H2SO4/KOH控制到pH7.6+/-0.5(即pH7.1和8.1之间不用控制)。100升用于100升1B12的培养基物质在70升水中灭菌，然后加入10升无菌的10％2-(羟丙基)-β-环糊精溶液，并接种20升的20升中间培养物。主要培养的持续时间是6-7天，条件是30℃,200rpm,0.5升空气每升液体每分钟，0.5巴超压，pH用H2SO4/KOH控制到pH7.6+/-0.5。100升发酵的接种链示意图如下维持培养物(500ml)G52培养基 500升用于500升1B12的培养基物质在350升水中灭菌，然后加入50升无菌的10％2-(羟丙基)-β-环糊精溶液，并接种100升的100升中间培养物。主要培养的持续时间是6-7天，条件是30℃,120rpm,0.5升空气每升液体每分钟，0.5巴超压，pH用H2SO4/KOH控制到pH7.6+/-0.5。产物分析样品的制备将50ml样品与2ml聚苯乙烯树脂Amberlite XAD16(Rohm+Haas,Frankfurt，德国)混和，并于30℃以180rpm振摇一小时。然后使用150μm尼龙筛过滤树脂，用少量水清洗然后与滤器一起加到15ml Nunc试管中。将产物从树脂上洗脱向盛有滤器和树脂的试管中加入10ml异丙醇(＞99％)。然后，将密封的试管于室温在Rota-Mixer(Labinco BV，荷兰)上振摇30分钟。然后，离心出2ml液体，并使用移液器将上清液加到HPLC管中。HPLC分析层析柱Waters-Symetry C18,100×4mm,3.5μmWAT066220+预备柱3.9×20mmWAT054225溶剂A0.02％磷酸B乙腈(HPLC级)梯度41％B0-7min100％B 7.2-7.8min41％B8-12min烤箱温度30℃检测250nm,UV-DAD检测加样体积10μl保持时间EpoA:4.30min EpoB:5.38minB加入环糊精和环糊精衍生物对得到的epothilone浓度的影响环糊精是环形(α-1,4)连接的α-D-吡喃型葡萄糖的寡糖，具有相对疏水的中央腔和亲水的外部表面区域。
具体区分下述物质(括号中的数字给出了每个分子的葡萄糖单元的数目)α-环糊精(6)、β-环糊精(7)、γ-环糊精(8)、δ-环糊精(9)、ε-环糊精(10)、ζ-环糊精(11)、η-环糊精(12)、和θ-环糊精(13)。尤其优选δ-环糊精，特别是α-环糊精、β-环糊精或γ-环糊精，或者它们的混合物。
环糊精衍生物主要是上述环糊精的衍生物，尤其是α-环糊精、β-环糊精或γ-环糊精的衍生物，主要是那些有一个或几个甚至多达所有羟基(每个葡萄糖自由基有3个)被醚化或酯化的衍生物。醚主要是烷基醚，尤其是低级烷基，诸如甲基或乙基醚，还有丙基或丁基醚；芳香基羟烷基醚，诸如苯基羟基低级烷基醚，尤其苯基羟乙基醚；羟烷基醚，特别是羟基低级烷基醚，尤其是2-羟乙基醚，羟丙基醚诸如2-羟丙基醚或羟丁基醚诸如2-羟丁基醚；羧基烷基醚，特别是羧基低级烷基醚，尤其是羧甲基或羧乙基醚；衍生化羧基烷基醚，特别是衍生化羧基低级烷基醚，其中衍生化羧基是醚化或酰胺化的羧基(主要是氨基羰基、单-或二-低级烷基氨基羰基、吗啉代-、哌啶子基-、吡咯烷子基(pyrrolidino)-或哌嗪子基(piperazino)-羰基、或烷氧基羰基)，特别是低级烷氧羰基-低级烷基醚，例如甲氧羰基丙基醚或乙氧羰基丙基醚；磺基烷基醚，特别是磺基低级烷基醚，尤其是磺基丁基醚；其中一个或几个OH基团被具有下式的基团醚化的环糊精-O-[alk-O-]n-H其中alk是烷基，尤其是低级烷基，而且n是2-12的整数，尤其是2-5，特别是2或3；其中一个或几个OH基团被下式的基团醚化的环糊精 其中R’是氢、羟基、-O-(alk-O)z-H、-O-(alk(-R)-O-)p-H或-O-(alk(-R)-O-)q-alk-CO-Y；alk在所有情况中是烷基，尤其是低级烷基；m、n、p、q和z是1-12的整数，优选1-5，特别是1-3；Y是OR1或NR2R3，其中R1、R2和R3互相独立地是氢或低级烷基，或R2和R3与连接氮一起表示吗啉代、哌啶子基、吡咯烷子基或哌嗪子基；或者分支环糊精，其中存在与其它糖分子的醚化或乙缩醛，尤其是葡糖基-、二葡糖基-、(G2-β-环糊精)、麦芽糖基-或二麦芽糖基环糊精，或N-乙酰氨基葡糖基、氨基葡糖基-、N-乙酰氨基半乳糖基-或氨基半乳糖基-环糊精。
酯主要是烷酰酯，特别是低级烷酰酯，诸如环糊精的乙酰酯。
还有可能环糊精中同时存在两种或更多种不同的所述醚和酯基团。
两种或更多种所述环糊精和/或环糊精衍生物的混合物也可以存在。
特别优选的是α-、β-或γ-环糊精或它们的低级烷基醚，诸如甲基-β-环糊精或特别是2,6-二-O-甲基-β-环糊精，或特别是它们的羟基低级烷基醚，诸如2-羟丙基-α-、2-羟丙基-β-或2-羟丙基-γ-环糊精。
向培养基中加入环糊精或环糊精衍生物，其浓度优选0.02-10，更优选0.05-5，尤其0.1-4，例如0.1-2重量百分比(w/v)。
环糊精或环糊精衍生物是已知的或可以通过已知的方法生产(参阅例如US3,459,731；US4,383,992；US4,535,152；US4,659,696；EP0094157；EP0149197；EP0197571；EP0300526；EP0320032；EP0499322；EP0503710；EP0818469；WO90/12035；WO91/11200；WO93/19061；WO95/08993；WO96/14090；GB2,189,245；DE3,118,218；DE3,317,064及其中提到的有关环糊精或环糊精衍生物合成的参考文献或还有T.Loftsson和M.E.Brewster(1996)，环糊精的药物应用药物溶解性和稳定性(PharmaceuticalApplications of Cyclodextrins:Drug Solubilization andStabilisation)，药物科学杂志(Journal of PharmaceuticalScience)85(10):1017-1025；R.A.Rajewski和V.J.Stella(1996)，环糊精的药物应用体内药物投递(Pharmaceutical Applications ofCyclodextrins:In Vivo Drug Delivery)，药物科学杂志85(11):1142-1169)。
这里测试的所有环糊精衍生物可以从Fluka公司，Buchs,CH购得。测试在具有50ml培养容量的200ml摇瓶中进行。作为对照，使用装有吸附剂树脂Amberlite XAD-16(Rohm和Haas,Frankfurt，德国)和无任何吸附剂添加的摇瓶。培养5天后，通过HPLC测定下列epothilone的浓度表2
1)除Amberlite(％v/v)之外，所有百分比均为重量百分比(％w/v)。少数测试的环糊精(2,6-二-O-甲基-β-环糊精、甲基-β-环糊精)显示在使用的浓度对epothilone产量没有影响或有负面影响。1-2％2-羟基-丙基-β-环糊精和β-环糊精在实施例中与不使用环糊精生产相比将epothilone产量提高6-8倍。C有1％2-(羟丙基)-β-环糊精的10升发酵发酵在15升玻璃发酵罐中进行。培养基含有10g/l的2-(羟丙基)-β-环糊精(购自Wacker Chemie,Munich，德国)。发酵过程列于表3。发酵在6天后停止并开始提取。
表310升发酵的过程
D有1％2-(羟丙基)-β-环糊精的100升发酵发酵在150升发酵罐中进行。培养基含有10g/l的2-(羟丙基)-β-环糊精。发酵过程列于表4。发酵液在7天后收获并提取。
表4100升发酵的过程
E有1％2-(羟丙基)-β-环糊精的500升发酵发酵在750升发酵罐中进行。培养基含有10g/l的2-(羟丙基)-β-环糊精。发酵过程列于表5。发酵液在7天后收获并提取。
表5500升发酵的过程
F不加吸附剂的10升发酵比较实施例发酵在15升玻璃发酵罐中进行。培养基不含有任何环糊精或其它吸附剂。发酵过程列于表6。没有收获并提取发酵液。
表6不会吸附剂的10升发酵的过程
G逐步得到epothilone从500升主要培养物中分离从实施例2D的500升主要培养收获的体积是450升，并使用Westfalia澄清分离器(clarifying separator)SA-20-06型(rpm=6500)分离成液相(离心液+冲洗水=650升)和固相(细胞=大约15kg)。发现epothilone的主要部分在离心液中。离心所得的细胞浆含有＜15％的测定的epothilone部分，不再进一步处理。然后将650升离心液置于4000升搅拌器中，与10升Amberlite XAD-16(离心液∶树脂=65∶1)混合并搅拌。接触大约2小时后，在Heine溢流式离心机(overflow centrifuge)(桶容量40升；rpm=2800)中离心下树脂。从离心机上卸下树脂，并用10-15升去离子水清洗。解吸附作用通过两次搅拌树脂进行，每次均将一部分与30升异丙醇在30升玻璃搅拌器中搅拌30分钟。使用抽滤器从树脂中分离出异丙醇相。然后在真空操作的循环蒸发器(Schmid-Verdampfer)中加入15-20升水将异丙醇从混合异丙醇相中除去，并将得到的大约10升水相提取3次，每次用10升乙酸乙酯抽提。提取在30升玻璃搅拌器中进行。在真空操作的循环蒸发器(Schmid-Verdampfer)中将乙酸乙酯提取液浓缩至3-5升，之后在真空下在旋转蒸发器(Büchi型)中浓缩至干燥。得到50.2g乙酸乙酯提取物。将乙酸乙酯提取物溶解于500ml甲醇中，使用折叠滤器将不溶部分滤掉，并将溶液加到10kg Sephadex LH 20层析柱(Pharmacia,Uppsala，瑞典)(层析柱直径20cm，填充高度大约1.2m)上。用甲醇作为洗脱液进行洗脱。epothiloneA和B主要存在于级分21-23中(每份级分1升)。在真空中在旋转蒸发器中将这些级分浓缩至干燥(总重量9.0g)。然后将这些Sephadex洗脱峰级分(9.0g)溶解于92ml乙腈∶水∶二氯甲烷=50∶40∶2中，将溶液用折叠滤器过滤并加到RP层析柱(配备Prepbar200,Merck；2.0kgLiChrospher RP-18,Merch，颗粒大小12μm，柱直径10cm，填充高度42cm；Merch,Darmstadt，德国)上。用乙腈∶水=3∶7进行洗脱(流速=500ml/min；epothiloneA的保持时间=大约51-59min；epothiloneB的保持时间=大约60-69min)。级分用UV检测仪于250nm监测。在真空下在Büchi-Rotavapor旋转蒸发器上将级分浓缩至干燥。epothiloneA洗脱峰级分的重量是700mg，而且根据HPLC(外部标准)含量是75.1％。epothiloneB洗脱峰级分的重量是1980mg，而且含量根据HPLC(外部标准)是86.6％。最后，epothiloneA收集部分(700mg)由5ml乙酸乙酯∶甲苯=2∶3结晶，并产生170mgepothiloneA的纯晶体[含量根据HPLC(面积％)=94.3％]。epothiloneB收集部分(1980mg)由18ml甲醇进行结晶并产生1440mgepothiloneB的纯晶体[含量根据HPLC(面积％)=99.2％]。m.p.(epothiloneB)例如124-125℃；epothiloneB的1H-NMR数据500MHz-NMR，溶剂DMSO-d6。化学取代δ以相对于TMS的ppm表示。
s=单峰；d=双峰；m=多重峰δ(峰裂数)integral(H的数目)7.34(s) 16.50(s) 15.28(d) 15.08(d) 14.46(d) 14.08(m) 13.47(m) 13.11(m) 12.83(dd) 12.64(s) 32.36(m) 22.09(s) 32.04(m) 11.83(m) 11.61(m) 11.47-1.24(m) 41.18(s) 6
1.13(m) 21.06(d) 30.89(d+s，重叠) 6∑=41实施例15重组生产的epothilone的医疗用途包含epothilone的药物制剂或组合物可被用于例如癌症(诸如各种人实体瘤)的治疗。这些抗癌制剂包含(例如)活性剂量的epothilone和一种或几种有机或无机液体或固体的适合药用的载体物质。这些制剂以例如肠道、鼻腔、直肠、口腔、或非肠道方式，尤其是肌肉内或静脉内方式给药。活性成分的剂量取决于病人的体重、年龄和体格和药物动力学状况并进一步取决于给药方式。因为epothilone模仿紫杉醇的生物学效应，故epothilone有可能在使用紫杉醇治疗癌症的组合物和方法中取代紫杉醇。见，例如，美国专利号5,496,804,5,565,478，和5,641,803，引用这些作为参考。
例如，用于治疗时，提供的epothiloneB是2ml玻璃瓶单独包装，配制成1mg/ml清亮的、无色静脉注射液浓缩物。该物质用聚乙二醇300(PEG300)配制并用50或100ml 0.9％氯化钠注射液USP稀释以达到药物输液需要的最终浓度。以每21天一次30分钟静脉输液(三周治疗一次)六个循环或者每7天一次30分钟静脉输液(一周治疗一次)进行。
优选的，对于每周一次的治疗，剂量在大约0.1-大约6mg/m2，优选大约0.1-大约5mg/m2，更优选大约0.1-大约3mg/m2，甚至更优选0.1-1.7mg/m2，最优选大约0.3-大约1mg/m2之间；对于三周一次的治疗(每三周一次或每个第三周治疗)，剂量在大约0.3-18mg/m2，优选大约0.3-大约15mg/m2，更优选大约0.3-12mg/m2，甚至更优选大约0.3-大约7.5mg/m2，还要更优选大约0.3-大约5mg/m2，最优选大约1.0-3.0mg/m2之间。优选这个剂量通过对人进行2-180min，优选2-120min，更优选大约5-大约30min，最优选大约10-大约30min(如大约30min)完成静脉内(i.v.)给药。
虽然本发明已经由它们的特定实施方案作为参考描述过，明显的，众多的变化、修饰和实施方案是可能的，相应的，所有这些变化、修饰和实施方案将被看作是在本发明的精神和范围之内。
序列表<110>Novartis AG<120>用于epothilone生物合成的基因<130>4-30582A<140><141><160>30<170>PatentIn Ver.2.0<210>1<211>68750<212>DNA<213>纤维堆囊菌<400>1aagcttcgct cgacgccctc ttcgcccgcg ccacctctgc ccgtgtgctc gatgatggcc 60acggccgggc cacggagcgg catgtgctcg ccgaggcgcg cgggatcgag gacctccgcg 120ccctccgaga gcacctccgc atccaggaag gggggccgtc ctttcactgc atgtgcctcg 180gcgacctgac ggtggagctc ctcgcgcacg accagcccct cgcgtccatc agcttccacc 240atgcccgcag cctgaggcac cccgactgga cctcggacgc gatgctcgtc gacggccccg 300cgctcgtccg gtggctcgcc gcgcgcggcg cgccgggtcc cctccgcgag tacgaagagg 360agcgcgagcg agcccgaacc gcgcaggagg cgaggcgcct gtggctcgcg gccgcgccgc 420cctgcttcgc gcccgatctg ccccgcttcg aggacgacgc caacgggctg ccgctcggcc 480cgatgtcgcc tgaagtcgcc gaggccgagc ggcgcctccg cgcctcgtac gcgactcctg 540agctcgcctg tgccgcgctg ctcgcctggc tcgggacggg cgcgggtccc tggtccggat 600atcccgccta cgagatgctg ccagagaatc tgctcctcgg gtttggcctc ccgaccgcga 660tcgccgcggc ctccgcgccc ggcacatcgg aggccgctct ccgcggcgca gcgcggctgt 720tcgcctcctg ggaggtcgta tcgagcaaga agagccagct cggcaacatc cccgaagccc 780tgtgggagcg gctccggacg atcgtccgcg cgatgggcaa tgccgacaac ctctctcgct 840tcgagcgcgc cgaggcgatc gcggcggagg tgcgccgcct gcgcgcacag ccggcgccct 900tcgcggcggg cgccggcctg gcggtcgctg gggtctcctc gagcggccgg ctctcgggcc 960tcgtgaccga cggagacgca ttgtactccg gcgacggcaa cgacatcgtc atgttccaac 1020ccggccggat ctcgccggtc gtgctgctcg ccggaaccga tcccttcttc gagctcgcac 1080cgcccctcag ccagatgctc ttcgtcgcgc acgccaacgc gggcaccatc tccaaggtcc 1140tgacggaagg cagccccctc atcgtgatgg caagaaacca ggcgcgaccg atgagcctcg 1200tccacgctcg cgggttcatg gcgtgggtca accaggccat ggtgcccgac cccgagcggg 1260gcgcgccctt cgtcgtccag cgctcgacca tcatggaatt cgagcacccc acgcctcgtt 1320gtctccacga gcccgccggc agcgctttct ccctcgcctg cgacgaggag cacctctact 1380ggtgcgagct ttcggctggc cggctcgagc tatggcgcca cccgcaccac cgccccggcg 1440ccccgagccg cttcgcgtac ctcggcgagc accccattgc ggcgacctgg tacccctcgc 1500tcaccctcaa tgcgacccac gtgctgtggg ccgaccctga tcgcagggcc atcctcgggg 1560tcgacaagcg caccggcgta gagcccatcg tcctcgcgga gacgcgccat 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2460cgcgaggatg cgctcgcgca cgaacgcatc gaagccctgg tcggccgcgc gccccacgag 2520caccccctgc accaggctcc ccgtgttgta catccactgc gcccccggct gatgcatgag 2580cggcagcgtc ccgagccgcc ggatccactc gtctggcccg tgcggcgtca tcggcaccgg 2640ctgcgcgttg acgagcccga gctcgtcgat ggcccgctgg atcggcgacg atgcgtcgaa 2700cgagattccg aagcccatcg tgaacgtcat caggtcgcgc accgtgatcg gccgctccgc 2760gggcaccgtc tcgtcgatcg gaccatcgat gcgcgccagc accttccggt tcgcgagctc 2820cggcaaccat cggtcgacgg gggagtcgag gtcgagcttg ccttcctcga cgagcatcat 2880caccgccgtc gcggtgaccg ccttcgtcat cgaggcgatc cggaagatcg tgtcccgccg 2940catgggcgcg ctgccgccga gctcggtcac gcccaccgcg tccacgtgca cgtcgtcgcc 3000gcgcgcgacc agccagaccg ctcccggcat ctgccccgcc gccacctccg ccgccatcac 3060ctcgcgcgcg ggcgccagcg cgccggcccc cgcgtcctgc cctggctgcc cctcctcctc 3120ggccccaccc aacgcgcacc ccggcgccgc cacgctgatc aaagctccca taaactcccg 3180ccttctcatg accgtcgatg cctctccgag cgggggcgcc tgcccctgcc gagagcactg 3240actgcccgcg cccgaaaaaa tcatcggtgc cccgtcacga tcgccgccgg gcgtggctcc 3300gcccggccgc ccgctcgggc gcccgcccct ggacgagcaa agctcgcccg cccgcgctca 3360gcacgccgct tgccatgtcc ggcctgcacc cacaccgagg agccacccac cctgatgcac 3420ggcctcaccg agcggcaggt cctgctctcg ctcgtcaccc tcgcgctcat cctcgtgacc 3480gcgcgcgcct ccggcgagct cgcgcggcgg ctgcgccagc ccgaggtgct cggggagctc 3540ttcggcggcg tcgtgctggg cccctccgtc gtcggcgcgc tcgcgcccgg gttccatcga 3600gccctcttcc aggagccggc ggtcggggtc gtgctctcgg gcatctcctg gataggcgcg 3660ctcctcctgc tgctgatggc gggcatcgag gtcgacgtgg gcatcctgcg caaggaggcg 3720cgccccgggg cgctctcggc gctcggcgcg atcgcgcccc cgctcgcggc gggcgccgcc 3780ttctcggcgc tcgtgctcga tcggcccctt ccgagcggcc tcttcctcgg gatcgtgctc 3840tcggtgacgg cggtcagcgt gatcgcgaag gtgctgatcg agcgcgagtc gatgcgccgc 3900agctatgcgc aggtgacgct cgcggcgggg gtggtcagcg aggtcgctgc ctgggtgctc 3960gtcgcgatga cgtcgtcgag ctacggcgcg tcgcccgcgc tggcggtcgc ccggagcgcg 4020ctcctggcga gcggattctt gctgttcatg gtgctcgtcg ggcggcggct cacccacctc 4080gcgatgcgct gggtggccga cgcgacgcgc gtctccaagg gacaggtgtc gctcgtcctc 4140gtcctcacgt tcctggccgc ggcgctgacg cagcggctcg gcctgcaccc gctgctcggc 4200gcgttcgcgc tcggcgtgct gctcaacagc gctcctcgca ccaaccgccc tctcctcgac 4260ggcgtgcaga cgctcgtggc gggcctcttc gcgcctgtgt tcttcgtcct cgcgggcatg 4320cgcgtcgacg tgtcgcagct gcgcacgccg gcggcgtggg ggacggtcgc gttgctgctg 4380gcgaccgcga cggcggcgaa ggtcgtcccc gccgcgctcg gcgcgcggct cggcgggctc 4440aggggcagcg aggcggcgct cgtggcggtg ggcctgaaca tgaagggcgg cacggacctc 4500atcgtcgcga tcgtcggcgt cgagctcggg ctcctctcca acgaggctta tacgatgtac 4560gccgtcgtcg cgctggtcac ggtgaccgcc tcacccgcgc tcctcatctg gctcgagaaa 4620agggcgcctc cgacgcagga ggagtcggct cgcctcgagc gcgaggaggc cgcgaggcgc 4680gcgtacatcc ccggggtcga gcggatcctc gtcccgatcg tggcgcacgc cctgcccggg 4740ttcgccacgg acatcgtgga gagcatcgtc gcctccaagc gaaagctcgg cgagacggtc 4800gacatcacgg agctctccgt ggagcagcag gcgcccggcc catcgcgcgc cgcgggggag 4860gcgagccggg ggctcgcgag gctcggcgcg cgcctccgcg tcggcatctg gcggcaaagg 4920cgcgagctgc gcggctcgat ccaggcgatc ctgcgcgcct cgcgggatca cgatctgctc 4980gtgatcggcg cgcgatcgcc ggcgcgcgcg cgcggaatgt cgttcggtcg cctgcaggac 5040gcgatcgtcc agcgggccga gtccaacgtg ctcgtcgtgg tgggcgaccc tccggcggcg 5100gagcgcgcct ccgcgcggcg gatcctcgtc ccgatcatcg gcctcgagta ctccttcgcc 5160gccgccgatc tcgcggccca cgtggcgctg gcgtgggacg ccgagctcgt gctgctcagc 5220agcgcgcaga ccgatccggg cgcggtcgtc tggcgcgatc gcgagccatc ccgggtgcgc 5280gcggtggcgc ggagcgtcgt cgacgaggcg gtcttccggg ggcgccggct cggcgtgcgc 5340gtctcgtcgc gcgtgcacgt gggcgcgcac ccgagcgacg agataacgcg ggagctcgcg 5400cgcgccccgt acgatctgct cgtgctcgga tgctacgacc atgggccgct cggccggctc 5460tacctcggca gcacggtcga gtcggtggtg gtccggagcc gggtgccggt cgcgttgctc 5520gtcgcgcatg gagggactcg agagcaggtg aggtgaggct tccaccgcgc tcgcccgtga 5580ggaagcgagc gcccggctct gccgacgatc gtcactcccg gtccgtgtag gcgatcgtgc 5640tgagcagcgc gttctccgcc tgacgcgagt cgagccgggt atgctgcacg acgatggggg 5700cgtccgattc gatcacgctg gcatagtccg tatcgcgcgg gatcggctcg ggttcggtca 5760gatcgttgaa ccggacgtgc cgggtgcgcc tcgctggaac ggtcacccgg taaggcccgg 5820cggggtcgcg gtcgctgaag taaacggtga tggcgacctg cgcgtcccgg tccgacgcat 5880tcaacaggca ggccgtctca tggctcgtca tctgcggctc aggtccgttg ctcccgcctg 5940ggatgtagcc ctctgcgatt gcacagcgcg tccgcccgat cggcttgtcc atgtgtcctc 6000cctcctggct cctctttggc agcctccctc tgctgtccag gagcgatggc ctcttcgctc 6060gacgcgctcg gggatccatg gctgaggatc ctcgccgagc gctccctgcc gaccggcgcg 6120ccgagcgccg acgggctttg aaagcgcgcg accggccagc ccggacgcgg gcccgagagg 6180gacagtgggt ccgccgtgaa gcagagaggc gatcgaggtg gtgagatgaa acacgtcgac 6240acgggccgac gattcggccg ccggacaggg cacacgctcg gtcttctcgc gagcatggcg 6300ctcgccggct gcggcggtcc gagcgagaaa accgtgcagg gcacgcggct cgcgcccggc 6360gccgatgcgc gcgtcaccgc cgacgtcgac cccgacgccg cgaccacgcg gccggcggtg 6420gacgccgttc acctctcgcc gcccgagcgg ctcgaggccg gcagcgagcg gttcgtcgtc 6480tggcagcgtc cgagccccga gcccccgtgg cgacgggccg gagtgctcga ctacaatgct 6540gacagccgaa gaggcaagct ggccgagacg accgtgccgt atgccaactt cgagctgctc 6600atcaccgccg agaagcagag cagccctcag tcgccatcgt ctgccgccgt catcgggccg 6660acgtctgtcg ggtgacatcg cgctatcagc agcgctgagc ccgccagcag gccccagggc 6720cctgcctcga tggccttccc catcacccct gcgcactcct ccagcgacgg ccgcgcagcg 6780acggccgcgt ccaagcaacc gccgtgccgg cgcggctcca cgcgcgcgac aggcgagcgt 6840cctggcgcgg cctgcgcatc gctggaagga tcggcggagc atggatagag aatcgaggat 6900cgcgatcttt gttgccatcg cagccaacgt ggcgatcgcg gcggtcaagt tcatcgccgc 6960cgccgtgacc ggcagctcgg cgaggcgttt gccgacttcg gcggcgtccc gcgcgtgctg 7020ctctacgaca acctcaagag cgccgtcgtc gagcgccacg gcgacgcgat ccggttccac 7080cccacgctgc tggctctgtc ggcgcattac cgcttcgagc cgcgccccgt cgccgtcgcc 7140cgcggcaacg agaagggccg cgtccagcgc gccatcacgg cgtggacgac atggcgcgga 7200aacgtcgtcg taaccgccca gcaatgtcat gggaatggcc ccttgaaatg gccccttgag 7260ggggctggcc ggggtcgacg atatcgcgcg atctccccgt caattcccga tggtaaaaga 7320aaaatttgtc atagatcgta agctgtgata gtggtctgtc ttacgttgcg tcttccgcac 7380ctcgagcgag ttctctcgga taactttcaa tttttccgag gggggcttgg tctctggttc 7440ctcaggaagc ctgatcggga cgagctaatt cccatccatt tttttgaggc tctgctcaaa 7500gggattagat cgagtgagac agttcttttg cagtgcgcga agaacctggg cctcgaccgg 7560aggacgatcg acgtccgcga gcgggtcagc cgctgaggat gtgcccgtcg tggcggatcg 7620tcccatcgag cgcgcagccg aagatccgat tgcgatcgtc ggagcgagtt gccgtctgcc 7680cggtggcgtg atcgatctga gcgggttctg gacgctcctc gagggctcgc gcgacaccgt 7740cgggcgagtc cccgccgaac gctgggatgc agcagcgtgg tttgatcccg accccgatgc 7800cccggggaag acgcccgtta cgcgcgcatc tttcctgagc gacgtagcct gcttcgacgc 7860ctccttcttc ggcatctcgc ctcgcgaagc gctgcggatg gaccctgcac atcgactctt 7920gctggaggtg tgctgggagg cgctggagaa cgccgcgatc gctccatcgg cgctcgtcgg 7980tacggaaacg ggagtgttca tcgggatcgg cccgtccgaa tatgaggccg cgctgccgca 8040agcgacggcg tccgcagaga tcgacgctca tggcgggctg gggacgatgc ccagcgtcgg 8100agcgggccga atctcgtatg ccctcgggct gcgagggccg tgtgtcgcgg tggatacggc 8160ctattcgtcc tcgctggtgg ccgttcatct ggcctgtcag agcttgcgct ccggggaatg 8220ctccacggcc ctggctggtg gggtatcgct gatgttgtcg ccgagcaccc tcgtgtggct 8280ctcgaagacc cgggcgctgg ccagggacgg tcgctgcaag gcattttcgg cggaggccga 8340tgggttcgga cgaggcgaag ggtgcgccgt cgtggtcctc aagcggctca gtggagcccg 8400cgcggacggc gatcggatat tggcggtgat tcgaggatcc gcgatcaatc acgacggtgc 8460gagcagcggt ctgaccgtgc cgaacgggag ctcccaagaa atcgtgctga aacgggccct 8520ggcggacgca ggctgcgccg cgtcttcggt gggttatgtc gaggcacacg gcacgggcac 8580gacgcttggt gaccccatcg aaatccaagc tctgaatgcg gtatacggcc tcgggcgaga 8640tgtcgccacg ccgctgctga tcgggtcggt gaagaccaac cttggccatc ctgagtatgc 8700gtcggggatc actgggctgc tgaaggtcgt cttgtccctt cagcacgggc agattcctgc 8760gcacctccac gcgcaggcgc tgaacccccg gatctcatgg ggtgatcttc ggctgaccgt 8820cacgcgcgcc cggacaccgt ggccggactg gaatacgccg cgacgggcgg gggtgagctc 8880gttcggcatg agcgggacca acgcgcacgt ggtgctggaa gaggcgccgg cggcgacgtg 8940cacaccgccg gcgccggagc gaccggcaga gctgctggtg ctgtcggcaa ggaccgcgtc 9000agccctggat gcacaggcgg cgcggctgcg cgaccatctg gagacctacc cttcgcagtg 9060tctgggcgat gtggcgttca gtctggcgac gacgcgcagc gcgatggagc accggctcgc 9120ggtggcggcg acgtcgaggg aggggctgcg ggcagccctg gacgctgcgg cgcagggaca 9180gacgtcgccc ggtgcggtgc gcagtatcgc cgattcctca cgcggcaagc tcgcctttct 9240cttcaccgga cagggggcgc agacgctggg catgggccgt gggctgtacg atgtatggtc 9300cgcgttccgc gaggcgttcg acctgtgcgt gaggctgttc aaccaggagc tcgaccggcc 9360gctccgcgag gtgatgtggg ccgaaccggc cagcgtcgac gccgcgctgc tcgaccagac 9420agccttcacc cagccggcgc tgttcacctt cgaatatgcg ctcgccgcgc tgtggcggtc 9480gtggggtgta gagccggagt tggtcgccgg ccatagcatc ggtgagctgg tggctgcctg 9540cgtggcgggc gtgttctcgc ttgaggacgc ggtgttcctg gtggctgcgc gcgggcgcct 9600gatgcaggcg ctgccggccg gcggggcgat ggtgtcgatc gaggcgccgg aggccgatgt 9660ggctgctgcg gtggcgccgc acgcagcgtc ggtgtcgatc gccgcggtca acgctccgga 9720ccaggtggtc atcgcgggcg ccgggcaacc cgtgcatgcg atcgcggcgg cgatggccgc 9780gcgcggggcg cgaaccaagg cgctccacgt ctcgcatgcg ttccactcac cgctcatggc 9840cccgatgctg gaggcgttcg ggcgtgtggc cgagtcggtg agctaccggc ggccgtcgat 9900cgtcctggtc agcaatctga gcgggaaggc ttgcacagac gaggtgagct cgccgggcta 9960ttgggtgcgc cacgcgcgag aggtggtgcg cttcgcggat ggagtgaagg cgctgcacgc 10020ggccggtgcg ggcaccttcg tcgaggtcgg tccgaaatcg acgctgctcg gcctggtgcc 10080tgcctgcatg ccggacgccc ggccggcgct gctcgcatcg tcgcgcgctg ggcgtgacga 10140gccggcgacc gtgctcgagg cgctcggcgg gctctgggcc gtcggtggcc tggtctcctg 10200ggccggcctc ttcccctcag gggggcggcg ggtgccgctg cccacgtacc cttggcagcg 10260cgagcgctac tggatcgaca cgaaagccga cgacgcggcg cgtggcgacc gccgtgctcc 10320gggagcgggt cacgacgagg tcgaggaggg gggcgcggtg cgcggcggcg accggcgcag 10380cgctcggctc gaccatccgc cgcccgagag cggacgccgg gagaaggtcg aggccgccgg 10440cgaccgtccg ttccggctcg agatcgatga gccaggcgtg cttgatcacc tcgtgcttcg 10500ggtcacggag cggcgcgccc ctggtctggg cgaggtcgag atcgccgtcg acgcggcggg 10560gctcagcttc aatgatgtcc agctcgcgct gggcatggtg cccgacgacc tgccgggaaa 10620gcccaaccct ccgctgctgc tcggaggcga gtgcgccggg cgcatcgtcg ccgtgggcga 10680gggcgtgaac ggcctcgtgg tgggccaacc ggtcatcgcc ctttcggcgg gagcgtttgc 10740tacccacgtc accacgtcgg ctgcgctggt gctgcctcgg cctcaggcgc tctcggcgat 10800cgaggcggcc gccatgcccg tcgcgtacct gacggcatgg tacgcgctcg acagaatagc 10860ccgccttcag ccgggggagc gggtgctgat ccatgcggcg accggcgggg tcggtctcgc 10920cgcggtgcag tgggcgcagc acgtgggagc cgaggtccat gcgacggccg gcacgcccga 10980gaaacgcgcc tacctggagt cgctgggcgt gcggtatgtg agcgattccc gctcggaccg 11040gttcgtcgcc gacgtgcgcg cgtggacggg cggcgaggga gtagacgtcg tgctcaactc 11100gctctcgggc gagctgatcg acaagagttt caatctcctg cgatcgcacg gccggtttgt 11160ggagctcggc aagcgcgact gttacgcgga taaccagctc gggctgcggc cgttcctgcg 11220caatctctcc ttctcgctgg tggatctccg ggggatgatg ctcgagcggc cggcgcgggt 11280ccgtgcgctc ttggaggagc tcctcggcct gatcgcggca ggcgtgttca cccctccccc 11340catcgcgacg ctcccgatcg cccgtgtcgc cgatgcgttc cggagcatgg cgcaggcgca 11400gcatcttggg aagctcgtac tcacgctggg tgacccggag gtccagatcc gtattccaac 11460ccacgcaggc gccggcccgt ccaccgggga tcgggacctg ctcgacaggc tcgcgtcagc 11520tgcgccggcc gcgcgcgcgg cggcgctgga ggcgttcctc cgtacgcagg tctcgcaggt 11580gctgcgcacg cccgaaatca aggtcggcgc ggaggcgctg ttcacccgcc tcggcatgga 11640ctcgctcatg gccgtggagc tgcgcaatcg tatcgaggcg agcctcaagc tgaagctgtc 11700gacgacgttc ctgtccacgt cccccaatat cgccttgttg gcccaaaacc tgttggatgc 11760tctcgccaca gctctctcct tggagcgggt ggcggcggag aacctacggg caggcgtgca 11820aaacgacttc gtctcatcgg gcgcagatca agactgggaa atcattgccc tatgacgatc 11880aatcagcttc tgaacgagct cgagcaccag ggtatcaagc tggcggccga tggggagcgc 11940ctccagatac aggcccccaa gaacgccctg aacccgaacc tgctcgctcg aatctccgag 12000cacaaaagca cgatcctgac gatgctccgt cagagactcc ccgcagaatc catcgtgccc 12060gccccagccg agcggcacgc tccgtttcct ctcacagaca tccaagaatc ctactggctg 12120ggccggacag gagcgtttac ggtccccagc gggatccacg cctatcgcga atacgactgt 12180acggatctcg acgtgccgag gctgagccgc gcctttcgga aagtcgtcgc gcggcacgac 12240atgcttcggg cccacacgct gcccgacatg atgcaggtga tcgagcctaa agtcgacgcc 12300gacatcgaga tcatcgatct gcgcgggctc gaccggagca cacgggaagc gaggctcgtg 12360tcgttgcgag atgcgatgtc gcaccgcatc tatgacaccg agcgccctcc gctctatcac 12420gtcgtcgccg ttcggctgga cgagcggcaa acccgtctcg tgctcagtat cgatctcatt 12480aacgttgacc taggcagcct gtccatcatc ttcaaggact ggctcagctt ctacgaagat 12540cccgagacct ctctccctgt cctggagctc tcgtaccgcg attatgtact cgcgctggag 12600tctcgcaaga agtctgaggc gcatcaacga tcgatggatt actggaagcg gcgcatcgcc 12660gagctcccac ctccgccgac gcttccgatg aaggccgatc catctaccct gaaggagatc 12720cgcttccggc acacggagca atggctgccg tcggactcct ggggtcgatt gaagcggcgt 12780gtcggggagc gcgggctgac cccgacgggc gtcatcctgg ctgcattttc cgaggtgatc 12840gggcgctgga gcgcgagccc ccggtttacg ctcaacataa cgctcttcaa ccggctcccc 12900gtccatccgc gcgtgaacga tatcaccggg gacttcacgt cgatggtcct cctggacatc 12960gacaccactc gcgacaagag cttcgaacag cgcgctaagc gtattcaaga gcagctgtgg 13020gaagcgatgg atcactgcga cgtaagcggt atcgaggtcc agcgagaggc cgcccgggtc 13080ctggggatcc aacgaggcgc attgttcccc gtggtgctca cgagcgcgct taaccagcaa 13140gtcgttggtg tcacctcgtt gcagaggctc ggaactccgg tgtacaccag cacgcagact 13200cctcagctgc tgctggatca tcagctctac gagcacgatg gggacctcgt cctcgcgtgg 13260gacatcgtcg acggagtgtt cccgcccgac cttctggacg acatgctcga agcgtacgtc 13320gtttttctcc ggcggctcac tgaggaacca tggggtgaac aggtgcgctg ttcgcttccg 13380cctgcccagc tagaagcgcg ggcgagcgca aacgcgacca acgcgctgct gagcgagcat 13440acgctgcacg gcctgttcgc ggcgcgggtc gagcagctgc ccatgcagct cgccgtggtg 13500tcggcgcgca agacgctcac gtacgaagag ctttcgcgcc gttcgcggcg acttggcgcg 13560cggctgcgcg agcagggggc acgcccgaac acattggtcg cggtggtgat ggagaaaggc 13620tgggagcagg ttgtcgcggt tctcgcggtg ctcgagtcag gcgcggccta cgtgccgatc 13680gatgccgacc taccggcgga gcgtatccac tacctcctcg atcatggtga ggtaaagctc 13740gtgctgacgc agccatggct ggatggcaaa ctgtcatggc cgccggggat ccagcggctg 13800ctcgtgagcg aggccggcgt cgaaggcgac ggcgaccagc ctccgatgat gcccattcag 13860acaccttcgg atctcgcgta tgtcatctac acctcgggat ccacagggtt gcccaagggg 13920gtgatgatcg atcatcgggg tgccgtcaac accatcctgg acatcaacga gcgcttcgaa 13980atagggcccg gagacagggt gctggcgctc tcctcgctga gcttcgatct ctcggtctat 14040gatgtgttcg ggatcctggc ggcgggcggt acgatcgtgg tgccggacgc gtccaagctg 14100cgcgatccgg cgcattgggc agagttgatc gaacgagaga aggtgacggt gtggaactcg 14160gtgccggcgc tgatgcggat gctcgtcgag cattttgagg gtcgccccga ttcgctcgct 14220aggtctctgc ggctttcgct gctgagcggc gactggatcc cggtgggcct gcctggcgag 14280ctccaggcca tcaggcccgg cgtgtcggtg atcagcctgg gcggggccac cgaagcgtcg 14340atctggtcca tcgggtaccc cgtgaggaac gtcgacctat cgtgggcgag catcccctac 14400ggccgtccgc tgcgcaacca gacgttccac gtgctcgatg aggcgctcga accgcgcccg 14460gtctgggttc cggggcaact ctacattggc ggggtcgggc tggcactggg ctactggcgc 14520gatgaagaga agacgcgcaa gagcttcctc gtgcaccccg agaccgggga gcgcctctac 14580aagaccggcg atctgggccg ctacctgccc gatggaaaca tcgagttcat ggggcgtgag 14640gacaaccaaa tcaagcttcg cggataccgc gttgagctcg gggaaatcga ggaaacgctc 14700aagtcgcatc cgaacgtacg cgacgcggtg attgtgcccg tcgggaacga cgcggcgaac 14760aagctccttc tagcctatgt ggtcccggag ggcacacgga gacgcgctgc cgagcaggac 14820gcgagcctca agaccgagcg gatcgacgcg agagcacacg ccgccgaagc ggacggcttg 14880agcgacggcg agagggtgca gttcaagctc gctcgacacg gactccggag ggacctggac 14940ggaaagcccg tcgtcgatct gaccgggcag gatccgcggg aggcggggct ggacgtctac 15000gcgcgtcgcc gtagcgtccg aacgttcctt gaggccccga ttccgtttgt tgagtttggt 15060cgattcctga gctgcttgag cagcgtggag cccgacggcg cgacccttcc caaattccgt 15120tatccatcgg cgggcagcac gtacccggtg caaacctacg cgtatgtcaa atccggccgc 15180atcgagggcg tggacgaggg cttctattat taccacccgt tcgagcaccg tttgctgaag 15240ctctccgatc acgggatcga gcgcggagcg cacgttcggc aaaacttcga cgtgttcgat 15300gaagcggcgt tcaacctcct gttcgtgggc aggatcgacg ccatcgagtc gctgtatgga 15360tcgtcgtcgc gagaattttg cctgctggag gccggatata tggcgcagct cctgatggag 15420caggcgcctt cctgcaacat cggcgtctgt ccggtggggc aattcaattt tgaacaggtt 15480cggccggttc tcgacctgcg acattcggac gtttacgtgc acggcatgct gggcgggcgg 15540gtagacccgc ggcagttcca ggtctgtacg ctcggtcagg attcctcacc gaggcgcgcc 15600acgacgcgcg gcgcccctcc cggccgcgag cagcacttcg ccgatatgct tcgcgacttc 15660ttgaggacca aactacccga gtacatggtg cctacagtct tcgtggagct cgatgcgttg 15720ccgctgacgt ccaacggcaa ggtcgatcgt aaggccctgc gcgagcggaa ggatacctcg 15780tcgccgcggc attcggggca cacggcgcca cgggacgcct tggaggagat cctcgtcgcg 15840gtcgtacggg aggtgctcgg gctggaggtg gtcgggctcc agcagagctt cgtcgatctt 15900ggtgcgacat cgattcacat cgttcgcatg aggagcctgt tgcagaagag gctggatagg 15960gagatcgcca tcaccgagtt gttccagtac ccgaacctcg gctcgctggc gtccggtttg 16020cgccgagact cgagagatct agatcagcgg ccgaacatgc aggaccgagt ggaggttcgg 16080cgcaagggca ggagacgtag ctaagagcgc cgaacaaaac caggccgagc gggccgatga 16140gccgcaagcc cgcctgcgtc accctgggac tcatctgatc tgatcgcggg tacgcgtcgc 16200gggtgtgcgc gttgagccgt gttgttcgaa cgctgaggaa cggtgagctc atggaagaac 16260aagagtcctc cgctatcgca gtcatcggca tgtcgggccg ttttccgggg gcgcgggatc 16320tggacgaatt ctggaggaac cttcgagacg gcacggaggc cgtgcagcgc ttctccgagc 16380aggagctcgc ggcgtccgga gtcgaccccg cgctggtgct ggacccgagc tacgtccggg 16440cgggcagcgt gctggaagac gtcgaccggt tcgacgctgc tttcttcggc atcagcccgc 16500gcgaggcaga gctcatggat ccgcagcacc ggatcttcat ggaatgcgcc tgggaggcgc 16560tggagaacgc cggatacgac ccgacggctt acgagggctc tatcggcgtg tacgccggcg 16620ccaacatgag ctcgtacttg acgtcgaacc tccacgagca cccagcgatg atgcggtggc 16680ccggctggtt tcagacgttg atcggcaacg acaaggatta cctcgcgacc cacgtctcct 16740acaggctgaa tctgagaggg ccgagcatct ccgttcaaac tgcctgctcc acctcgctcg 16800tggcggttca cttggcgtgc atgagcctcc tggaccgcga gtgcgacatg gcgctggccg 16860gcgggattac cgtccggatc ccccatcgag ccggctatgt atatgctgag gggggcatct 16920tctctcccga cggccattgc cgggccttcg acgccaaggc gaacggcacg atcatgggca 16980acggctgcgg cgttgtcctc ctgaagccgc tggaccgggc gctctccgat ggtgatcccg 17040tccgcgcggt tatccttggg tctgccacaa acaacgacgg agcgaggaag atcgggttca 17100ctgcgcccag tgaggtgggc caggcgcaag cgatcatgga ggcgctggcg ctggcagggg 17160tcgaggcccg gtccatccaa tacatcgaga cccacgggac cggcacgctg ctcggagacg 17220ccatcgagac ggcggcgctg cggcgggtgt tcggtcgcga cgcttcggcc cggaggtctt 17280gcgcgatcgg ctccgtgaag accggcatcg gacacctcga atcggcggct ggcatcgccg 17340gtttgatcaa gacggtcttg gcgctggagc accggcagct gccgcccagc ctgaacttcg 17400agtctcctaa cccatcgatc gatttcgcga gcagcccgtt ctacgtcaat acctctctta 17460aggattggaa taccggctcg actccgcggc gggccggcgt cagctcgttc gggatcggcg 17520gcaccaacgc ccatgtcgtg ctggaggaag cgcccgcggc gaagcttcca gccgcggcgc 17580cggcgcgctc tgccgagctc ttcgtcgtct cggccaagag cgcagcggcg ctggatgccg 17640cggcggcacg gctacgagat catctgcagg cgcaccaggg gatttcgttg ggcgacgtcg 17700ccttcagcct ggcgacgacg cgcagcccca tggagcaccg gctcgcgatg gcggcgccgt 17760cgcgcgaggc gttgcgagag gggctcgacg cagcggcgcg aggccagacc ccgccgggcg 17820ccgtgcgtgg ccgctgctcc ccaggcaacg tgccgaaggt ggtcttcgtc tttcccggcc 17880agggctctca gtgggtcggc atgggccggc agctcctggc tgaggaaccc gtcttccacg 17940cggcgctttc ggcgtgcgac cgggccatcc aggccgaagc tggttggtcg ctgctcgcgg 18000agctcgccgc cgacgaaggg tcctcccagc tcgagcgcat cgacgtggtg cagccggtgc 18060tgttcgccct cgcggtggca tttgcggcgc tgtggcggtc gtggggtgtc gcgcccgacg 18120tcgtgatcgg ccacagcatg ggcgaggtag ccgccgcgca tgtggccggg gcgctgtcgc 18180tcgaggatgc ggtggcgatc atctgccggc gcagccggct gctccggcgc atcagcggtc 18240agggcgagat ggcggtgacc gagctgtcgc tggccgaggc cgaggcggcg ctccgaggct 18300acgaggatcg ggtgagcgtg gccgtgagca acagcccgcg ctcgacggtg ctctcgggcg 18360agccggcagc gatcggcgag gtgctgtcgt ccctgaacgc gaagggggtg ttctgccgtc 18420gggtgaaggt ggatgtcgcc agccacagcc cgcaggtcga cccgctgcgc gaggacctct 18480tggcagccct gggcgggctc cggccgggtg cggctgcggt gccgatgcgc tcgacggtga 18540cgggcgccat ggtagcgggc ccggagctcg gagcgaatta ctggatgaac aacctcaggc 18600agccagtgcg cttcgccgag gtagtccagg cgcagctcca aggcggccac ggtctgttcg 18660tggagatgag cccgcatccg atcctaacga cttcggtcga ggagatgcgg cgcgcggccc 18720agcgggcggg cgcagcggtg ggctcgctgc ggcgggggca ggacgagcgc ccggcgatgc 18780tggaggcgct gggcacgctg tgggcgcagg gctaccctgt accctggggg cggctgtttc 18840ccgcgggggg gcggcgggta ccgctgccga cctatccctg gcagcgcgag cggtactgga 18900tcgaagcgcc ggccaagagc gccgcgggcg atcgccgcgg cgtgcgtgcg ggcggtcacc 18960cgctcctcgg tgaaatgcag accctgtcaa cccagacgag cacgcggctg tgggagacga 19020cgctggatct caagcggctg ccgtggctcg gcgaccaccg ggtgcaggga gcggtcgtgt 19080ttccgggcgc ggcgtacctg gagatggcga tttcgtcggg ggccgaggct ttgggcgatg 19140gccctttgca gataactgac gtggtgctcg ccgaggcgct ggccttcgcg ggcgacgcgg 19200cggtgttggt ccaggtggtg acgacggagc agccgtcggg gcggctgcag ttccagatcg 19260cgagccgggc gccgggcgct ggccacgcgt ccttccgggt ccacgctcgc ggcgcgttgc 19320tccgagtgga gcgcaccgag gtcccggctg ggcttacgct ttccgctgtg cgcgcgcggc 19380tccaggccag catacccgcc gcggccacct acgcggagct gaccgagatg gggctgcagt 19440acggccctgc cttccagggg attgctgagc tatggcgggg tgaaggcgag gcgctgggac 19500gggtacgcct gcccgacgcg gccggctcgg cagcggagta tcggttgcat cctgcgctgc 19560tggacgcgtg cttccagatc gtcggcagcc tcttcgcccg cagtggcgag gcgacgccgt 19620gggtgcccgt ggagttgggc tcgctgcggc tcttgcagcg gccttcgggg gagctgtggt 19680gccatgcgcg cgtcgtgaac catgggcacc aaacccccga tcggcagggc gccgactttt 19740gggtggtcga cagctcgggt gcagtggtcg ccgaagtttg cgggctcgtg gcgcagcggc 19800ttccgggagg ggtgcgccgg cgcgaagaag acgattggtt cctggagctc gagtgggaac 19860ccgcagcggt cggcacagcc aaggtcaacg cgggccggtg gctgctcgtc ggcggcggcg 19920gtgggctcgg cgccgcgttg cgcgcgatgc tggaggccgg cggccatgcc gtcgtgcatg 19980cggcagagaa caacacgagc gctgccggcg tacgcgcgct cctggcaaag gcctttgacg 20040gccaggctcc gacggcggtg gtgcacctcg gcagcctcga tgggggtggc gagctcgacc 20100cagggctcgg ggcgcaaggc gcattggacg cgccccggag cgccgacgtc agtcccgatg 20160ccctcgatcc ggcgctggta cgtggctgcg acagcgtgct ctggaccgtg caggccctgg 20220ccggcatggg ctttcgagac gccccgcgat tgtggctttt gacccgcggc gcacaggccg 20280tcggcgccgg cgacgtctcc gtgacacagg caccgctgct ggggctgggc cgcgtcatcg 20340ccatggagca cgcggatctg cgctgcgctc gggtcgacct cgatccagcc cggcccgagg 20400gggagctcgc tgccctgctg gccgagctgc tggccgacga cgccgaagcg gaagtcgcgt 20460tgcgcggtgg cgagcgatgc gtcgctcgga tcgtccgccg gcagcccgag acccggcccc 20520gggggaggat cgagagctgc gttccgaccg acgtcaccat ccgcgcggac agcacctacc 20580ttgtgaccgg cggtctgggt gggctcggtc tgagcgtggc cggatggctg gccgagcgcg 20640gcgctggtca cctggtgctg gtgggccgct ccggcgcggc gagcgtggag caacgggcag 20700ccgtcgcggc gctcgaggcc cgcggcgcgc gcgtcaccgt ggcgaaggcg gatgtcgccg 20760atcgggcgca gctcgagcgg atcctccgcg aggttaccac gtcggggatg ccgctgcggg 20820gcgtcgtcca tgcggccggc atcttggacg acgggctgct gatgcagcag actcccgcgc 20880ggtttcgtaa ggtgatggcg cccaaggtcc agggggcctt gcacctgcac gcgttgacgc 20940gcgaagcgcc gctttccttc ttcgtgctgt acgcttcggg agtagggctc ttgggctcgc 21000cgggccaggg caactacgcc gcggccaaca cgttcctcga cgctctggcg caccaccgga 21060gggcgcaggg gctgccagcg ttgagcgtcg actggggcct gttcgcggag gtgggcatgg 21120cggccgcgca ggaagatcgc ggcgcgcggc tggtctcccg cggaatgcgg agcctcaccc 21180ccgacgaggg gctgtccgct ctggcacggc tgctcgaaag cggccgcgct caggtggggg 21240tgatgccggt gaacccgcgg ctgtgggtgg agctctaccc cgcggcggcg tcttcgcgaa 21300tgttgtcgcg cctggtgacg gcgcatcgcg cgagcgccgg cgggccagcc ggggacgggg 21360acctgctccg ccgcctcgcc gctgccgagc cgagcgcgcg gagcgcgctc ctggagccgc 21420tcctccgcgc gcagatctcg caggtgctgc gcctccccga gggcaagatc gaggtggacg 21480ccccgctcac gagcctgggc atgaactcgc tgatggggct cgagctgcgc aaccgcatcg 21540aggccatgct gggcatcacc gtaccggcaa cgctgttgtg gacctgtccc acggtggcgg 21600cgctgagcgg gcatctggcg cgggaggcat gcgaagccgc tcctgtggag tcaccgcaca 21660ccaccgccga ctctgccgtc gagatcgagg agatgtcgca ggacgatctg acgcagttga 21720tcgcagcaaa attcaaggcg cttacatgac tactcgcggt cctacggcac agcagaatcc 21780gctgaaacaa gcggccatca tcattcagcg gctggaggag cggctcgctg ggctcgcaca 21840ggcggagctg gaacggaccg agccgatcgc catcgtcggt atcggctgcc gcttccctgg 21900cggtgcggac gctccggaag cgttttggga gctgctcgac gcggagcgcg acgcggtcca 21960gccgctcgac atgcgctggg cgctggtggg tgtcgctccc gtcgaggccg tgccgcactg 22020ggcggggctg ctcaccgagc cgatagattg cttcgatgct gcgttcttcg gcatctcgcc 22080tcgggaggcg cgatcgctcg acccgcagca tcgtctgttg ctggaggtcg cttgggaggg 22140gctcgaggac gccggtatcc cgccccggtc catcgacggg agccgcaccg gtgtgttcgt 22200cggcgctttc acggcggact acgcgcgcac ggtcgctcgg ctgccgcgcg aggagcgaga 22260cgcgtacagc gccaccggca acatgctcag catcgccgcc ggacggctgt cgtacacgct 22320ggggttgcag ggaccttgcc tgaccgtcga cacggcgtgc tcgtcatcgc tggtggcgat 22380tcacctcgcc tgccgcagcc tgcgcgcagg agagagcgat ctcgcgttgg cgggaggggt 22440cagcgcgctc ctctcccccg acatgatgga agccgcggcg cgcacgcaag cgctgtcgcc 22500cgatggtcgt tgccggacct tcgatgcttc ggccaacggg ttcgtccgtg gcgagggctg 22560tggcctggtc gtcctcaaac ggctctccga cgcgcaacgg gatggcgacc gcatctgggc 22620gctgatccgg ggctcggcca tcaaccatga tggccggtcg accgggttga ccgcgcccaa 22680cgtgctggct caggagacgg tcttgcgcga ggcgctgcgg agcgcccacg tcgaagctgg 22740ggccgtcgat tacgtcgaga cccacggaac agggacctcg ctgggcgatc ccatcgaggt 22800cgaggcgctg cgggcgacgg tggggccggc gcgctccgac ggcacacgct gcgtgctggg 22860cgcggtgaag accaacatcg gccatctcga ggccgcggca ggcgtagcgg gcctgatcaa 22920ggcagcgctt tcgctgacgc acgagcgcat cccgagaaac ctcaacttcc gcacgctcaa 22980tccgcggatc cggctcgagg gcagcgcgct cgcgttggcg accgagccgg tgccgtggcc 23040gcgcacggac cgcccgcgct tcgcgggggt gagctcgttc gggatgagcg gaacgaacgc 23100gcatgtggtg ctggaagagg cgccggcggt ggagctgtgg cctgccgcgc cggagcgctc 23160ggcggagctt ttggtgctgt cgggcaagag cgagggggcg ctcgatgcgc aggcggcgcg 23220gctgcgcgag cacctggaca tgcacccgga gctcgggctc ggggacgtgg cgttcagcct 23280ggcgacgacg cgcagcgcga tgagccaccg gctcgcggtg gcggtgacgt cgcgcgaggg 23340gctgctggcg gcgctctcgg ccgtggcgca ggggcagacg ccggcggggg cggcgcgctg 23400catcgcgagc tcctcgcgcg gcaagctggc gttcctgttc accggacagg gcgcgcagac 23460gccgggcatg ggccgggggc tttgcgcggc gtggccagcg ttccgggagg cgttcgaccg 23520gtgcgtggcg ctgttcgacc gggagctgga ccgcccgctg cgcgaggtga tgtgggcgga 23580ggcggggagc gccgagtcgt tgttgctcga ccagacggcg ttcacccagc ccgcgctctt 23640cgcggtggag tacgcgctga cggcgctgtg gcggtcgtgg ggcgtagagc cggagctcct 23700ggttgggcat agcatcgggg agctggtggc ggcgtgcgtg gcgggggtgt tctcgctgga 23760agatggggtg aggctcgtgg cggcgcgcgg gcggctgatg caggggctct cggcgggcgg 23820cgcgatggtg tcgctcggag cgccggaggc ggaggtggcg gcggcggtgg cgccgcacgc 23880ggcgtcggtg tcgatcgcgg cggtcaatgg gccggagcag gtggtgatcg cgggcgtgga 23940gcaagcggtg caggcgatcg cggcggggtt cgcggcgcgc ggcgcgcgca ccaagcggct 24000gcatgtctcg cacgcgttcc actcgccgct gatggaaccg atgctggagg agttcgggcg 24060ggtggcggcg tcggtgacgt accggcggcc aagcgtttcg ctggtgagca acctgagcgg 24120gaaggtggtc acggacgagc tgagcgcgcc ggggtactgg gtgcggcacg tgcgggaggc 24180ggtgcgcttc gcggacgggg tgaaggcgct gcacgaagcc ggcgcgggga cgttcgtcga 24240agtgggcccg aagccgacgc tgctcgggct gttgccagcc tgcctgccgg aggcggagcc 24300gacgctgctg gcgtcgttgc gcgccgggcg cgaggaggct gcgggggtgc tcgaggcgct 24360gggcaggctg tgggccgccg gcggctcggt cagctggccg ggcgtcttcc ccacggctgg 24420gcggcgggtg ccgctgccga cctatccgtg gcagcggcag cggtactgga tcgaggcgcc 24480ggccgaaggg ctcggagcca cggccgccga tgcgctggcg cagtggttct accgggtgga 24540ctggcccgag atgcctcgct catccgtgga ttcgcggcga gcccggtccg gcgggtggct 24600ggtgctggcc gaccggggtg gagtcgggga ggcggccgcg gcggcgcttt cgtcgcaggg 24660atgttcgtgc gccgtgctcc atgcgcccgc cgaggcctcc gcggttgccg agcaggtgac 24720ccaggccctc ggtggccgca acgactggca gggggtgctg tacctgtggg gtctggacgc 24780cgtcgtggag gcgggggcat cggccgaaga ggtcgccaaa gtcacccatc ttgccgcggc 24840gccggtgctc gcgctgattc aggcgctcgg cacggggccg cgctcacccc ggctctggat 24900cgtgacccga ggggcctgca cggtgggcgg cgagcctgac gctgccccct gtcaggcggc 24960gctgtggggt atgggccggg tcgcggcgct agagcatccc ggctcctggg gcgggctcgt 25020ggacctggat ccggaggaga gcccgacgga ggtcgaggcc ctggtggccg agctgctttc 25080gccggacgcc gaggatcagc tggcattccg ccaggggcgc cggcgcgcag cgcggcttgt 25140ggccgcccca ccggagggaa acgcagcgcc ggtgtcgctg tctgcggagg ggagttactt 25200ggtgacgggt gggctgggcg cccttggcct cctcgttgcg cggtggttgg tggagcgcgg 25260ggcggggcac cttgtgctga tcagccggca cggattgccc gaccgcgagg aatggggccg 25320agatcagccg ccagaggtgc gcgcgcgcat tgcggcgatc gaggcgctgg aggcgcaggg 25380cgcgcgggtc accgtggcgg cggtcgacgt ggccgatgcc gaaggcatgg cggcgctctt 25440ggcggccgtc gagccgccgc tgcggggggt agtgcacgcc gcgggtctgc tcgacgacgg 25500gctgctggcc caccaggacg ctggtcggct cgcccgggtg ttgcgcccca aggtggaggg 25560ggcatgggtg ctgcacaccc ttacccgcga gcagccgctg gacctcttcg tactgttttc 25620ctcggcgtcg ggcgtcttcg gctcgatcgg ccagggcagc tacgcggcag gcaatgcctt 25680tttggacgcg ctggcggacc tccgccgaac gcaggggctc gccgccctga gcatcgcctg 25740gggcctgtgg gcggaggggg ggatgggctc gcaggcgcag cgccgggaac acgaggcatc 25800gggaatctgg gcgatgccga cgagtcgggc cctggcggcg atggaacggc tgctcggtac 25860gcgcgcgacg cagcgcgtgg tcatccagat ggattgggcc catgcgggag cggcgccgcg 25920cgacgcgagc cgaggccgct tctgggatcg gctggtaact gccacgaaag aggcctcctc 25980ctcggccgtg ccagctgcgg agcgctggcg caacgcgtct gttgtggaga cccgctcggc 26040gctctacgag cttgtgcgcg gcgtggtcgc cggggtgatg ggctttaccg accagggcac 26100gctcgacgtg cgacgaggct tcgccgagca gggcctcgac tccctgatgg ccgtggagat 26160ccgcaaacgg cttcagggtg agctgggtat gccgctgtcg gcgacgctag cgttcgacca 26220tccgaccgtg gagcggctgg tggaatactt gctgagccag gcgctggagc tgcaggaccg 26280caccgacgtg cggagcgttc ggttgccggc gacagaggac ccgatcgcca tcgtgggtgc 26340cgcctgccgc ttcccgggcg gggtcgagga cctggagtcc tactggcagc tgttgaccga 26400gggcgtggtg gtcagcaccg aggtgccggc cgaccggtgg aatggggcag acgggcgcgt 26460ccccggctcg ggagaggcac agagacagac ctacgtgccc aggggtggct ttctgcgcga 26520ggtggagacg ttcgatgcgg cgttcttcca catctcgcct cgggaggcga tgagcctgga 26580cccgcaacag cggctgctgc tggaagtgag ctgggaggcg atcgagcgcg cgggccagga 26640cccgtcggcg ctgcgcgaga gccccacggg cgtgttcgtg ggcgcgggcc ccaacgaata 26700tgccgagcgg gtgcaggaac tcgccgatga ggcggcgggg ctctacagcg gcaccggcaa 26760catgctcagc gttgcggcgg gacggctatc atttttcctg ggcctgcacg ggccgaccct 26820ggctgtggat acggcgtgct cctcgtcgct ggtggcgctg cacctcggct gccagagctt 26880gcgacggggc gagtgcgacc aagccctggt tggcggggtc aacatgctgc tctcgccgaa 26940gaccttcgcg ctgctctcac ggatgcacgc actttcgccc ggcgggcggt gcaagacgtt 27000ctcggccgac gcggacggct acgcgcgggc cgagggctgc gccgtggtgg tgctcaagcg 27060gctctccgac gcgcagcgcg accgcgaccc catcctggcg gtgatccggg gtacggcgat 27120caatcatgat ggcccgagca gcgggctgac agtgcccagc ggccctgccc aggaggcgct 27180gttacgccag gcgctggcgc acgcaggggt ggttccggcc gacgtcgatt tcgtggaatg 27240ccacgggacc gggacggcgc tgggcgaccc gatcgaggtg cgtgcgctga gcgacgtgta 27300cgggcaagcc cgccctgcgg accgaccgct gatcctggga gccgccaagg ccaaccttgg 27360gcacatggag cccgcggcgg gcctggccgg cttgctcaag gcggtgctcg cgctggggca 27420agagcaaata ccagcccagc cggagctggg cgagctcaac ccgctcttgc cgtgggaggc 27480gctgccggtg gcggtggccc gcgcagcggt gccgtggccg cgcacggacc gcccgcgctt 27540cgcgggggtg agctcgttcg ggatgagcgg aacgaacgcg catgtggtgc tggaagaggc 27600gccggcggtg gagctgtggc ctgccgcgcc ggagcgctcg gcggagcttt tggtgctgtc 27660gggcaagagc gagggggcgc tcgatgcgca ggcggcgcgg ctgcgcgagc acctggacat 27720gcacccggag ctcgggctcg gggacgtggc gttcagcctg gcgacgacgc gcagcgcgat 27780gaaccaccgg ctcgcggtgg cggtgacgtc gcgcgagggg ctgctggcgg cgctttcggc 27840cgtggcgcag gggcagacgc cgccgggggc ggcgcgctgc atcgcgagct cgtcgcgcgg 27900caagctggcg ttcctgttca ccggacaggg cgcgcagacg ccgggcatgg gccgggggct 27960ttgcgcggcg tggccagcgt tccgggaggc gttcgaccgg tgcgtggcgc tgttcgaccg 28020ggagctggac cgcccgctgc gcgaggtgat gtgggcggag ccggggagcg ccgagtcgtt 28080gttgctcgac cagacggcgt tcacccagcc cgcgctcttc acggtggagt acgcgctgac 28140ggcgctgtgg cggtcgtggg gcgtagagcc ggagctggtg gctgggcata gcgccgggga 28200gctggtggcg gcgtgcgtgg cgggggtgtt ctcgctggaa gatggggtga ggctcgtggc 28260ggcgcgcggg cggctgatgc aggggctctc ggcgggcggc gcgatggtgt cgctcggagc 28320gccggaggcg gaggtggcgg cggcggtggc gccgcacgcg gcgtcggtgt cgatcgcggc 28380ggtcaatggg ccggagcagg tggtgatcgc gggcgtggag caagcggtgc aggcgatcgc 28440ggcggggttc gcggcgcgcg gcgcgcgcac caagcggctg catgtctcgc acgcgtccca 28500ctcgccgctg atggaaccga tgctggagga gttcgggcgg gtggcggcgt cggtgacgta 28560ccggcggcca agcgtttcgc tggtgagcaa cctgagcggg aaggtggtcg cggacgagct 28620gagcgcgccg gggtactggg tgcggcacgt gcgggaggcg gtgcgcttcg cggacggggt 28680gaaggcgctg cacgaagccg gtgcgggcac gttcgtcgaa gtgggcccga agccgacgct 28740gctcgggctg ttgccagcct gcctgccgga ggcggagccg acgctgctgg cgtcgttgcg 28800cgccgggcgc gaggaggctg cgggggtgct cgaggcgctg ggcaggccgt gggccgccgg 28860cggctcggtc agctggccgg gcgtcttccc cacggctggg cggcgggtgc cgctgccgac 28920ctatccgtgg cagcggcagc ggtactggcc cgacatcgag cctgacagcc gtcgccacgc 28980agccgcggat ccgacccaag gctggttcta tcgcgtggac tggccggaga tacctcgcag 29040cctccagaaa tcagaggagg cgagccgcgg gagctggctg gtattggcgg ataagggtgg 29100agtcggcgag gcggtcgctg cagcgctgtc gacacgtgga cttccatgcg tcgtgctcca 29160tgcgccggca gagacatccg cgaccgccga gctggtgacc gaggctgccg gcggtcgaag 29220cgattggcag gtagtgctct acctgtgggg tctggacgcc gtcgtcggtg cggaggcgtc 29280gatcgatgag atcggcgacg cgacccgtcg tgctaccgcg ccggtgctcg gcttggctcg 29340gtttctgagc accgtgtctt gtccgccccg actctgggcc gtgacccggg gggcacgcat 29400cgttggcgac gagcccgcga tcgccccctg tcaggcggcg ttacggggca tgggccgggc 29460ggcggcgccc gagcatcccg gggcccgggg cgggctcgtg gacctggatc cccgagcgag 29520cccgccccaa gccagcccga tcgacggcga gatgctcgtc accgagctat tgtcgcagga 29580gaccgaggat cagctcgcct tccgccatgg gcgccggcac gcggcacggc tggtggccgc 29640cccgccacag gggcaagcgg caccggtgtc gctgtctgcg gaggcgagct acctggtgac 29700gggaggcccc ggcgggctgg gcctgatcgc ggcccagtgg ctggcggagc tgggagcgcg 29760gcacttggtg ctgaccagcc ggcgcgggtt gcccgaccgg caggcgtggt gcgagcagca 29820gccgcctgag atccgcgcgc ggatcgcagc ggtcgaggcg ctggaggcgc ggggtgcacg 29880ggtgaccgtg gcagcggtgg acgtggccga cgtcgaaccg atgacagcgc tggtttcgtc 29940ggtcgagccc ccgctgcgag gggtggtgca cgccgctggc gtcagcgtca tgcgtccact 30000ggcggagacg gacgagaccc tgctcgagtc ggtgctccgt cccaaggtgg ccgggagctg 30060gctgctgcac cggctgctgc acggccggcc tctcgacctg ttcgtgctgt tctcgtcggg 30120cgcagcggtg tggggtagcc atagccaggg tgcgtacgcg gcggccaacg ctttcctcga 30180cgggctcgcg catcttcggc gttcgcaatc gctgcctgcg ttgagcgtcg cgtggggtct 30240gtgggccgag ggaggcatgg cggacgcgga ggctcatgca cgtctgagcg acatcggggt 30300tctgcccatg tcgacgtcgg cagcgttgtc ggcgctccag cgcctggtgg agaccggcgc 30360ggctcagcgc acggtgaccc ggatggactg ggcgcgcttc gcgccggtgt acaccgctcg 30420agggcgtcgc aacctgcttt cggcgctggt cgcagggcgc gacatcatcg cgccttcccc 30480tccggcggca gcaacccgga actggcgtgg cctgtccgtt gcggaagccc gcgtggctct 30540gcacgagatc gtccatgggg ccgtcgctcg ggtgctgggc ttcctcgacc cgagcgcgct 30600cgatcctggg atggggttca atgagcaggg cctcgactcg ttgatggcgg tggagatccg 30660caacctcctt caggctgagc tggacgtgcg gctttcgacg acgctggcct ttgatcatcc 30720gacggtacag cggctggtgg agcatctgct cgtcgatgta ctgaagctgg aggatcgcag 30780cgacacccag catgttcggt cgttggcgtc agacgagccc atcgccatcg tgggagccgc 30840ctgccgcttc ccgggcgggg tggaggacct ggagtcctac tggcagctat tggccgaggg 30900cgtggtggtc agcgccgagg tgccggccga ccggtgggat gcggcggact ggtacgaccc 30960tgatccggag atcccaggcc ggacttacgt gaccaaaggc gccttcctgc gcgatttgca 31020gagattggat gcgaccttct tccgcatctc gcctcgcgag gcgatgagcc tcgacccgca 31080gcagcggttg ctcctggagg taagctggga agcgctcgag agcgcgggta tcgctccgga 31140tacgctgcga gatagcccca ccggggtgtt cgtgggtgcg gggcccaatg agtactacac 31200gcagcggctg cgaggcttca ccgacggagc ggcagggttg tacggcggca ccgggaacat 31260gctcagcgtt acggctggac ggctgtcgtt tttcctgggt ctgcacggcc cgacgctggc 31320catggatacg gcgtgctcgt catccctggt cgcgctgcac ctcgcctgcc agagcctgcg 31380actgggcgag tgcgatcaag cgctggttgg cggggtcaac gtgctgctcg cgccggagac 31440cttcgtgctg ctctcacgga tgcgcgcgct ttcgcccgac gggcggtgca agacgttctc 31500ggccgacgcg gacggctacg cgcggggcga ggggtgcgcc gtggtggtgc tcaagcggct 31560gcgcgatgcg cagcgcgccg gcgactccat cctggcgctg atccggggaa gcgcggtgaa 31620ccacgacggc ccgagcagcg ggctgaccgt acccaacgga cccgcccagc aagcattgct 31680gcgccaggcg ctttcgcaag caggcgtgtc tccggtcgac gttgattttg tggagtgtca 31740cgggacaggg acggcgctgg gcgacccgat cgaggtgcag gcgctgagcg aggtgtatgg 31800tccagggcgc tccggggacc gaccgctggt gctgggggcc gccaaggcca acgtcgcgca 31860tctggaggcg gcatctggct tggccagcct gctcaaggcc gtgcttgcgc tgcggcacga 31920gcagatcccg gcccagccgg agctggggga gctcaacccg cacttgccgt ggaacacgct 31980gccggtggcg gtgccacgta aggcggtgcc gtgggggcgc ggcgcacgcc cgcgtcgggc 32040cggcgtgagc gcgttcgggt tgagcggaac caacgtgcat gtcgtgctgg aggaggcacc 32100ggaggtggag ccggcgcccg cggcgccggc gcgaccggtg gagctggtcg tgctatcggc 32160caagagcgcg gcggcgctgg acgccgcggc ggcacggctc tcggcgcacc tgtccgcgca 32220cccggagctg agcctcggcg acgtggcgtt cagcctggcg acgacgcgca gcccgatgga 32280gcaccggctc gccatcgcga cgacctcgcg cgaggccctg cgaggcgcgc tggacgccgc 32340ggcgcagcaa aagacgccgc agggcgcggt gcgcggcaag gccgtgtcct cacgcggtaa 32400gctggctttc ctgttcaccg gacagggcgc gcaaatgccg ggcatgggcc gtgggctgta 32460cgaaacgtgg cctgcgttcc gggaggcgtt cgaccggtgc gtggcgctct tcgatcggga 32520gatcgaccag cctctgcgcg aggtgatgtg ggctgcgccg ggcctcgctc aggcggcgcg 32580gctcgatcag accgcgtacg cgcagccggc tctctttgcg ctggagtacg cgctggctgc 32640cctgtggcgt tcgtggggcg tggagccgca cgtactgctc ggtcatagca tcggcgagct 32700ggtcgccgcc tgcgtggcgg gcgtgttctc gctcgaagat gcggtgaggt tggtggccgc 32760gcgcgggcgg ctgatgcagg cgctacccgc cggcggtgcc atggtagcca tcgcagcgtc 32820cgaggccgag gtggccgcct ccgtggcgcc ccacgccgcc acggtgtcga tcgccgcggt 32880caacggtcct gacgccgtcg tgatcgccgg cgccgaggta caggtgctcg ccctcggcgc 32940gacgttcgcg gcgcgtggga tacgcacgaa gaggctcgcc gtctcccatg cgttccactc 33000gccgctcatg gatccgatgc tggaagactt ccagcgggtc gctgcgacga tcgcgtaccg 33060cgcgccagac cgcccggtgg tgtcgaatgt caccggccac gtcgcaggcc ccgagatcgc 33120cacgcccgag tattgggtcc ggcatgtgcg aagcgccgtg cgcttcggcg acggggcaaa 33180ggcgttgcat gccgcgggtg ccgccacgtt cgtcgaggtt ggcccgaagc cggtcctgct 33240cgggctgttg ccagcgggcc tcggggaagc ggacgcggtc ctcgtgccgt cgctacgcgc 33300ggaccgctcg gaatgcgagg tggtcctcgc ggcgctcggg gcttggtatg cctggggggg 33360tgcgctcgac tggaagggcg ggggccccga tggcgcgcgc cgcgtggctc tgcccatgta 33420tccatggcag cgtgagcgcc attggatgga cctcaccccg cgaagcgccg cgcctgcagg 33480gatcgcaggt cgctggccgc tggctggtgt cgggctctgc atgcccggcg ctgtgttgca 33540ccacgtgctc tcgatcggac cacgccatca gcccttcctc ggtgatcacc tcgtgtttgg 33600caaggtggtg gtgcccggcg cctttcatgt cgcggtgatc ctcagcatcg ccgccgagcg 33660ctggcccgag cgggcgatcg agctgacagg cgtggagttc ctgaaggcca tcgcgatgga 33720gcccgaccag gaggtcgagc tccacgccgt gctcaccccc gaagccgccg gggatggcta 33780cctgttcgag ctggcgaccc tggcggcgcc ggagaccgaa cgccgatgga cgacccacgc 33840ccgcggtcgg gtgcagccga cagacggcgc gcccggcgcg ttgccgcgcc tcgaggtgct 33900ggaggaccgc gcgatccagc ccctcgactt cgccggattc ctcgacaggt tatcggcggt 33960gcggatcggc tggggtccgc tttggcgatg gctgcaggac gggcgcgtcg gcgacgaggc 34020ctcgcttgcc accctcgtgc cgacctatcc gaacgcccac gacgtggcgc ccttgcaccc 34080gatcctgctg gacaacggct ttgcggtgag cctgctgtca acccggagcg agccggagga 34140cgacgggacg cccccgctgc cgttcgccgt ggaacgggtg cggtggtggc gggcgccggt 34200tggaagggtg cggtgtggcg gcgtgccgcg gtcgcaggca ttcggtgtct cgagcttcgt 34260gctggtcgac gaaactggcg aggtggtcgc cgaggtggag ggatttgttt gccgccgggc 34320gccgcgagag gtgttcctgc ggcaggagtc gggcgcgtcg actgcagcct tgtaccgcct 34380cgactggccc gaagcgccct tgcccgatgc gcctgcggaa cggatcgagg agagctgggt 34440cgtggtggca gcacctggct cggagatggc cgcggcgctc gcaacacggc tcaaccgctg 34500cgtcctcgcc gaacccaaag gcctcgaggc ggccctcgcg ggggtgtctc ccgcaggtgt 34560gatctgcctc tgggaggctg gagcccacga ggaagctccg gcggcggcgc agcgtgtggc 34620gaccgagggc ctctcggtgg tgcaggcgct cagggaccgc gcggtgcgcc tgtggtgggt 34680gaccatgggc gcagtggccg tcgaggccgg tgagcgggtg caggtcgcca cagcgccggt 34740atggggcctc ggccggacag tgatgcagga gcgcccggag ctcagctgca ctctggtgga 34800tttggagccg gaggccgatg cagcgcgctc agctgacgtt ctgttgcggg agctcggtcg 34860cgctgacgac gagacacagg tggctttccg ttccggaaag cgccgcgtag cgcggctggt 34920caaagcgacg acccccgaag ggctcctggt ccctgacgca gagtcctatc gactggaggc 34980tgggcagaag ggcacattgg accagctccg cctcgcgccg gcacagcgcc gggcacctgg 35040cccgggcgag gtcgagatca aggtaaccgc ctcggggctc aacttccgga ccgtcctcgc 35100tgtgctggga atgtatccgg gcgacgccgg gccgatgggc ggagattgtg ccggtgtcgc 35160cacggcggtg ggccaggggg tgcgccacgt cgcggtcggc gatgctgtca tgacgctggg 35220gacgttgcat cgattcgtca cggtcgacgc gcggctggtg gtccggcagc ctgcagggct 35280gactcccgcg caggcagcta cggtgccggt cgcgttcctg acggcctggc tcgctctgca 35340cgacctgggg aatctgcggc gcggcgagcg ggtgctgatc catgctgcgg ccggcggtgt 35400gggcatggcc gcggtgcaaa tcgcccgatg gataggggcc gaggtgttcg ccacggcgag 35460cccgtccaag tgggcagcgg ttcaggccat gggcgtgccg cgcacgcaca tcgccagctc 35520gcggacgctg gagtttgctg agacgttccg gcaggtcacc ggcggccggg gcgtggacgt 35580ggtgctcaac gcgctggccg gcgagttcgt ggacgcgagc ctgtccctgc tgtcgacggg 35640cgggcggttc ctcgagatgg gcaagaccga catacgggat cgagccgcgg tcgcggcggc 35700gcatcccggt gttcgctatc gggtattcga catcctggag ctcgctccgg atcgaactcg 35760agagatcctc gagcgcgtgg tcgagggctt tgctgcggga catctgcgcg cattgccggt 35820gcatgcgttc gcgatcacca aggccgaggc agcgtttcgg ttcatggcgc aagcgcggca 35880tcagggcaag gtcgtgctgc tgccggcgcc ctccgcagcg cccttggcgc cgacgggcac 35940cgtactgctg accggtgggc tgggagcgtt ggggctccac gtggcccgct ggctcgccca 36000gcagggcgtg ccgcacatgg tgctcacagg tcggcggggc ctggatacgc cgggcgctgc 36060caaagccgtc gcggagatcg aagcgctcgg cgctcgggtg acgatcgcgg cgtcggatgt 36120cgccgatcgg aatgcgctgg aggctgtgct ccaggccatt ccggcggagt ggccgttaca 36180gggcgtgatc catgcagccg gagcgctcga tgatggtgtg cttgatgagc agaccaccga 36240ccgcttctcg cgggtgctgg caccgaaggt gactggcgcc tggaatctgc atgagctcac 36300ggcgggcaac gatctcgctt tcttcgtgct gttctcctcc atgtcggggc tcttgggctc 36360ggccgggcag tccaactatg cggcggccaa caccttcctc gacgcgctgg ccgcgcatcg 36420gcgggccgaa ggcctggcgg cgcagagcct cgcgtggggc ccatggtcgg acggaggcat 36480ggcagcgggg ctcagcgcgg cgctgcaggc gcggctcgct cggcatggga tgggagctct 36540gtcgccggct cagggcaccg cgctgctcgg gcaggcgctg gctcggccgg aaacgcagct 36600cggggcgatg tcgctcgacg tgcgtgcggc aagccaagct tcgggagcgg cagtgccgcc 36660tgtgtggcgc gcgttggtgc gcgcggaggc gcgccatacg gcggctgggg cgcagggggc 36720attggccgcg cgtcttgggg cgctgcccga ggcgcgtcgc gccgacgagg tgcgcaaggt 36780cgtgcaggcc gagatcgcgc gcgtgctttc atggagcgcc gcgagcgccg tgcccgtcga 36840tcggccgctg tcggacttgg gcctcgactc gctcacggcg gtggagctgc gcaacgtgct 36900cggccagcgg gtgggtgcga cgctgccggc gacgctggca ttcgatcacc cgacggtcga 36960cgcgctcacg cgctggctgc tcgataaggt cctggccgtg gccgagccga gcgtatcgtc 37020cgcaaagtcg tcgccgcagg tcgccctcga cgagcccatt gccatcatcg gcatcggctg 37080ccgtttccca ggcggcgtgg ccgatccgga gtcgttttgg cggctgctcg aagagggcag 37140cgatgccgtc gtcgaggtgc cgcatgagcg atgggacatc gacgcgttct atgatccgga 37200tccggatgtg cgcggcaaga tgacgacacg ctttggcggc ttcctgtccg atatcgaccg 37260gttcgatccg gccttcttcg gcatctcgcc gcgcgaagcg acgaccatgg atccgcagca 37320gcggctgctc ctggagacga gctgggaggc gttcgagcgc gccgggattt tgcccgagcg 37380gctgatgggc agcgataccg gcgtgttcgt ggggctcttc taccaggagt acgctgcgct 37440cgccggcggc atcgaggcgt tcgatggcta tctaggcacc ggcaccacgg ccagcgtcgc 37500ctcgggcagg atctcttatg tgctcgggct aaaggggccg agcctgacgg tggacaccgc 37560gtgctcctcg tcgctggtcg cggtgcacct ggcctgccag gcgctgcggc ggggcgagtg 37620ttcggtggcg ctggccggcg gcgtggcgct gatgctcacg ccggcgacgt tcgtggagtt 37680cagccggctg cgaggcctgg ctcccgacgg acggtgcaag agcttctcgg ccgcagccga 37740cggcgtgggg tggagcgaag gctgcgccat gctcctgctc aaaccgcttc gcgatgcgca 37800gcgcgatggg gatccgatcc tggcggtgat ccgcggcacc gcggtgaacc aggatgggcg 37860cagcaacggg ctgacggcgc ccaacgggtc gtcgcagcaa gaggtgatcc gtcgggccct 37920ggagcaggcg gggctggctc cggcggacgt cagctacgtc gagtgccacg gcaccggcac 37980gacgttgggc gaccccatcg aagtgcaggc cctgggcgcc gtgctggcac aggggcgacc 38040ctcggaccgg ccgctcgtga tcgggtcggt gaagtccaat atcggacata cgcaggctgc 38100ggcgggcgtg gccggtgtca tcaaggtggc gctggcgctc gagcgcgggc ttatcccgag 38160gagcctgcat ttcgacgcgc ccaatccgca cattccgtgg tcggagctcg ccgtgcaggt 38220ggccgccaaa cccgtcgaat ggacgagaaa cggcgtgccg cgacgagccg gggtgagctc 38280gtttggcgtc agcgggacca acgcgcacgt ggtgctggag gaggcgccag cggcggcgtt 38340cgcgcccgcg gcggcgcgtt cagcggagct tttcgtgctg tcggcgaaga gcgccgcggc 38400gctggacgcg caggcggcgc ggctttcggc gcacgtcgtt gcgcacccgg agctcggcct 38460cggcgacctg gcgttcagcc tggcgacgac ccgcagcccg atgacgtacc ggctcgcggt 38520ggcggcgacc tcgcgcgagg cgctgtctgc cgcgctcgac acagcggcgc aggggcaggc 38580gccgcccgca gcggctcgcg gccacgcttc cacaggcagc gccccaaagg tggttttcgt 38640ctttcctggc cagggctccc agtggctggg catgggccaa aagctcctct cggaggagcc 38700cgtcttccgc gacgcgctct cggcgtgtga ccgagcgatt caggccgaag ccggctggtc 38760gctgctcgcc gagctcgcgg ccgatgagac cacctcgcag ctcggccgca tcgacgtggt 38820gcagccggcg ctgttcgcga tcgaggtcgc gctgtcggcg ctgtggcggt cgtggggcgt 38880cgagccggat gcagtggtag gccacagcat gggcgaagtg gcggccgcgc acgtcgccgg 38940cgccctgtcg ctcgaggatg ctgtagcgat catctgccgg cgcagcctgc tgctgcggcg 39000gatcagcggc caaggcgaga tggcggtcgt cgagctttcc ctggccgagg ccgaggcagc 39060gctcctgggc tacgaagacc ggctcagcgt ggcggtgagc aacagcccgc gctcgacggt 39120gctggcgggc gagccggcag cgctcgcaga ggtgctggcg atccttgcgg caaagggggt 39180gttctgccgt cgagtcaagg tggacgtcgc cagccacagc ccacagatcg acccgctgcg 39240cgacgagcta ttggcagcat tgggcgagct cgagccgcga caagcgaccg tgtcgatgcg 39300ctcgacggtg acgagcacga tcatggcggg cccggagctc gtggcgagct actgggcgga 39360caacgttcga cagccggtgc gcttcgccga agcggtgcaa tcgttgatgg aagacggtca 39420tgggctgttc gtggagatga gcccgcatcc gatcctgacg acatcggtcg aggagatccg 39480acgggcgacg aagcgggagg gagtcgcggt gggctcgttg cggcgtggac aggacgagcg 39540cctgtccatg ttggaggcgc tgggagcgct ctgggtacac ggccaggcgg tgggctggga 39600gcggctgttc tccgcgggcg gcgcgggcct ccgtcgcgtg ccgctgccga cctatccctg 39660gcagcgcgag cggtactggg tcgatgcgcc gaccggcggc gcggcgggcg gcagccgctt 39720tgctcatgcg ggcagtcacc cgctcctggg tgaaatgcag accctgtcga cccagaggag 39780cacgcgcgtg tgggagacga cgctggatct caaacggctg ccgtggctcg gcgatcaccg 39840ggtgcagggg gcggtcgtgt tcccgggcgc ggcgtacctg gagatggcgc tttcgtccgg 39900ggccgaggcc ttgggtgacg gtccgctcca ggtcagcgat gtggtgctcg ccgaggcgct 39960ggccttcgcg gatgatacgc cggcggcggt gcaggtcatg gcgaccgagg agcgaccagg 40020ccgcctgcaa ttccacgttg cgagccgggt gccgggccac ggcggtgctg cctttcgaag 40080ccatgcccgc ggggtgctgc gccagatcga gcgcgccgag gtcccggcga ggctggatct 40140ggccgcgctt cgtgcccggc ttcaggccag cgcacccgct gcggctacct atgcggcgct 40200ggccgagatg gggctcgagt acggcccagc gttccagggg cttgtcgagc tgtggcgggg 40260ggagggcgag gcgctgggac gtgtgcggct ccccgaggcc gccggctccc cagccgcgtg 40320ccggctccac cccgcgctct tggatgcgtg cttccacgtg agcagcgcct tcgctgaccg 40380cggcgaggcg acgccatggg tacccgtgga aatcggctcg ctgcggtggt tccagcggcc 40440gtcgggggag ctgtggtgtc atgcgcggag tgtgagccac ggaaagccaa cacccgaccg 40500gcggagtacc gacttctggg tggtcgacag cacgggcgcg atcgtcgccg agatctccgg 40560gctcgtggcg cagcggctcg cgggaggtgt acgccggcgc gaagaagacg actggttcat 40620ggagccggct tgggaaccga ccgcggtccc cggatccgag gtcatggcgg gccggtggct 40680gctcatcggc tcgggcggcg ggctcggcgc tgcgctccac tcggcgctga cggaagctgg 40740ccattccgtc gtccacgcga cagggcgcgg cacgagcgcc gccgggttgc aggcactctt 40800gacggcgtcc ttcgacggcc aggccccgac gtcggtggtg cacctcggca gcctcgatga 40860gcgtggcgtg ctcgacgcgg atgccccctt cgacgccgat gcgcttgagg agtcgctggt 40920gcgcggctgc gacagcgtgc tctggaccgt gcaggccgtg gccggggcgg gcttccgaga 40980tcctccgcgg ttgtggctcg tgacacgcgg cgctcaggcc atcggcgccg gcgacgtctc 41040tgtggcgcaa gcgccgctcc tggggctggg ccgcgttatc gccttggagc acgccgagct 41100gcgctgcgct cggatcgacc tcgatccagc gcggcgcgac ggagaagtcg atgagctgct 41160tgccgagctg ttggccgacg acgccgagga ggaagtcgcg tttcgcggcg gtgagcggcg 41220cgtggcccgg ctcgtccgaa ggctgcccga gaccgactgc cgagagaaaa tcgagcccgc 41280ggaaggccgg ccgttccggc tggagatcga tgggtccggc gtgctcgacg acctggtgct 41340ccgagccacg gagcggcgcc ctcctggccc gggcgaggtc gagatcgccg tcgaggcggc 41400ggggctcaac tttctcgacg tgatgagggc catggggatc taccctgggc ccggggacgg 41460tccggttgcg ctgggcgccg agtgctccgg ccgaattgtc gcgatgggcg aaggtgtcga 41520gagccttcgt atcggccagg acgtcgtggc cgtcgcgccc ttcagtttcg gcacccacgt 41580caccatcgac gcccggatgc tcgcacctcg ccccgcggcg ctgacggccg cgcaggcagc 41640cgcgctgccc gtcgcattca tgacggcctg gtacggtctc gtccatctgg ggaggctccg 41700ggccggcgag cgcgtgctca tccactcggc gacggggggc accgggctcg ctgctgtgca 41760gatcgcccgc cacctcggcg cggagatatt tgcgaccgct ggtacaccgg agaagcgggc 41820gtggctgcgc gagcagggga tcgcgcacgt gatggactcg cggtcgctgg acttcgccga 41880gcaagtgctg gccgcgacga agggcgaggg ggtcgacgtc gtgttgaact cgctgtctgg 41940cgccgcgatc gacgcgagcc tttcgaccct cgtgccggac ggccgcttca tcgagctcgg 42000caagacggac atctatgcag atcgctcgct ggggctcgct cacttcagga agagcctgtc 42060ctacagcgcc gtcgatcttg cgggcttggc cgtgcgtcgg cccgagcgcg tcgcagcgct 42120gctggcggag gtggtggacc tgctcgcacg gggagcgctg cagccgcttc cggtagagat 42180cttccccctc tcgcgggccg cggacgcgtt ccggaaaatg gcgcaagcgc agcatctcgg 42240gaagctcgtg ctcgcgctgg aggacccgga cgtgcggatc cgcgttccgg gcgaatccgg 42300cgtcgccatc cgcgcggacg gcgcctacct cgtgaccggc ggtctggggg ggctcggtct 42360gagcgtggct ggatggctgg ccgagcaggg ggctgggcat ctggtgctgg tgggccgctc 42420cggcgcggtg agcgcggagc agcagacggc tgtcgccgcg ctcgaggcgc acggcgcgcg 42480tgtcacggta gcgagggcag acgtcgccga tcgggcgcag atggagcgga tcctccgcga 42540ggttaccgcg tcggggatgc cgctccgcgg cgtcgttcat gcggccggaa tcctggacga 42600cgggctgctg atgcagcaaa cccccgcgcg gttccgcgcg gtcatggcgc ccaaggtccg 42660aggggccttg cacctgcatg cgttgacacg cgaagcgccg ctctccttct tcgtgctgta 42720cgcttcggga gcagggctct tgggctcgcc gggccagggc aactacgccg cggccaacac 42780gttcctcgac gcactggcac accaccggag ggcgcagggg ctgccagcat tgagcatcga 42840ctggggcctg ttcgcggacg tgggtttggc cgccgggcag caaaatcgcg gcgcacggct 42900ggtcacccgc gggacgcgga gcctcacccc cgacgaaggg ctgtgggcgc tcgagcgcct 42960gctcgacggc gatcgcaccc aggccggggt catgccgttc gacgtgcggc agtgggtgga 43020gttctacccg gcggcggcat cttcgcggag gttgtcgcgg ctcatgacgg cacggcgcgt 43080ggcttccggt cggctcgccg gggatcggga cctgctcgaa cggctcgcca ccgccgaggc 43140gggcgcgcgg gcagggatgc tgcaggaggt cgtgcgcgcg caggtctcgc aggtgctgcg 43200cctctccgaa ggcaagctcg acgtggatgc gccgctcacg agcctgggaa tggactcgct 43260gatggggcta gagctgcgca accgcatcga ggccgtgctc ggcatcacca tgccggcgac 43320cctgctgtgg acctacccca cggtggcagc gctgagtgcg catctggctt ctcatgtcgt 43380ctctacgggg gatggggaat ccgcgcgccc gccggataca gggagcgtgg ctccaacgac 43440ccacgaagtc gcttcgctcg acgaagacgg gttgttcgcg ttgattgatg cgtcactcgc 43500gcgcgcggga aagaggtgat tgcgtgacag accgagaagg ccagctcctg gagcgcttgc 43560gtgaggttac tctggccctt cgcaagacgc tgaacgagcg cgataccctg gagctcgaga 43620agaccgagcc gatcgccatc gtggggatcg gctgccgctt ccccggcgga gcgggcactc 43680cggaggcgtt ctgggagctg ctcgacgacg ggcgcgacgc gatccggccg ctcgaggagc 43740gctgggcgct cgtaggtgtc gacccaggcg acgacgtacc gcgctgggcg gggctgctca 43800ccgaggccat cgacggcttc gacgccgcgt tcttcggtat cgccccccgg gaggcacggt 43860cgctcgaccc gcagcatcgc ctgctgctgg aggtcgcctg ggaggggttc gaagacgccg 43920gcatcccgcc caggtccctc gtcgggagcc gcaccggcgt gttcgtcggc gtctgcgcca 43980cggagtacct ccacgccgcc gtcgcgcacc agccgcgcga agagcgggac gcgtacagca 44040ccaccggcaa catgctcagc atcgccgccg gacggctatc gtacacgctg gggctgcagg 44100gaccttgcct gaccgtcgat acggcgtgct cgtcatcgct ggtggccatt cacctcgcct 44160gccgcagcct gcgcgctcga gagagcgatc tcgcgctggc gggaggggtc aacatgcttc 44220tctcccccga cacgatgcga gctctggcgc gcacccaggc gctgtcgccc aatggccgtt 44280gccagacctt cgacgcgtcg gccaacgggt tcgtccgtgg ggagggctgc ggtctgatcg 44340tgctcaagcg attgagcgac gcgcggcggg atggggaccg gatctgggcg ctgatccgag 44400gatcggccat caatcaggac ggccggtcga cggggttgac ggcgcccaac gtgctcgccc 44460agggggcgct cttgcgcgag gcgctgcgga acgccggcgt cgaggccgag gccatcggtt 44520acatcgagac ccacggggcg gcaacctcgc tgggcgaccc catcgagatc gaagcgctgc 44580gcgctgtggt ggggccggcg cgagccgacg gagcgcgctg cgtgctgggc gcggtgaaga 44640ccaacctcgg ccacctggag ggcgctgccg gcgtggcggg cctgatcaag gcgacgcttt 44700cgctacatca cgagcgcatc ccgaggaacc tcaactttcg tacgctcaat ccgcggatcc 44760ggatcgaggg gaccgcgctc gcgttggcga ccgaaccggt gccctggccg cggacgggcc 44820ggacgcgctt cgcgggagtg agctcgttcg ggatgagcgg gaccaacgcg catgtggtgt 44880tggaggaggc gccggcggtg gagcctgagg ccgcggcccc cgagcgcgca gcggagctgt 44940tcgtcctgtc ggcgaagagc gcggcggcgc tggatgcgca ggcagcccgg ctgcgggacc 45000acctggagaa gcacgtcgag cttggcctcg gcgatgtggc gttcagcctg gcgacgacgc 45060gcagcgcgat ggagcaccgg ctggcggtgg ccgcgagctc gcgcgaggcg ctgcgagggg 45120cgctttcggc cgcagcgcag gggcacacgc cgccgggagc cgtgcgtggg cgggcctcgg 45180gcggcagcgc gccgaaggtg gtcttcgtgt ttcccggtca gggctcgcag tgggtgggca 45240tgggccgaaa gctcatggcc gaagagccgg tcttccgggc ggcgctggag ggttgcgacc 45300gggccatcga ggcggaagcg ggctggtcgc tgctcgggga gctctccgcc gacgaggccg 45360cctcgcagct cgggcgcatc gacgtggttc agccggtgct cttcgccatg gaagtagcgc 45420tttctgcgct gtggcggtcg tggggagtgg agccggaagc ggtggtgggc cacagcatgg 45480gcgaggttgc ggcggcgcac gtggccggcg cgctgtcgct cgaggacgcg gtggcgatca 45540tctgccggcg cagccggctg ctgcggcgga tcagcggtca gggggagatg gcgctggtcg 45600agctgtcgct ggaggaggcc gaggcggcgc tgcgtggcca tgagggtcgg ctgagcgtgg 45660cggtgagcaa cagcccgcgc tcgaccgtgc tcgccggcga gccggcggcg ctctcggagg 45720tgctggcggc gctgacggcc aagggggtgt tctggcggca ggtgaaggtg gacgtcgcca 45780gccatagccc gcaggtcgac ccgctgcgcg aagagctgat cgcggcgctg ggagcgatcc 45840ggccgcgagc ggctgcggtg ccgatgcgct cgacggtgac gggcggggtg atcgcgggtc 45900cggagctcgg tgcgagctac tgggcggaca accttcggca gccggtgcgc ttcgctgcgg 45960cggcgcaagc gctgctggag ggtggccccg cgctgttcat cgagatgagc ccgcacccga 46020tcctggtgcc gcccctggac gagatccaga cggcggccga gcaagggggc gctgcggtgg 46080gctcgctgcg gcgagggcag gacgagcgcg cgacgctgct ggaggcgctg gggacgctgt 46140gggcgtccgg ctatccggtg agctgggctc ggctgttccc cgcgggcggc aggcgggttc 46200cgctgccgac ctatccctgg cagcacgagc ggtgctggat cgaggtcgag cctgacgccc 46260gccgcctcgc cgcagccgac cccaccaagg actggttcta ccgaacggac tggcccgagg 46320tgccccgcgc cgccccgaaa tcggagacag ctcatgggag ctggctgctg ttggccgaca 46380ggggtggggt cggtgaggcg gtcgctgcag cgctgtcgac gcgcggactt tcctgcaccg 46440tgcttcatgc gtcggctgac gcctccaccg tcgccgagca ggtatccgaa gctgccagtc 46500gccgaaacga ctggcaggga gtcctctacc tgtggggcct cgacgccgtc gtcgatgctg 46560gggcatcggc cgacgaagtc agcgaggcta cccgccgtgc caccgcaccc gtccttgggc 46620tggttcgatt cctgagcgct gcgccccatc ctcctcgctt ctgggtggtg acccgcgggg 46680catgcacggt gggcggcgag ccagaggcct ctctttgcca agcggcgttg tggggcctcg 46740cgcgcgtcgc ggcgctggag caccccgctg cctggggtgg cctcgtggac ctggatcctc 46800agaagagccc gacggagatc gagcccctgg tggccgagct gctttcgccg gacgccgagg 46860atcaactggc gttccgcagc ggtcgcaggc acgcagcacg ccttgtagcc gccccgccgg 46920agggcgacgt cgcaccgata tcgctgtccg cggaggggag ctacctggtg acgggcgggc 46980tgggtggcct tggtctgctc gtggctcggt ggctggtgga gcggggagct cgacatctgg 47040tgctcaccag ccggcacggg ctgccagagc gacaggcgtc gggcggagag cagccgccgg 47100aggcccgcgc gcgcatcgca gcggtcgagg ggctggaagc gcagggcgcg cgggtgaccg 47160tggcagcggt ggatgtcgcc gaggccgatc ccatgacggc gctgctggcc gccatcgagc 47220ccccgttgcg cggggtggtg cacgccgccg gcgtcttccc cgtgcgtcac ctggcggaga 47280cggacgaggc cctgctggag tcggtgctcc gtcccaaggt ggccgggagc tggctgctgc 47340accggctgct gcgcgaccgg cctctcgacc tgttcgtgct gttctcgtcg ggcgcggcgg 47400tgtggggtgg caaaggccaa ggcgcatacg ccgcggccaa tgcgttcctc gacgggctcg 47460cgcaccatcg ccgcgcgcac tcgctgccgg cgttgagcct cgcctggggc ttatgggccg 47520agggaggcat ggttgatgca aaggctcatg cacgtctgag cgacatcggg gtcctgccca 47580tggccacggg gccggccttg tcggcgctgg agcgcctggt gaacaccagc gctgtccagc 47640gttcggtcac acggatggac tgggcgcgct tcgcgccggt ctatgccgcg cgagggcggc 47700gcaacttgct ttcggctctg gtcgcggagg acgagcgcgc tgcgtctccc ccggtgccga 47760cggcaaaccg gatctggcgc ggcctgtccg ttgcggagag ccgctcagcc ctctacgagc 47820tcgttcgcgg catcgtcgcc cgggtgctgg gcttctccga cccgggcgcg ctcgacgtcg 47880gccgaggctt cgccgagcag gggctcgact ccctgatggc tctggagatc cgtaaccgcc 47940ttcagcgcga gctgggcgaa cggctgtcgg cgactctggc cttcgaccac ccgacggtgg 48000agcggctggt ggcgcatctc ctcaccgacg tgctgaagct ggaggaccgg agcgacaccc 48060ggcacatccg gtcggtggcg gcggatgacg acatcgccat cgtcggtgcc gcctgccggt 48120tcccaggtgg ggatgagggc ctggagacat actggcggca tctggccgag ggcatggtgg 48180tcagcaccga ggtgccagcc gaccggtggc gcgcggcgga ctggtacgac cccgatccgg 48240aggttccggg ccggacctat gtggccaagg gtgccttcct ccgcgatgtg cgcagcttgg 48300atgcggcgtt cttcgccatt tcccctcgtg aggcgatgag cctggacccg caacagcggc 48360tgttgctgga ggtgagctgg gaggcgatcg agcgcgctgg ccaggacccg atggcgctgc 48420gcgagagcgc cacgggcgtg ttcgtgggca tgatcgggag cgagcacgcc gagcgggtgc 48480agggcctcga cgacgacgcg gcgttgctgt acggcaccac cggcaacctg ctcagcgtcg 48540ccgctggacg gctgtcgttc ttcctgggtc tgcacggccc gacgatgacg gtggacaccg 48600cctgctcgtc gtcgctggtg gcgttgcacc tcgcctgcca gagcctgcga ttgggcgagt 48660gcgaccaggc cctggccggc gggtccagcg tgcttttgtc gccgcggtca ttcgtcgcgg 48720cgtcgcgcat gcgtttgctt tcgccagatg ggcggtgcaa gacgttctcg gccgctgcag 48780acggctttgc gcgggccgag ggctgcgccg tggtggtgct caagcggctc cgtgacgcgc 48840agcgcgaccg cgaccccatc ctggcggtgg tcaggagcac ggcgatcaac cacgatggcc 48900cgagcagcgg gctcacggtg cccagcggtc ctgcccagca ggcgttgcta cgccaggcgc 48960tggcgcaagc gggcgtggcg ccggccgagg tcgatttcgt ggagtgccac gggacgggga 49020cagcgctggg tgacccgatc gaggtgcagg cgctgggcgc ggtgtacggg cggggccgcc 49080ccgcggagcg gccgctctgg ctgggcgctg tcaaggccaa cctcggccac ctggaggccg 49140cggcgggctt ggccggcgtg ctcaaggtgc tcttggcgct ggagcacgag cagattccgg 49200ctcaaccgga gctcgacgag ctcaacccgc acatcccgtg ggcagagctg ccagtggccg 49260ttgtccgcag ggcggtcccc tggccgcgcg gcgcgcgccc gcgtcgtgca ggcgtgagcg 49320ctttcggcct gagcgggacc aacgcgcatg tggtgttgga ggaggcgccg gcggtggagc 49380ctgtggccgc ggcccccgag cgcgcagcgg agctgttcgt cctgccggcg aagagcgcgg 49440cggcgctgga tgcgcaggca gcccggctgc gggaccacct ggagaagcat gtcgagcttg 49500gcctcggcga tgtggcgttc agcctggcga cgacgcgcag cgcgatggag caccggctgg 49560cggtggccgc gagctcgcgc gaggcgctgc gaggggcgct ttcggccgca gcgcaggggc 49620acacgccgcc gggagccgtg cgtgggcggg cctcgggcgg cagcgcgccg aaggtggtct 49680tcgtgtttcc cggccagggc tcgcagtggg tgggcatggg ccgaaagctc atggccgaag 49740agccggtctt ccgggcggcg ctggagggtt gcgaccgggc catcgaggcg gaagcgggct 49800ggtcgctgct cggggagctc tccgccgacg aggccgcctc gcagctcggg cgcatcgacg 49860tggttcagcc ggtgctgttc gccatggaag tagcgctttc tgcgctgtgg cggtcgtggg 49920gagtggagcc ggaagcggtg gtgggccaca gcatgggcga ggttgcggcg gcgcacgtgg 49980ccggcgcgct gtcgctcgag gacgcggtgg cgatcatctg ccggcgcagc cggctgctgc 50040ggcggatcag cggtcagggg gagatggcgc tggtcgagct gtcgctggag gaggccgagg 50100cggcgctgcg tggccatgag ggtcggctga gcgtggcggt gagcaacagc ccgcgctcga 50160ccgtgctcgc cggcgagccg gcggcgctct cggaggtgct ggcggcgctg acggccaagg 50220gggtgttctg gcggcaggtg aaggtggacg tcgccagcca tagcccgcag gtcgacccgc 50280tgcgcgaaga gctgatcgcg gcgctgggag cgatccggcc gcgagcggct gcggtgccga 50340tgcgctcgac ggtgacgggc ggggtgatcg cgggtccgga gctcggtgcg agctactggg 50400cggacaacct tcggcagccg gtgcgcttcg ctgcggcggc gcaagcgctg ctggagggtg 50460gccccgcgct gttcatcgag atgagcccgc acccgatcct ggtgccgccc ctggacgaga 50520tccagacggc ggccgagcaa gggggcgctg cggtgggctc gctgcggcga gggcaggacg 50580agcgcgcgac gctgctggag gcgctgggga cgctgtgggc gtccggctat ccggtgagct 50640gggctcggct gttccccgcg ggcggcaggc gggttccgct gccgacctat ccctggcagc 50700acgagcggta ctggatcgag gacagcgtgc atgggtcgaa gccctcgctg cggcttcggc 50760agcttcgcaa cggcgccacg gaccatccgc tgctcggggc tccattgctc gtctcggcgc 50820gacccggagc tcacttgtgg gagcaagcgc tgagcgacga gaggctatcc tacctttcgg 50880aacatagggt ccatggcgaa gccgtgttgc ccagcgcggc gtatgtagag atggcgctcg 50940ccgccggcgt agatctctat ggcacggcga cgctggtgct ggagcagctg gcgctcgagc 51000gagccctcgc cgtgccctcc gaaggcggac gcatcgtgca agtggccctc agcgaagaag 51060gtcccggtcg ggcctcattc caggtatcga gtcgtgagga ggcaggtagg agctgggtgc 51120ggcacgccac ggggcacgtg tgtagcggcc agagctcagc ggtgggagcg ttgaaggaag 51180ctccgtggga gattcaacgg cgatgtccga gcgtcctgtc gtcggaggcg ctctatccgc 51240tgctcaacga gcacgccctc gactatggtc cctgcttcca gggcgtggag caggtgtggc 51300tcggcacggg ggaggtgctc ggccgggtac gcttgccagg agacatggca tcctcaagtg 51360gcgcctaccg gattcatccc gccttgttgg atgcatgttt tcaggtgctg acagcgctgc 51420tcaccacgcc ggaatccatc gagattcgga ggcggctgac ggatctccac gaaccggatc 51480tcccgcggtc cagggctccg gtgaatcaag cggtgagtga cacctggctg tgggacgccg 51540cgctggacgg tggacggcgc cagagcgcga gcgtgcccgt cgacctggtg ctcggcagct 51600tccatgcgaa gtgggaggtc atggagcgcc tcgcgcaggc gtacatcatc ggcactctcc 51660gcatatggaa cgtcttctgc gctgctggag agcgtcacac gatagacgag ttgctcgtca 51720ggcttcaaat ctctgtcgtc tacaggaagg tcatcaagcg atggatggaa caccttgtcg 51780cgatcggcat ccttgtaggg gacggagagc attttgtgag ctctcagccg ctgccggagc 51840ctgatttggc ggcggtgctc gaggaggccg ggagggtgtt cgccgacctc ccagtcctat 51900ttgagtggtg caagtttgcc ggggaacggc tcgcggacgt attgaccggt aagacgctcg 51960cgctcgagat cctcttccct ggtggctcgt tcgatatggc ggagcgaatc tatcgagatt 52020cgcccatcgc ccgttactcg aacggcatcg tgcgcggtgt cgtcgagtcg gcggcgcggg 52080tggtagcacc gtcgggaatg ttcagcatct tggagatcgg agcagggacg ggcgcgacca 52140ccgccgccgt cctcccggtg ttgctgcctg accggacgga gtaccatttc accgatgttt 52200ctccgctctt ccttgctcgc gcggagcaaa gatttcgaga ttatccattc ctgaagtatg 52260gcattctgga tgtcgaccag gagccagctg gccagggata cgcacatcag aggtttgacg 52320tcatcgtcgc ggccaatgtc atccatgcga cccgcgatat aagagccacg gcgaagcgtc 52380tcctgtcgtt gctcgcgccc ggaggccttc tggtgctggt cgagggcaca gggcatccga 52440tctggttcga tatcaccacg ggattgattg aggggtggca gaagtacgaa gatgatcttc 52500gtatcgacca tccgctcctg cctgctcgga cctggtgtga cgtcctgcgc cgggtaggct 52560ttgcggacgc cgtgagtctg ccaggcgacg gatctccggc ggggatcctc ggacagcacg 52620tgatcctctc gcgcgcgccg ggcatagcag gagccgcttg tgacagctcc ggtgagtcgg 52680cgaccgaatc gccggccgcg cgtgcagtac ggcaggaatg ggccgatggc tccgctgacg 52740tcgtccatcg gatggcgttg gagaggatgt acttccaccg ccggccgggc cggcaggttt 52800gggtccacgg tcgattgcgt accggtggag gcgcgttcac gaaggcgctc gctggagatc 52860tgctcctgtt cgaagacacc gggcaggtcg tggcagaggt tcaggggctc cgcctgccgc 52920agctcgaggc ttctgctttc gcgccgcggg acccgcggga agagtggttg tacgctttgg 52980aatggcagcg caaagaccct ataccagagg ctccggcagc cgcgtcttct tcctccgcgg 53040gggcttggct cgtgctgatg gaccagggcg ggacaggcgc tgcgctcgta tcgctgctgg 53100aagggcgagg cgaggcgtgc gtgcgcgtca tcgcgggtac ggcatacgcc tgcctcgcgc 53160cggggctgta tcaagtcgat ccggcgcagc cagatggctt tcataccctg ctccgcgatg 53220cattcggcga ggaccggatt tgtcgcgcgg tagtgcatat gtggagcctt gatgcgacgg 53280cagcagggga gagggcgaca gcggagtcgc ttcaggccga tcaactcctg gggagcctga 53340gcgcgctttc tctggtgcag gcgctggtgc gccggaggtg gcgcaacatg ccgcggcttt 53400ggctcttgac ccgcgccgtg catgcggtgg gcgcggagga cgcagcggcc tcggtggcgc 53460aggcgccggt gtggggcctc ggtcggacgc tcgcgctcga gcatccagag ctgcggtgca 53520cgctcgtgga cgtgaacccg gcgccgtctc cagaggacgc agccgcactg gcggtggagc 53580tcggggcgag cgacagagag gaccaggtcg cattgcgctc ggatggccgc tacgtggcgc 53640gcctcgtgcg gagctccttt tccggcaagc ctgctacgga ttgcggcatc cgggcggacg 53700gcagctatgt gatcaccgat ggcatgggga gagtggggct ctcggtcgcg caatggatgg 53760tgatgcaggg ggcccgccat gtggtgctcg tggatcgcgg cggcgcttcc gaggcatccc 53820gggatgccct ccggtccatg gccgaggctg gcgcggaggt gcagatcgtg gaggccgacg 53880tggctcggcg cgacgatgtc gctcggctcc tctcgaagat cgaaccgtcg atgccgccgc 53940ttcgggggat cgtgtacgtg gacgggacct tccagggcga ctcctcgatg ctggagctgg 54000atgcccgtcg cttcaaggag tggatgtatc ccaaggtgct cggagcgtgg aacctgcacg 54060cgctgaccag ggatagatcg ctggacttct tcgtcctgta ttcctcgggc acctcgcttc 54120tgggcttgcc aggacagggg agccgcgccg ccggtgacgc cttcttggac gccatcgcgc 54180atcaccggtg caaggtgggc cttacagcga tgagcatcaa ctggggattg ctctccgaag 54240catcatcgcc ggcgaccccg aacgacggcg gagcacggct cgaataccgg gggatggaag 54300gcctcacgct ggagcaggga gcggcggcgc tcgggcgctt gctcgcacga cccagggcgc 54360aggtaggggt gatgcggctg aatctgcgcc agtggttgga gttctatccc aacgcggccc 54420gattggcgct gtgggcggag ctgctgaagg agcgtgaccg cgccgaccga ggcgcgtcga 54480acgcgtcgaa cctgcgcgag gcgctgcaga gcgccaggcc cgaagatcgt cagttgattc 54540tggagaagca cttgagcgag ctgttggggc gggggctgcg ccttccgccg gagaggatcg 54600agcggcacgt gccgttcagc aatctcggca tggactcgct gataggcctg gagctccgca 54660accgcatcga ggccgcgctc ggcatcaccg tgccggcgac cctgctatgg acctacccta 54720acgtagcagc tctgagcggg agcttgctag acattctgtt tccgaatgcc ggcgcgaccc 54780acgctccggc caccgagcgg gagaagagct tcgagaacga tgccgcagat ctcgaggctc 54840tgcggggcat gacggacgag cagaaggacg cgttgctcgc cgaaaagctg gcgcagctcg 54900cgcagatcgt tggtgagtaa gggaccgagg gagtatggcg accacgaatg ccgggaagct 54960tgagcatgcc cttctgctca tggacaagct tgcgaaaaag aacgcgtctt tggagcaaga 55020gcggaccgag ccgatcgcca tcgtaggcat tggctgccgc ttccccggcg gagcggacac 55080tccggaggca ttctgggagc tgctcgactc aggccgagac gcggtccagc cgctcgaccg 55140gcgctgggcg ctggtcggcg tccatcccag cgaggaggtg ccgcgctggg ccggactgct 55200caccgaggcg gtggacggct tcgacgccgc gttctttggc acctcgcctc gggaggcgcg 55260gtcgctcgat cctcagcaac gcctgctgct ggaggtcacc tgggaagggc tcgaggacgc 55320cggcatcgca ccccagtccc tcgacggcag ccgcaccggg gtgttcctgg gcgcatgcag 55380cagcgactac tcgcataccg ttgcgcaaca gcggcgcgag gagcaggacg catacgacat 55440caccggcaat acgctcagcg tcgccgccgg acggttgtct tatacgctag ggctgcaggg 55500accctgcctg accgtcgaca cggcctgctc gtcgtcgctc gtggccatcc accttgcctg 55560ccgcagcctg cgcgctcgcg agagcgatct cgcgctggcg ggaggcgtca acatgctcct 55620ttcgtccaag acgatgataa tgctggggcg catccaggcg ctgtcgcccg atggccactg 55680ccggacattc gacgcctcgg ccaacgggtt cgtccgtggg gagggctgcg gtatggtcgt 55740gctcaaacgg ctctccgacg cccagcgaca cggcgatcgg atctgggctc tgatccgggg 55800ttcggccatg aatcaggatg gccggtcgac agggttgatg gcacccaatg tgctcgctca 55860ggaggcgctc ttgcgcgagg cgctgcagag cgctcgcgtc gacgccgggg ccatcggtta 55920tgtcgagacc cacggaacgg ggacctcgct cggcgacccg atcgaggtcg aggcgctgcg 55980tgccgtgttg gggccggcgc gggccgatgg gagccgctgc gtgctgggcg cagtgaagac 56040aaacctcggc cacctggagg gcgctgcagg cgtggcgggt ttgatcaagg cggcgctggc 56100tctgcaccac gaactgatcc cgcgaaacct ccatttccac acgctcaatc cgcggatccg 56160gatcgagggg accgcgctcg cgctggcgac ggagccggtg ccgtggccgc gggcgggccg 56220accgcgcttc gcgggggtga gcgcgttcgg cctcagcggc accaacgtcc atgtcgtgct 56280ggaggaggcg ccggccacgg tgctcgcacc ggcgacgccg gggcgctcag cggagctttt 56340ggtgctgtcg gcgaagagcg ccgccgcgct ggacgcacag gcggcgcggc tctcagcgca 56400catcgccgcg tacccggagc agggtctcgg agacgtcgcg ttcagcctgg tatcgacgcg 56460tagcccgatg gagcaccggc tcgcggtggc ggcgacctcg cgcgaggcgc tgcgaagcgc 56520gctggaggtt gcggcgcagg ggcagacccc ggcaggcgcg gcgcgcggca gggccgcttc 56580ctcgcccggc aagctcgcct tcctgttcgc cgggcagggc gcgcaggtgc cgggcatggg 56640ccgtgggttg tgggaggcgt ggccggcgtt ccgcgagacc ttcgaccggt gcgtcacgct 56700cttcgaccgg gagctccatc agccgctctg cgaggtgatg tgggccgagc cgggcagcag 56760caggtcgtcg ttgctggacc agacggcgtt cacccagccg gcgctctttg cgctggagta 56820cgcgctggcc gcgctcttcc ggccgtgggg cgtggagccg gagctcgtcg ccggccatag 56880cctcggcgag ctggtggccg cctgcgtggc gggtgtgttc tccctcgagg acgccgtgcg 56940cttggtggtc gcgcgcggcc ggttgatgca ggcgctgccg gccggcggcg cgatggtatc 57000gatcgccgcg ccggaggccg acgtggctgc cgcggtggcg ccgcacgcag cgttggtgtc 57060gatcgcggca gtcaatgggc cggagcaggt ggtgatcgcg ggcgccgaga aattcgtgca 57120gcagatcgcg gcggcgttcg cggcgcgggg ggcgcgaacc aaaccgctgc atgtctcgca 57180cgcgttccac tcgccgctca tggatccgat gctggaggcg ttccggcggg tgactgagtc 57240ggtgacgtac cggcggcctt cgatcgcgct ggtgagcaac ctgagcggga agccctgcac 57300cgatgaggtg agcgcgccgg gttactgggt gcgtcacgcg cgagaggcgg tgcgcttcgc 57360ggacggagtg aaggcgctgc acgcggccgg tgcgggcctc ttcgtcgagg tggggccgaa 57420gccgacgctg ctcggccttg tgccggcctg cctgccggat gccaggccgg tgctgctccc 57480agcgtcgcgc gccgggcgtg acgaggctgc gagcgcgcta gaggcgctgg gtgggttctg 57540ggtcgtcggt ggatcggtca cctggtcggg tgtcttccct tcgggcggac ggcgggtacc 57600gctgccaacc tatccctggc agcgcgagcg ttactggatc gaagcgccgg tcgatcgtga 57660ggcggacggc accggccgtg ctcgggcggg gggccacccc cttctgggtg aagtcttttc 57720cgtgtcgacc catgccggtc tgcgcctgtg ggagacgacg ctggaccgaa agcggctgcc 57780gtggctcggc gagcaccggg cgcaggggga ggtcgtgttt cctggcgccg ggtacctgga 57840gatggcgctg tcgtcggggg ccgagatctt gggcgatgga ccgatccagg tcacggatgt 57900ggtgctcatc gagacgctga ccttcgcggg cgatacggcg gtaccggtcc aggtggtgac 57960gaccgaggag cgaccgggac ggctgcggtt ccaggtagcg agtcgggagc cgggggaacg 58020tcgcgcgccc ttccggatcc acgcccgcgg cgtgctgcgc cggatcgggc gcgtcgagac 58080cccggcgagg tcgaacctcg ccgccctgcg cgcccggctt catgccgccg tgcccgctgc 58140ggctatctat ggtgcgctcg ccgagatggg gcttcaatac ggcccggcgt tgcgggggct 58200cgccgagctg tggcggggtg agggcgaggc gctgggcagg gtgagactgc ctgaggccgc 58260cggctccgcg acagcctacc agctgcatcc ggtgctgctg gacgcgtgcg tccaaatgat 58320tgttggcgcg ttcgccgatc gcgatgaggc gacgccgtgg gcgccggtgg aggtgggctc 58380ggtgcggctg ttccagcggt ctcctgggga gctatggtgc catgcgcgcg tcgtgagcga 58440tggtcaacag gcctccagcc ggtggagcgc cgactttgag ttgatggacg gtacgggcgc 58500ggtggtcgcc gagatctccc ggctggtggt ggagcggctt gcgagcggtg tacgccggcg 58560cgacgcagac gactggttcc tggagctgga ttgggagccc gcggcgctcg gtgggcccaa 58620gatcacagcc ggccggtggc tgctgctcgg cgagggtggt gggctcgggc gctcgttgtg 58680ctcggcgctg aaggccgccg gccatgtcgt cgtccacgcc gcgggggacg acacgagcac 58740tgcaggaatg cgcgcgctcc tggccaacgc gttcgacggc caggccccga cggccgtggt 58800gcacctcagc agcctcgacg ggggcggcca gctcggcccg gggctcgggg cgcagggcgc 58860gctcgacgcg ccccggagcc cagatgtcga tgccgatgcc ctcgaatcgg cgctgatgcg 58920tggttgcgac agcgtgctct ccctggtgca agcgctggtc ggcatggacc tccgaaacgc 58980gccgcggctg tggctcttga cccgcggggc tcaggcggcc gccgccggcg atgtctccgt 59040ggtgcaagcg ccgctgttgg ggctgggccg caccatcgcc ttggagcacg ccgagctgcg 59100ctgtatcagc gtcgacctcg atccagccga gcctgaaggg gaagccgatg ctttgctggc 59160cgagctactt gcagatgatg ccgaggagga ggtcgcgctg cgcggtggcg accggctcgt 59220tgcgcggctc gtccaccggc tgcccgacgc tcagcgccgg gagaaggtcg agcccgccgg 59280tgacaggccg ttccggctag agatcgatga acccggcgcg ctggaccaac tggtgctccg 59340agccacgggg cggcgcgctc ctggtccggg cgaggtcgag atctccgtcg aagcggcggg 59400gctcgactcc atcgacatcc agctggcgtt gggcgttgct cccaatgatc tgcctggaga 59460agaaatcgag ccgttggtgc tcggaagcga gtgcgccggg cgcatcgtcg ctgtgggcga 59520gggcgtgaac ggccttgtgg tgggccagcc ggtgatcgct cttgcggcgg gagtatttgc 59580tacccatgtc accacgtcgg ccacgctggt gttgcctcgg cctctggggc tctcggcgac 59640cgaggcggcc gcgatgcccc tcgcgtattt gacggcctgg tacgccctcg acaaggtcgc 59700ccacctgcag gcgggggagc gggtgctgat ccatgcggag gccggtggtg tcggtctttg 59760cgcggtgcga tgggcgcagc gcgtgggcgc cgaggtgtat gcgaccgccg acacgcccga 59820gaaccgtgcc tacctggagt cgctgggcgt gcggtacgtg agcgattccc gctcgggccg 59880gttcgtcaca gacgtgcatg catggacgga cggcgagggt gtggacgtcg tgctcgactc 59940gctttcgggc gagcgcatcg acaagagcct catggtcctg cgcgcctgtg gtcgccttgt 60000gaagctgggc aggcgcgacg actgcgccga cacgcagcct gggctgccgc cgctcctacg 60060gaatttttcc ttctcgcagg tggacttgcg gggaatgatg ctcgatcaac cggcgaggat 60120ccgtgcgctc ctcgacgagc tgttcgggtt ggtcgcagcc ggtgccatca gcccactggg 60180gtcggggttg cgcgttggcg gatccctcac gccaccgccg gtcgagacct tcccgatctc 60240tcgcgcagcc gaggcattcc ggaggatggc gcaaggacag catctcggga agctcgtgct 60300cacgctggac gacccggagg tgcggatccg cgctccggcc gaatccagcg tcgccgtccg 60360cgcggacggc acctaccttg tgaccggcgg tctgggtggc ctcggtctgc gcgtggccgg 60420atggctggcc gagcggggcg cggggcaact ggtgctggtg ggccgctccg gtgcggcgag 60480cgcagagcag cgagccgccg tggcggcgct ggaggcccac ggcgcgcgcg tcacggtggc 60540gaaagcggac gtcgccgatc ggtcacagat cgagcgggtc ctccgcgagg ttaccgcgtc 60600ggggatgccg ctgcggggtg tcgtgcatgc ggcaggtctc gtggacgacg ggctgctgat 60660gcagcagact ccggcgcggt tccgcacggt gatgggacct aaggtccagg gggccttgca 60720cttgcacacg ctgacacgcg aagcgcctct ttccttcttc gtgctgtacg cttctgcagc 60780tgggcttttc ggctcgccag gccagggcaa ctacgccgca gccaacgcgc tcctcgacgc 60840cctttcgcat caccgaaggg cgcagggcct gccggcgctg agcatcgact ggggcatgtt 60900cacggaggtg gggatggccg tcgcgcaaga aaaccgtggc gcgcggcaga tctctcgcgg 60960gatgcggggc accacccccg atgagggtct gtcagctctg gcgcgcttgc tcgagggtga 61020tcgcgtgcag acgggggtga taccgatcac tccgcggcag tgggtggagt tctacccggc 61080aacagcggcc tcacggaggt tgtcgcggct ggtgaccacg cagcgcgcgg tcgctgatcg 61140gaccgccggg gatcgggacc tgctcgaaca gcttgcgtcg gctgagccga gcgcgcgggc 61200ggggctgctg caggacgtcg tgcgcgtgca ggtctcgcat gtgctgcgtc tccctgaaga 61260caagatcgag gtggatgccc cgctctcgag catgggcatg gactcgctga tgagcctgga 61320gctgcgcaac cgcatcgagg ctgcgctggg cgtcgccgcg cctgcagcct tggggtggac 61380gtacccaacg gtagcagcga taacgcgctg gctgctcgac gacgccctcg tcgtccggct 61440tggcggcggg tcggacacgg acgaatcgac ggcgagcgcc ggttcgttcg tccacgtcct 61500ccgctttcgt cctgtcgtca agccgcgggc tcgtctcttc tgttttcacg gttctggcgg 61560ctcgcccgag ggcttccgtt cctggtcgga gaagtctgag tggagcgatc tggaaatcgt 61620ggccatgtgg cacgatcgca gcctcgcctc cgaggacgcg cctggtaaga agtacgtcca 61680agaggcggcc tcgctgattc agcactatgc agacgcaccg tttgcgttag tagggttcag 61740cctgggtgtc cggttcgtca tggggacagc cgtggagctc gccagtcgtt ccggcgcacc 61800ggctccgctg gccgtcttca cgttgggcgg cagcttgatc tcttcttcag agatcacccc 61860ggagatggag accgatataa tagccaagct cttcttccga aatgccgcgg gtttcgtgcg 61920atccacccaa caagtccagg ccgatgctcg cgcagacaag gtcatcacag acaccatggt 61980ggctccggcc cccggggact cgaaggagcc gcccgtgaag atcgcggtcc ctatcgtcgc 62040catcgccggc tcggacgatg tgatcgtgcc tccgagcgac gttcaggatc tacaatctcg 62100caccacggag cgcttctata tgcatctcct tcccggagat cacgaatttc tcgtcgatcg 62160agggcgcgag atcatgcaca tcgtcgactc gcatctcaat ccgctgctcg ccgcgaggac 62220gacgtcgtca ggccccgcgt tcgaggcaaa atgatggcag cctccctcgg gcgcgcgaga 62280tggttgggag cagcgtgggc gctggcggcc ggcggcaggc cgcggaggcg catgagcctt 62340cctggacgtt tgcagtatag gagattttat gacacaggag caagcgaatc agagtgagac 62400gaagcctgct ttcgacttca agccgttcgc gcctgggtac gcggaggacc cgttccccgc 62460gatcgagcgc ctgagagagg caacccccat cttctactgg gatgaaggcc gctcctgggt 62520cctcacccga taccacgacg tgtcggcggt gttccgcgac gaacgcttcg cggtcagtcg 62580agaagagtgg gaatcgagcg cggagtactc gtcggccatt cccgagctca gcgatatgaa 62640gaagtacgga ttgttcgggc tgccgccgga ggatcacgct cgggtccgca agctcgtcaa 62700cccgtcgttt acgtcacgcg ccatcgacct gctgcgcgcc gaaatacagc gcaccgtcga 62760ccagctgctc gatgctcgct ccggacaaga ggagttcgac gttgtgcggg attacgcgga 62820gggaatcccg atgcgcgcga tcagcgctct gttgaaggtt ccggccgagt gtgacgagaa 62880gttccgtcgc ttcggctcgg cgactgcgcg cgcgctcggc gtgggtttgg tgccccaggt 62940cgatgaggag accaagaccc tggtcgcgtc cgtcaccgag gggctcgcgc tgctccatga 63000cgtcctcgat gagcggcgca ggaacccgct cgaaaatgac gtcttgacga tgctgcttca 63060ggccgaggcc gacggcagca ggctgagcac gaaggagctg gtcgcgctcg tgggtgcgat 63120tatcgctgct ggcaccgata ccacgatcta ccttatcgcg ttcgctgtgc tcaacctgct 63180gcggtcgccc gaggcgctcg agctggtgaa ggccgagccc gggctcatga ggaacgcgct 63240cgatgaggtg ctccgcttcg acaatatcct cagaatagga actgtgcgtt tcgccaggca 63300ggacctggag tactgcgggg catcgatcaa gaaaggggag atggtctttc tcctgatccc 63360gagcgccctg agagatggga ctgtattctc caggccagac gtgtttgatg tgcgacggga 63420cacgggcgcg agcctcgcgt acggtagagg cccccatgtc tgccccgggg tgtcccttgc 63480tcgcctcgag gcggagatcg ccgtgggcac catcttccgt aggttccccg agatgaagct 63540gaaagaaact cccgtgtttg gataccaccc cgcgttccgg aacatcgaat cactcaacgt 63600catcttgaag ccctccaaag ctggatagct cgcgggggta tcgcttcccg aacctcattc 63660cctcatgata cagctcgcgc gcgggtgctg tctgccgcgg gtgcgattcg atccagcgga 63720caagcccatt gtcagcgcgc gaagatcgaa tccacggccc ggagaagagc ccgtccgggt 63780gacgtcggaa gaagtgccgg gcgccgccct gggagcgcaa agctcgctcg ttcgcgctca 63840gcacgccgct cgtcatgtcc ggccctgcac ccgcgccgag gagccgcccg ccctgatgca 63900cggcctcacc gagcggcagg ttctgctctc gctcgtcgcc ctcgcgctcg tcctcctgac 63960cgcgcgcgcc ttcggcgagc tcgcgcggcg gctgcgccag cccgaggtgc tcggcgagct 64020cttcggcggc gtggtgctgg gcccgtccgt cgtcggcgcg ctcgctcctg ggttccatcg 64080agtcctcttc caggatccgg cggtcggggt cgtgctctcc ggcatctcct ggataggcgc 64140gctcgtcctg ctgctcatgg cgggtatcga ggtcgatgtg agcatcctgc gcaaggaggc 64200gcgccccggg gcgctctcgg cgctcggcgc gatcgcgccc ccgctgcgca cgccggggcc 64260gctggtgcag cgcatgcagg gcgcgttcac gtgggatctc gacgtctcgc cgcgacgctc 64320tgcgcaagcc tgagcctcgg cgcctgctcg tacacctcgc cggtgctcgc tccgcccgcg 64380gacatccggc cgcccgccgc ggcccagctc gagccggact cgccggatga cgaggccgac 64440gaggccgacg aggcgctccg cccgttccgc gacgcgatcg ccgcgtactc ggaggccgtt 64500cggtgggcgg aggcggcgca gcggccgcgg ctggagagcc tcgtgcggct cgcgatcgtg 64560cggctgggca aggcgctcga caaggtccct ttcgcgcaca cgacggccgg cgtctcccag 64620atcgccggca gactccagaa cgatgcggtc tggttcgatg tcgccgcccg gtacgcgagc 64680ttccgcgcgg cgacggagca cgcgctccgc gacgcggcgt cggccatgga ggcgctcgcg 64740gccggcccgt accgcggatc gagccgcgtg tccgctgccg taggggagtt tcggggggag 64800gcggcgcgcc ttcaccccgc ggaccgtgta cccgcgtccg accagcagat cctgaccgcg 64860ctgcgcgcag ccgagcgggc gctcatcgcg ctctacactg cgttcgcccg tgaggagtga 64920gcctctctcg ggcgcagccg agcggcggcg tgccggtggt tccctcttcg caaccatgac 64980cggagccgcg ctcggtccgc gcagcggcta gcgcgcgtcg cggcagagat cgctggagcg 65040acaggcgacg acccgcccga gggtgtcgaa cggattgccg cagccctcat tgcggatccc 65100ctccagacac tcgttcagct gcttggcgtc gatgccgcct gggcactcgc cgaaggtcag 65160ctcgtcgcgc cactcggatc ggatcttgtt cgagcacgcg tccttgctcg aatactcccg 65220gtcttgtccg atgttgttgc accgcgcctc gcggtcgcac cgcgccgcca cgatgctatc 65280gacggcgctg ccgactggca ccggcgcctc gccctgcgcg ccacccgggg tttgcgcctc 65340cccgcctgac cgcttttcgc cgccgcacgc cgcgagcagg ctcattcccg acaccgagat 65400caggcccacg accagcttcc cagcaatctt ttgcatggct tcccctccct cacgacacgt 65460cacatcagag actctccgct cggctcgtcg gttcgacagc cggcgacggc cacgagcaga 65520accgtccccg accagaacag ccgcatgcgg gtttctcgca acatgccccg acatccttgc 65580gactagcgtg cctccgctcg tgccgagatc ggctgtcctg tgcgacggca atatcctgcg 65640atcggccggg caggaggtac cgacacgggc gccgggcggg aggtgccgcc acgggctcga 65700aatgtgctgc ggcaggcgcc tccatgcccg cagccgggaa cgcggcgccc ggccagcctc 65760ggggtgacgc cgcaaacggg agatgctccc ggagaggcgc cgggcacagc cgagcgccgt 65820caccaccgtg cgcactcgtg agctccagct cctcggcata gaagagaccg tcactcccgg 65880tccgtgtagg cgatcgtgct gatcagcgcg ttctccgcct gacgcgagtc gagccgggta 65940tgctgcacga caatgggaac gtccgattcg atcacgctgg catagtccgt atcgcgcggg 66000atcggctcgg gttcggtcag atcgttgaac cggacgtgcc gggtgcgcct cgctgggacg 66060gtcacccggt acggcccggc ggggtcgcgg tcgctgaagt agacggtgat ggcgacctgc 66120gcgtcccggt ccgacgcatt caacaggcag gccgtctcat ggctcgtcat ctgcggctcg 66180ggtccgttgc tccggcctgg gatgtagccc tctgcgattg cccagcgcgt ccgcccgatc 66240ggcttctcca tatgtcctcc ctgctggctc ctctttggct gcctccctct gctgtccagg 66300agcgacggcc tcttctcccg acgcgctcgg ggatccatgg ctgaggatcc tcgccgagcg 66360ctccttgccg accggcgcgc cgagcgccga cgggctttga aagcacgcga ccggacacgt 66420gatgccggcg cgacgaggcc gccccgcgtc tgatcccgat cgtgacatcg cgacgtccgc 66480cggcgcctct gcaggccggc ctgagcgttg cgcggtcatg gtcgtcctcg cgtcaccgcc 66540acccgccgat tcacatccca ccgcggcacg acgcttgctc aaaccgcggc gagacggccg 66600ggcggctgtg gtaccggcca gcccggacgc gaggcccgag agggacagtg ggtccgccgt 66660gaagcagtga ggcgatcgag gtggcagatg aaacacgttg acacgggccg acgagtcggc 66720cgccggatag ggctcacgct cggtctcctc gcgagcatgg cgctcgccgg ctgtggcggc 66780ccgagcgaga aaatcgtgca gggcacgcgg ctcgcgcccg gcgccgatgc gcacgtcgcc 66840gccgacgtcg accccgacgc cgcgaccacg cggctggcgg tggacgtcgt tcacctctcg 66900ccgcccgagc gcatcgaggc cggcagcgag cggttcgtcg tctggcagcg tccgagctcc 66960gagtccccgt ggcaacgggt cggagtgctc gactacaacg ctgccagccg aagaggcaag 67020ctggccgaga cgaccgtgcc gcatgccaac ttcgagctgc tcatcaccgt cgagaagcag 67080agcagccctc agtctccatc ttctgccgcc gtcatcgggc cgacgtccgt cgggtaacat 67140cgcgctatca gcagcgctga gcccgccagc aggccccaga gccctgcctc gatcgccttc 67200tccatcatat catccctgcg tactcctcca gcgacggccg cgtcgaagca accgccgtgc 67260cggcgcggct ctacgtgcgc gacaggagag cgtcctggcg cggcctgcgc atcgctggaa 67320ggatcggcgg agcatggaga aagaatcgag gatcgcgatc tacggcgcca tcgcagccaa 67380cgtggcgatc gcggcggtca agttcatcgc cgccgccgtg accggcagct cggcgatgct 67440ctccgagggc gtgcactccc tcgtcgatac tgcagacggg ctcctcctcc tgctcggcaa 67500gcaccggagc gcacgcccgc ccgacgccga gcatccgttc ggccacggca aggagctcta 67560tttctggacg ctgatcgtcg ccatcatgat cttcgccgcg ggcggcggcg tctcgatcta 67620cgaagggatc ttgcacctct tgcacccgcg ccagatcgag gatccgacgt ggaactacgt 67680cgtcctcggc gcagcggccg tcttcgaggg gacgtcgctc atcatctcga tccacgagtt 67740caagaagaag gacggacagg gctacctcgc ggcgatgcgg tccagcaagg acccgacgac 67800gttcacgatc gtcctggagg actccgcggc gctcgccggg ctcaccatcg ccttcctcgg 67860cgtctggctc gggcaccgcc tgggaaaccc ctacctcgac ggcgcggcgt cgatcggcat 67920cggcctcgtg ctcgccgcgg tcgcggtctt cctcgccagc cagagccgtg ggctcctcgt 67980gggggagagc gcggacaggg agctcctcgc cgcgatccgc gcgctcgcca gcgcagatcc 68040tggcgtgtcg gcggtggggc ggcccctgac gatgcacttc ggtccgcacg aagtcctggt 68100cgtgctgcgc atcgagttcg acgccgcgct cacggcgtcc ggggtcgcgg aggcgatcga 68160gcgcatcgag acccggatac ggagcgagcg acccgacgtg aagcacatct acgtcgaggc 68220caggtcgctc caccagcgcg cgagggcgtg acgcgccgtg gagagaccgc gcgcggcctc 68280cgccatcctc cgcggcgccc gggctcaggt ggccctcgca gcagggcgcg cctggcgggc 68340aaaccgtgca gacgtcgtcc ttcgacgcga ggtacgctgg ttgcaagtcg tcacgccgta 68400tcgcgaggtc cggcagcgcc ggagcccggg cgggccgggc gcacgaaggc gcggcgagcg 68460caggcttcga ggggggcgac gtcatgagga aggccagggc gcatggggcg atgctcggcg 68520ggcgagatga cggctggcgt cgcggcctcc ccggcgccgg cgcgcttcgc gccgcgctcc 68580agcgcggtcg ctcgcgcgat ctcgcccggc gccggctcat cgcctccgtg tccctcgccg 68640gcggcgccag catggcggtc gtctcgctgt tccagctcgg gatcatcgag cgcctgcccg 68700atcctccgct tccagggttc gattcggcca aggtgacgag ctccgatatc68750<210>2<211>1421<212>PRT<213>纤维堆囊菌<400>2Val Ala Asp Arg Pro Ile Glu Arg Ala Ala Glu Asp Pro Ile Ala Ile1 5 10 15Val Gly Ala Ser Cys Arg Leu Pro Gly Gly Val Ile Asp Leu Ser Gly20 25 30Phe Trp Thr Leu Leu Glu Gly Ser Arg Asp Thr Val Gly Arg Val Pro35 40 45Ala Glu Arg Trp Asp Ala Ala Ala Trp Phe Asp Pro Asp Pro Asp Ala50 55 60Pro Gly Lys Thr Pro Val Thr Arg Ala Ser Phe Leu Ser Asp Val Ala65 70 75 80Cys Phe Asp Ala Ser Phe Phe Gly Ile Ser Pro Arg Glu Ala Leu Arg85 90 95Met Asp Pro Ala His Arg Leu Leu Leu Glu Val Cys Trp Glu Ala Leu100 105 110Glu Asn Ala Ala Ile Ala Pro Ser Ala Leu Val Gly Thr Glu Thr Gly115 120 125Val Phe Ile Gly Ile Gly Pro Ser Glu Tyr Glu Ala Ala Leu Pro Gln130 135 140Ala Thr Ala Ser Ala Glu Ile Asp Ala His Gly Gly Leu Gly Thr Met145 150 155 160Pro Ser Val Gly Ala Gly Arg Ile Ser Tyr Ala Leu Gly Leu Arg Gly165 170 175Pro Cys Val Ala Val Asp Thr Ala Tyr Ser Ser Ser Leu Val Ala Val180 185 190His Leu Ala Cys Gln Ser Leu Arg Ser Gly Glu Cys Ser Thr Ala Leu195 200 205Ala Gly Gly Val Ser Leu Met Leu Ser Pro Ser Thr Leu Val Trp Leu210 215 220Ser Lys Thr Arg Ala Leu Ala Arg Asp Gly Arg Cys Lys Ala Phe Ser225 230 235 240Ala Glu Ala Asp Gly Phe Gly Arg Gly Glu Gly Cys Ala Val Val Val245 250 255Leu Lys Arg Leu Ser Gly Ala Arg Ala Asp Gly Asp Arg Ile Leu Ala260 265 270Val Ile Arg Gly Ser Ala Ile Asn His Asp Gly Ala Ser Ser Gly Leu275 280 285Thr Val Pro Asn Gly Ser Ser Gln Glu Ile Val Leu Lys Arg Ala Leu290 295 300Ala Asp Ala Gly Cys Ala Ala Ser Ser Val Gly Tyr Val Glu Ala His305 310 315 320Gly Thr Gly Thr Thr Leu Gly Asp pro Ile Glu Ile Gln Ala Leu Asn325 330 335Ala Val Tyr Gly Leu Gly Arg Asp Val Ala Thr Pro Leu Leu Ile Gly340 345 350Ser Val Lys Thr Asn Leu Gly His Pro Glu Tyr Ala Ser Gly Ile Thr355 360 365Gly Leu Leu Lys Val Val Leu Ser Leu Gln His Gly Gln Ile Pro Ala370 375 380His Leu His Ala Gln Ala Leu Asn Pro Arg Ile Ser Trp Gly Asp Leu385 390 395 400Arg Leu Thr Val Thr Arg Ala Arg Thr Pro Trp Pro Asp Trp Asn Thr405 410 415Pro Arg Arg Ala Gly Val Ser Ser Phe Gly Met Ser Gly Thr Asn Ala420 425 430His Val Val Leu Glu Glu Ala Pro Ala Ala Thr Cys Thr Pro Pro Ala435 440 445Pro Glu Arg Pro Ala Glu Leu Leu Val Leu Ser Ala Arg Thr Ala Ser450 455 460Ala Leu Asp Ala Gln Ala Ala Arg Leu Arg Asp His Leu Glu Thr Tyr465 470 475 480Pro Ser Gln Cys Leu Gly Asp Val Ala Phe Ser Leu Ala Thr Thr Arg485 490 495Ser Ala Met Glu His Arg Leu Ala Val Ala Ala Thr Ser Arg Glu Gly500 505 510Leu Arg Ala Ala Leu Asp Ala Ala Ala Gln Gly Gln Thr Ser Pro Gly515 520 525Ala Val Arg Ser Ile Ala Asp Ser Ser Arg Gly Lys Leu Ala Phe Leu530 535 540Phe Thr Gly Gln Gly Ala Gln Thr Leu Gly Met Gly Arg Gly Leu Tyr545 550 555 560Asp Val Trp Ser Ala Phe Arg Glu Ala Phe Asp Leu Cys Val Arg Leu565 570 575Phe Asn Gln Glu Leu Asp Arg Pro Leu Arg Glu Val Met Trp Ala Glu580 585 590Pro Ala Ser Val Asp Ala Ala Leu Leu Asp Gln Thr Ala Phe Thr Gln595 600 605Pro Ala Leu Phe Thr Phe Glu Tyr A la Leu Ala Ala Leu Trp Arg Ser610 615 620Trp Gly Val Glu Pro Glu Leu Val Ala Gly His Ser Ile Gly Glu Leu625 630 635 640Val Ala Ala Cys Val Ala Gly Val Phe Ser Leu Glu Asp Ala Val Phe645 650 655Leu Val Ala Ala Arg Gly Arg Leu Met Gln Ala Leu Pro Ala Gly Gly660 665 670Ala Met Val Ser Ile Glu Ala Pro Glu Ala Asp Val Ala Ala Ala Val675 680 685Ala Pro His Ala Ala Ser Val Ser Ile Ala Ala Val Asn Ala Pro Asp690 695 700Gln Val Val Ile Ala Gly Ala Gly Gln Pro Val His Ala Ile Ala Ala705 710 715 720Ala Met Ala Ala Arg Gly Ala Arg Thr Lys Ala Leu His Val Ser His725 730 735Ala Phe His Ser Pro Leu Met Ala Pro Met Leu Glu Ala Phe Gly Arg740 745 750Val Ala Glu Ser Val Ser Tyr Arg Arg Pro Ser Ile Val Leu Val Ser755 760 765Ash Leu Ser Gly Lys Ala Cys Thr Asp Glu Val Ser Ser Pro Gly Tyr770 775 780Trp Val Arg His Ala Arg Glu Val Val Arg Phe Ala Asp Gly Val Lys785 790 795 800Ala Leu His Ala Ala Gly Ala Gly Thr Phe Val Glu Val Gly Pro Lys805 810 815Ser Thr Leu Leu Gly Leu Val Pro Ala Cys Met Pro Asp Ala Arg Pro820 825 830Ala Leu Leu Ala Ser Ser Arg Ala Gly Arg Asp Glu Pro Ala Thr Val835 840 845Leu Glu Ala Leu Gly Gly Leu Trp Ala Val Gly Gly Leu Val Ser Trp850 855 860Ala Gly Leu Phe Pro Ser Gly Gly Arg Arg Val Pro Leu Pro Thr Tyr865 870 875 880Pro Trp Gln Arg Glu Arg Tyr Trp Ile Asp Thr Lys Ala Asp Asp Ala885 890 895Ala Arg Gly Asp Arg Arg Ala Pro Gly Ala Gly His Asp Glu Val Glu900 905 910Glu Gly Gly Ala Val Arg Gly Gly Asp Arg Arg Ser Ala Arg Leu Asp915 920 925His Pro Pro Pro Glu Ser Gly Arg Arg Glu Lys Val Glu Ala Ala Gly930 935 940Asp Arg Pro Phe Arg Leu Glu Ile Asp Glu Pro Gly Val Leu Asp His945 950 955 960Leu Val Leu Arg Val Thr Glu Arg Arg Ala Pro Gly Leu Gly Glu Val965 970 975Glu Ile Ala Val Asp Ala Ala Gly Leu Ser Phe Asn Asp Val Gln Leu980 985 990Ala Leu Gly Met Val Pro Asp Asp Leu Pro Gly Lys Pro Asn Pro Pro99510001005Leu Leu Leu GlyGly Glu Cys Ala GlyArg Ile Val Ala Val Gly Glu1010 1015 1020Gly Val Asn Gly Leu Val Val Gly Gln Pro Val Ile Ala Leu Ser Ala1025 103010351040Gly Ala Phe Ala Thr His Val Thr Thr Ser Ala Ala Leu Val Leu Pro104510501055Arg Pro Gln Ala Leu Ser Ala Ile Glu Ala Ala Ala Met Pro Val Ala106010651070Tyr Leu Thr Ala Trp Tyr Ala Leu Asp Arg Ile Ala Arg Leu Gln Pro107510801085Gly Glu Arg Val Leu Ile His Ala Ala Thr Gly Gly Val Gly Leu Ala109010951100Ala Val Gln Trp Ala Gln His Val Gly Ala Glu Val His Ala Thr Ala1105 111011151120Gly Thr Pro Glu Lys Arg Ala Tyr Leu Glu Ser Leu Gly Val Arg Tyr112511301135Val Ser Asp Ser Arg Ser Asp Arg Phe Val Ala Asp Val Arg Ala Trp114011451150Thr Gly Gly Glu Gly Val Asp Val Val Leu Asn Ser Leu Ser Gly Glu115511601165Leu Ile Asp Lys Ser Phe Asn Leu Leu Arg Ser His Gly Arg Phe Val117011751180Glu Leu Gly Lys Arg Asp Cys Tyr Ala Asp Asn Gln Leu Gly Leu Arg1185 119011951200Pro Phe Leu Arg Asn Leu Ser Phe Ser Leu Val Asp Leu Arg Gly Met120512101215Met Leu Glu Arg Pro Ala Arg Val Arg Ala Leu Leu Glu Glu Leu Leu122012251230Gly Leu Ile Ala Ala Gly Val Phe Thr Pro Pro Pro Ile Ala Thr Leu123512401245Pro Ile Ala Arg Val Ala Asp Ala Phe Arg Ser Met Ala Gln Ala Gln125012551260His Leu Gly Lys Leu Val Leu Thr Leu Gly Asp Pro Glu Val Gln Ile1265 127012751280Arg Ile Pro Thr His Ala Gly Ala Gly Pro Ser Thr Gly Asp Arg Asp128512901295Leu Leu Asp Arg Leu Ala Ser Ala Ala Pro Ala Ala Arg Ala Ala Ala130013051310Leu Glu Ala Phe Leu Arg Thr Gln Val Ser Gln Va1 Leu Arg Thr Pro131513201325Glu Ile Lys Val Gly Ala Glu Ala Leu Phe Thr Arg Leu Gly Met Asp133013351340Ser Leu Met Ala Val Glu Leu Arg Asn Arg Ile Glu Ala Ser Leu Lys1345 135013551360Leu Lys Leu Ser Thr Thr Phe Leu Ser Thr Ser Pro Asn Ile Ala Leu136513701375Leu Ala Gln Asn Leu Leu Asp Ala Leu Ala Thr Ala Leu Ser Leu Glu138013851390Arg Val Ala Ala Glu Asn Leu Arg Ala Gly Val Gln Asn Asp Phe Val139514001405Ser Ser Gly Ala Asp Gln Asp Trp Glu Ile Ile Ala Leu141014151420<210>3<211>1410<212>PRT<213>纤维堆囊菌<400>3Met Thr Ile Asn Gln Leu Leu Asn Glu Leu Glu His Gln Gly Ile Lys1 5 10 15Leu Ala Ala Asp Gly Glu Arg Leu Gln Ile Gln Ala Pro Lys Asn Ala20 25 30Leu Asn Pro Asn Leu Leu Ala Arg Ile Ser Glu His Lys Ser Thr Ile35 40 45Leu Thr Met Leu Arg Gln Arg Leu Pro Ala Glu Ser Ile Val Pro Ala50 55 60Pro Ala Glu Arg His Ala Pro Phe Pro Leu Thr Asp Ile Gln Glu Ser65 70 75 80Tyr Trp Leu Gly Arg Thr Gly Ala Phe Thr Val Pro Ser Gly Ile His85 90 95Ala Tyr Arg Glu Tyr Asp Cys Thr Asp Leu Asp Val Pro Arg Leu Ser100 105 110Arg Ala Phe Arg Lys Val Val Ala Arg His Asp Met Leu Arg Ala His115 120 125Thr Leu Pro Asp Met Met Gln Val Ile Glu Pro Lys Val Asp Ala Asp130 135 140Ile Glu Ile Ile Asp Leu Arg Gly Leu Asp Arg Ser Thr Arg Glu Ala145 150 155 160Arg Leu Val Ser Leu Arg Asp Ala Met Ser His Arg Ile Tyr Asp Thr165 170 175Glu Arg Pro Pro Leu Tyr His Val Val Ala Val Arg Leu Asp Glu Arg180 185 190Gln Thr Arg Leu Val Leu Ser Ile Asp Leu Ile Asn Val Asp Leu Gly195 200 205Ser Leu Ser Ile Ile Phe Lys Asp Trp Leu Ser Phe Tyr Glu Asp Pro210 215 220Glu Thr Ser Leu Pro Val Leu Glu Leu Ser Tyr Arg Asp Tyr Val Leu225 230 235 240Ala Leu Glu Ser Arg Lys Lys Ser Glu Ala His Gln Arg Ser Met Asp245 250 255Tyr Trp Lys Arg Arg Ile Ala Glu Leu Pro Pro Pro Pro Thr Leu Pro260 265 270Met Lys Ala Asp Pro Ser Thr Leu Lys Glu Ile Arg Phe Arg His Thr275 280 285Glu Gln Trp Leu Pro Ser Asp Ser Trp Gly Arg Leu Lys Arg Arg Val290 295 300Gly Glu Arg Gly Leu Thr Pro Thr Gly Val Ile Leu Ala Ala Phe Ser305 310 315 320Glu Val Ile Gly Arg Trp Ser Ala Ser Pro Arg Phe Thr Leu Asn Ile325 330 335Thr Leu Phe Asn Arg Leu Pro Val His Pro Arg Val Asn Asp Ile Thr340 345 350Gly Asp Phe Thr Ser Met Val Leu Leu Asp Ile Asp Thr Thr Arg Asp355 360 365Lys Ser Phe Glu Gln Arg Ala Lys Arg Ile Gln Glu Gln Leu Trp Glu370 375 380Ala Met Asp His Cys Asp Val Ser Gly Ile Glu Val Gln Arg Glu Ala385 390 395 400Ala Arg Val Leu Gly Ile Gln Arg Gly Ala Leu Phe Pro Val Val Leu405 410 415Thr Ser Ala Leu Asn Gln Gln Val Val Gly Val Thr Ser Leu Gln Arg420 425 430Leu Gly Thr Pro Val Tyr Thr Ser Thr Gln Thr Pro Gln Leu Leu Leu435 440 445Asp His Gln Leu Tyr Glu His Asp Gly Asp Leu Val Leu Ala Trp Asp450 455 460Ile Val Asp Gly Val Phe Pro Pro Asp Leu Leu Asp Asp Met Leu Glu465 470 475 480Ala Tyr Val Val Phe Leu Arg Arg Leu Thr Glu Glu Pro Trp Gly Glu485 490 495Gln Val Arg Cys Ser Leu Pro Pro Ala Gln Leu Glu Ala Arg Ala Ser500 505 510Ala Asn Ala Thr Asn Ala Leu Leu Ser Glu His Thr Leu His Gly Leu515 520 525Phe Ala Ala Arg Val Glu Gln Leu Pro Met Gln Leu Ala Val Val Ser530 535 540Ala Arg Lys Thr Leu Thr Tyr Glu Glu Leu Ser Arg Arg Ser Arg Arg545 550 555 560Leu Gly Ala Arg Leu Arg Glu Gln Gly Ala Arg Pro Asn Thr Leu Val565 570 575Ala Val Val Met Glu Lys Gly Trp Glu Gln Val Val Ala Val Leu Ala580 585 590Val Leu Glu Ser Gly Ala Ala Tyr Val Pro Ile Asp Ala Asp Leu Pro595 600 605Ala Glu Arg Ile His Tyr Leu Leu Asp His Gly Glu Val Lys Leu Val610 615 620Leu Thr Gln Pro Trp Leu Asp Gly Lys Leu Ser Trp Pro Pro Gly Ile625 630 635 640Gln Arg Leu Leu Val Ser Glu Ala Gly Val Glu Gly Asp Gly Asp Gln645 650 655Pro Pro Met Met Pro Ile Gln Thr Pro Ser Asp Leu Ala Tyr Val Ile660 665 670Tyr Thr Ser Gly Ser Thr Gly Leu Pro Lys Gly Val Met Ile Asp His675 680 685Arg Gly Ala Val Asn Thr Ile Leu Asp Ile Asn Glu Arg Phe Glu Ile690 695 700Gly Pro Gly Asp Arg Val Leu Ala Leu Ser Ser Leu Ser Phe Asp Leu705 710 715 720Ser Val Tyr Asp Val Phe Gly Ile Leu Ala Ala Gly Gly Thr Ile Val725 730 735Val Pro Asp Ala Ser Lys Leu Arg Asp Pro Ala His Trp Ala Glu Leu740 745 750Ile Glu Arg Glu Lys Val Thr Val Trp Asn Ser Val Pro Ala Leu Met755 760 765Arg Met Leu Val Glu His Phe Glu Gly Arg Pro Asp Ser Leu Ala Arg770 775 780Ser Leu Arg Leu Ser Leu Leu Ser Gly Asp Trp Ile Pro Val Gly Leu785 790 795 800Pro Gly Glu Leu Gln Ala Ile Arg Pro Gly Val Ser Val Ile Ser Leu805 810 815Gly Gly Ala Thr Glu Ala Ser Ile Trp Ser Ile Gly Tyr Pro Val Arg820 825 830Asn Val Asp Leu Ser Trp Ala Ser Ile Pro Tyr Gly Arg Pro Leu Arg835 840 845Asn Gln Thr Phe His Val Leu Asp Glu A la Leu Glu Pro Arg Pro Val850 855 860Trp Val Pro Gly Gln Leu Tyr Ile Gly Gly Val Gly Leu Ala Leu Gly865 870 875 880Tyr Trp Arg Asp Glu Glu Lys Thr Arg Lys Ser Phe Leu Val His Pro885 890 895Glu Thr Gly Glu Arg Leu Tyr Lys Thr Gly Asp Leu Gly Arg Tyr Leu900 905 910Pro Asp Gly Asn Ile Glu Phe Met Gly Arg Glu Asp Asn Gln Ile Lys915 920 925Leu Arg Gly Tyr Arg Val Glu Leu Gly Glu Ile Glu Glu Thr Leu Lys930 935 940Ser His Pro Asn Val Arg Asp Ala Val Ile Val Pro Val Gly Asn Asp945 950 955 960Ala Ala Asn Lys Leu Leu Leu Ala Tyr Val Val Pro Glu Gly Thr Arg965 970 975Arg Arg Ala Ala Glu Gln Asp Ala Ser Leu Lys Thr Glu Arg Ile Asp980 985 990Ala Arg Ala His Ala Ala Glu Ala Asp Gly Leu Ser Asp Gly Glu Arg99510001005Val Gln Phe Lys Leu Ala Arg His Gly Leu Arg Arg Asp Leu Asp Gly101010151020Lys Pro Val Val Asp Leu Thr Gly Gln Asp Pro Arg Glu Ala Gly Leu1025 103010351040Asp Val Tyr Ala Arg Arg Arg Ser Val Arg Thr Phe Leu Glu Ala Pro104510501055Ile Pro Phe Val Glu Phe Gly Arg Phe Leu Ser Cys Leu Ser Ser Val106010651070Glu Pro Asp Gly Ala Thr Leu Pro Lys Phe Arg Tyr Pro Ser Ala Gly107510801085Ser Thr Tyr Pro Val Gln Thr Tyr Ala Tyr Val Lys Ser Gly Arg Ile109010951100Glu Gly Val Asp Glu Gly Phe Tyr Tyr Tyr His Pro Phe Glu His Arg1105 111011151120Leu Leu Lys Leu Ser Asp His Gly Ile Glu Arg Gly Ala His Val Arg112511301135Gln Asn Phe Asp Val Phe Asp Glu Ala Ala Phe Asn Leu Leu Phe Val114011451150Gly Arg Ile Asp Ala Ile Glu Ser Leu Tyr Gly Ser Ser Ser Arg Glu115511601165Phe Cys Leu Leu Glu Ala Gly Tyr Met Ala Gln Leu Leu Met Glu Gln117011751180Ala Pro Ser Cys Asn Ile Gly Val Cys Pro Val Gly Gln Phe Asn Phe1185 119011951200Glu Gln Val Arg Pro Val Leu Asp Leu Arg His Ser Asp Val Tyr Val120512101215His Gly Met Leu Gly Gly Arg Val Asp Pro Arg Gln Phe Gln Val Cys122012251230Thr Leu Gly Gln Asp Ser Ser Pro Arg Arg Ala Thr Thr Arg Gly Ala123512401245Pro Pro Gly Arg Glu Gln His Phe Ala Asp Met Leu Arg Asp Phe Leu125012551260Arg Thr Lys Leu Pro Glu Tyr Met Val Pro Thr Val Phe Val Glu Leu1265 127012751280Asp Ala Leu Pro Leu Thr Ser Asn Gly Lys Val Asp Arg Lys Ala Leu128512901295Arg Glu Arg Lys Asp Thr Ser Ser Pro Arg His Ser Gly His Thr Ala130013051310Pro Arg Asp Ala Leu Glu Glu Ile Leu Val Ala Val Val Arg Glu Val131513201325Leu Gly Leu Glu Val Val Gly Leu Gln Gln Ser Phe Val Asp Leu Gly133013351340Ala Thr Ser Ile His Ile Val Arg Met Arg Ser Leu Leu Gln Lys Arg1345 135013551360Leu Asp Arg Glu Ile Ala Ile Thr Glu Leu Phe Gln Tyr Pro Asn Leu136513701375Gly Ser Leu Ala Ser Gly Leu Arg Arg Asp Ser Arg Asp Leu Asp Gln138013851390Arg Pro Asn Met Gln Asp Arg Val Glu Val Arg Arg Lys Gly Arg Arg139514001405Arg Ser1410<210>4<211>1832<212>PRT<213>纤维堆囊菌<400>4Met Glu Glu Gln Glu Ser Ser Ala Ile Ala Val Ile Gly Met Ser Gly1 5 10 15Arg Phe Pro Gly Ala Arg Asp Leu Asp Glu Phe Trp Arg Asn Leu Arg20 25 30Asp Gly Thr Glu Ala Val Gln Arg Phe Ser Glu Gln Glu Leu Ala Ala35 40 45Ser Gly Val Asp Pro Ala Leu Val Leu Asp Pro Ser Tyr Val Arg Ala50 55 60Gly Ser Val Leu Glu Asp Val Asp Arg Phe Asp Ala Ala Phe Phe Gly65 70 75 80Ile Ser Pro Arg Glu Ala Glu Leu Met Asp Pro Gln His Arg Ile Phe85 90 95Met Glu Cys Ala Trp Glu Ala Leu Glu Asn Ala Gly Tyr Asp Pro Thr100 105 110Ala Tyr Glu Gly Ser Ile Gly Val Tyr Ala Gly Ala Asn Met Ser Ser115 120 125Tyr Leu Thr Ser Asn Leu His Glu His Pro Ala Met Met Arg Trp Pro130 135 140Gly Trp Phe Gln Thr Leu Ile Gly Asn Asp Lys Asp Tyr Leu Ala Thr145 150 155 160His Val Ser Tyr Arg Leu Asn Leu Arg Gly Pro Ser Ile Ser Val Gln165 170 175Thr Ala Cys Ser Thr Ser Leu Val Ala Val His Leu Ala Cys Met Ser180 185 190Leu Leu Asp Arg Glu Cys Asp Met Ala Leu Ala Gly Gly Ile Thr Val195 200 205Arg Ile Pro His Arg Ala Gly Tyr Val Tyr Ala Glu Gly Gly Ile Phe210 215 220Ser Pro Asp Gly His Cys Arg Ala Phe Asp Ala Lys Ala Asn Gly Thr225 230 235 240Ile Met Gly Asn Gly Cys Gly Val Val Leu Leu Lys Pro Leu Asp Arg245 250 255Ala Leu Ser Asp Gly Asp Pro Val Arg Ala Val Ile Leu Gly Ser Ala260 265 270Thr Asn Asn Asp Gly Ala Arg Lys Ile Gly Phe Thr Ala Pro Ser Glu275 280 285Val Gly Gln Ala Gln Ala Ile Met Glu Ala Leu Ala Leu Ala Gly Val290 295 300Glu Ala Arg Ser Ile Gln Tyr Ile Glu Thr His Gly Thr Gly Thr Leu305 310 315 320Leu Gly Asp Ala Ile Glu Thr Ala Ala Leu Arg Arg Val Phe Gly Arg325 330 335Asp Ala Ser Ala Arg Arg Ser Cys Ala Ile Gly Ser Val Lys Thr Gly340 345 350Ile Gly His Leu Glu Ser Ala Ala Gly Ile Ala Gly Leu Ile Lys Thr355 360 365Val Leu Ala Leu Glu His Arg Gln Leu Pro Pro Ser Leu Asn Phe Glu370 375 380Ser Pro Asn Pro Ser Ile Asp Phe Ala Ser Ser Pro Phe Tyr Val Asn385 390 395 400Thr Ser Leu Lys Asp Trp Asn Thr Gly Ser Thr Pro Arg Arg Ala Gly405 410 415Val Ser Ser Phe Gly Ile Gly Gly Thr Asn Ala His Val Val Leu Glu420 425 430Glu Ala Pro Ala Ala Lys Leu Pro Ala Ala Ala Pro Ala Arg Ser Ala435 440 445Glu Leu Phe Val Val Ser Ala Lys Ser Ala Ala Ala Leu Asp Ala Ala450 455 460Ala Ala Arg Leu Arg Asp His Leu Gln Ala His Gln Gly Ile Ser Leu465 470 475 480Gly Asp Val Ala Phe Ser Leu Ala Thr Thr Arg Ser Pro Met Glu His485 490 495Arg Leu Ala Met Ala Ala Pro Ser Arg Glu Ala Leu Arg Glu Gly Leu500 505 510Asp Ala Ala Ala Arg Gly Gln Thr Pro Pro Gly Ala Val Arg Gly Arg515 520 525Cys Ser Pro Gly Asn Val Pro Lys Val Val Phe Val Phe Pro Gly Gln530 535 540Gly Ser Gln Trp Val Gly Met Gly Arg Gln Leu Leu Ala Glu Glu Pro545 550 555 560Val Phe His Ala Ala Leu Ser Ala Cys Asp Arg Ala Ile Gln Ala Glu565 570 575Ala Gly Trp Ser Leu Leu Ala Glu Leu Ala Ala Asp Glu Gly Ser Ser580 585 590Gln Leu Glu Arg Ile Asp Val Val Gln Pro Val Leu Phe Ala Leu Ala595 600 605Val Ala Phe Ala Ala Leu Trp Arg Ser Trp Gly Val Ala Pro Asp Val610 615 620Val Ile Gly His Ser Met Gly Glu Val Ala Ala Ala His Val Ala Gly625 630 635 640Ala Leu Ser Leu Glu Asp Ala Val Ala Ile Ile Cys Arg Arg Ser Arg645 650 655Leu Leu Arg Arg Ile Ser Gly Gln Gly Glu Met Ala Val Thr Glu Leu660 665 670Ser Leu Ala Glu Ala Glu Ala Ala Leu Arg Gly Tyr Glu Asp Arg Val675 680 685Ser Val Ala Val Ser Asn Ser Pro Arg Ser Thr Val Leu Ser Gly Glu690 695 700Pro Ala Ala Ile Gly Glu Val Leu Ser Ser Leu Asn Ala Lys Gly Val705 710 715 720Phe Cys Arg Arg Val Lys Val Asp Val Ala Ser His Ser Pro Gln Val725 730 735Asp Pro Leu Arg Glu Asp Leu Leu Ala Ala Leu Gly Gly Leu Arg Pro740 745 750Gly Ala Ala Ala Val Pro Met Arg Ser Thr Val Thr Gly Ala Met Val755 760 765Ala Gly Pro Glu Leu Gly Ala Asn Tyr Trp Met Asn Asn Leu Arg Gln770 775 780Pro Val Arg Phe Ala Glu Val Val Gln Ala Gln Leu Gln Gly Gly His785 790 795 800Gly Leu Phe Val Glu Met Ser Pro His Pro Ile Leu Thr Thr Ser Val805 810 815Glu Glu Met Arg Arg Ala Ala Gln Arg Ala Gly Ala Ala Val Gly Ser820 825 830Leu Arg Arg Gly Gln Asp Glu Arg Pro Ala Met Leu Glu Ala Leu Gly835 840 845Thr Leu Trp Ala Gln Gly Tyr Pro Val Pro Trp Gly Arg Leu Phe Pro850 855 860Ala Gly Gly Arg Arg Val Pro Leu Pro Thr Tyr Pro Trp Gln Arg Glu865 870 875 880Arg Tyr Trp Ile Glu Ala Pro Ala Lys Ser Ala Ala Gly Asp Arg Arg885 890 895Gly Val Arg Ala Gly Gly His Pro Leu Leu Gly Glu Met Gln Thr Leu900 905 910Ser Thr Gln Thr Ser Thr Arg Leu Trp Glu Thr Thr Leu Asp Leu Lys915 920 925Arg Leu Pro Trp Leu Gly Asp His Arg Val Gln Gly Ala Val Val Phe930 935 940Pro Gly Ala Ala Tyr Leu Glu Met Ala Ile Ser Ser Gly Ala Glu Ala945 950 955 960Leu Gly Asp Gly Pro Leu Gln Ile Thr Asp Val Val Leu Ala Glu Ala965 970 975Leu Ala Phe Ala Gly Asp Ala Ala Val Leu Val Gln Val Val Thr Thr980 985 990Glu Gln Pro Ser Gly Arg Leu Gln Phe Gln Ile Ala Ser Arg Ala Pro99510001005Gly Ala Gly His Ala Ser Phe Arg Val His Ala Arg Gly Ala Leu Leu101010151020Arg Val Glu Arg Thr Glu Val Pro Ala Gly Leu Thr Leu Ser Ala Val1025 103010351040Arg Ala Arg Leu Gln Ala Ser Ile Pro Ala Ala Ala Thr Tyr Ala Glu104510501055Leu Thr Glu Met Gly Leu Gln Tyr Gly Pro Ala Phe Gln Gly Ile Ala106010651070Glu Leu Trp Arg Gly Glu Gly Glu Ala Leu Gly Arg Val Arg Leu Pro107510801085Asp Ala Ala Gly Ser Ala Ala Glu Tyr Arg Leu His Pro Ala Leu Leu109010951100Asp Ala Cys Phe Gln Ile Val Gly Ser Leu Phe Ala Arg Ser Gly Glu1105 111011151120Ala Thr Pro Trp Val Pro Val Glu Leu Gly Ser Leu Arg Leu Leu Gln112511301135Arg Pro Ser Gly Glu Leu Trp Cys His Ala Arg Val Val Asn His Gly114011451150His Gln Thr Pro Asp Arg Gln Gly Ala Asp Phe Trp Val Val Asp Ser115511601165Ser Gly Ala Val Val Ala Glu Val Cys Gly Leu Val Ala Gln Arg Leu117011751180Pro Gly Gly Val Arg Arg Arg Glu Glu Asp Asp Trp Phe Leu Glu Leu1185 119011951200Glu Trp Glu Pro Ala Ala Val Gly Thr Ala Lys Val Asn Ala Gly Arg120512101215Trp Leu Leu Leu Gly Gly Gly Gly Gly Leu Gly Ala Ala Leu Arg Ala122012251230Met Leu Glu Ala Gly Gly His Ala Val Val His Ala Ala Glu Asn Asn123512401245Thr Ser Ala Ala Gly Val Arg Ala Leu Leu Ala Lys Ala Phe Asp Gly125012551260Gln Ala Pro Thr Ala Val Val His Leu Gly Ser Leu Asp Gly Gly Gly1265 127012751280Glu Leu Asp Pro Gly Leu Gly Ala Gln Gly Ala Leu Asp Ala Pro Arg128512901295Ser Ala Asp Val Ser Pro Asp Ala Leu Asp Pro Ala Leu Val Arg Gly130013051310Cys Asp Ser Val Leu Trp Thr Val Gln Ala Leu Ala Gly Met Gly Phe131513201325Arg Asp Ala Pro Arg Leu Trp Leu Leu Thr Arg Gly Ala Gln Ala Val133013351340Gly Ala Gly Asp Val Ser Val Thr Gln Ala Pro Leu Leu Gly Leu Gly1345 135013551360Arg Val Ile Ala Met Glu His Ala Asp Leu Arg Cys Ala Arg Val Asp136513701375Leu Asp Pro Ala Arg Pro Glu Gly Glu Leu Ala Ala Leu Leu Ala Glu138013851390Leu Leu Ala Asp Asp Ala Glu Ala Glu Val Ala Leu Arg Gly Gly Glu139514001405Arg Cys Val Ala Arg Ile Val Arg Arg Gln Pro Glu Thr Arg Pro Arg141014151420Gly Arg Ile Glu Ser Cys Val Pro Thr Asp Val Thr Ile Arg Ala Asp1425 143014351440Ser Thr Tyr Leu Val Thr Gly Gly Leu Gly Gly Leu Gly Leu Ser Val144514501455Ala Gly Trp Leu Ala Glu Arg Gly Ala Gly His Leu Val Leu Val GlyArg Ser Gly Ala Ala Ser Val Glu Gln Arg Ala Ala Val Ala Ala Leu147514801485Glu Ala Arg Gly Ala Arg Val Thr Val Ala Lys Ala Asp Val Ala Asp149014951500Arg Ala Gln Leu Glu Arg Ile Leu Arg Glu Val Thr Thr Ser Gly Met1505 151015151520Pro Leu Arg Gly Val Val His Ala Ala Gly Ile Leu Asp Asp Gly Leu152515301535Leu Met Gln Gln Thr Pro Ala Arg Phe Arg Lys Val Met Ala Pro Lys154015451550Val Gln Gly Ala Leu His Leu His Ala Leu Thr Arg Glu Ala Pro Leu155515601565Ser Phe Phe Val Leu Tyr Ala Ser Gly Val Gly Leu Leu Gly Ser Pro157015751580Gly Gln Gly Asn Tyr Ala Ala Ala Asn Thr Phe Leu Asp Ala Leu Ala1585 159015951600His His Arg Arg Ala Gln Gly Leu Pro Ala Leu Ser Val Asp Trp Gly160516101615Leu Phe Ala Glu Val Gly Met Ala Ala Ala Gln Glu Asp Arg Gly Ala162016251630Arg Leu Val Ser Arg Gly Met Arg Ser Leu Thr Pro Asp Glu Gly Leu163516401645Ser Ala Leu Ala Arg Leu Leu Glu Ser Gly Arg Ala Gln Val Gly Val165016551660Met Pro Val Asn Pro Arg Leu Trp Val Glu Leu Tyr Pro Ala Ala Ala1665 167016751680Ser Ser Arg Met Leu Ser Arg Leu Val Thr Ala His Arg Ala Ser Ala168516901695Gly Gly Pro Ala Gly Asp Gly Asp Leu Leu Arg Arg Leu Ala Ala Ala170017051710Glu Pro Ser Ala Arg Ser Ala Leu Leu Glu Pro Leu Leu Arg Ala Gln171517201725Ile Ser Gln Val Leu Arg Leu Pro Glu Gly Lys Ile Glu Val Asp Ala173017351740Pro Leu Thr Ser Leu Gly Met Asn Ser Leu Met Gly Leu Glu Leu Arg1745 175017551760Asn Arg Ile Glu Ala Met Leu Gly Ile Thr Val Pro A la Thr Leu Leu17651770 1775Trp Thr Tyr Pro Thr Val Ala Ala Leu Ser Gly His Leu Ala Arg Glu178017851790Ala Cys Glu Ala Ala Pro Val Glu Ser Pro His Thr Thr Ala Asp Ser179518001805Ala Val Glu Ile Glu Glu Met Ser Gln Asp Asp Leu Thr Gln Leu Ile181018151820Ala Ala Lys Phe Lys Ala Leu Thr1825 1830<210>5<211>7257<212>PRT<213>纤维堆囊菌<400>5Met Thr Thr Arg Gly Pro Thr Ala Gln Gln Asn Pro Leu Lys Gln Ala1 5 10 15Ala Ile Ile Ile Gln Arg Leu Glu Glu Arg Leu Ala Gly Leu Ala Gln20 25 30Ala Glu Leu Glu Arg Thr Glu Pro Ile Ala Ile Val Gly Ile Gly Cys35 40 45Arg Phe Pro Gly Gly Ala Asp Ala Pro Glu Ala Phe Trp Glu Leu Leu50 55 60Asp Ala Glu Arg Asp Ala Val Gln Pro Leu Asp Met Arg Trp Ala Leu65 70 75 80Val Gly Val Ala Pro Val Glu Ala Val Pro His Trp Ala Gly Leu Leu85 90 95Thr Glu Pro Ile Asp Cys Phe Asp Ala Ala Phe Phe Gly Ile Ser Pro100 105 110Arg Glu Ala Arg Ser Leu Asp Pro Gln His Arg Leu Leu Leu Glu Val115 120 125Ala Trp Glu Gly Leu Glu Asp Ala Gly Ile Pro Pro Arg Ser Ile Asp130 135 140Gly Ser Arg Thr Gly Val Phe Val Gly Ala Phe Thr Ala Asp Tyr Ala145 150 155 160Arg Thr Val Ala Arg Leu Pro Arg Glu Glu Arg Asp Ala Tyr Ser Ala165 170 175Thr Gly Asn Met Leu Ser Ile Ala Ala Gly Arg Leu Ser Tyr Thr Leu180 185 190Gly Leu Gln Gly Pro Cys Leu Thr Val Asp Thr Ala Cys Ser Ser Ser195 200 205Leu Val Ala Ile His Leu Ala Cys Arg Ser Leu Arg Ala Gly Glu Ser210 215 220Asp Leu Ala Leu Ala Gly Gly Val Ser Ala Leu Leu Ser Pro Asp Met225 230 235 240Met Glu Ala Ala Ala Arg Thr Gln Ala Leu Ser Pro Asp Gly Arg Cys245 250 255Arg Thr Phe Asp Ala Ser Ala Asn Gly Phe Val Arg Gly Glu Gly Cys260 265 270Gly Leu Val Val Leu Lys Arg Leu Ser Asp Ala Gln Arg Asp Gly Asp275 280 285ArG Ile Trp Ala Leu Ile Arg Gly Ser Ala Ile Asn His Asp Gly Arg290 295 300Ser Thr Gly Leu Thr Ala Pro Asn Val Leu Ala Gln Glu Thr Val Leu305 310 315 320Arg Glu Ala Leu Arg Ser Ala His Val Glu Ala Gly Ala Val Asp Tyr325 330 335Val Glu Thr His Gly Thr Gly Thr Ser Leu Gly Asp Pro Ile Glu Val340 345 350Glu Ala Leu Arg Ala Thr Val Gly Pro Ala Arg Ser Asp Gly Thr Arg355 360 365Cys Val Leu Gly Ala Val Lys Thr Asn Ile Gly His Leu Glu Ala Ala370 375 380Ala Gly Val Ala Gly Leu Ile Lys Ala Ala Leu Ser Leu Thr His Glu385 390 395 400Arg Ile Pro Arg Asn Leu Asn Phe Arg Thr Leu Asn Pro Arg Ile Arg405 410 415Leu Glu Gly Ser Ala Leu Ala Leu Ala Thr Glu Pro Val Pro Trp Pro420 425 430Arg Thr Asp Arg Pro Arg Phe Ala Gly Val Ser Ser Phe Gly Met Ser435 440 445Gly Thr Asn Ala His Val Val Leu Glu Glu Ala Pro Ala Val Glu Leu450 455 460Trp Pro Ala Ala Pro Glu Arg Ser Ala Glu Leu Leu Val Leu Ser Gly465 470 475 480Lys Ser Glu Gly Ala Leu Asp Ala Gln Ala Ala Arg Leu Arg Glu His485 490 495Leu Asp Met His Pro Glu Leu Gly Leu Gly Asp Val Ala Phe Ser Leu500 505 510Ala Thr Thr Arg Ser Ala Met Ser His Arg Leu Ala Val Ala Val Thr515 520 525Ser Arg Glu Gly Leu Leu Ala Ala Leu Ser Ala Val Ala Gln Gly Gln530 535 540Thr Pro Ala Gly Ala Ala Arg Cys Ile Ala Ser Ser Ser Arg Gly Lys545 550 555 560Leu Ala Phe Leu Phe Thr Gly Gln Gly Ala Gln Thr Pro Gly Met Gly565 570 575Arg Gly Leu Cys Ala Ala Trp Pro Ala Phe Arg Glu Ala Phe Asp Arg580 585 590Cys Val Ala Leu Phe Asp Arg Glu Leu Asp Arg Pro Leu Arg Glu Val595 600 605Met Trp Ala Glu Ala Gly Ser Ala Glu Ser Leu Leu Leu Asp Gln Thr610 615 620Ala Phe Thr Gln Pro Ala Leu Phe Ala Val Glu Tyr Ala Leu Thr Ala625 630 635 640Leu Trp Arg Ser Trp Gly Val Glu Pro Glu Leu Leu Val Gly His Ser645 650 655Ile Gly Glu Leu Val Ala Ala Cys Val Ala Gly Val Phe Ser Leu Glu660 665 670Asp Gly Val Arg Leu Val Ala Ala Arg Gly Arg Leu Met Gln Gly Leu675 680 685Ser Ala Gly Gly Ala Met Val Ser Leu Gly Ala Pro Glu Ala Glu Val690 695 700Ala Ala Ala Val Ala Pro His Ala Ala Ser Val Ser Ile Ala Ala Val705 710 715 720Asn Gly Pro Glu Gln Val Val Ile Ala Gly Val Glu Gln Ala Val Gln725 730 735Ala Ile Ala Ala Gly Phe Ala Ala Arg Gly Ala Arg Thr Lys Arg Leu740 745 750His Val Ser His Ala Phe His Ser Pro Leu Met Glu Pro Met Leu Glu755 760 765Glu Phe Gly Arg Val Ala Ala Ser Val Thr Tyr Arg Arg Pro Ser Val770 775 780Ser Leu Val Ser Asn Leu Ser Gly Lys Val Val Thr Asp Glu Leu Ser785 790 795 800Ala Pro Gly Tyr Trp Val Arg His Val Arg Glu Ala Val Arg Phe Ala805 810 815Asp Gly Val Lys Ala Leu His Glu Ala Gly Ala Gly Thr Phe Val Glu820 825 830Val Gly Pro Lys Pro Thr Leu Leu Gly Leu Leu Pro Ala Cys Leu Pro835 840 845Glu Ala Glu Pro Thr Leu Leu Ala Ser Leu Arg Ala Gly Arg Glu Glu850 855 860Ala Ala Gly Val Leu Glu Ala Leu Gly Arg Leu Trp Ala Ala Gly Gly865 870 875 880Ser Val Ser Trp Pro Gly Val Phe Pro Thr Ala Gly Arg Arg Val Pro885 890 895Leu Pro Thr Tyr Pro Trp Gln Arg Gln Arg Tyr Trp Ile Glu Ala Pro900 905 910Ala Glu Gly Leu Gly Ala Thr Ala Ala Asp Ala Leu Ala Gln Trp Phe915 920 925Tyr Arg Val Asp Trp Pro Glu Met Pro Arg Ser Ser Val Asp Ser Arg930 935 940Arg Ala Arg Ser Gly Gly Trp Leu Val Leu Ala Asp Arg Gly Gly Val945 950 955 960Gly Glu Ala Ala Ala Ala Ala Leu Ser Ser Gln Gly Cys Ser Cys Ala965 970 975Val Leu His Ala Pro Ala Glu Ala Ser Ala Val Ala Glu Gln Val Thr980 985 990Gln Ala Leu Gly Gly Arg Asn Asp Trp Gln Gly Val Leu Tyr Leu Trp99510001005Gly Leu Asp Ala Val Val Glu Ala Gly Ala Ser Ala Glu Glu Val Ala101010151020Lys Val Thr His Leu Ala Ala Ala Pro Val Leu Ala Leu Ile Gln Ala1025 103010351040Leu Gly Thr Gly Pro Arg Ser Pro Arg Leu Trp Ile Val Thr Arg Gly104510501055Ala Cys Thr Val Gly Gly Glu Pro Asp Ala Ala Pro Cys Gln Ala Ala106010651070Leu Trp Gly Met Gly Arg Val Ala Ala Leu Glu His Pro Gly Ser Trp107510801085Gly Gly Leu Val Asp Leu Asp Pro Glu Glu Ser Pro Thr Glu Val Glu109010951100Ala Leu Val Ala Glu Leu Leu Ser Pro Asp Ala Glu Asp Gln Leu Ala1105 111011151120Phe Arg Gln Gly Arg Arg Arg Ala Ala Arg Leu Val Ala Ala Pro Pro112511301135Glu Gly Asn Ala Ala Pro Val Ser Leu Ser Ala Glu Gly Ser Tyr Leu114011451150Val Thr Gly Gly Leu Gly Ala Leu Gly Leu Leu Val Ala Arg Trp Leu115511601165Val Glu Arg Gly Ala Gly His Leu Val Leu Ile Ser Arg His Gly Leu117011751180Pro Asp Arg Glu Glu Trp Gly Arg Asp Gln Pro Pro Glu Val Arg Ala1185 119011951200Arg Ile Ala Ala Ile Glu Ala Leu Glu Ala Gln Gly Ala Arg Val Thr120512101215Val Ala Ala Val Asp Val Ala Asp Ala Glu Gly Met Ala Ala Leu Leu122012251230Ala Ala Val Glu Pro Pro Leu Arg Gly Val Val His Ala Ala Gly Leu123512401245Leu Asp Asp Gly Leu Leu Ala His Gln Asp Ala Gly Arg Leu Ala Arg125012551260Val Leu Arg Pro Lys Val Glu Gly Ala Trp Val Leu His Thr Leu Thr1265 127012751280Arg Glu Gln Pro Leu Asp Leu Phe Val Leu Phe Ser Ser Ala Ser Gly128512901295Val Phe Gly Ser Ile Gly Gln Gly Ser Tyr Ala Ala Gly Asn Ala Phe130013051310Leu Asp Ala Leu Ala Asp Leu Arg Arg Thr Gln Gly Leu Ala Ala Leu131513201325Ser Ile Ala Trp Gly Leu Trp Ala Glu Gly Gly Met Gly Ser Gln Ala133013351340Gln Arg Arg Glu His Glu Ala Ser Gly Ile Trp Ala Met Pro Thr Ser1345 135013551360Arg Ala Leu Ala Ala Met Glu Trp Leu Leu Gly Thr Arg Ala Thr Gln136513701375Arg Va1 Val Ile Gln Met Asp Trp Ala His Ala Gly Ala Ala Pro Arg138013851390Asp Ala Ser Arg Gly Arg Phe Trp Asp Arg Leu Val Thr Ala Thr Lys139514001405Glu Ala Ser Ser Ser Ala Val Pro Ala Val Glu Arg Trp Arg Asn Ala141014151420Ser Val Val Glu Thr Arg Ser Ala Leu Tyr Glu Leu Val Arg Gly Val1425 143014351440Val Ala Gly Val Met Gly Phe Thr Asp Gln Gly Thr Leu Asp Val Arg144514501455Arg Gly Phe Ala Glu Gln Gly Leu Asp Ser Leu Met Ala Val Glu Ile146014651470Arg Lys Arg Leu Gln Gly Glu Leu Gly Met Pro Leu Ser Ala Thr Leu147514801485Ala Phe Asp His Pro Thr Val Glu Arg Leu Val Glu Tyr Leu Leu Ser149014951500Gln Ala Leu Glu Leu Gln Asp Arg Thr Asp Val Arg Ser Val Arg Leu1505 151015151520Pro Ala Thr Glu Asp Pro Ile Ala Ile Val Gly Ala Ala Cys Arg Phe152515301535Pro Gly Gly Val Glu Asp Leu Glu Ser Tyr Trp Gln Leu Leu Thr Glu154015451550Gly Val Val Val Ser Thr Glu Val Pro Ala Asp Arg Trp Asn Gly Ala155515601565Asp Gly Arg Val Pro Gly Ser Gly Glu Ala Gln Arg Gln Thr Tyr Val157015751580Pro Arg Gly Gly Phe Leu Arg Glu Val Glu Thr Phe Asp Ala Ala Phe1585 159015951600Phe His Ile Ser Pro Arg Glu Ala Met Ser Leu Asp Pro Gln Gln Arg160516101615Leu Leu Leu Glu Val Ser Trp Glu Ala Ile Glu Arg Ala Gly Gln Asp162016251630Pro Ser Ala Leu Arg Glu Ser Pro Thr Gly Val Phe Val Gly Ala Gly163516401645Pro Asn Glu Tyr Ala Glu Arg Val Gln Glu Leu Ala Asp Glu Ala Ala165016551660Gly Leu Tyr Ser Gly Thr Gly Asn Met Leu Ser Val Ala Ala Gly Arg1665 167016751680Leu Ser Phe Phe Leu Gly Leu His Gly Pro Thr Leu Ala Val Asp Thr168516901695Ala Cys Ser Ser Ser Leu Val Ala Leu His Leu Gly Cys Gln Ser Leu170017051710Arg Arg Gly Glu Cys Asp Gln Ala Leu Val Gly Gly Val Asn Met Leu1715 17201725Leu Ser Pro Lys Thr Phe Ala Leu Leu Ser Arg Met His Ala Leu Ser173017351740Pro Gly Gly Arg Cys Lys Thr Phe Ser Ala Asp Ala Asp Gly Tyr Ala1745 175017551760Arg Ala Glu Gly Cys Ala Val Val Val Leu Lys Arg Leu Ser Asp Ala176517701775Gln Arg Asp Arg Asp Pro Ile Leu Ala Val Ile Arg Gly Thr Ala Ile178017851790Asn His Asp Gly Pro Ser Ser Gly Leu Thr Val Pro Ser Gly Pro Ala179518001805Gln Glu Ala Leu Leu Arg Gln Ala Leu Ala His Ala Gly Val Val Pro181018151820Ala Asp Val Asp Phe Val Glu Cys His Gly Thr Gly Thr Ala Leu Gly1825 183018351840Asp Pro Ile Glu Val Arg Ala Leu Ser Asp Val Tyr Gly Gln Ala Arg184518501855Pro Ala Asp Arg Pro Leu Ile Leu Gly Ala Ala Lys Ala Asn Leu Gly186018651870His Met Glu Pro Ala Ala Gly Leu Ala Gly Leu Leu Lys Ala Val Leu187518801885Ala Leu Gly Gln Glu Gln Ile Pro Ala Gln Pro Glu Leu Gly Glu Leu189018951900Asn Pro Leu Leu Pro Trp Glu Ala Leu Pro Val Ala Val Ala Arg Ala1905 191019151920Ala Val Pro Trp Pro Arg Thr Asp Arg Pro Arg Phe Ala Gly Val Ser192519301935Ser Phe Gly Met Ser Gly Thr Asn Ala His Val Val Leu Glu Glu Ala194019451950Pro Ala Val Glu Leu Trp Pro Ala Ala Pro Glu Arg Ser Ala Glu Leu195519601965Leu Val Leu Ser Gly Lys Ser Glu Gly Ala Leu Asp Ala Gln Ala Ala197019751980Arg Leu Arg Glu His Leu Asp Met His Pro Glu Leu Gly Leu Gly Asp1985 199019952000Val Ala Phe Ser Leu Ala Thr Thr Arg Ser Ala Met Asn His Arg Leu200520102015Ala Val Ala Val Thr Ser Arg Glu Gly Leu Leu Ala Ala Leu Ser Ala202020252030Val Ala Gln Gly Gln Thr Pro Pro Gly Ala Ala Arg Cys Ile Ala Ser203520402045Ser Ser Arg Gly Lys Leu Ala Phe Leu Phe Thr Gly Gln Gly Ala Gln205020552060Thr Pro Gly Met Gly Arg Gly Leu Cys Ala Ala Trp Pro Ala Phe Arg2065 207020752080Glu Ala Phe Asp Arg Cys Val Ala Leu Phe Asp Arg Glu Leu Asp Arg208520902095Pro Leu Arg Glu Val Met Trp Ala Glu Pro Gly Ser Ala Glu Ser Leu210021052110Leu Leu Asp Gln Thr Ala Phe Thr Gln Pro Ala Leu Phe Thr Val Glu211521202125Tyr Ala Leu Thr Ala Leu Trp Arg Ser Trp Gly Val Glu Pro Glu Leu213021352140Val Ala Gly His Ser Ala Gly Glu Leu Val Ala Ala Cys Val Ala Gly2145 215021552160Val Phe Ser Leu Glu Asp Gly Val Arg Leu Val Ala Ala Arg Gly Arg216521702175Leu Met Gln Gly Leu Ser Ala Gly Gly Ala Met Val Ser Leu Gly Ala218021852190Pro Glu Ala Glu Val Ala Ala Ala Val Ala Pro His Ala Ala Ser Val219522002205Ser Ile Ala Ala Val Asn Gly Pro Glu Gln Val Val Ile Ala Gly Val221022152220Glu Gln Ala Val Gln Ala Ile Ala Ala Gly Phe Ala Ala Arg Gly Ala2225 223022352240Arg Thr Lys Arg Leu His Val Ser His Ala Ser His Ser Pro Leu Met224522502255Glu Pro Met Leu Glu Glu Phe Gly Arg Val Ala Ala Ser Val Thr Tyr226022652270Arg Arg Pro Ser Val Ser Leu Val Ser Asn Leu Ser Gly Lys Val Val227522802285Ala Asp Glu Leu Ser Ala Pro Gly Tyr Trp Val Arg His Val Arg Glu229022952300Ala Val Arg Phe Ala Asp Gly Val Lys Ala Leu His Glu Ala Gly Ala2305 231023152320Gly Thr Phe Val Glu Val Gly Pro Lys Pro Thr Leu Leu Gly Leu Leu232523302335Pro Ala Cys Leu Pro Glu Ala Glu Pro Thr Leu Leu Ala Ser Leu Arg234023452350Ala Gly Arg Glu Glu Ala Ala Gly Val Leu Glu Ala Leu Gly Arg Leu235523602365Trp Ala Ala Gly Gly Ser Val Ser Trp Pro Gly Val Phe Pro Thr Ala237023752380Gly Arg Arg Val Pro Leu Pro Thr Tyr Pro Trp Gln Arg Gln Arg Tyr2385 239023952400Trp Pro Asp Ile Glu Pro Asp Ser Arg Arg His Ala Ala Ala Asp Pro240524102415Thr Gln Gly Trp Phe Tyr Arg Val Asp Trp Pro Glu Ile Pro Arg Ser242024252430Leu Gln Lys Ser Glu Glu Ala Ser Arg Gly Ser Trp Leu Val Leu Ala243524402445Asp Lys Gly Gly Val Gly Glu Ala Val Ala Ala Ala Leu Ser Thr Arg245024552460Gly Leu Pro Cys Val Val Leu His Ala Pro Ala Glu Thr Ser Ala Thr2465 247024752480Ala Glu Leu Val Thr Glu Ala Ala Gly Gly Arg Ser Asp Trp Gln Val248524902495Val Leu Tyr Leu Trp Gly Leu Asp Ala Val Val Gly Ala Glu Ala Ser250025052510Ile Asp Glu Ile Gly Asp Ala Thr Arg Arg Ala Thr Ala Pro Val Leu251525202525Gly Leu Ala Arg Phe Leu Ser Thr Val Ser Cys Ser Pro Arg Leu Trp253025352540Val Val Thr Arg Gly Ala Cys Ile Val Gly Asp Glu Pro Ala Ile Ala2545 255025552560Pro Cys Gln Ala Ala Leu Trp Gly Met Gly Arg Val Ala Ala Leu Glu256525702575His Pro Gly Ala Trp Gly Gly Leu Val Asp Leu Asp Pro Arg Ala Ser258025852590Pro Pro Gln Ala Ser Pro Ile Asp Gly Glu Met Leu Val Thr Glu Leu259526002605Leu Ser Gln Glu Thr Glu Asp Gln Leu Ala Phe Arg His Gly Arg Arg261026152620His Ala Ala Arg Leu Val Ala Ala Pro Pro Gln Gly Gln Ala Ala Pro2625 263026352640Val Ser Leu Ser Ala Glu Ala Ser Tyr Leu Val Thr Gly Gly Leu Gly264526502655Gly Leu Gly Leu Ile Val Ala Gln Trp Leu Val Glu Leu Gly Ala Arg266026652670His Leu Val Leu Thr Ser Arg Arg Gly Leu Pro Asp Arg Gln Ala Trp267526802685Cys Glu Gln Gln Pro Pro Glu Ile Arg Ala Arg Ile Ala Ala Val Glu269026952700Ala Leu Glu Ala Arg Gly Ala Arg Val Thr Val Ala Ala Val Asp Val2705 271027152720Ala Asp Val Glu Pro Met Thr Ala Leu Val Ser Ser Val Glu Pro Pro272527302735Leu Arg Gly Val Val His Ala Ala Gly Val Ser Val Met Arg Pro Leu274027452750Ala Glu Thr Asp Glu Thr Leu Leu Glu Ser Val Leu Arg Pro Lys Val275527602765Ala Gly Ser Trp Leu Leu His Arg Leu Leu His Gly Arg Pro Leu Asp277027752780Leu Phe Val Leu Phe Ser Ser Gly Ala Ala Val Trp Gly Ser His Ser2785 279027952800Gln Gly Ala Tyr Ala Ala Ala Asn Ala Phe Leu Asp Gly Leu Ala His280528102815Leu Arg Arg Ser Gln Ser Leu Pro Ala Leu Ser Val Ala Trp Gly Leu282028252830Trp Ala Glu Gly Gly Met Ala Asp Ala Glu Ala His Ala Arg Leu Ser283528402845Asp Ile Gly Val Leu Pro Met Ser Thr Ser Ala Ala Leu Ser Ala Leu285028552860Gln Arg Leu Val Glu Thr Gly Ala Ala Gln Arg Thr Val Thr Arg Met2865 287028752880Asp Trp Ala Arg Phe Ala Pro Val Tyr Thr Ala Arg Gly Arg Arg Asn288528902895Leu Leu Ser Ala Leu Val Ala Gly Arg Asp Ile Ile Ala Pro Ser Pro290029052910Pro Ala Ala Ala Thr Arg Asn Trp Arg Gly Leu Ser Val Ala Glu Ala291529202925Arg Val Ala Leu His Glu Ile Val His Gly Ala Val Ala Arg Val Leu293029352940Gly Phe Leu Asp Pro Ser Ala Leu Asp Pro Gly Met Gly Phe Asn Glu2945 295029552960Gln Gly Leu Asp Ser Leu Met Ala Val Glu Ile Arg Asn Leu Leu Gln296529702975Ala Glu Leu Asp Val Arg Leu Ser Thr Thr Leu Ala Phe Asp His Pro298029852990Thr Val Gln Arg Leu Val Glu His Leu Leu Val Asp Val Leu Lys Leu299530003005Glu Asp Arg Ser Asp Thr Gln His Val Arg Ser Leu Ala Ser Asp Glu3010 30153020Pro Ile Ala Ile Val Gly Ala Ala Cys Arg Phe Pro Gly Gly Val Glu3025 303030353040Asp Leu Glu Ser Tyr Trp Gln Leu Leu Ala Glu Gly Val Val Val Ser304530503055Ala Glu Val Pro Ala Asp Arg Trp Asp Ala Ala Asp Trp Tyr Asp Pro306030653070Asp Pro Glu Ile Pro Gly Arg Thr Tyr Val Thr Lys Gly Ala Phe Leu307530803085Arg Asp Leu Gln Arg Leu Asp Ala Thr Phe Phe Arg Ile Ser Pro Arg309030953100Glu Ala Met Ser Leu Asp Pro Gln Gln Arg Leu Leu Leu Glu Val Ser3105 311031153120Trp Glu Ala Leu Glu Ser Ala Gly Ile Ala Pro Asp Thr Leu Arg Asp312531303135Ser pro Thr Gly Val Phe Val Gly Ala Gly Pro Asn Glu Tyr Tyr Thr314031453150Gln Arg Leu Arg Gly Phe Thr Asp Gly A la Ala Gly Leu Tyr Gly Gly31553160 3165Thr Gly Asn Met Leu Ser Val Thr Ala Gly Arg Leu Ser Phe Phe Leu317031753180Gly Leu His Gly Pro Thr Leu Ala Met Asp Thr Ala Cys Ser Ser Ser3185 319031953200Leu Val Ala Leu His Leu Ala Cys Gln Ser Leu Arg Leu Gly Glu Cys320532103215Asp Gln Ala Leu Val Gly Gly Val Asn Val Leu Leu Ala Pro Glu Thr322032253230Phe Val Leu Leu Ser Arg Met Arg Ala Leu Ser Pro Asp Gly Arg Cys323532403245Lys Thr Phe Ser Ala Asp Ala Asp Gly Tyr Ala Arg Gly Glu Gly Cys325032553260Ala Val Val Val Leu Lys Arg Leu Arg Asp Ala Gln Arg Ala Gly Asp3265 327032753280Ser Ile Leu Ala Leu Ile Arg Gly Ser Ala Val Asn His Asp Gly Pro328532903295Ser Ser Gly Leu Thr Val Pro Asn Gly Pro Ala Gln Gln Ala Leu Leu330033053310Arg Gln Ala Leu Ser Gln Ala Gly Val Ser Pro Val Asp Val Asp Phe331533203325Val Glu Cys His Gly Thr Gly Thr Ala Leu Gly Asp Pro Ile Glu Val333033353340Gln Ala Leu Ser Glu Val Tyr Gly Pro Gly Arg Ser Gly Asp Arg Pro3345 335033553360Leu Val Leu Gly Ala Ala Lys Ala Asn Val Ala His Leu Glu Ala Ala336533703375Ser Gly Leu Ala Ser Leu Leu Lys Ala Val Leu Ala Leu Arg His Glu338033853390Gln Ile Pro Ala Gln Pro Glu Leu Gly Glu Leu Asn Pro His Leu Pro339534003405Trp Asn Thr Leu Pro Val Ala Val Pro Arg Lys Ala Val Pro Trp Gly341034153420Arg Gly Ala Arg Pro Arg Arg Ala Gly Val Ser Ala Phe Gly Leu Ser3425 343034353440Gly Thr Asn Val His Val Val Leu Glu Glu Ala Pro Glu Val Glu Pro344534503455Ala Pro Ala Ala Pro Ala Arg Pro Val Glu Leu Val Val Leu Ser Ala346034653470Lys Ser Ala Ala Ala Leu Asp Ala Ala Ala Ala Arg Leu Ser Ala His347534803485Leu Ser Ala His Pro Glu Leu Ser Leu Gly Asp Val Ala Phe Ser Leu3490 34953500Ala Thr Thr Arg Ser Pro Met Glu His Arg Leu Ala Ile Ala Thr Thr3505 351035153520Ser Arg Glu Ala Leu Arg Gly Ala Leu Asp Ala Ala Ala Gln Gln Lys352535303535Thr Pro Gln Gly Ala Val Arg Gly Lys Ala Val Set Ser Arg Gly Lys354035453550Leu Ala Phe Leu Phe Thr Gly Gln Gly Ala Gln Met Pro Gly Met Gly355535603565Arg Gly Leu Tyr Glu Thr Trp Pro Ala Phe Arg Glu Ala Phe Asp Arg357035753580Cys Val Ala Leu Phe Asp Arg Glu Ile Asp Gln Pro Leu Arg Glu Val3585 359035953600Met Trp Ala Ala Pro Gly Leu Ala Gln Ala Ala Arg Leu Asp Gln Thr360536103615Ala Tyr Ala Gln Pro Ala Leu Phe Ala Leu Glu Tyr Ala Leu Ala Ala362036253630Leu Trp Arg Ser Trp Gly Val Glu Pro His Val Leu Leu Gly His Ser363536403645Ile Gly Glu Leu Val Ala Ala Cys Val Ala Gly Val Phe Ser Leu Glu365036553660Asp Ala Val Arg Leu Val Ala Ala Arg Gly Arg Leu Met Gln Ala Leu3665 367036753680Pro Ala Gly Gly Ala Met Val Ala Ile Ala Ala Ser Glu Ala Glu Val368536903695Ala Ala Ser Val Ala Pro His Ala Ala Thr Val Ser Ile Ala Ala Val370037053710Asn Gly Pro Asp Ala Val Val Ile Ala Gly Ala Glu Val Gln Val Leu371537203725Ala Leu Gly Ala Thr Phe Ala Ala Arg Gly Ile Arg Thr Lys Arg Leu373037353740Ala Val Ser His Ala Phe His Ser Pro Leu Met Asp Pro Met Leu Glu3745 375037553760Asp Phe Gln Arg Val Ala Ala Thr Ile Ala Tyr Arg Ala Pro Asp Arg376537703775Pro Val Val Ser Asn Val Thr Gly His Val Ala Gly Pro Glu Ile Ala378037853790Thr Pro Glu Tyr Trp Val Arg His Val Arg Ser Ala Val Arg Phe Gly379538003805Asp Gly Ala Lys Ala Leu His Ala Ala Gly Ala Ala Thr Phe Val Glu381038153820Val Gly Pro Lys Pro Val Leu Leu Gly Leu Leu Pro Ala Cys Leu Gly3825 383038353840Glu Ala Asp Ala Val Leu Val Pro Ser Leu Arg Ala Asp Arg Ser Glu384538503855Cys Glu Val Val Leu Ala Ala Leu Gly Ala Trp Tyr Ala Trp Gly Gly386038653870Ala Leu Asp Trp Lys Gly Val Phe Pro Asp Gly Ala Arg Arg Val Ala387538803885Leu Pro Met Tyr Pro Trp Gln Arg Glu Arg His Trp Met Asp Leu Thr389038953900Pro Arg Ser Ala Ala Pro Ala Gly Ile Ala Gly Arg Trp Pro Leu Ala3905 391039153920Gly Val Gly Leu Cys Met Pro Gly Ala Val Leu His His Val Leu Ser392539303935Ile Gly Pro Arg His Gln Pro Phe Leu Gly Asp His Leu Val Phe Gly394039453950Lys Val Val Val Pro Gly Ala Phe His Val Ala Val Ile Leu Ser Ile395539603965Ala Ala Glu Arg Trp Pro Glu Arg Ala Ile Glu Leu Thr Gly Val Glu397039753980Phe Leu Lys Ala Ile Ala Met Glu Pro Asp Gln Glu Val Glu Leu His3985 3990 3995 4000Ala Val Leu Thr Pro Glu Ala Ala Gly Asp Gly Tyr Leu Phe Glu Leu400540104015Ala Thr Leu Ala Ala Pro Glu Thr Glu Arg Arg Trp Thr Thr His Ala402040254030Arg Gly Arg Val Gln Pro Thr Asp Gly Ala Pro Gly Ala Leu Pro Arg403540404045Leu Glu Val Leu Glu Asp Arg Ala Ile Gln Pro Leu Asp Phe Ala Gly405040554060Phe Leu Asp Arg Leu Ser Ala Val Arg Ile Gly Trp Gly Pro Leu Trp4065 407040754080Arg Trp Leu Gln Asp Gly Arg Val Gly Asp Glu Ala Ser Leu Ala Thr408540904095Leu Val Pro Thr Tyr Pro Asn Ala His Asp Val Ala Pro Leu His Pro410041054110Ile Leu Leu Asp Asn Gly Phe Ala Val Ser Leu Leu Ser Thr Arg Ser411541204125Glu Pro Glu Asp Asp Gly Thr Pro Pro Leu Pro Phe Ala Val Glu Arg413041354140Val Arg Trp Trp Arg Ala Pro Val Gly Arg Val Arg Cys Gly Gly Val4145 415041554160Pro Arg Ser Gln Ala Phe Gly Val Ser Ser Phe Val Leu Val Asp Glu416541704175Thr Gly Glu Val Val Ala Glu Val Glu Gly Phe Val Cys Arg Arg Ala418041854190Pro Arg Glu Val Phe Leu Arg Gln Glu Ser Gly Ala Ser Thr Ala Ala419542004205Leu Tyr Arg Leu Asp Trp Pro Glu Ala Pro Leu Pro Asp Ala Pro Ala4210 42154220Glu Arg Ile Glu Glu Ser Trp Val Val Val Ala Ala Pro Gly Ser Glu4225 423042354240Met Ala Ala Ala Leu Ala Thr Arg Leu Asn Arg Cys Val Leu Ala Glu424542504255Pro Lys Gly Leu Glu Ala Ala Leu Ala Gly Val Ser Pro Ala Gly Val426042654270Ile Cys Leu Trp Glu Ala Gly Ala His Glu Glu Ala Pro Ala Ala Ala427542804285Gln Arg Val Ala Thr Glu Gly Leu Ser Val Val Gln Ala Leu Arg Asp429042954300Arg Ala Val Arg Leu Trp Trp Val Thr Met Gly Ala Val Ala Val Glu4305 431043154320Ala Gly Glu Arg Val Gln Val Ala Thr Ala Pro Val Trp Gly Leu Gly432543304335Arg Thr Val Met Gln Glu Arg Pro Glu Leu Ser Cys Thr Leu Val Asp434043454350Leu Glu Pro Glu Ala Asp Ala Ala Arg Ser Ala Asp Val Leu Leu Arg435543604365Glu Leu Gly Arg Ala Asp Asp Glu Thr Gln Val Ala Phe Arg Ser Gly437043754380Lys Arg Arg Val Ala Arg Leu Val Lys Ala Thr Thr Pro Glu Gly Leu4385 439043954400Leu Val Pro Asp Ala Glu Ser Tyr Arg Leu Glu Ala Gly Gln Lys Gly440544104415Thr Leu Asp Gln Leu Arg Leu Ala Pro Ala Gln Arg Arg Ala Pro Gly442044254430Pro Gly Glu Val Glu Ile Lys Val Thr Ala Ser Gly Leu Asn Phe Arg443544404445Thr Val Leu Ala Val Leu Gly Met Tyr Pro Gly Asp Ala Gly Pro Met445044554460Gly Gly Asp Cys Ala Gly Val Ala Thr Ala Val Gly Gln Gly Val Arg4465 447044754480His Val Ala Val Gly Asp Ala Val Met Thr Leu Gly Thr Leu His Arg448544904495Phe Val Thr Val Asp Ala Arg Leu Val val Arg Gln Pro Ala Gly Leu450045054510Thr Pro Ala Gln Ala Ala Thr Val Pro val Ala Phe Leu Thr Ala Trp451545204525Leu Ala Leu His Asp Leu Gly Asn Leu Arg Arg Gly Glu Arg Val Leu453045354540Ile His Ala Ala Ala Gly Gly Val Gly Met Ala Ala Val Gln Ile Ala4545 455045554560Arg Trp Ile Gly Ala Glu Val Phe Ala Thr Ala Ser Pro Ser Lys Trp456545704575Ala Ala Val Gln Ala Met Gly Val Pro Arg Thr His Ile Ala Ser Ser458045854590Arg Thr Leu Glu Phe Ala Glu Thr Phe Arg Gln Val Thr Gly Gly Arg459546004605Gly Val Asp Val Val Leu Asn Ala Leu Ala Gly Glu Phe Val Asp Ala461046154620Ser Leu Ser Leu Leu Ser Thr Gly Gly Arg Phe Leu Glu Met Gly Lys4625 4630 4635 4640Thr Asp Ile Arg Asp Arg Ala Ala Val Ala Ala Ala His Pro Gly Val464546504655Arg Tyr Arg Val Phe Asp Ile Leu Glu Leu Ala Pro Asp Arg Thr Arg466046654670Glu Ile Leu Glu Arg Val Val Glu Gly Phe Ala Ala Gly His Leu Arg467546804685Ala Leu Pro Val His Ala Phe Ala Ile Thr Lys Ala Glu Ala Ala Phe469046954700Arg Phe Met Ala Gln Ala Arg His Gln Gly Lys Val Val Leu Leu Pro4705 471047154720Ala Pro Ser Ala Ala Pro Leu Ala Pro Thr Gly Thr Val Leu Leu Thr472547304735Gly Gly Leu Gly Ala Leu Gly Leu His Val Ala Arg Trp Leu Ala Gln474047454750Gln Gly Val Pro His Met Val Leu Thr Gly Arg Arg Gly Leu Asp Thr475547604765Pro Gly Ala Ala Lys Ala Val Ala Glu Ile Glu Ala Leu Gly Ala Arg4770 4775 4780Val Thr Ile Ala Ala Ser Asp Val Ala Asp Arg Asn Ala Leu Glu Ala4785 479047954800Val Leu Gln Ala Ile Pro Ala Glu Trp Pro Leu Gln Gly Val Ile His480548104815Ala Ala Gly Ala Leu Asp Asp Gly Val Leu Asp Glu Gln Thr Thr Asp482048254830Arg Phe Ser Arg Val Leu Ala Pro Lys Val Thr Gly Ala Trp Asn Leu483548404845His Glu Leu Thr Ala Gly Asn Asp Leu Ala Phe Phe Val Leu Phe Ser485048554860Ser Met Ser Gly Leu Leu Gly Ser Ala Gly Gln Ser Asn Tyr Ala Ala4865 487048754880Ala Asn Thr Phe Leu Asp Ala Leu Ala Ala His Arg Arg Ala Glu Gly488548904895Leu Ala Ala Gln Ser Leu Ala Trp Gly Pro Trp Ser Asp Gly Gly Met490049054910Ala Ala Gly Leu Ser Ala Ala Leu Gln Ala Arg Leu Ala Arg His Gly491549204925Met Gly Ala Leu Ser Pro Ala Gln Gly Thr Ala Leu Leu Gly Gln Ala493049354940Leu Ala Arg Pro Glu Thr Gln Leu Gly Ala Met Ser Leu Asp Val Arg4945 495049554960Ala Ala Ser Gln Ala Ser Gly Ala Ala Val Pro Pro Val Trp Arg Ala496549704975Leu Val Arg Ala Glu Ala Arg His Thr Ala Ala Gly Ala Gln Gly Ala498049854990Leu Ala Ala Arg Leu Gly Ala Leu Pro Glu Ala Arg Arg Ala Asp Glu499550005005Val Arg Lys Val Val Gln Ala Glu Ile Ala Arg Val Leu Ser Trp Ser501050155020Ala Ala Ser Ala Val Pro Val Asp Arg Pro Leu Ser Asp Leu Gly Leu5025 503050355040Asp Ser Leu Thr Ala Val Glu Leu Arg Asn Val Leu Gly Gln Arg Val504550505055Gly Ala Thr Leu Pro Ala Thr Leu Ala Phe Asp His Pro Thr Val Asp506050655070Ala Leu Thr Arg Trp Leu Leu Asp Lys Val Leu Ala Val Ala Glu Pro507550805085Ser Val Ser Ser Ala Lys Ser Ser Pro Gln Val Ala Leu Asp Glu Pro509050955100Ile Ala Ile Ile Gly Ile Gly Cys Arg Phe Pro Gly Gly Val Ala Asp5105 511051155120Pro Glu Ser Phe Trp Arg Leu Leu Glu Glu Gly Ser Asp Ala Val Val512551305135Glu Val Pro His Glu Arg Trp Asp Ile Asp Ala Phe Tyr Asp Pro Asp514051455150Pro Asp Val Arg Gly Lys Met Thr Thr Arg Phe Gly Gly Phe Leu Ser515551605165Asp Ile Asp Arg Phe Asp Pro Ala Phe Phe Gly Ile Ser Pro Arg Glu517051755180Ala Thr Thr Met Asp Pro Gln Gln Arg Leu Leu Leu Glu Thr Ser Trp5185 519051955200Glu Ala Phe Glu Arg Ala Gly Ile Leu Pro Glu Arg Leu Met Gly Ser520552105215Asp Thr Gly Val Phe Val Gly Leu Phe Tyr Gln Glu Tyr Ala Ala Leu522052255230Ala Gly Gly Ile Glu Ala Phe Asp Gly Tyr Leu Gly Thr Gly Thr Thr523552405245Ala Ser Val Ala Ser Gly Arg Ile Ser Tyr Val Leu Gly Leu Lys Gly525052555260Pro Ser Leu Thr Val Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Val5265 527052755280His Leu Ala Cys Gln Ala Leu Arg Arg Gly Glu Cys Ser Val Ala Leu528552905295Ala Gly Gly Val Ala Leu Met Leu Thr Pro Ala Thr Phe Val Glu Phe530053055310Ser Arg Leu Arg Gly Leu Ala Pro Asp Gly Arg Cys Lys Ser Phe Ser531553205325Ala Ala Ala Asp Gly Val Gly Trp Ser Glu Gly Cys Ala Met Leu Leu533053355340Leu Lys Pro Leu Arg Asp Ala Gln Arg Asp Gly Asp Pro Ile Leu Ala5345 535053555360Val Ile Arg Gly Thr Ala Val Asn Gln Asp Gly Arg Ser Asn Gly Leu536553705375Thr Ala Pro Asn Gly Ser Ser Gln Gln Glu Val Ile Arg Arg Ala Leu538053855390Glu Gln Ala Gly Leu Ala Pro Ala Asp Val Ser Tyr Val Glu Cys His539554005405Gly Thr Gly Thr Thr Leu Gly Asp Pro Ile Glu Val Gln Ala Leu Gly541054155420Ala Val Leu Ala Gln Gly Arg Pro Ser Asp Arg Pro Leu Val Ile Gly5425 543054355440Ser Val Lys Ser Asn Ile Gly His Thr Gln Ala Ala Ala Gly Val Ala544554505455Gly Val Ile Lys Val Ala Leu Ala Leu Glu Arg Gly Leu Ile Pro Arg546054655470Ser Leu His Phe Asp Ala Pro Asn Pro His Ile Pro Trp Ser Glu Leu547554805485Ala Val Gln val Ala Ala Lys Pro Val Glu Trp Thr Arg Asn Gly Val549054955500Pro Arg Arg Ala Gly Val Ser Ser Phe Gly Val Ser Gly Thr Asn Ala5505 551055155520His Val Val Leu Glu Glu Ala Pro Ala Ala Ala Phe Ala Pro Ala Ala552555305535Ala Arg Ser Ala Glu Leu Phe Val Leu Ser Ala Lys Ser Ala Ala Ala554055455550Leu Asp Ala Gln Ala Ala Arg Leu Ser Ala His Val Val Ala His Pro555555605565Glu Leu Gly Leu Gly Asp Leu Ala Phe Ser Leu Ala Thr Thr Arg Ser557055755580Pro Met Thr Tyr Arg Leu Ala Val Ala Ala Thr Ser Arg Glu Ala Leu5585 559055955600Ser Ala Ala Leu Asp Thr Ala Ala Gln Gly Gln Ala Pro Pro Ala Ala560556105615Ala Arg Gly His Ala Ser Thr Gly Ser Ala Pro Lys Val Val Phe Val562056255630Phe Pro Gly Gln Gly Ser Gln Trp Leu Gly Met Gly Gln Lys Leu Leu563556405645Ser Glu Glu Pro Val Phe Arg Asp Ala Leu Ser Ala Cys Asp Arg Ala565056555660Ile Gln Ala Glu Ala Gly Trp Ser Leu Leu Ala Glu Leu Ala Ala Asp5665 567056755680Glu Thr Thr Ser Gln Leu Gly Arg Ile Asp Val Val Gln Pro Ala Leu568556905695Phe Ala Ile Glu Val Ala Leu Ser Ala Leu Trp Arg Ser Trp Gly Val570057055710Glu Pro Asp Ala Val Val Gly His Ser Met Gly Glu Val Ala Ala Ala571557205725His Val Ala Gly Ala Leu Ser Leu Glu Asp Ala Val Ala Ile Ile Cys573057355740Arg Arg Ser Leu Leu Leu Arg Arg Ile Ser Gly Gln Gly Glu Met Ala5745 575057555760Val Val Glu Leu Ser Leu Ala Glu Ala Glu Ala Ala Leu Leu Gly Tyr576557705775Glu Asp Arg Leu Ser Val Ala Val Ser ASn Ser Pro Arg Ser Thr Val578057855790Leu Ala Gly Glu Pro Ala Ala Leu Ala Glu Val Leu Ala Ile Leu Ala579558005805Ala Lys Gly Val Phe Cys Arg Arg Val Lys Val Asp Val Ala Ser His581058155820Ser Pro Gln Ile Asp Pro Leu Arg Asp Glu Leu Leu Ala Ala Leu Gly5825 583058355840Glu Leu Glu Pro Arg Gln Ala Thr Val Ser Met Arg Ser Thr Val Thr584558505855Ser Thr Ile Met Ala Gly Pro Glu Leu Val Ala Ser Tyr Trp Ala Asp586058655870Asn Val Arg Gln Pro Val Arg Phe Ala Glu Ala Val Gln Ser Leu Met587558805885Glu Asp Gly His Gly Leu Phe Val Glu Met Ser Pro His Pro Ile Leu589058955900Thr Thr Ser Val Glu Glu Ile Arg Arg Ala Thr Lys Arg Glu Gly Val5905 591059155920Ala Val Gly Ser Leu Arg Arg GlY Gln Asp Glu Arg Leu Ser Met Leu592559305935Glu Ala Leu Gly Ala Leu Trp Val His Gly Gln Ala Val Gly Trp Glu594059455950Arg Leu Phe Ser Ala Gly Gly Ala Gly Leu Arg Arg Val Pro Leu Pro595559605965Thr Tyr Pro Trp Gln Arg Glu Arg Tyr Trp Val Asp Ala Pro Thr Gly597059755980Gly Ala Ala Gly Gly Ser Arg Phe Ala His Ala Gly Ser His Pro Leu5985 599059956000Leu Gly Glu Met Gln Thr Leu Ser Thr Gln Arg Ser Thr Arg Val Trp600560106015Glu Thr Thr Leu Asp Leu Lys Arg Leu Pro Trp Leu Gly Asp His Arg602060256030Val Gln Gly Ala Val Val Phe Pro Gly Ala Ala Tyr Leu Glu Met Ala603560406045Leu Ser Ser Gly Ala Glu Ala Leu Gly Asp Gly Pro Leu Gln Val Ser605060556060Asp Val Val Leu Ala Glu Ala Leu Ala Phe Ala Asp Asp Thr Pro Ala6065 607060756080Ala Val Gln Val Met Ala Thr Glu Glu Arg Pro Gly Arg Leu Gln Phe608560906095His Val Ala Ser Arg Val Pro Gly His Gly Gly Ala Ala Phe Arg Ser610061056110His Ala Arg Gly Val Leu Arg Gln Ile Glu Arg Ala Glu Val Pro Ala611561206125Arg Leu Asp Leu Ala Ala Leu Arg Ala Arg Leu Gln Ala Ser Ala Pro613061356140Ala Ala Ala Thr Tyr Ala Ala Leu Ala Glu Met Gly Leu Glu Tyr Gly6145 615061556160Pro Ala Phe Gln Gly Leu Val Glu Leu Trp Arg Gly Glu Gly Glu Ala616561706175Leu Gly Arg Val Arg Leu Pro Glu Ala Ala Gly Ser Pro Ala Ala Cys618061856190Arg Leu His Pro Ala Leu Leu Asp Ala Cys Phe His Val Ser Ser Ala619562006205Phe Ala Asp Arg Gly Glu Ala Thr Pro Trp Val Pro Val Glu Ile Gly621062156220Ser Leu Arg Trp Phe Gln Arg Pro Ser Gly Glu Leu Trp Cys His Ala6225 623062356240Arg Ser Val Ser His Gly Lys Pro Thr Pro Asp Arg Arg Ser Thr Asp624562506255Phe Trp Val Val Asp Ser Thr Gly Ala Ile Val Ala Glu Ile Ser Gly626062656270Leu Val Ala Gln Arg Leu Ala Gly Gly Val Arg Arg Arg Glu Glu Asp627562806285Asp Trp Phe Met Glu Pro Ala Trp Glu Pro Thr Ala Val Pro Gly Ser629062956300Glu Val Met Ala Gly Arg Trp Leu Leu Ile Gly Ser Gly Gly Gly Leu6305 631063156320Gly Ala Ala Leu His Ser Ala Leu Thr Glu Ala Gly His Ser Val Val632563306335His Ala Thr Gly Arg Gly Thr Ser Ala Ala Gly Leu Gln Ala Leu Leu634063456350Thr Ala Ser Phe Asp Gly Gln Ala Pro Thr Ser Val Val His Leu Gly635563606365Ser Leu Asp Glu Arg Gly Val Leu Asp Ala Asp Ala Pro Phe Asp Ala637063756380Asp Ala Leu Glu Glu Ser Leu Val Arg Gly Cys Asp Ser Val Leu Trp6385 639063956400Thr Val Gln Ala Val Ala Gly Ala Gly Phe Arg Asp Pro Pro Arg Leu640564106415Trp Leu Val Thr Arg Gly Ala Gln Ala Ile Gly Ala Gly Asp Val Ser642064256430Val Ala Gln Ala Pro Leu Leu Gly Leu Gly Arg Val Ile Ala Leu Glu643564406445His Ala Glu Leu Arg Cys Ala Arg Ile Asp Leu Asp Pro Ala Arg Arg645064556460Asp Gly Glu Val Asp Glu Leu Leu Ala Glu Leu Leu Ala Asp Asp Ala6465 647064756480Glu Glu Glu Val Ala Phe Arg Gly Gly Glu Arg Arg Val Ala Arg Leu648564906495Val Arg Arg Leu Pro Glu Thr Asp Cys Arg Glu Lys Ile Glu Pro Ala650065056510Glu Gly Arg Pro Phe Arg Leu Glu Ile Asp Gly Ser Gly Val Leu Asp651565206525Asp Leu Val Leu Arg Ala Thr Glu Arg Arg Pro Pro Gly Pro Gly Glu653065356540Val Glu Ile Ala Val Glu Ala Ala Gly Leu Asn Phe Leu Asp Val Met6545 655065556560Arg Ala Met Gly Ile Tyr Pro Gly Pro Gly Asp Gly Pro Val Ala Leu656565706575Gly Ala Glu Cys Ser Gly Arg Ile Val Ala Met Gly Glu Gly Val Glu658065856590Ser Leu Arg Ile Gly Gln Asp Val Val Ala Val Ala Pro Phe Ser Phe659566006605Gly Thr His Val Thr Ile Asp Ala Arg Met Leu Ala Pro Arg Pro Ala661066156620Ala Leu Thr Ala Ala Gln Ala Ala Ala Leu Pro Val Ala Phe Met Thr6625 663066356640Ala Trp Tyr Gly Leu Val His Leu Gly Arg Leu Arg Ala Gly Glu Arg664566506655Val Leu Ile His Ser Ala Thr Gly Gly Thr Gly Leu Ala Ala Val Gln666066656670Ile Ala Arg His Leu Gly Ala Glu Ile Phe Ala Thr Ala Gly Thr Pro667566806685Glu Lys Arg Ala Trp Leu Arg Glu Gln Gly Ile Ala His Val Met Asp669066956700Ser Arg Ser Leu Asp Phe Ala Glu Gln Val Leu Ala Ala Thr Lys Gly6705 671067156720Glu Gly Val Asp Val Val Leu Asn Ser Leu Ser Gly Ala Ala Ile Asp672567306735Ala Ser Leu Ser Thr Leu Val Pro Asp Gly Arg Phe Ile Glu Leu Gly674067456750Lys Thr Asp Ile Tyr Ala Asp Arg Ser Leu Gly Leu Ala His Phe Arg675567606765Lys Ser Leu Ser Tyr Ser Ala Val Asp Leu Ala Gly Leu Ala Val Arg677067756780Arg Pro Glu Arg Val Ala Ala Leu Leu Ala Glu Val Val Asp Leu Leu6785 679067956800Ala Arg Gly Ala Leu Gln Pro Leu Pro Val Glu Ile Phe Pro Leu Ser680568106815Arg Ala Ala Asp Ala Phe Arg Lys Met Ala Gln Ala Gln His Leu Gly682068256830Lys Leu Val Leu Ala Leu Glu Asp Pro Asp Val Arg Ile Arg Val Pro683568406845Gly Glu Ser Gly Val Ala Ile Arg Ala Asp Gly Ala Tyr Leu Val Thr685068556860Gly Gly Leu Gly Gly Leu Gly Leu Ser Val Ala Gly Trp Leu Ala Glu6865 687068756880Gln Gly Ala Gly His Leu Val Leu Val Gly Arg Ser Gly Ala Val Ser688568906895Ala Glu Gln Gln Thr Ala Val Ala Ala Leu Glu Ala His Gly Ala Arg690069056910Val Thr Val Ala Arg Ala Asp Val Ala Asp Arg Ala Gln Met Glu Arg691569206925Ile Leu Arg Glu Val Thr Ala Ser Gly Met Pro Leu Arg Gly Val Val693069356940His Ala Ala Gly Ile Leu Asp Asp Gly Leu Leu Met Gln Gln Thr Pro6945 695069556960Ala Arg Phe Arg Ala Val Met Ala Pro Lys Val Arg Gly Ala Leu His696569706975Leu His Ala Leu Thr Arg Glu Ala Pro Leu Ser Phe Phe Val Leu Tyr698069856990Ala Ser Gly Ala Gly Leu Leu Gly Ser Pro Gly Gln Gly Asn Tyr Ala699570007005Ala Ala Asn Thr Phe Leu Asp Ala Leu Ala His His Arg Arg Ala Gln701070157020Gly Leu Pro Ala Leu Ser Ile Asp Trp Gly Leu Phe Ala Asp Val Gly7025 703070357040Leu Ala Ala Gly Gln Gln Asn Arg Gly Ala Arg Leu Val Thr Arg Gly704570507055Thr Arg Ser Leu Thr Pro Asp Glu Gly Leu Trp Ala Leu Glu Arg Leu706070657070Leu Asp Gly Asp Arg Thr Gln Ala G1y Val Met Pro Phe Asp Val Arg707570807085Gln Trp Val Glu Phe Tyr Pro Ala Ala Ala Ser Ser Arg Arg Leu Ser709070957100Arg Leu Met Thr Ala Arg Arg Val Ala Ser Gly Arg Leu Ala Gly Asp7105 711071157120Arg Asp Leu Leu Glu Arg Leu Ala Thr Ala Glu Ala Gly Ala Arg Ala712571307135Gly Met Leu Gln Glu Val Val Arg Ala Gln Val Ser Gln Val Leu Arg714071457150Leu Ser Glu Gly Lys Leu Asp Val Asp Ala Pro Leu Thr Ser Leu Gly715571607165Met Asp Ser Leu Met Gly Leu Glu Leu Arg Asn Arg Ile Glu Ala Val717071757180Leu Gly Ile Thr Met Pro Ala Thr Leu Leu Trp Thr Tyr Pro Thr Val7185 719071957200Ala Ala Leu Ser Ala His Leu Ala Ser His Val Val Ser Thr Gly Asp720572107215Gly Glu Ser Ala Arg Pro Pro Asp Thr Gly Ser Val Ala Pro Thr Thr722072257230His Glu Val Ala Ser Leu Asp Glu Asp Gly Leu Phe Ala Leu Ile Asp723572407245Glu Ser Leu Ala Arg Ala Gly Lys Arg72507255<210>6<211>3798<212>PRT<213>纤维堆囊菌<400>6Val Thr Asp Arg Glu Gly Gln Leu Leu Glu Arg Leu Arg Glu Val Thr1 5 10 15Leu Ala Leu Arg Lys Thr Leu Asn Glu Arg Asp Thr Leu Glu Leu Glu20 25 30Lys Thr Glu Pro Ile Ala Ile Val Gly Ile Gly Cys Arg Phe Pro Gly35 40 45Gly Ala Gly Thr Pro Glu Ala Phe Trp Glu Leu Leu Asp Asp Gly Arg50 55 60Asp Ala Ile Arg Pro Leu Glu Glu Arg Trp Ala Leu Val Gly Val Asp65 70 75 80Pro Gly Asp Asp Val Pro Arg Trp Ala Gly Leu Leu Thr Glu Ala Ile85 90 95Asp Gly Phe Asp Ala Ala Phe Phe Gly Ile Ala Pro Arg Glu Ala Arg100 105 110Ser Leu Asp Pro Gln His Arg Leu Leu Leu Glu Val Ala Trp Glu Gly115 120 125Phe Glu Asp Ala Gly Ile Pro Pro Arg Ser Leu Val Gly Ser Arg Thr130 135 140Gly Val Phe Val Gly Val Cys Ala Thr Glu Tyr Leu His Ala Ala Val145 150 155 160Ala His Gln Pro Arg Glu Glu Arg Asp Ala Tyr Ser Thr Thr Gly Asn165 170 175Met Leu Ser Ile Ala Ala Gly Arg Leu Ser Tyr Thr Leu Gly Leu Gln180 185 190Gly Pro Cys Leu Thr Val Asp Thr Ala Cys Ser Ser Ser Leu Val Ala195 200 205Ile His Leu Ala Cys Arg Ser Leu Arg Ala Arg Glu Ser Asp Leu Ala210 215 220Leu Ala Gly Gly Val Asn Met Leu Leu Ser Pro Asp Thr Met Arg Ala225 230 235 240Leu Ala Arg Thr Gln Ala Leu Ser Pro Asn Gly Arg Cys Gln Thr Phe245 250 255Asp Ala Ser Ala Asn Gly Phe Val Arg Gly Glu Gly Cys Gly Leu Ile260 265 270Val Leu Lys Arg Leu Ser Asp Ala Arg Arg Asp Gly Asp Arg Ile Trp275 280 285Ala Leu Ile Arg Gly Ser Ala Ile Asn Gln Asp Gly Arg Ser Thr Gly290 295 300Leu Thr Ala Pro Asn Val Leu Ala Gln Gly Ala Leu Leu Arg Glu Ala305 310 315 320Leu Arg Asn Ala Gly Val Glu Ala Glu Ala Ile Gly Tyr Ile Glu Thr325 330 335His Gly Ala Ala Thr Ser Leu Gly Asp Pro Ile Glu Ile Glu Ala Leu340 345 350Arg Ala Val Val Gly Pro Ala Arg Ala Asp Gly Ala Arg Cys Val Leu355 360 365Gly Ala Val Lys Thr Asn Leu Gly His Leu Glu Gly Ala Ala Gly Val370 375 380Ala Gly Leu Ile Lys Ala Thr Leu Ser Leu His His Glu Arg Ile Pro385 390 395 400Arg Asn Leu Asn Phe Arg Thr Leu Asn Pro Arg Ile Arg Ile Glu Gly405 410 415Thr Ala Leu Ala Leu Ala Thr Glu Pro Val Pro Trp Pro Arg Thr Gly420 425 430Arg Thr Arg Phe Ala Gly Val Ser Ser Phe Gly Met Ser Gly Thr Asn435 440 445Ala His Val Val Leu Glu Glu Ala Pro Ala Val Glu Pro Glu Ala Ala450 455 460Ala Pro Glu Arg Ala Ala Glu Leu Phe Val Leu Ser Ala Lys Ser Ala465 470 475 480Ala Ala Leu Asp Ala Gln Ala Ala Arg Leu Arg Asp His Leu Glu Lys485 490 495His Val Glu Leu Gly Leu Gly Asp Val Ala Phe Ser Leu Ala Thr Thr500 505 510Arg Ser Ala Met Glu His Arg Leu Ala Val Ala Ala Ser Ser Arg Glu515 520 525Ala Leu Arg Gly Ala Leu Ser Ala Ala Ala Gln Gly His Thr Pro Pro530 535 540Gly Ala Val Arg Gly Arg Ala Ser Gly Gly Ser Ala Pro Lys Val Val545 550 555 560Phe Val Phe Pro Gly Gln Gly Ser Gln Trp Val Gly Met Gly Arg Lys565 570 575Leu Met Ala Glu Glu Pro Val Phe Arg Ala Ala Leu Glu Gly Cys Asp580 585 590Arg Ala Ile Glu Ala Glu Ala Gly Trp Ser Leu Leu Gly Glu Leu Ser595 600 605Ala Asp Glu Ala Ala Ser Gln Leu Gly Arg Ile Asp Val Val Gln Pro610 615 620Val Leu Phe Ala Met Glu Val Ala Leu Ser Ala Leu Trp Arg Ser Trp625 630 635 640Gly Val Glu Pro Glu Ala Val Val Gly His Ser Met Gly Glu Val Ala645 650 655Ala Ala His Val Ala Gly Ala Leu Ser Leu Glu Asp Ala Val Ala Ile660 665 670Ile Cys Arg Arg Ser Arg Leu Leu Arg Arg Ile Ser Gly Gln Gly Glu675 680 685Met Ala Leu Val Glu Leu Ser Leu Glu Glu Ala Glu Ala Ala Leu Arg690 695 700Gly His Glu Gly Arg Leu Ser Val Ala Val Ser Asn Ser Pro Arg Ser705 710 715 720Thr Val Leu Ala Gly Glu Pro Ala Ala Leu Ser Glu Val Leu Ala Ala725 730 735Leu Thr Ala Lys Gly Val Phe Trp Arg Gln Val Lys Val Asp Val Ala740 745 750Ser His Ser Pro Gln Val Asp Pro Leu Arg Glu Glu Leu Ile Ala Ala755 760 765Leu Gly Ala Ile Arg Pro Arg Ala Ala Ala Val Pro Met Arg Ser Thr770 775 780Val Thr Gly Gly Val Ile Ala Gly Pro Glu Leu Gly Ala Ser Tyr Trp785 790 795 800Ala Asp Asn Leu Arg Gln Pro Val Arg Phe Ala Ala Ala Ala Gln Ala805 810 815Leu Leu Glu Gly Gly Pro Ala Leu Phe Ile Glu Met Ser Pro His Pro820 825 830Ile Leu Val Pro Pro Leu Asp Glu Ile Gln Thr Ala Ala Glu Gln Gly835 840 845Gly Ala Ala Val Gly Ser Leu Arg Arg Gly Gln Asp Glu Arg Ala Thr850 855 860Leu Leu Glu Ala Leu Gly Thr Leu Trp Ala Ser Gly Tyr Pro Val Ser865 870 875 880Trp Ala Arg Leu Phe Pro Ala Gly Gly Arg Arg Val Pro Leu Pro Thr885 890 895Tyr Pro Trp Gln His Glu Arg Cys Trp Ile Glu Val Glu Pro Asp Ala900 905 910Arg Arg Leu Ala Ala Ala Asp Pro Thr Lys Asp Trp Phe Tyr Arg Thr915 920 925Asp Trp Pro Glu Val Pro Arg Ala Ala Pro Lys Ser Glu Thr Ala His930 935 940Gly Ser Trp Leu Leu Leu Ala Asp Arg Gly Gly Val Gly Glu Ala Val945 950 955 960Ala Ala Ala Leu Ser Thr Arg Gly Leu Ser Cys Thr Val Leu His Ala965 970 975Ser Ala Asp Ala Ser Thr Val Ala Glu Gln Val Ser Glu Ala Ala Ser980 985 990Arg Arg Asn Asp Trp Gln Gly Val Leu Tyr Leu Trp Gly Leu Asp Ala99510001005Val Val Asp Ala Gly Ala Ser Ala Asp Glu Val Ser Glu Ala Thr Arg101010151020Arg Ala Thr Ala Pro Val Leu Gly Leu Val Arg Phe Leu Ser Ala Ala1025 103010351040Pro His Pro Pro Arg Phe Trp Val Val Thr Arg Gly Ala Cys Thr Val104510501055Gly Gly Glu Pro Glu Ala Ser Leu Cys Gln Ala Ala Leu Trp Gly Leu106010651070Ala Arg Val Ala Ala Leu Glu His Pro Ala Ala Trp Gly Gly Leu Val107510801085Asp Leu Asp Pro Gln Lys Ser Pro Thr Glu Ile Glu Pro Leu Val Ala109010951100Glu Leu Leu Ser Pro Asp Ala Glu Asp Gln Leu Ala Phe Arg Ser Gly1105 111011151120Arg Arg His Ala Ala Arg Leu Val Ala Ala Pro Pro Glu Gly Asp Val112511301135Ala Pro Ile Ser Leu Ser Ala Glu Gly Ser Tyr Leu Val Thr Gly Gly114011451150Leu Gly Gly Leu Gly Leu Leu Val Ala Arg Trp Leu Val Glu Arg Gly115511601165Ala Arg His Leu Val Leu Thr Ser Arg His Gly Leu Pro Glu Arg Gln117011751180Ala Ser Gly Gly Glu Gln Pro Pro Glu Ala Arg Ala Arg Ile Ala Ala1185 119011951200Val Glu Gly Leu Glu Ala Gln Gly Ala Arg Val Thr Val Ala Ala Val120512101215Asp Val Ala Glu Ala Asp Pro Met Thr Ala Leu Leu Ala Ala Ile Glu122012251230Pro Pro Leu Arg Gly Val Val His Ala Ala Gly Val Phe Pro Val Arg123512401245His Leu Ala Glu Thr Asp Glu Ala Leu Leu Glu Ser Val Leu Arg Pro125012551260Lys Val Ala Gly Ser Trp Leu Leu His Arg Leu Leu Arg Asp Arg Pro1265 127012751280Leu Asp Leu Phe Val Leu Phe Ser Ser Gly Ala Ala Val Trp Gly Gly128512901295Lys Gly Gln Gly Ala Tyr Ala Ala Ala Asn Ala Phe Leu Asp Gly Leu130013051310Ala His His Arg Arg Ala His Ser Leu Pro Ala Leu Ser Leu Ala Trp131513201325Gly Leu Trp Ala Glu Gly Gly Met Val Asp Ala Lys Ala His Ala Arg133013351340Leu Ser Asp Ile Gly Val Leu Pro Met Ala Thr Gly Pro Ala Leu Ser1345 135013551360Ala Leu Glu Arg Leu Val Asn Thr Ser Ala Val Gln Arg Ser Val Thr136513701375Arg Met Asp Trp Ala Arg Phe Ala Pro Val Tyr Ala Ala Arg Gly Arg138013851390Arg Asn Leu Leu Ser Ala Leu Val Ala Glu Asp Glu Arg Ala Ala Ser139514001405Pro Pro Val Pro Thr Ala Asn Arg Ile Trp Arg Gly Leu Ser Val Ala141014151420Glu Ser Arg Ser Ala Leu Tyr Glu Leu Val Arg Gly Ile Val Ala Arg1425 143014351440Val Leu Gly Phe Ser Asp Pro Gly Ala Leu Asp Val Gly Arg Gly Phe1445 14501455Ala Glu Gln Gly Leu Asp Ser Leu Met Ala Leu Glu Ile Arg Asn Arg146014651470Leu Gln Arg Glu Leu Gly Glu Arg Leu Ser Ala Thr Leu Ala Phe Asp147514801485His Pro Thr Val Glu Arg Leu Val Ala His Leu Leu Thr Asp Val Leu149014951500Lys Leu Glu Asp Arg Ser Asp Thr Arg His Ile Arg Ser Val Ala Ala1505 151015151520Asp Asp Asp Ile Ala Ile Val Gly Ala Ala Cys Arg Phe Pro Gly Gly152515301535Asp Glu Gly Leu Glu Thr Tyr Trp Arg His Leu Ala Glu Gly Met Val154015451550Val Ser Thr Glu Val Pro Ala Asp Arg Trp Arg Ala Ala Asp Trp Tyr155515601565Asp Pro Asp Pro Glu Val Pro Gly Arg Thr Tyr Val Ala Lys Gly Ala157015751580Phe Leu Arg Asp Val Arg Ser Leu Asp Ala Ala Phe Phe Ala Ile Ser1585 159015951600Pro Arg Glu Ala Met Ser Leu Asp Pro Gln Gln Arg Leu Leu Leu Glu160516101615Val Ser Trp Glu Ala Ile Glu Arg Ala Gly Gln Asp Pro Met Ala Leu162016251630Arg Glu Ser Ala Thr Gly Val Phe Val Gly Met Ile Gly Ser Glu His163516401645Ala Glu Arg Val Gln Gly Leu Asp Asp Asp Ala Ala Leu Leu Tyr Gly165016551660Thr Thr Gly Asn Leu Leu Ser Val Ala Ala Gly Arg Leu Ser Phe Phe1665 167016751680Leu Gly Leu His Gly Pro Thr Met Thr Val Asp Thr Ala Cys Ser Ser168516901695Ser Leu Val Ala Leu His Leu Ala Cys Gln Ser Leu Arg Leu Gly Glu170017051710Cys Asp Gln Ala Leu Ala Gly Gly Ser Ser Val Leu Leu Ser Pro Arg171517201725Ser Phe Val Ala Ala Ser Arg Met Arg Leu Leu Ser Pro Asp Gly Arg173017351740Cys Lys Thr Phe Ser Ala Ala Ala Asp Gly Phe Ala Arg Ala Glu Gly1745 175017551760Cys Ala Val Val Val Leu Lys Arg Leu Arg Asp Ala Gln Arg Asp Arg176517701775Asp Pro Ile Leu Ala Val Val Arg Ser Thr Ala Ile Asn His Asp Gly178017851790Pro Ser Ser Gly Leu Thr Val Pro Ser Gly Pro Ala Gln Gln Ala Leu179518001805Leu Arg Gln Ala Leu Ala Gln Ala Gly Val Ala Pro Ala Glu Val Asp181018151820Phe Val Glu Cys His Gly Thr Gly Thr Ala Leu Gly Asp Pro Ile Glu1825 183018351840Val Gln Ala Leu Gly Ala Val Tyr Gly Arg Gly Arg Pro Ala Glu Arg184518501855Pro Leu Trp Leu Gly Ala Val Lys Ala Asn Leu Gly His Leu Glu Ala186018651870Ala Ala Gly Leu Ala Gly Val Leu Lys Val Leu Leu Ala Leu Glu His187518801885Glu Gln Ile Pro Ala Gln Pro Glu Leu Asp Glu Leu Asn Pro His Ile189018951900Pro Trp Ala Glu Leu Pro Val Ala Val Val Arg Arg Ala Val Pro Trp1905 191019151920Pro Arg Gly Ala Arg Pro Arg Arg Ala Gly Val Ser Ala Phe Gly Leu192519301935Ser Gly Thr Asn Ala His Val Val Leu Glu Glu Ala Pro Ala Val Glu194019451950Pro Val Ala Ala Ala Pro Glu Arg Ala Ala Glu Leu Phe Val Leu Ser195519601965Ala Lys Ser Ala Ala Ala Leu Asp Ala Gln Ala Ala Arg Leu Arg Asp197019751980His Leu Glu Lys His Val Glu Leu Gly Leu Gly Asp Val Ala Phe Ser1985 199019952000Leu Ala Thr Thr Arg Ser Ala Met Glu His Arg Leu Ala Val Ala Ala200520102015Ser Ser Arg Glu Ala Leu Arg Gly Ala Leu Ser Ala Ala Ala Gln Gly202020252030His Thr Pro Pro Gly Ala Val Arg Gly Arg Ala Ser Gly Gly Ser Ala203520402045Pro Lys Val Val Phe Val Phe Pro Gly Gln Gly Ser Gln Trp Val Gly205020552060Met Gly Arg Lys Leu Met Ala Glu Glu Pro Val Phe Arg Ala Ala Leu2065 207020752080Glu Gly Cys Asp Arg Ala Ile Glu Ala Glu Ala Gly Trp Ser Leu Leu208520902095Gly Glu Leu Ser Ala Asp Glu Ala Ala Ser Gln Leu Gly Arg Ile Asp210021052110Val Val Gln Pro Val Leu Phe Ala Met Glu Val Ala Leu Ser Ala Leu211521202125Trp Arg Ser Trp Gly Val Glu Pro Glu Ala Val Val Gly His Ser Met213021352140Gly Glu Val Ala Ala Ala His Val Ala Gly Ala Leu Ser Leu Glu Asp2145 215021552160Ala Val Ala Ile Ile Cys Arg Arg Ser Arg Leu Leu Arg Arg Ile Ser216521702175Gly Gln Gly Glu Met Ala Leu Val Glu Leu Ser Leu Glu Glu Ala Glu218021852190Ala Ala Leu Arg Gly His Glu Gly Arg Leu Ser Val Ala Val Ser Asn219522002205Ser Pro Arg Ser Thr Val Leu Ala Gly Glu Pro Ala Ala Leu Ser Glu221022152220Val Leu Ala Ala Leu Thr Ala Lys Gly Val Phe Trp Arg Gln Val Lys2225 223022352240Val Asp Val Ala Ser His Ser Pro Gln Val Asp Pro Leu Arg Glu Glu224522502255Leu Ile Ala Ala Leu Gly Ala Ile Arg Pro Arg Ala Ala Ala Val Pro226022652270Met Arg Ser Thr Val Thr Gly Gly Val Ile Ala Gly Pro Glu Leu Gly227522802285Ala Ser Tyr Trp Ala Asp Asn Leu Arg Gln Pro Val Arg Phe Ala Ala229022952300Ala Ala Gln Ala Leu Leu Glu Gly Gly Pro Ala Leu Phe Ile Glu Met2305 231023152320Ser Pro His Pro Ile Leu Val Pro Pro Leu Asp Glu Ile Gln Thr Ala232523302335Ala Glu Gln Gly Gly Ala Ala Val Gly Ser Leu Arg Arg Gly Gln Asp234023452350Glu Arg Ala Thr Leu Leu Glu Ala Leu Gly Thr Leu Trp Ala Ser Gly235523602365Tyr Pro Val Ser Trp Ala Arg Leu Phe Pro Ala Gly Gly Arg Arg Val237023752380Pro Leu Pro Thr Tyr Pro Trp Gln His Glu Arg Tyr Trp Ile Glu Asp2385 239023952400Ser Val His Gly Ser Lys Pro Ser Leu Arg Leu Arg Gln Leu Arg Asn240524102415Gly Ala Thr Asp His Pro Leu Leu Gly Ala Pro Leu Leu Val Ser Ala242024252430Arg Pro Gly Ala His Leu Trp Glu Gln Ala Leu Ser Asp Glu Arg Leu243524402445Ser Tyr Leu Ser Glu His Arg Val His Gly Glu Ala Val Leu Pro Ser245024552460Ala Ala Tyr Val Glu Met Ala Leu Ala Ala Gly Val Asp Leu Tyr Gly2465 247024752480Thr Ala Thr Leu Val Leu Glu Gln Leu Ala Leu Glu Arg Ala Leu Ala248524902495Val Pro Ser Glu Gly Gly Arg Ile Val Gln Val Ala Leu Ser Glu Glu250025052510Gly Pro Gly Arg Ala Ser Phe Gln Val Ser Ser Arg Glu Glu Ala Gly251525202525Arg Ser Trp Val Arg His Ala Thr Gly His Val Cys Ser Gly Gln Ser2530 2535 2540Ser Ala Val Gly Ala Leu Lys Glu Ala Pro Trp Glu Ile Gln Arg Arg2545 255025552560Cys Pro Ser Val Leu Ser Ser Glu Ala Leu Tyr Pro Leu Leu Asn Glu256525702575His Ala Leu Asp Tyr Gly Pro Cys Phe Gln Gly Val Glu Gln Val Trp258025852590Leu Gly Thr Gly Glu Val Leu Gly Arg Val Arg Leu Pro Gly Asp Met259526002605Ala Ser Ser Ser Gly Ala Tyr Arg Ile His Pro Ala Leu Leu Asp Ala261026152620Cys Phe Gln Val Leu Thr Ala Leu Leu Thr Thr Pro Glu Ser Ile Glu2625 263026352640Ile Arg Arg Arg Leu Thr Asp Leu His Glu Pro Asp Leu Pro Arg Ser264526502655Arg Ala Pro Val Asn Gln Ala Val Ser Asp Thr Trp Leu Trp Asp Ala266026652670Ala Leu Asp Gly Gly Arg Arg Gln Ser Ala Ser Val Pro Val Asp Leu267526802685Val Leu Gly Ser Phe His Ala Lys Trp Glu Val Met Glu Arg Leu Ala269026952700Gln Ala Tyr Ile Ile Gly Thr Leu Arg Ile Trp Asn Val Phe Cys Ala2705 271027152720Ala Gly Glu Arg His Thr Ile Asp Glu Leu Leu Val Arg Leu Gln Ile272527302735Ser Val Val Tyr Arg Lys Val Ile Lys Arg Trp Met Glu His Leu Val274027452750Ala Ile Gly Ile Leu Val Gly Asp Gly Glu His Phe Val Ser Ser Gln275527602765Pro Leu Pro Glu Pro Asp Leu Ala Ala Val Leu Glu Glu Ala Gly Arg277027752780Val Phe Ala Asp Leu Pro Val Leu Phe Glu Trp Cys Lys Phe Ala Gly2785 279027952800Glu Arg Leu Ala Asp Val Leu Thr Gly Lys Thr Leu Ala Leu Glu Ile280528102815Leu Phe Pro Gly Gly Ser Phe Asp Met Ala Glu Arg Ile Tyr Arg Asp282028252830Ser Pro Ile Ala Arg Tyr Ser Asn Gly Ile Val Arg Gly Val Val Glu283528402845Ser Ala Ala Arg Val Val Ala Pro Ser Gly Met Phe Ser Ile Leu Glu285028552860Ile Gly Ala Gly Thr Gly Ala Thr Thr Ala Ala Val Leu Pro Val Leu2865 287028752880Leu Pro Asp Arg Thr Glu Tyr His Phe Thr Asp Val Ser Pro Leu Phe288528902895Leu Ala Arg Ala Glu Gln Arg Phe Arg Asp Tyr Pro Phe Leu Lys Tyr290029052910Gly Ile Leu Asp Val Asp Gln Glu Pro Ala Gly Gln Gly Tyr Ala His291529202925Gln Arg Phe Asp Val Ile Val Ala Ala Asn Val Ile His Ala Thr Arg293029352940Asp Ile Arg Ala Thr Ala Lys Arg Leu Leu Ser Leu Leu Ala Pro Gly2945 295029552960Gly Leu Leu Val Leu Val Glu Gly Thr Gly His Pro Ile Trp Phe Asp296529702975Ile Thr Thr Gly Leu Ile Glu Gly Trp Gln Lys Tyr Glu Asp Asp Leu298029852990Arg Ile Asp His Pro Leu Leu Pro Ala Arg Thr Trp Cys Asp Val Leu299530003005Arg Arg Val Gly Phe Ala Asp Ala Val Ser Leu Pro Gly Asp Gly Ser301030153020Pro Ala Gly Ile Leu Gly Gln His Val Ile Leu Ser Arg Ala Pro Gly3025 303030353040Ile Ala Gly Ala Ala Cys Asp Ser Ser Gly Glu Ser Ala Thr Glu Ser304530503055Pro Ala Ala Arg Ala Val Arg Gln Glu Trp Ala Asp Gly Ser Ala Asp306030653070Val Val His Arg Met Ala Leu Glu Arg Met Tyr Phe His Arg Arg Pro307530803085Gly Arg Gln Val Trp Val His Gly Arg Leu Arg Thr Gly Gly Gly Ala309030953100Phe Thr Lys Ala Leu Ala Gly Asp Leu Leu Leu Phe Glu Asp Thr Gly3105 311031153120Gln Val Val Ala Glu Val Gln Gly Leu Arg Leu Pro Gln Leu Glu Ala312531303135Ser Ala Phe Ala Pro Arg Asp Pro Arg Glu Glu Trp Leu Tyr Ala Leu314031453150Glu Trp Gln Arg Lys Asp Pro Ile Pro Glu Ala Pro Ala Ala Ala Ser315531603165Ser Ser Ser Ala Gly Ala Trp Leu Val Leu Met Asp Gln Gly Gly Thr317031753180Gly Ala Ala Leu Val Ser Leu Leu Glu Gly Arg Gly Glu Ala Cys Val3185 319031953200Arg Val Ile Ala Gly Thr Ala Tyr Ala Cys Leu Ala Pro Gly Leu Tyr320532103215Gln Val Asp Pro Ala Gln Pro Asp Gly Phe His Thr Leu Leu Arg Asp322032253230Ala Phe Gly Glu Asp Arg Ile Cys Arg Ala Val Val His Met Trp Ser323532403245Leu Asp Ala Thr Ala Ala Gly Glu Arg Ala Thr Ala Glu Ser Leu Gln325032553260Ala Asp Gln Leu Leu Gly Ser Leu Ser Ala Leu Ser Leu Val Gln Ala3265 327032753280Leu Val Arg Arg Arg Trp Arg Asn Met Pro Arg Leu Trp Leu Leu Thr328532903295Arg Ala Val His Ala Val Gly Ala Glu Asp Ala Ala Ala Ser Val Ala330033053310Gln Ala Pro Val Trp Gly Leu Gly Arg Thr Leu Ala Leu Glu His Pro331533203325Glu Leu Arg Cys Thr Leu Val Asp Val Asn Pro Ala Pro Ser Pro Glu333033353340Asp Ala Ala Ala Leu Ala Val Glu Leu Gly Ala Ser Asp Arg Glu Asp3345 335033553360Gln Val Ala Leu Arg Ser Asp Gly Arg Tyr Val Ala Arg Leu Val Arg336533703375Ser Ser Phe Ser Gly Lys Pro Ala Thr Asp Cys Gly Ile Arg Ala Asp338033853390Gly Ser Tyr Val Ile Thr Asp Gly Met Gly Arg Val Gly Leu Ser Val339534003405Ala Gln Trp Met Val Met Gln Gly Ala Arg His Val Val Leu Val Asp341034153420Arg Gly Gly Ala Ser Glu Ala Ser Arg Asp Ala Leu Arg Ser Met Ala3425 343034353440Glu Ala Gly Ala Glu Val Gln Ile Val Glu Ala Asp Val Ala Arg Arg344534503455Asp Asp Val Ala Arg Leu Leu Ser Lys Ile Glu Pro Ser Met Pro Pro346034653470Leu Arg Gly Ile Val Tyr Val Asp Gly Thr Phe Gln Gly Asp Ser Ser347534803485Met Leu Glu Leu Asp Ala Arg Arg Phe Lys Glu Trp Met Tyr Pro Lys349034953500Val Leu Gly Ala Trp Asn Leu His Ala Leu Thr Arg Asp Arg Ser Leu3505 351035153520Asp Phe Phe Val Leu Tyr Ser Ser Gly Thr Ser Leu Leu Gly Leu Pro352535303535Gly Gln Gly Ser Arg Ala Ala Gly Asp Ala Phe Leu Asp Ala Ile Ala354035453550His His Arg Cys Lys Val Gly Leu Thr Ala Met Ser Ile Asn Trp Gly355535603565Leu Leu Ser Glu Ala Ser Ser Pro Ala Thr Pro Asn Asp Gly Gly Ala357035753580Arg Leu Glu Tyr Arg Gly Met Glu Gly Leu Thr Leu Glu Gln Gly Ala3585 359035953600Ala Ala Leu Gly Arg Leu Leu Ala Arg Pro Arg Ala Gln Val Gly Val360536103615Met Arg Leu Asn Leu Arg Gln Trp Leu Glu Phe Tyr Pro Asn Ala Ala362036253630Arg Leu Ala Leu Trp Ala Glu Leu Leu Lys Glu Arg Asp Arg Ala Asp363536403645Arg Gly Ala Ser Asn Ala Ser Asn Leu Arg Glu Ala Leu Gln Ser Ala365036553660Arg Pro Glu Asp Arg Gln Leu Ile Leu Glu Lys His Leu Ser Glu Leu3665 367036753680Leu Gly Arg Gly Leu Arg Leu Pro Pro Glu Arg Ile Glu Arg His Val368536903695Pro Phe Ser Asn Leu Gly Met Asp Ser Leu Ile Gly Leu Glu Leu Arg370037053710Asn Arg Ile Glu Ala Ala Leu Gly Ile Thr Val Pro Ala Thr Leu Leu371537203725Trp Thr Tyr Pro Asn Val Ala Ala Leu Ser Gly Ser Leu Leu Asp Ile373037353740Leu Phe Pro Asn Ala Gly Ala Thr His Ala Pro Ala Thr Glu Arg Glu3745 375037553760Lys Ser Phe Glu Asn Asp Ala Ala Asp Leu Glu Ala Leu Arg Gly Met376537703775Thr Asp Glu Gln Lys Asp Ala Leu Leu Ala Glu Lys Leu Ala Gln Leu378037853790Ala Gln Ile Val Gly Glu3795<210>7<211>2439<212>PRT<213>纤维堆囊菌<400>7Met Ala Thr Thr Asn Ala Gly Lys Leu Glu His Ala Leu Leu Leu Met1 5 10 15Asp Lys Leu Ala Lys Lys Asn Ala Ser Leu Glu Gln Glu Arg Thr Glu20 25 30Pro Ile Ala Ile Val Gly Ile Gly Cys Arg Phe Pro Gly Gly Ala Asp35 40 45Thr Pro Glu Ala Phe Trp Glu Leu Leu Asp Ser Gly Arg Asp Ala Val50 55 60Gln Pro Leu Asp Arg Arg Trp Ala Leu Val Gly Val His Pro Ser Glu65 70 75 80Glu Val Pro Arg Trp Ala Gly Leu Leu Thr Glu Ala Val Asp Gly Phe85 90 95Asp Ala Ala Phe Phe Gly Thr Ser Pro Arg Glu Ala Arg Ser Leu Asp100 105 110Pro Gln Gln Arg Leu Leu Leu Glu Val Thr Trp Glu Gly Leu Glu Asp115 120 125Ala Gly Ile Ala Pro Gln Ser Leu Asp Gly Ser Arg Thr Gly Val Phe130 135 140Leu Gly Ala Cys Ser Ser Asp Tyr Ser His Thr Val Ala Gln Gln Arg145 150 155 160Arg Glu Glu Gln Asp Ala Tyr Asp Ile Thr Gly Asn Thr Leu Ser Val165 170 175Ala Ala Gly Arg Leu Ser Tyr Thr Leu Gly Leu Gln Gly Pro Cys Leu180 185 190Thr Val Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Ile His Leu Ala195 200 205Cys Arg Ser Leu Arg Ala Arg Glu Ser Asp Leu Ala Leu Ala Gly Gly210 215 220Val Asn Met Leu Leu Ser Ser Lys Thr Met Ile Met Leu Gly Arg Ile225 230 235 240Gln Ala Leu Ser Pro Asp Gly His Cys Arg Thr Phe Asp Ala Ser Ala245 250 255Asn Gly Phe Val Arg Gly Glu Gly Cys Gly Met Val Val Leu Lys Arg260 265 270Leu Ser Asp Ala Gln Arg His Gly Asp Arg Ile Trp Ala Leu Ile Arg275 280 285Gly Ser Ala Met Asn Gln Asp Gly Arg Ser Thr Gly Leu Met Ala Pro290 295 300Asn Val Leu Ala Gln Glu Ala Leu Leu Arg Glu Ala Leu Gln Ser Ala305 310 315 320Arg Val Asp Ala Gly Ala Ile Gly Tyr Val Glu Thr His Gly Thr Gly325 330 335Thr Ser Leu Gly Asp Pro Ile Glu Val Glu Ala Leu Arg Ala Val Leu340 345 350Gly Pro Ala Arg Ala Asp Gly Ser Arg Cys Val Leu Gly Ala Val Lys355 360 365Thr Asn Leu Gly His Leu Glu Gly Ala Ala Gly Val Ala Gly Leu Ile370 375 380Lys Ala Ala Leu Ala Leu His His Glu Leu Ile Pro Arg Asn Leu His385 390 395 400Phe His Thr Leu Asn Pro Arg Ile Arg Ile Glu Gly Thr Ala Leu Ala405 410 415Leu Ala Thr Glu Pro Val Pro Trp Pro Arg Ala Gly Arg Pro Arg Phe420 425 430Ala Gly Val Ser Ala Phe Gly Leu Ser Gly Thr Asn Val His Val Val435 440 445Leu Glu Glu Ala Pro Ala Thr Val Leu Ala Pro Ala Thr Pro Gly Arg450 455 460Ser Ala Glu Leu Leu Val Leu Ser Ala Lys Ser Ala Ala Ala Leu Asp465 470 475 480Ala Gln Ala Ala Arg Leu Ser Ala His Ile Ala Ala Tyr Pro Glu Gln485 490 495Gly Leu Gly Asp Val Ala Phe Ser Leu Val Ser Thr Arg Ser Pro Met500 505 510Glu His Arg Leu Ala Val Ala Ala Thr Ser Arg Glu Ala Leu Arg Ser515 520 525Ala Leu Glu Val Ala Ala Gln Gly Gln Thr Pro Ala Gly Ala Ala Arg530 535 540Gly Arg Ala Ala Ser Ser Pro Gly Lys Leu Ala Phe Leu Phe Ala Gly545 550 555 560Gln Gly Ala Gln Val Pro Gly Met Gly Arg Gly Leu Trp Glu Ala Trp565 570 575Pro Ala Phe Arg Glu Thr Phe Asp Arg Cys Val Thr Leu Phe Asp Arg580 585 590Glu Leu His Gln Pro Leu Cys Glu Val Met Trp Ala Glu Pro Gly Ser595 600 605Ser Arg Ser Ser Leu Leu Asp Gln Thr Ala Phe Thr Gln Pro Ala Leu610615 620Phe Ala Leu Glu Tyr Ala Leu Ala Ala Leu Phe Arg Ser Trp Gly Val625 630 635 640Glu Pro Glu Leu Val Ala Gly His Ser Leu Gly Glu Leu Val Ala Ala645 650 655Cys Val Ala Gly Val Phe Ser Leu Glu Asp Ala Val Arg Leu Val Val660 665 670Ala Arg Gly Arg Leu Met Gln Ala Leu Pro Ala Gly Gly Ala Met Val675 680 685Ser Ile Ala Ala Pro Glu Ala Asp Val Ala Ala Ala Val Ala Pro His690 695 700Ala Ala Leu Val Ser Ile Ala Ala Val Asn Gly Pro Glu Gln Val Val705 710 715 720Ile Ala Gly Ala Glu Lys Phe Val Gln Gln Ile Ala Ala Ala Phe Ala725 730 735Ala Arg Gly Ala Arg Thr Lys Pro Leu His Val Ser His Ala Phe His740 745 750Ser Pro Leu Met Asp Pro Met Leu Glu Ala Phe Arg Arg Val Thr Glu755 760 765Ser Val Thr Tyr Arg Arg Pro Ser Ile Ala Leu Val Ser Asn Leu Ser770 775 780Gly Lys Pro Cys Thr Asp Glu Val Ser Ala Pro Gly Tyr Trp Val Arg785 790 795 800His Ala Arg Glu Ala Val Arg Phe Ala Asp Gly Val Lys Ala Leu His805 810 815Ala Ala Gly Ala Gly Leu Phe Val Glu Val Gly Pro Lys Pro Thr Leu820 825 830Leu Gly Leu Val Pro Ala Cys Leu Pro Asp Ala Arg Pro Val Leu Leu835 840 845Pro Ala Ser Arg Ala Gly Arg Asp Glu Ala Ala Ser Ala Leu Glu Ala850 855 860Leu Gly Gly Phe Trp Val Val Gly Gly Ser Val Thr Trp Ser Gly Val865 870 875 880Phe Pro Ser Gly Gly Arg Arg Val Pro Leu Pro Thr Tyr Pro Trp Gln885 890 895Arg Glu Arg Tyr Trp Ile Glu Ala Pro Val Asp Arg Glu Ala Asp Gly900 905 910Thr Gly Arg Ala Arg Ala Gly Gly His Pro Leu Leu Gly Glu Val Phe915 920 925Ser Val Ser Thr His Ala Gly Leu Arg Leu Trp Glu Thr Thr Leu Asp930 935 940Arg Lys Arg Leu Pro Trp Leu Gly Glu His Arg Ala Gln Gly Glu Val945 950 955 960Val Phe Pro Gly Ala Gly Tyr Leu Glu Met Ala Leu Ser Ser Gly Ala965 970 975Glu Ile Leu Gly Asp Gly Pro Ile Gln Val Thr Asp Val Val Leu Ile980 985 990Glu Thr Leu Thr Phe Ala Gly Asp Thr Ala Val Pro Val Gln Val Val99510001005Thr Thr Glu Glu Arg Pro Gly Arg Leu Arg Phe Gln Val Ala Ser Arg101010151020Glu Pro Gly Glu Arg Arg Ala Pro Phe Arg Ile His Ala Arg Gly Val1025 103010351040Leu Arg Arg Ile Gly Arg Val Glu Thr Pro Ala Arg Ser Asn Leu Ala104510501055Ala Leu Arg Ala Arg Leu His Ala Ala Val Pro Ala Ala Ala Ile Tyr106010651070Gly Ala Leu Ala Glu Met Gly Leu Gln Tyr Gly Pro Ala Leu Arg Gly107510801085Leu Ala Glu Leu Trp Arg Gly Glu Gly Glu Ala Leu Gly Arg Val Arg109010951100Leu Pro Glu Ala Ala Gly Ser Ala Thr Ala Tyr Gln Leu His Pro Val1105 111011151120Leu Leu Asp Ala Cys Val Gln Met Ile Val GlyAla Phe Ala Asp Arg11251130 1135Asp Glu Ala Thr Pro Trp Ala Pro Val Glu Val Gly Ser Val Arg Leu114011451150Phe Gln Arg Ser Pro Gly Glu Leu Trp Cys His Ala Arg Val Val Ser115511601165Asp Gly Gln Gln Ala Ser Ser Arg Trp Ser Ala Asp Phe Glu Leu Met117011751180Asp Gly Thr Gly Ala Val Val Ala Glu Ile Ser Arg Leu Val Val Glu1185 119011951200Arg Leu Ala Ser Gly Val Arg Arg Arg Asp Ala Asp Asp Trp Phe Leu120512101215Glu Leu Asp Trp Glu Pro Ala Ala Leu Gly Gly Pro Lys Ile Thr Ala122012251230Gly Arg Trp Leu Leu Leu Gly Glu Gly Gly Gly Leu Gly Arg Ser Leu123512401245Cys Ser Ala Leu Lys Ala Ala Gly His Val Val Val His Ala Ala Gly125012551260Asp Asp Thr Ser Thr Ala Gly Met Arg Ala Leu Leu Ala Asn Ala Phe1265 127012751280Asp Gly Gln Ala Pro Thr Ala Val Val His Leu Ser Ser Leu Asp Gly128512901295Gly Gly Gln Leu Gly Pro Gly Leu Gly Ala Gln Gly Ala Leu Asp Ala130013051310Pro Arg Ser Pro Asp Val Asp Ala Asp Ala Leu Glu Ser Ala Leu Met131513201325Arg Gly Cys Asp Ser Val Leu Ser Leu Val Gln Ala Leu Val Gly Met133013351340Asp Leu Arg Asn Ala Pro Arg Leu Trp Leu Leu Thr Arg Gly Ala Gln1345 1350 1355 1360Ala Ala Ala Ala Gly Asp Val Ser Val Val Gln Ala Pro Leu Leu Gly136513701375Leu Gly Arg Thr Ile Ala Leu Glu His Ala Glu Leu Arg Cys Ile Ser138013851390Val Asp Leu Asp Pro Ala Glu Pro Glu Gly Glu Ala Asp Ala Leu Leu139514001405Ala Glu Leu Leu Ala Asp Asp Ala Glu Glu Glu Val Ala Leu Arg Gly141014151420Gly Asp Arg Leu Val Ala Arg Leu Val His Arg Leu Pro Asp Ala Gln1425 143014351440Arg Arg Glu Lys Val Glu Pro Ala Gly Asp Arg Pro Phe Arg Leu Glu144514501455Ile Asp Glu Pro Gly Ala Leu Asp Gln Leu Val Leu Arg Ala Thr Gly146014651470Arg Arg Ala Pro Gly Pro Gly Glu Val Glu Ile Ser Val Glu Ala Ala147514801485Gly Leu Asp Ser Ile Asp Ile Gln Leu Ala Leu Gly Val Ala Pro Asn149014951500Asp Leu Pro Gly Glu Glu Ile Glu Pro Leu Val Leu Gly Ser Glu Cys1505 151015151520Ala Gly Arg Ile Val Ala Val Gly Glu Gly Val Asn Gly Leu Val Val152515301535Gly Gln Pro Val Ile Ala Leu Ala Ala Gly Val Phe Ala Thr His Val154015451550Thr Thr Ser Ala Thr Leu Val Leu Pro Arg Pro Leu Gly Leu Ser Ala155515601565Thr Glu Ala Ala Ala Met Pro Leu Ala Tyr Leu Thr Ala Trp Tyr Ala157015751580Leu Asp Lys Val Ala His Leu Gln Ala Gly Glu Arg Val Leu Ile His1585 159015951600Ala Glu Ala Gly Gly Val Gly Leu Cys Ala Val Arg Trp Ala Gln Arg160516101615Val Gly Ala Glu Val Tyr Ala Thr Ala Asp Thr Pro Glu Asn Arg Ala162016251630Tyr Leu Glu Ser Leu Gly Val Arg Tyr Val Ser Asp Ser Arg Ser Gly163516401645Arg Phe Val Thr Asp Val His Ala Trp Thr Asp Gly Glu Gly Val Asp165016551660Val Val Leu Asp Ser Leu Ser Gly Glu Arg Ile Asp Lys Ser Leu Met1665 167016751680Val Leu Arg Ala Cys Gly Arg Leu Val Lys Leu Gly Arg Arg Asp Asp168516901695Cys Ala Asp Thr Gln Pro Gly Leu Pro Pro Leu Leu Arg Asn Phe Ser170017051710Phe Ser Gln Val Asp Leu Arg Gly Met Met Leu Asp Gln Pro Ala Arg171517201725Ile Arg Ala Leu Leu Asp Glu Leu Phe Gly Leu Val Ala Ala Gly Ala173017351740Ile Ser Pro Leu Gly Ser Gly Leu Arg Val Gly Gly Ser Leu Thr Pro1745 175017551760Pro Pro Val Glu Thr Phe Pro Ile Ser Arg Ala Ala Glu Ala Phe Arg176517701775Arg Met Ala Gln Gly Gln His Leu Gly Lys Leu Va1 Leu Thr Leu Asp178017851790Asp Pro Glu Val Arg Ile Arg Ala Pro Ala Glu Ser Ser Val Ala Val179518001805Arg Ala Asp Gly Thr Tyr Leu Val Thr Gly Gly Leu Gly Gly Leu Gly181018151820Leu Arg Val Ala Gly Trp Leu Ala Glu Arg Gly Ala Gly Gln Leu Val1825 183018351840Leu val Gly Arg Ser Gly Ala Ala Ser Ala Glu Gln Arg Ala Ala Val184518501855Ala Ala Leu Glu Ala His Gly Ala Arg Val Thr Val Ala Lys Ala Asp186018651870Val Ala Asp Arg Ser Gln Ile Glu Arg Val Leu Arg Glu Val Thr Ala187518801885Ser Gly Met Pro Leu Arg Gly Val Val His Ala Ala Gly Leu Val Asp189018951900Asp Gly Leu Leu Met Gln Gln Thr Pro Ala Arg Phe Arg Thr Val Met1905 191019151920Gly Pro Lys Val Gln Gly Ala Leu His Leu His Thr Leu Thr Arg Glu192519301935Ala Pro Leu Ser Phe Phe Val Leu Tyr Ala Ser Ala Ala Gly Leu Phe194019451950Gly Ser Pro Gly Gln Gly Asn Tyr Ala Ala Ala Asn Ala Phe Leu Asp195519601965Ala Leu Ser His His Arg Arg Ala Gln Gly Leu Pro Ala Leu Ser Ile197019751980Asp Trp Gly Met Phe Thr Glu Val Gly Met Ala Val Ala Gln Glu Asn1985 199019952000Arg Gly Ala Arg Gln Ile Ser Arg Gly Met Arg Gly Ile Thr Pro Asp200520102015Glu Gly Leu Ser Ala Leu Ala Arg Leu Leu Glu Gly Asp Arg Val Gln202020252030Thr Gly Val Ile Pro Ile Thr Pro Arg Gln Trp Val Glu Phe Tyr Pro203520402045Ala Thr Ala Ala Ser Arg Arg Leu Ser Arg Leu Val Thr Thr Gln Arg205020552060Ala Val Ala Asp Arg Thr Ala Gly Asp Arg Asp Leu Leu Glu Gln Leu2065 207020752080Ala Ser Ala Glu Pro Ser Ala Arg Ala Gly Leu Leu Gln Asp Val Val208520902095Arg Val Gln Val Ser His Val Leu Arg Leu Pro Glu Asp Lys Ile Glu210021052110Val Asp Ala Pro Leu Ser Ser Met Gly Met Asp Ser Leu Met Ser Leu211521202125Glu Leu Arg Asn Arg Ile Glu Ala A la Leu Gly Val Ala Ala Pro Ala21302135 2140Ala Leu Gly Trp Thr Tyr Pro Thr Val Ala Ala Ile Thr Arg Trp Leu2145 215021552160Leu Asp Asp Ala Leu Val Val Arg Leu Gly Gly Gly Ser Asp Thr Asp216521702175Glu Ser Thr Ala Ser Ala Gly Ser Phe Val His Val Leu Arg Phe Arg218021852190Pro Val Val Lys Pro Arg Ala Arg Leu Phe Cys Phe His Gly Ser Gly219522002205Gly Ser Pro Glu Gly Phe Arg Ser Trp Ser Glu Lys Ser Glu Trp Ser221022152220Asp Leu Glu Ile Val Ala Met Trp His Asp Arg Ser Leu Ala Ser Glu2225 223022352240Asp Ala Pro Gly Lys Lys Tyr Val Gln Glu Ala Ala Ser Leu Ile Gln224522502255His Tyr Ala Asp Ala Pro Phe Ala Leu Val Gly Phe Ser Leu Gly Val226022652270Arg Phe Val Met Gly Thr Ala Val Glu Leu Ala Ser Arg Ser Gly Ala227522802285Pro Ala Pro Leu Ala Val Phe Thr Leu Gly Gly Ser Leu Ile Ser Ser229022952300Ser Glu Ile Thr Pro Glu Met Glu Thr Asp Ile Ile Ala Lys Leu Phe2305 231023152320Phe Arg Asn Ala Ala Gly Phe Val Arg Ser Thr Gln Gln Val Gln Ala232523302335Asp Ala Arg Ala Asp Lys Val Ile Thr Asp Thr Met Val Ala Pro Ala234023452350Pro Gly Asp Ser Lys Glu Pro Pro Val Lys Ile Ala Val Pro Ile Val235523602365Ala Ile Ala Gly Ser Asp Asp Val Ile Val Pro Pro Ser Asp Val Gln237023752380Asp Leu Gln Ser Arg Thr Thr Glu Arg Phe Tyr Met His Leu Leu Pro2385 239023952400Gly Asp His Glu Phe Leu Val Asp Arg Gly Arg Glu Ile Met His Ile240524102415Val Asp Ser His Leu Asn Pro Leu Leu Ala Ala Arg Thr Thr Ser Ser242024252430Gly Pro Ala Phe Glu Ala Lys2435<210>8<211>419<212>PRT<213>纤维堆囊菌<400>8Met Thr Gln Glu Gln Ala Asn Gln ser Glu Thr Lys Pro Ala Phe Asp1 5 10 15Phe Lys Pro Phe Ala Pro Gly Tyr Ala Glu Asp Pro Phe Pro Ala Ile20 25 30Glu Arg Leu Arg Glu Ala Thr Pro Ile Phe Tyr Trp Asp Glu Gly Arg35 40 45Ser Trp Val Leu Thr Arg Tyr His Asp Val Ser Ala Val Phe Arg Asp50 55 60Glu Arg Phe Ala Val Ser Arg Glu Glu Trp Glu Ser Ser Ala Glu Tyr65 70 75 80Ser Ser Ala Ile Pro Glu Leu Ser Asp Met Lys Lys Tyr Gly Leu Phe85 90 95Gly Leu Pro Pro Glu Asp His Ala Arg Val Arg Lys Leu Val Asn Pro100 105 110Ser Phe Thr Ser Arg Ala Ile Asp Leu Leu Arg Ala Glu Ile Gln Arg115 120 125Thr Val Asp Gln Leu Leu Asp Ala Arg Ser Gly Gln Glu Glu Phe Asp130 135 140Val Val Arg Asp Tyr Ala Glu Gly Ile Pro Met Arg Ala Ile Ser Ala145 150 155 160Leu Leu Lys Val Pro Ala Glu Cys Asp Glu Lys Phe Arg Arg Phe Gly165 170 175Ser Ala Thr Ala Arg Ala Leu Gly Val Gly Leu Val Pro Gln Val Asp180 185 190Glu Glu Thr Lys Thr Leu Val Ala Ser Val Thr Glu Gly Leu Ala Leu195 200 205Leu His Asp Val Leu Asp Glu Arg Arg Arg Asn Pro Leu Glu Asn Asp210 215 220Val Leu Thr Met Leu Leu Gln Ala Glu Ala Asp Gly Ser Arg Leu Ser225 230 235 240Thr Lys Glu Leu Val Ala Leu Val Gly Ala Ile Ile Ala Ala Gly Thr245 250 255Asp Thr Thr Ile Tyr Leu Ile Ala Phe Ala Val Leu Asn Leu Leu Arg260 265 270Ser Pro Glu Ala Leu Glu Leu Val Lys Ala Glu Pro Gly Leu Met Arg275 280 285Asn Ala Leu Asp Glu Val Leu Arg Phe Asp Asn Ile Leu Arg Ile Gly290 295 300Thr Val Arg Phe Ala Arg Gln Asp Leu Glu Tyr Cys Gly Ala Ser Ile305 310 315 320Lys Lys Gly Glu Met Val Phe Leu Leu Ile Pro Ser Ala Leu Arg Asp325 330 335Gly Thr Val Phe Ser Arg Pro Asp Val Phe Asp Val Arg Arg Asp Thr340 345 350Gly Ala Ser Leu Ala Tyr Gly Arg Gly Pro His Val Cys Pro Gly Val355 360 365Ser Leu Ala Arg Leu Glu Ala Glu Ile Ala Val Gly Thr Ile Phe Arg370 375 380Arg Phe Pro Glu Met Lys Leu Lys Glu Thr Pro Val Phe Gly Tyr His385 390 395 400Pro Ala Phe Arg Asn Ile Glu Ser Leu Asn Val Ile Leu Lys Pro Ser405 410 415Lys Ala Gly<210>9<211>607<212>PRT<213>纤维堆囊菌<400>9Ala Ser Leu Asp Ala Leu Phe Ala Arg Ala Thr Ser Ala Arg Val Leu1 5 10 15Asp Asp Gly His Gly Arg Ala Thr Glu Arg His Val Leu Ala Glu Ala20 25 30Arg Gly Ile Glu Asp Leu Arg Ala Leu Arg Glu His Leu Arg Ile Gln35 40 45Glu Gly Gly Pro Ser Phe His Cys Met Cys Leu Gly Asp Leu Thr Val50 55 60Glu Leu Leu Ala His Asp Gln Pro Leu Ala Ser Ile Ser Phe His His65 70 75 80Ala Arg Ser Leu Arg His Pro Asp Trp Thr Ser Asp Ala Met Leu Val85 90 95Asp Gly Pro Ala Leu Val Arg Trp Leu Ala Ala Arg Gly Ala Pro Gly100 105 110Pro Leu Arg Glu Tyr Glu Glu Glu Arg Glu Arg Ala Arg Thr Ala Gln115 120 125Glu Ala Arg Arg Leu Trp Leu Ala Ala Ala Pro Pro Cys Phe Ala Pro130 135 140Asp Leu Pro Arg Phe Glu Asp Asp Ala Asn Gly Leu Pro Leu Gly Pro145 150 155 160Met Ser Pro Glu Val Ala Glu Ala Glu Arg Arg Leu Arg Ala Ser Tyr165 170 175Ala Thr Pro Glu Leu Ala Cys Ala Ala Leu Leu Ala Trp Leu Gly Thr180 185 190Gly Ala Gly Pro Trp Ser Gly Tyr Pro Ala Tyr Glu Met Leu Pro Glu195 200 205Asn Leu Leu Leu Gly Phe Gly Leu Pro Thr Ala Ile Ala Ala Ala Ser210 215 220Ala Pro Gly Thr Ser Glu Ala Ala Leu Arg Gly Ala Ala Arg Leu Phe225 230 235 240Ala Ser Trp Glu Val Val Ser Ser Lys Lys Ser Gln Leu Gly Asn Ile245 250 255Pro Glu Ala Leu Trp Glu Arg Leu Arg Thr Ile Val Arg Ala Met Gly260 265 270Asn Ala Asp Asn Leu Ser Arg Phe Glu Arg Ala Glu Ala Ile Ala Ala275 280 285Glu Val Arg Arg Leu Arg Ala Gln Pro Ala Pro Phe Ala Ala Gly Ala290 295 300Gly Leu Ala Val Ala Gly Val Ser Ser Ser Gly Arg Leu Ser Gly Leu305 310 315 320Val Thr Asp Gly Asp Ala Leu Tyr Ser Gly Asp Gly Asn Asp Ile Val325 330 335Met Phe Gln Pro Gly Arg Ile Ser Pro Val Val Leu Leu Ala Gly Thr340 345 350Asp Pro Phe Phe Glu Leu Ala Pro Pro Leu Ser Gln Met Leu Phe Val355 360 365Ala His Ala Asn Ala Gly Thr Ile Ser Lys Val Leu Thr Glu Gly Ser370 375 380Pro Leu Ile Val Met Ala Arg Asn Gln Ala Arg Pro Met Ser Leu Val385 390 395 400His Ala Arg Gly Phe Met Ala Trp Val Asn Gln Ala Met Val Pro Asp405 410 415Pro Glu Arg Gly Ala Pro Phe Val Val Gln Arg Ser Thr Ile Met Glu420 425 430Phe Glu His Pro Thr Pro Arg Cys Leu His Glu Pro Ala Gly Ser Ala435 440 445Phe Ser Leu Ala Cys Asp Glu Glu His Leu Tyr Trp Cys Glu Leu Ser450 455 460Ala Gly Arg Leu Glu Leu Trp Arg His Pro His His Arg Pro Gly Ala465 470 475 480Pro Ser Arg Phe Ala Tyr Leu Gly Glu His Pro Ile Ala Ala Thr Trp485 490 495Tyr Pro Ser Leu Thr Leu Asn Ala Thr His Val Leu Trp Ala Asp Pro500 505 510Asp Arg Arg Ala Ile Leu Gly Val Asp Lys Arg Thr Gly Val Glu Pro515 520 525Ile Val Leu Ala Glu Thr Arg His Pro Pro Ala His Val Val Ser Glu530 535 540Asp Arg Asp Ile Phe Ala Leu Thr Gly Gln Pro Asp Ser Arg Asp Trp545 550 555 560His Val Glu His Ile Arg Ser Gly Ala Ser Thr Val Val Ala Asp Tyr565 570 575Gln Arg Gln Leu Trp Asp Arg Pro Asp Met Val Leu Asn Arg Arg Gly580 585 590Leu Phe Phe Thr Thr Asn Asp Arg Ile Leu Thr Leu Ala Arg Ser595 600 605<210>l0<211>423<212>PRT<213>纤维堆囊菌<400>10Met Gly Ala Leu Ile Ser Val Ala Ala Pro Gly Cys Ala Leu Gly Gly1 5 10 15Ala Glu Glu Glu Gly Gln Pro Gly Gln Asp Ala Gly Ala Gly Ala Leu20 25 30Ala Pro Ala Arg Glu Val Met Ala Ala Glu Val Ala Ala Gly Gln Met35 40 45Pro Gly Ala Val Trp Leu Val Ala Arg Gly Asp Asp Val His Val Asp50 55 60Ala Val Gly Val Thr Glu Leu Gly Gly Ser Ala Pro Met Arg Arg Asp65 70 75 80Thr Ile Phe Arg Ile Ala Ser Met Thr Lys Ala Val Thr Ala Thr Ala85 90 95Val Met Met Leu Val Glu Glu Gly Lys Leu Asp Leu Asp Ser Pro Val100 105 110Asp Arg Trp Leu Pro Glu Leu Ala Asn Arg Lys Val Leu Ala Arg Ile115 120 125Asp Gly Pro Ile Asp Glu Thr Val Pro Ala Glu Arg Pro Ile Thr Val130 135 140Arg Asp Leu Met Thr Phe Thr Met Gly Phe Gly Ile Ser Phe Asp Ala145 150 155 160Ser Ser Pro Ile Gln Arg Ala Ile Asp Glu Leu Gly Leu Val Asn Ala165 170 175Gln Pro Val Pro Met Thr Pro His Gly Pro Asp Glu Trp Ile Arg Arg180 185 19065 70 75 80Gly Ala Leu Leu Leu Leu Leu Met Ala Gly Ile Glu Val Asp Val Gly85 90 95Ile Leu Arg Lys Glu Ala Arg Pro Gly Ala Leu Ser Ala Leu Gly Ala100 105 110Ile Ala Pro Pro Leu Ala Ala Gly Ala Ala Phe Ser Ala Leu Val Leu115 120 125Asp Arg Pro Leu Pro Ser Gly Leu Phe Leu Gly Ile Val Leu Ser Val130 135 140Thr Ala Val Ser Val Ile Ala Lys Val Leu Ile Glu Arg Glu Ser Met145 150 155 160Arg Arg Ser Tyr Ala Gln Val Thr Leu Ala Ala Gly Val Val Ser Glu165 170 175Val Ala Ala Trp Val Leu Val Ala Met Thr Ser Ser Ser Tyr Gly Ala180 185 190Ser Pro Ala Leu Ala Val Ala Arg Ser Ala Leu Leu Ala Ser Gly Phe195 200 205Leu Leu Phe Met Val Leu Val Gly Arg Arg Leu Thr His Leu Ala Met210 215 220Arg Trp Val Ala Asp Ala Thr Arg Val Ser Lys Gly Gln Val Ser Leu225 230 235 240Val Leu Val Leu Thr Phe Leu Ala Ala Ala Leu Thr Gln Arg Leu Gly245 250 255Leu His Pro Leu Leu Gly Ala Phe Ala Leu Gly Val Leu Leu Asn Ser260 265 270Ala Pro Arg Thr Asn Arg Pro Leu Leu Asp Gly Val Gln Thr Leu Val275 280 285Ala Gly Leu Phe Ala Pro Val Phe Phe Val Leu Ala Gly Met Arg Val290 295 300Asp Val Ser Gln Leu Arg Thr Pro Ala Ala Trp Gly Thr Val Ala Leu305 310 315 320Leu Leu Ala Thr Ala Thr Ala Ala Lys Val Val Pro Ala Ala Leu Gly325 330 335Ala Arg Leu Gly Gly Leu Arg Gly Ser Glu Ala Ala Leu Val Ala Val340 345 350Gly Leu Asn Met Lys Gly Gly Thr Asp Leu Ile Val Ala Ile Val Gly355 360 365Val Glu Leu Gly Leu Leu Ser Asn Glu Ala Tyr Thr Met Tyr Ala Val370 375 380Val Ala Leu Val Thr Val Thr Ala Ser Pro Ala Leu Leu Ile Trp Leu385 390 395 400Glu Lys Arg Ala Pro Pro Thr Gln Glu Glu Ser Ala Arg Leu Glu Arg405 410 415Leu Gly Thr Leu Pro Leu Met His Gln Pro Gly Ala Gln Trp Met Tyr195 200 205Asn Thr Gly Ser Leu Val Gln Gly Val Leu Val Gly Arg Ala Ala Asp210 215 220Gln Gly Phe Asp Ala Phe Val Arg Glu Arg Ile Leu Ala Pro Leu Gly225 230 235 240Met Arg Asp Thr Asp Phe His Val Pro Ala Asp Lys Leu Ala Arg Phe245 250 255Ala Gly Cys Gly Tyr Phe Thr Asp Glu Gln Thr Gly Glu Lys Thr Arg260 265 270Met Asp Arg Asp Gly Ala Glu Ser Ala Tyr Ala Ser Pro Pro Ala Phe275 280 285Pro Ser Gly Ala A la Gly Leu Val Ser Thr Val Asp Asp Tyr Leu Leu290 295 300Phe Ala Arg Met Leu Met Asn Gly Gly Val His Glu Gly Arg Arg Leu305 310 315 320Leu Ser Ala Ala Ser Val Arg Glu Met Thr Ala Asp His Leu Thr Pro325 330 335Ala Gln Lys Ala Ala Ser Ser Phe Phe Pro Gly Phe Phe Glu Thr His340 345 350Gly Trp Gly Tyr Gly Met Ala Val Val Thr Ala Pro Asp Ala Val Ser355 360 365Glu Val Pro Gly Arg Tyr Gly Trp Asp Gly Gly Phe Gly Thr Ser Trp370 375 380Ile Asn Asp Pro Gly Arg Glu Leu Ile Gly Ile Val Met Thr Gln Ser385 390 395 400Ala Gly Phe Leu Phe Ser Gly Ala Leu Glu Arg Phe Trp Arg Ser Val405 410 415Tyr Val Ala Thr Glu Ser Ala420<210>11<211>713<212>PRT<213>纤维堆囊菌<400>11Met His Gly Leu Thr Glu Arg Gln Val Leu Leu Ser Leu Val Thr Leu1 5 10 15Ala Leu Ile Leu Val Thr Ala Arg Ala Ser GlyGlu Leu Ala Arg Arg20 25 30Leu Arg Gln Pro Glu Val Leu Gly Glu Leu Phe Gly Gly Val Val Leu35 40 45Gly Pro Ser Val Val Gly Ala Leu Ala Pro Gly Phe His Arg Ala Leu50 55 60Phe Gln Glu Pro Ala Val Gly Val Val Leu Ser Gly Ile Ser Trp IleGlu Glu Ala Ala Arg Arg Ala Tyr Ile Pro Gly Val Glu Arg Ile Leu420 425 430Val Pro Ile Val Ala His Ala Leu Pro Gly Phe Ala Thr Asp Ile Val435 440 445Glu Ser Ile Val Ala Ser Lys Arg Lys Leu Gly Glu Thr Val Asp Ile450 455 460Thr Glu Leu Ser Val Glu Gln Gln A la Pro Gly Pro Ser Arg Ala Ala465 470 475 480Gly Glu Ala Ser Arg Gly Leu Ala Arg Leu Gly Ala Arg Leu Arg Val485 490 495Gly Ile Trp Arg Gln Arg Arg Glu Leu Arg Gly Ser Ile Gln Ala Ile500 505 510Leu Arg Ala Ser Arg Asp His Asp Leu Leu Val Ile Gly Ala Arg Ser515 520 525Pro Ala Arg Ala Arg Gly Met Ser Phe Gly Arg Leu Gln Asp Ala Ile530 535 540Val Gln Arg Ala Glu Ser Asn Val Leu Val Val Val Gly Asp Pro Pro545 550 555 560Ala Ala Glu Arg Ala Ser Ala Arg Arg Ile Leu Val Pro Ile Ile Gly565 570 575Leu Glu Tyr Ser Phe Ala Ala Ala Asp Leu Ala Ala His Val Ala Leu580 585 590Ala Trp Asp Ala Glu Leu Val Leu Leu Ser Ser Ala Gln Thr Asp Pro595 600 605Gly Ala Val Val Trp Arg Asp Arg Glu Pro Ser Arg Val Arg Ala Val610 615 620Ala Arg Ser Val Val Asp Glu Ala Val Phe Arg Gly Arg Arg Leu Gly625 630 635 640Val Arg Val Ser Ser Arg Val His Val Gly Ala His Pro Ser Asp Glu645 650 655Ile Thr Arg Glu Leu Ala Arg Ala Pro Tyr Asp Leu Leu Val Leu Gly660 665 670Cys Tyr Asp His Gly Pro Leu Gly Arg Leu Tyr Leu Gly Ser Thr Val675 680 685Glu Ser Val Val Val Arg Ser Arg Val Pro Val Ala Leu Leu Val Ala690 695 700His Gly Gly Thr Arg Glu Gln Val Arg705 710<210>12<211>126<212>PRT<213>纤维堆囊菌<400>12Met Asp Lys Pro Ile Gly Arg Thr Arg Cys Ala Ile Ala Glu Gly Tyr1 5 10 15Ile Pro Gly Gly Ser Asn Gly Pro Glu Pro Gln Met Thr Ser His Glu20 25 30Thr Ala Cys Leu Leu Asn Ala Ser Asp Arg Asp Ala Gln Val Ala Ile35 40 45Thr Val Tyr Phe Ser Asp Arg Asp Pro Ala Gly Pro Tyr Arg Val Thr50 55 60Val Pro Ala Arg Arg Thr Arg His Val Arg Phe Asn Asp Leu Thr Glu65 70 75 80Pro Glu Pro Ile Pro Arg Asp Thr Asp Tyr Ala Ser Val Ile Glu Ser85 90 95Asp Ala Pro Ile Val Val Gln His Thr Arg Leu Asp Ser Arg Gln Ala100 105 110Glu Asn Ala Leu Leu Ser Thr Ile Ala Tyr Thr Asp Arg Glu115 120 125<210>13<211>149<212>PRT<213>纤维堆囊菌<400>13Met Lys His Val Asp Thr Gly Arg Arg Phe Gly Arg Arg Ile Gly His1 5 10 15Thr Leu Gly Leu Leu Ala Ser Met Ala Leu Ala Gly Cys Gly Gly Pro20 25 30Ser Glu Lys Thr Val Gln Gly Thr Arg Leu Ala Pro Gly Ala Asp Ala35 40 45Arg Val Thr Ala Asp Val Asp Pro Asp Ala Ala Thr Thr Arg Leu Ala50 55 60Val Asp Val Val His Leu Ser Pro Pro Glu Arg Leu Glu Ala Gly Ser65 70 75 80Glu Arg Phe Val Val Trp Gln Arg Pro Ser Pro Glu Ser Pro Trp Arg85 90 95Arg Val Gly Val Leu Asp Tyr Asn Ala Asp Ser Arg Arg Gly Lys Leu100 105 110Ala Glu Thr Thr Val Pro Tyr Ala Asn Phe Glu Leu Leu Ile Thr Ala115 120 125Glu Lys Gln Ser Ser Pro Gln Ser Pro Ser Ser Ala Ala Val Ile Gly130 135 140Pro Thr Ser Val Gly145<210>14<211>184<212>PRT<213>纤维堆囊菌<400>14Val Thr Ser Glu Glu Val Pro Gly Ala Ala Leu Gly Ala Gln Ser Ser1 5 10 15Leu Val Arg Ala Gln His Ala Ala Arg His Val Arg Pro Cys Thr Arg20 25 30Ala Glu Glu Pro Pro Ala Leu Met His Gly Leu Thr Glu Arg Gln Val35 40 45Leu Leu Ser Leu Val Ala Leu Ala Leu Val Leu Leu Thr Ala Arg Ala50 55 60Phe Gly Glu Leu Ala Arg Arg Leu Arg Gln Pro Glu Val Leu Gly Glu65 70 75 80Leu Phe Gly Gly Val Val Leu Gly Pro Ser Val Val Gly Ala Leu Ala85 90 95Pro Gly Phe His Arg Val Leu Phe Gln Asp Pro Ala Val Gly Val Val100 105 110Leu Ser Gly Ile Ser Trp Ile Gly Ala Leu Val Leu Leu Leu Met Ala115 120 125Gly Ile Glu Val Asp Val Ser Ile Leu Arg Lys Glu Ala Arg Pro Gly130 135 140Ala Leu Ser Ala Leu Gly Ala Ile Ala Pro Pro Leu Arg Thr Pro Gly145 150 155 160Pro Leu Val Gln Arg Met Gln Gly Ala Phe Thr Trp Asp Leu Asp Val165 170 175Ser Pro Arg Arg Ser Ala Gln Ala180<210>15<211>145<212>PRT<213>纤维堆囊菌<400>15Val Asn Ala Pro Cys Met Arg Cys Thr Ser Gly Pro Gly Val Arg Ser1 5 10 15Gly Gly Ala Ile Ala Pro Ser Ala Glu Ser Ala Pro Gly Arg Ala Ser20 25 30Leu Arg Arg Met Leu Thr Ser Thr Ser Ile Pro Ala Met Ser Ser Arg35 40 45Thr Ser Ala Pro Ile Gln Glu Met Pro Glu Ser Thr Thr Pro Thr Ala50 55 60Gly Ser Trp Lys Arg Thr Arg Trp Asn Pro Gly Ala Ser Ala Pro Thr65 70 75 80Thr Asp Gly Pro Ser Thr Thr Pro Pro Lys Ser Ser Pro Ser Thr Ser85 90 95Gly Trp Arg Ser Arg Arg Ala Ser Ser Pro Lys Ala Arg Ala Val Arg100 105 110Arg Thr Ser Ala Arg Ala Thr Ser Glu Ser Arg Thr Cys Arg Ser Val115 120 125Arg Pro Cys Ile Arg Ala Gly Gly Ser Ser Ala Arg Val Gln Gly Arg130 135 140Thr145<210>16<211>185<212>PRT<213>纤维堆囊菌<400>16Val Leu Ala Pro Pro Ala Asp Ile Arg Pro Pro Ala Ala Ala Gln Leu1 5 10 15Glu Pro Asp Ser Pro Asp Asp Glu Ala Asp Glu A la Asp Glu Ala Leu20 25 30Arg Pro Phe Arg Asp Ala Ile Ala Ala Tyr Ser Glu Ala Val Arg Trp35 40 45Ala Glu Ala Ala Gln Arg Pro Arg Leu Glu Ser Leu Val Arg Leu Ala50 55 60Ile Val Arg Leu Gly Lys Ala Leu Asp Lys Val Pro Phe Ala His Thr65 70 75 80Thr Ala Gly Val Ser Gln Ile Ala Gly Arg Leu Gln Asn Asp Ala Val85 90 95Trp Phe Asp Val Ala Ala Arg Tyr Ala Ser Phe Arg Ala Ala Thr Glu100 105 110His Ala Leu Arg Asp Ala Ala Ser Ala Met Glu Ala Leu Ala Ala Gly115 120 125Pro Tyr Arg Gly Ser Ser Arg Val Ser Ala Ala Val Gly Glu Phe Arg130 135 140Gly Glu Ala Ala Arg Leu His Pro Ala Asp Arg Val Pro Ala Ser Asp145 150 155 160Gln Gln Ile Leu Thr Ala Leu Arg Ala Ala Glu Arg Ala Leu Ile Ala165 170 175Leu Tyr Thr Ala Phe Ala Arg Glu Glu180 185<210>17<211>146<212>PRT<213>纤维堆囊菌<400>17Met Ala Asp Ala Ala Ser Arg Ser Ala Cys Ser Val Ala Ala Arg Lys1 5 10 15Leu Ala Tyr Arg Ala Ala Thr Ser Asn Gln Thr Ala Ser Phe Trp Ser20 25 30Leu Pro Ala Ile Trp Glu Thr Pro Ala Val Val Cys Ala Lys Gly Thr35 40 45Leu Ser Ser Ala Leu Pro Ser Arg Thr Ile Ala Ser Arg Thr Arg Leu50 55 60Ser Ser Arg Gly Arg Cys Ala Ala Ser Ala His Arg Thr Ala Ser Glu65 70 75 80Tyr Ala Ala Ile Ala Ser Arg Asn Gly Arg Ser Ala Ser Ser Ala Ser85 90 95Ser Ala Ser Ser Ser Gly Glu Ser Gly Ser Ser Trp Ala Ala Ala Gly100 105 110Gly Arg Met Ser Ala Gly Gly Ala Ser Thr Gly Glu Val Tyr Glu Gln115 120 125Ala Pro Arg Leu Arg Leu Ala Gln Ser Val Ala Ala Arg Arg Arg Asp130 135 140Pro Thr145<210>18<211>288<212>PRT<213>纤维堆囊菌<400>18Val Thr Val Ser Ser Met Pro Arg Ser Trp Ser Ser Arg Val Arg Thr1 5 10 15Val Val Thr Ala Leu Gly Cys Ala Arg Arg Leu Ser Gly Ser Ile Ser20 25 30Arg Leu Arg Arg His Pro Glu Ala Gly Arg Ala Pro Arg Ser Arg Leu35 40 45Arg Ala Trp Arg Arg Leu Pro Gln His Ile Ser Ser Pro Trp Arg His50 55 60Leu Pro Pro Gly Ala Arg Val Gly Thr Ser Cys Pro Ala Asp Arg Arg65 70 75 80Ile Leu Pro Ser His Arg Thr Ala Asp Leu Gly Thr Ser Gly Gly Thr85 90 95Leu Val Ala Arg Met Ser Gly His Val Ala Arg Asn Pro His Ala Ala100 105 110Val Leu Val Gly Asp Gly Ser Ala Arg Gly Arg Arg Arg Leu Ser Asn115 120 125Arg Arg Ala Glu Arg Arg Val Ser Asp Val Thr Cys Arg Glu Gly Gly130 135 140Glu Ala Met Gln Lys Ile Ala Gly Lys Leu Val Val Gly Leu Ile Ser145 150 155 160Val Ser Gly Met Ser Leu Leu Ala Ala Cys Gly Gly Glu Lys Arg Ser165 170 175Gly Gly Glu Ala Gln Thr Pro Gly Gly Ala Gln Gly Glu Ala Pro Val180 185 190Pro Val Gly Ser Ala Val Asp Ser Ile Val Ala Ala Arg Cys Asp Arg195 200 205Glu Ala Arg Cys Asn Asn Ile Gly Gln Asp Arg Glu Tyr Ser Ser Lys210 215 220Asp Ala Cys Ser Asn Lys Ile Arg Ser Glu Trp Arg Asp Glu Leu Thr225 230 235 240Phe Gly Glu Cys Pro Gly Gly Ile Asp Ala Lys Gln Leu Asn Glu Cys245 250 255Leu Glu Gly Ile Arg Asn Glu Gly Cys Gly Asn Pro Phe Asp Thr Leu260 265 270Gly Arg Val Val Ala Cys Arg Ser Ser Asp Leu Cys Arg Asp Ala Arg275 280 285<210>19<211>288<212>PRT<213>纤维堆囊菌<400>19Val Thr Val Ser Ser Met Pro Arg Ser Trp Ser Ser Arg Val Arg Thr1 5 10 15Val Val Thr Ala Leu Gly Cys Ala Arg Arg Leu Ser Gly Ser Ile Ser20 25 30Arg Leu Arg Arg His Pro Glu Ala Gly Arg Ala Pro Arg Ser Arg Leu35 40 45Arg Ala Trp Arg Arg Leu Pro Gln His Ile Ser Ser Pro Trp Arg His50 55 60Leu Pro Pro Gly Ala Arg Val Gly Thr Ser Cys Pro Ala Asp Arg Arg65 70 75 80Ile Leu Pro Ser His Arg Thr Ala Asp Leu Gly Thr Ser Gly Gly Thr85 90 95Leu Val Ala Arg Met Ser Gly His Val Ala Arg Asn Pro His Ala Ala100 105 110Val Leu Val Gly Asp Gly Ser Ala Arg Gly Arg Arg Arg Leu Ser Asn115 120 125Arg Arg Ala Glu Arg Arg Val Ser Asp Val Thr Cys Arg Glu Gly Gly130 135 140Glu Ala Met Gln Lys Ile Ala Gly Lys Leu Val Val Gly Leu Ile Ser145 150 155 160Val Ser Gly Met Ser Leu Leu Ala Ala Cys Gly Gly Glu Lys Arg Ser165 170 175Gly Gly Glu Ala Gln Thr Pro Gly Gly Ala Gln Gly Glu Ala Pro Val180 185 190Pro Val Gly Ser Ala Val Asp Ser Ile Val Ala Ala Arg Cys Asp Arg195 200 205Glu Ala Arg Cys Asn Asn Ile Gly Gln Asp Arg Glu Tyr Ser Ser Lys210 215 220Asp Ala Cys Ser Asn Lys Ile Arg Ser Glu Trp Arg Asp Glu Leu Thr225 230 235 240Phe Gly Glu Cys Pro Gly Gly Ile Asp Ala Lys Gln Leu Asn Glu Cys245 250 255Leu Glu Gly Ile Arg Asn Glu Gly Cys Gly Asn Pro Phe Asp Thr Leu260 265 270Gly Arg Val Val Ala Cys Arg Ser Ser Asp Leu Cys Arg Asp Ala Arg275 280 285<210>20<211>155<212>PRT<213>纤维堆囊菌<400>20Met Asp Pro Arg Ala Arg Arg Glu Lys Arg Pro Ser Leu Leu Asp Ser1 5 10 15Arg Gly Arg Gln Pro Lys Arg Ser Gln Gln Gly Gly His Met Glu Lys20 25 30Pro Ile Gly Arg Thr Arg Trp Ala Ile Ala Glu Gly Tyr Ile Pro Gly35 40 45Arg Ser Asn Gly Pro Glu Pro Gln Met Thr Ser His Glu Thr Ala Cys50 55 60Leu Leu Asn Ala Ser Asp Arg Asp Ala Gln Val Ala Ile Thr Val Tyr65 70 75 80Phe Ser Asp Arg Asp Pro Ala Gly Pro Tyr Arg Val Thr Val Pro Ala85 90 95Arg Arg Thr Arg His Val Arg Phe Asn Asp Leu Thr Glu Pro Glu Pro100 105 110Ile Pro Arg Asp Thr Asp Tyr Ala Ser Val Ile Glu Ser Asp Val Pro115 120 125Ile Val Val Gln His Thr Arg Leu Asp Ser Arg Gln Ala Glu Asn Ala130 135 140Leu Ile Ser Thr Ile Ala Tyr Thr Asp Arg Glu145 150 155<210>21<211>156<212>PRT<213>纤维堆囊菌<400>21Val Arg Arg Ser Arg Trp Gln Met Lys His Val Asp Thr Gly Arg Arg1 5 10 15Val Gly Arg Arg Ile Gly Leu Thr Leu Gly Leu Leu Ala Ser Met Ala20 25 30Leu Ala Gly Cys Gly Gly Pro Ser Glu Lys Ile Val Gln Gly Thr Arg35 40 45Leu Ala Pro Gly Ala Asp Ala His Val Ala Ala Asp Val Asp Pro Asp50 55 60Ala Ala Thr Thr Arg Leu Ala Val Asp Val Val His Leu Ser Pro Pro65 70 75 80Glu Arg Ile Glu Ala Gly Ser Glu Arg Phe Val Val Trp Gln Arg Pro85 90 95Ser Ser Glu Ser Pro Trp Gln Arg Val Gly Val Leu Asp Tyr Asn Ala100 105 110Ala Ser Arg Arg Gly Lys Leu Ala Glu Thr Thr Val Pro His Ala Asn115 120 125Phe Glu Leu Leu Ile Thr Val Glu Lys Gln Ser Ser Pro Gln Ser Pro130 135 140Ser Ser Ala Ala Val Ile Gly Pro Thr Ser Val Gly145 150 155<210>22<211>305<212>PRT<2l3>纤维堆囊菌<400>22Met Glu Lys Glu Ser Arg Ile Ala Ile Tyr Gly Ala Ile Ala Ala Asn1 5 10 15Val Ala Ile Ala Ala Val Lys Phe Ile Ala Ala Ala Val Thr Gly Ser20 25 30Ser Ala Met Leu Ser Glu Gly Val His Ser Leu Val Asp Thr Ala Asp35 40 45Gly Leu Leu Leu Leu Leu Gly Lys His Arg Ser Ala Arg Pro Pro Asp50 55 60Ala Glu His Pro Phe Gly His Gly Lys Glu Leu Tyr Phe Trp Thr Leu65 70 75 80Ile Val Ala Ile Met Ile Phe Ala Ala Gly Gly Gly Val Ser Ile Tyr85 90 95Glu Gly Ile Leu His Leu Leu His Pro Arg Gln Ile Glu Asp Pro Thr100 105 110Trp Asn Tyr Val Val Leu Gly Ala Ala Ala Val Phe Glu Gly Thr Ser115 120 125Leu Ile Ile Ser Ile His Glu Phe Lys Lys Lys Asp Gly Gln Gly Tyr130 135 140Leu Ala Ala Met Arg Ser Ser Lys Asp Pro Thr Thr Phe Thr Ile Val145 150 155 160Leu Glu Asp Ser Ala Ala Leu Ala Gly Leu Thr Ile Ala Phe Leu Gly165 170 175Val Trp Leu Gly His Arg Leu Gly Asn Pro Tyr Leu Asp Gly Ala Ala180 185 190Ser Ile Gly Ile Gly Leu Val Leu Ala Ala Val Ala Val Phe Leu Ala195 200 205Ser Gln Ser Arg Gly Leu Leu Val Gly Glu Ser Ala Asp Arg Glu Leu210 215 220Leu Ala Ala Ile Arg Ala Leu Ala Ser Ala Asp Pro Gly Val Ser Ala225 230 235 240Val Gly Arg Pro Leu Thr Met His Phe Gly Pro His Glu Val Leu Val245 250 255Val Leu Arg Ile Glu Phe Asp Ala Ala Leu Thr Ala Ser Gly Val Ala260 265 270Glu Ala Ile Glu Arg Ile Glu Thr Arg Ile Arg Ser Glu Arg Pro Asp275 280 285Val Lys His Ile Tyr Val Glu Ala Arg Ser Leu His Gln Arg Ala Arg290 295 300Ala305<210>23<211>135<212>PRT<213>纤维堆囊菌<400>23Val Gln Thr Ser Ser Phe Asp Ala Arg Tyr Ala Gly Cys Lys Ser Ser1 5 10 15Arg Arg Ile Ala Arg Ser Gly Ser Ala Gly Ala Arg Ala Gly Arg Ala20 25 30His Glu Gly Ala Ala Ser Ala Gly Phe Glu Gly Gly Asp Val Met Arg35 40 45Lys Ala Arg Ala His Gly Ala Met Leu Gly Gly Arg Asp Asp Gly Trp50 55 60Arg Arg Gly Leu Pro Gly Ala Gly Ala Leu Arg Ala Ala Leu Gln Arg65 70 75 80Gly Arg Ser Arg Asp Leu Ala Arg Arg Arg Leu Ile Ala Ser Val Ser85 90 95Leu Ala Gly Gly Ala Ser Met Ala Val Val Ser Leu Phe Gln Leu Gly100 105 110Ile Ile Glu Arg Leu Pro Asp Pro Pro Leu Pro Gly Phe Asp Ser Ala115 120 125Lys Val Thr Ser Ser Asp Ile130 135<210>24<211>19<212>DNA<213>人工序列<220><223>人工序列描述通用反向引物<400>24ggaaacagct atgaccatg 19<210>25<211>17<212>DNA<213>人工序列<220><223>人工序列描述通用正向引物<400>25gtaaaacgac ggccagt 17<210>26<211>28<212>DNA<213>人工序列<220><223>人工序列描述PCR引物NH24末端”B”<400>26gtgactggcg cctggaatct gcatgagc 28<210>27<211>28<212>DNA<213>人工序列<220><223>人工序列描述PCR引物NH2末端”A”<400>27agcgggagct tgctagacat tctgtttc 28<210>28<211>24<212>DNA<213>人工序列<220><223>人工序列描述PCR引物NH2末端”B”<400>28gacgcgcctc gggcagcgcc ccaa 24<210>29<211>25<212>DNA<213>人工序列<220><223>人工序列描述PCR引物pEPO15-NH6末端”B”<400>29caccgaagcg tcgatctggt ccatc 25<210>30<211>25<212>DNA<213>人工序列<220><223>人工序列描述PCR引物pEPO15H2.7末端”A”<400>30cggtcagatc gacgacgggc tttcc 2权利要求
1．一种分离的核酸分子，其包含编码至少一种涉及epothilone生物合成的多肽的核苷酸序列。
2．权利要求1的分离的核酸分子，其中所述核苷酸序列是由粘细菌分离的。
3．权利要求2的分离的核酸分子，其中所述粘细菌是纤维堆囊菌。
4．包含与权利要求1的核酸分子可操作连接的异源启动子序列的嵌合基因。
5．包含权利要求4的嵌合基因的重组载体。
6．包含权利要求4的嵌合基因的重组宿主细胞。
7．权利要求6的重组宿主细胞，其是细菌。
8．权利要求7的重组宿主细胞，其是放线菌。
9．权利要求8的重组宿主细胞，其是链霉菌。
10．包含权利要求1的核酸分子的Bac克隆。
11．权利要求10的Bac克隆，其是pEPO15。
12．权利要求1的分离的核酸分子，其中所述多肽包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2,SEQ ID NO:2的氨基酸11-437,SEQ ID NO:2的氨基酸543-864,SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:3,SEQ ID NO:3的氨基酸72-81,SEQ ID NO:3的氨基酸118-125,SEQ ID NO:3的氨基酸199-212,SEQ ID NO:3的氨基酸353-363,SEQ ID NO:3的氨基酸549-565,SEQ ID NO:3的氨基酸588-603,SEQ ID NO:3的氨基酸669-684,SEQ ID NO:3的氨基酸815-821,SEQ ID NO:3的氨基酸868-892,SEQ ID NO:3的氨基酸903-912,SEQ ID NO:3的氨基酸918-940,SEQ ID NO:3的氨基酸1268-1274,SEQ ID NO:3的氨基酸1285-1297,SEQ ID NO:3的氨基酸973-1256,SEQ ID NO:3的氨基酸1344-1351,SEQ ID NO:4,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5,SEQ ID NO:5的氨基酸39-457,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸3555-3876,SEQ IDNO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸4433-4719,SEQID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸5010-5082,SEQ ID NO:5的氨基酸5103-5525,SEQ ID NO:5的氨基酸5631-5951,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:5的氨基酸6542-6837,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:5的氨基酸7140-7211,SEQ ID NO:6,SEQ ID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸561-881,SEQ ID NO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸1522-1946,SEQ ID NO:6的氨基酸2053-2373,SEQ ID NO:6的氨基酸2383-2551,SEQ ID NO:6的氨基酸2671-3045,SEQ ID NO:6的氨基酸3392-3636,SEQ ID NO:6的氨基酸3673-3745,SEQ ID NO:7,SEQ ID NO:7的氨基酸32-450,SEQ ID NO:7的氨基酸556-877,SEQ ID NO:7的氨基酸887-1051,SEQ ID NO:7的氨基酸1478-1790,SEQ ID NO:7的氨基酸1810-2055,SEQ ID NO:7的氨基酸2093-2164,SEQ ID NO:7的氨基酸2165-2439,SEQ ID NO:8,SEQ ID NO:10,SEQ ID NO:11，和SEQ ID NO:22。
13．权利要求12的分离的核酸分子，其中所述多肽包含选自下组的氨基酸序列:SEQ ID NO:2,SEQ ID NO:2的氨基酸11-437,SEQID NO:2的氨基酸543-864,SEQ ID NO:2的氨基酸974-1273,SEQID NO:2的氨基酸1314-1385,SEQ ID NO:3,SEQ ID NO:3的氨基酸72-81,SEQ ID NO:3的氨基酸118-125,SEQ ID NO:3的氨基酸199-212,SEQ ID NO:3的氨基酸353-363,SEQ ID NO:3的氨基酸549-565,SEQ ID NO:3的氨基酸588-603,SEQ ID NO:3的氨基酸669-684,SEQ ID NO:3的氨基酸815-821,SEQ ID NO:3的氨基酸868-892,SEQ ID NO:3的氨基酸903-912,SEQ ID NO:3的氨基酸918-940,SEQ ID NO:3的氨基酸1268-1274,SEQ ID NO:3的氨基酸1285-1297,SEQ ID NO:3的氨基酸973-1256,SEQ ID NO:3的氨基酸1344-1351,SEQ ID NO:4,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5,SEQ ID NO:5的氨基酸39-457,SEQID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸3555-3876,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸4433-4719,SEQ IDNO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸5010-5082,SEQID NO:5的氨基酸5103-5525,SEQ ID NO:5的氨基酸5631-5951,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:5的氨基酸6542-6837,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:5的氨基酸7140-7211,SEQ ID NO:6,SEQ ID NO:6的氨基酸35-454,SEQ IDNO:6的氨基酸561-881,SEQ ID NO:6的氨基酸1143-1393,SEQ IDNO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸1522-1946,SEQID NO:6的氨基酸2053-2373,SEQ ID NO:6的氨基酸2383-2551,SEQ ID NO:6的氨基酸2671-3045,SEQ ID NO:6的氨基酸3392-3636,SEQ ID NO:6的氨基酸3673-3745,SEQ ID NO:7,SEQ ID NO:7的氨基酸32-450,SEQ ID NO:7的氨基酸556-877,SEQ ID NO:7的氨基酸887-1051,SEQ ID NO:7的氨基酸1478-1790,SEQ ID NO:7的氨基酸1810-2055,SEQ ID NO:7的氨基酸2093-2164,SEQ IDNO:7的氨基酸2165-2439,SEQ ID NO:8,SEQ ID NO:10,SEQ IDNO:11，和SEQ ID NO:22。
14．权利要求12的分离的核酸分子，其中所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸1900-3171的互补序列，SEQ ID NO:1的核苷酸3415-5556,SEQ ID NO:1的核苷酸7610-11875,SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸10529-11428,SEQID NO:1的核苷酸11549-11764,SEQ ID NO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQID NO:1的核苷酸14313-14334,SEQ ID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639,SEQ ID NO:1的核苷酸15901-15924,SEQ ID NO:1的核苷酸16251-21749,SEQID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸21746-43519,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸25184-25942,SEQID NO:1的核苷酸26045-26263,SEQ ID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸35042-35902,SEQID NO:1的核苷酸35930-36667,SEQ ID NO:I的核苷酸36773-36991,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸41369-42256,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸43524-54920,SEQID NO:1的核苷酸43626-44885,SEQ ID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸48087-49361,SEQ ID NO:1的核苷酸49680-50642,SEQ ID NO:1的核苷酸50670-51176,SEQ ID NO:1的核苷酸51534-52657,SEQ ID NO:1的核苷酸53697-54431,SEQID NO:1的核苷酸54540-54758,SEQ ID NO:1的核苷酸54935-62254,SEQ ID NO:1的核苷酸55028-56284,SEQ ID NO:1的核苷酸56600-57565,SEQ ID NO:1的核苷酸57593-58087,SEQ ID NO:1的核苷酸59366-60304,SEQ ID NO:1的核苷酸60362-61099,SEQ ID NO:1的核苷酸61211-61426,SEQ ID NO:1的核苷酸61427-62254,SEQID NO:1的核苷酸62369-63628,SEQ ID NO:1的核苷酸67334-68251,和SEQ ID NO:1的核苷酸1-68750。
15．权利要求12的核酸分子，其中所述核苷酸序列选自下组:SEQID NO:1的核苷酸1900-3171的互补序列，SEQ ID NO:1的核苷酸3415-5556,SEQ ID NO:1的核苷酸7610-11875,SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:1的核苷酸11549-11764,SEQID NO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:1的核苷酸14313-14334,SEQID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639,SEQ ID NO:1的核苷酸15901-15924,SEQ ID NO:1的核苷酸16251-21749,SEQ ID NO:1的核苷酸16269-17546,SEQID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸21746-43519,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸26045-26263,SEQID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸35930-36667,SEQID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸41369-42256,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸43524-54920,SEQ ID NO:1的核苷酸43626-44885,SEQID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸48087-49361,SEQ ID NO:1的核苷酸49680-50642,SEQ ID NO:1的核苷酸50670-51176,SEQ ID NO:1的核苷酸51534-52657,SEQ ID NO:1的核苷酸53697-54431,SEQ ID NO:1的核苷酸54540-54758,SEQID NO:1的核苷酸54935-62254,SEQ ID NO:1的核苷酸55028-56284,SEQ ID NO:1的核苷酸56600-57565,SEQ ID NO:1的核苷酸57593-58087,SEQ ID NO:1的核苷酸59366-60304,SEQ ID NO:1的核苷酸60362-61099,SEQ ID NO:1的核苷酸61211-61426,SEQ ID NO:1的核苷酸61427-62254,SEQ ID NO:1的核苷酸62369-63628,SEQID NO:1的核苷酸67334-68251，和SEQ ID NO:1的核苷酸1-68750。
16．包含与权利要求12的核酸分子可操作连接的异源启动子序列的嵌合基因。
17．包含权利要求16的嵌合基因的重组载体。
18．包含权利要求16的嵌合基因的重组宿主细胞。
19．权利要求18的重组宿主细胞，其是细菌。
20．权利要求19的重组宿主细胞，其是放线菌。
21．权利要求20的重组宿主细胞，其是链霉菌。
22．权利要求1的分离的核酸分子，其中所述核苷酸序列包含与选自下组的核苷酸序列中连续的20个碱基对的部分序列相同的连续20个碱基对核苷酸部分SEQ ID NO:1的核苷酸1900-3171的互补序列，SEQ ID NO:1的核苷酸3415-5556,SEQ ID NO:1的核苷酸7610-11875,SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸10529-11428,SEQ IDNO:1的核苷酸11549-11764,SEQ ID NO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQID NO:1的核苷酸14313-14334,SEQ ID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639,SEQ ID NO:1的核苷酸15901-15924,SEQ ID NO:1的核苷酸16251-21749,SEQID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸21746-43519,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸25184-25942,SEQID NO:1的核苷酸26045-26263,SEQ ID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸35042-35902,SEQID NO:1的核苷酸35930-36667,SEQ ID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸41369-42256,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸43524-54920,SEQID NO:1的核苷酸43626-44885,SEQ ID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸48087-49361,SEQ ID NO:1的核苷酸49680-50642,SEQ ID NO:1的核苷酸50670-51176,SEQ ID NO:1的核苷酸51534-52657,SEQ ID NO:1的核苷酸53697-54431,SEQID NO:1的核苷酸54540-54758,SEQ ID NO:1的核苷酸54935-62254,SEQ ID NO:1的核苷酸55028-56284,SEQ ID NO:1的核苷酸56600-57565,SEQ ID NO:1的核苷酸57593-58087,SEQ ID NO:1的核苷酸59366-60304,SEQ ID NO:1的核苷酸60362-61099,SEQ ID NO:1的核苷酸61211-61426,SEQ ID NO:1的核苷酸61427-62254,SEQID NO:1的核苷酸62369-63628,SEQ ID NO:1的核苷酸67334-68251，和SEQ ID NO:1的核苷酸1-68750。
23．包含与权利要求22的核酸分子可操作连接的异源启动子序列的嵌合基因。
24．包含权利要求23的嵌合基因的重组载体。
25．包含权利要求23的嵌合基因的重组宿主细胞。
26．权利要求25的重组宿主细胞，其是细菌。
27．权利要求26的重组宿主细胞，其是放线菌。
28．权利要求27的重组宿主细胞，其是链霉菌。
29．一种分离的核酸分子，其包含编码至少一个epothilone合酶结构域的核苷酸序列。
30．权利要求29的分离的核酸分子，其中所述epothilone合酶结构域是β-酮脂酰合酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸11-437,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:5的氨基酸39-457,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸5103-5525,SEQ ID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸1522-1946，和SEQ ID NO:7的氨基酸32-450。
31．权利要求30的分离的核酸分子，其中所述β-酮脂酰合酶结构域包含选自下组的氨基酸序列:SEQ ID NO:2的氨基酸11-437,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:5的氨基酸39-457,SEQID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸5103-5525,SEQ ID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸1522-1946，和SEQ ID NO:7的氨基酸32-450。
32．权利要求30的分离的核酸分子，其中所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸7643-8920,SEQID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸43626-44885,SEQ ID NO:1的核苷酸48087-49361，和SEQ IDNO:1的核苷酸55028-56284。
33．权利要求30的分离的核酸分子，其中所述核苷酸序列包含与选自下组的核苷酸序列中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸21860-23116,SEQID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸43626-44885,SEQ ID NO:1的核苷酸48087-49361，和SEQ ID NO:1的核苷酸55028-56284。
34．权利要求30的分离的核酸分子，其中所述核苷酸序列选自下组SEQ ID NO:1的核苷酸7643-8920,SEQ ID NO:1的核苷酸16269-17546,SEQ ID NO:1的核苷酸21860-23116,SEQ ID NO:1的核苷酸26318-27595,SEQ ID NO:1的核苷酸30815-32092,SEQ ID NO:1的核苷酸37052-38320,SEQ ID NO:1的核苷酸43626-44885,SEQID NO:1的核苷酸48087-49361，和SEQ ID NO:1的核苷酸55028-56284。
35．权利要求29的分离的核酸分子，其中所述epothilone合酶结构域是酰基转移酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸543-864,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸3555-3876,SEQ IDNO:5的氨基酸5631-5951,SEQ ID NO:6的氨基酸561-881,SEQ IDNO:6的氨基酸2053-2373，和SEQ ID NO:7的氨基酸556-877。
36．权利要求35的分离的核酸分子，其中所述酰基转移酶结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸543-864,SEQ IDNO:4的氨基酸539-859,SEQ ID NO:5的氨基酸563-884,SEQ IDNO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸3555-3876,SEQID NO:5的氨基酸5631-5951,SEQ ID NO:6的氨基酸561-881,SEQ ID NO:6的氨基酸2053-2373，和SEQ ID NO:7的氨基酸556-877。
37．权利要求35的分离的核酸分子，其中所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸49680-50642，和SEQ ID NO:1的核苷酸56600-57565。
38．权利要求35的分离的核酸分子，其中所述核苷酸序列包含与选自下组的核苷酸序列中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸23431-24397,SEQID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸45204-46166,SEQ ID NO:1的核苷酸49680-50642，和SEQ ID NO:1的核苷酸56600-57565。
39．权利要求35的分离的核酸分子，其中所述核苷酸序列选自下组SEQ ID NO:1的核苷酸9236-10201,SEQ ID NO:1的核苷酸17865-18827,SEQ ID NO:1的核苷酸23431-24397,SEQ ID NO:1的核苷酸27911-28876,SEQ ID NO:1的核苷酸32408-33373,SEQ ID NO:1的核苷酸38636-39598,SEQ ID NO:1的核苷酸45204-46166,SEQID NO:1的核苷酸49680-50642，和SEQ ID NO:1的核苷酸56600-57565。
40．权利要求29的分离的核酸分子，其中所述epothilone合酶结构域是烯酰基还原酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸6542-6837，和SEQID NO:7的氨基酸1478-1790。
41．权利要求40的分离的核酸分子，其中所述烯酰基还原酶结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸6542-6837，和SEQ ID NO:7的氨基酸1478-1790。
42．权利要求40的分离的核酸分子，其中所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸41369-42256，和SEQ ID NO:1的核苷酸59366-60304。
43．权利要求40的分离的核酸分子，其中所述核苷酸序列包含与选自下组的核苷酸序列中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分SEQ ID NO:1的核苷酸10529-11428,SEQ IDNO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸41369-42256,和SEQ ID NO:1的核苷酸59366-60304。
44．权利要求40的分离的核酸分子，其中所述核苷酸序列选自下组SEQ ID NO:1的核苷酸10529-11428,SEQ ID NO:1的核苷酸35042-35902,SEQ ID NO:1的核苷酸41369-42256，和SEQ ID NO:1的核苷酸59366-60304。
45．权利要求29的分离的核酸分子，其中所述epothilone合酶结构域是酰基载体蛋白结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5的氨基酸1434-1506,SEQ IDNO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸5010-5082,SEQID NO:5的氨基酸7140-7211,SEQ ID NO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸3673-3745，和SEQ ID NO:7的氨基酸2093-2164。
46．权利要求45的分离的核酸分子，其中所述酰基载体蛋白结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5的氨基酸1434-1506,SEQ ID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸5010-5082,SEQ ID NO:5的氨基酸7140-7211,SEQ ID NO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸3673-3745，和SEQ ID NO:7的氨基酸2093-2164。
47．权利要求45的分离的核酸分子，其中所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸11549-11764,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸26045-26263,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸54540-54758，和SEQ ID NO:1的核苷酸61211-61426。
48．权利要求45的分离的核酸分子，其中所述核苷酸序列包含与选自下组的核苷酸序列中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分SEQ ID NO:1的核苷酸11549-11764,SEQ IDNO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸26045-26263,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸36773-36991,SEQ ID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸54540-54758，和SEQ IDNO:1的核苷酸61211-61426。
49．权利要求45的分离的核酸分子，其中所述核苷酸序列选自下组SEQ ID NO:1的核苷酸11549-11764,SEQ ID NO:1的核苷酸21414-21626,SEQ ID NO:1的核苷酸26045-26263,SEQ ID NO:1的核苷酸30539-30759,SEQ ID NO:1的核苷酸36773-36991,SEQID NO:1的核苷酸43163-43378,SEQ ID NO:1的核苷酸47811-48032,SEQ ID NO:1的核苷酸54540-54758，和SEQ ID NO:1的核苷酸61211-61426。
50．权利要求29的分离的核酸分子，其中所述epothilone合酶结构域是脱水酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:6的氨基酸2383-2551，和SEQ ID NO:7的氨基酸887-1051。
51．权利要求50的分离的核酸分子，其中所述脱水酶结构域包含选自下组的氨基酸序列SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸5964-6132,SEQ IDNO:6的氨基酸2383-2551，和SEQ ID NO:7的氨基酸887-1051。
52．权利要求50的分离的核酸分子，其中所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸50670-51176，和SEQ ID NO:1的核苷酸57593-58087。
53．权利要求50的分离的核酸分子，其中所述核苷酸序列包含与选自下组的核苷酸序列中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分SEQ ID NO:1的核苷酸18855-19361,SEQ IDNO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸50670-51176，和SEQ ID NO:1的核苷酸57593-58087。
54．权利要求50的分离的核酸分子，其中所述核苷酸序列选自下组SEQ ID NO:1的核苷酸18855-19361,SEQ ID NO:1的核苷酸33401-33889,SEQ ID NO:1的核苷酸39635-40141,SEQ ID NO:1的核苷酸50670-51176，和SEQ ID NO:1的核苷酸57593-58087。
55．权利要求29的分离的核酸分子，其中所述epothilone合酶结构域是β-酮还原酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸6857-7101,SEQ IDNO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸3392-3636，和SEQ ID NO:7的氨基酸1810-2055。
56．权利要求55的分离的核酸分子，其中所述β-酮还原酶结构域包含选自下组的氨基酸序列SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸3392-3636，和SEQ ID NO:7的氨基酸1810-2055。
57．权利要求55的分离的核酸分子，其中所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸35930-36667,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸53697-54431，和SEQ ID NO:1的核苷酸60362-61099。
58．权利要求55的分离的核酸分子，其中所述核苷酸序列包含与选自下组的核苷酸序列中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分SEQ ID NO:1的核苷酸20565-21302,SEQ IDNO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸35930-36667,SEQ ID NO:1的核苷酸42314-43048,SEQ ID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸53697-54431，和SEQ ID NO:1的核苷酸60362-61099。
59．权利要求55的分离的核酸分子，其中所述核苷酸序列选自下组SEQ ID NO:1的核苷酸20565-21302,SEQ ID NO:1的核苷酸25184-25942,SEQ ID NO:1的核苷酸29678-30429,SEQ ID NO:1的核苷酸35930-36667,SEQ ID NO:1的核苷酸42314-43048,SEQID NO:1的核苷酸46950-47702,SEQ ID NO:1的核苷酸53697-54431,和SEQ ID NO:1的核苷酸60362-61099。
60．权利要求29的分离的核酸分子，其中所述epothilone合酶结构域是甲基转移酶结构域，其包含与SEQ ID NO:6的氨基酸2671-3045基本相似的氨基酸序列。
61．权利要求60的分离的核酸分子，其中所述甲基转移酶结构域包含SEQ ID NO:6的氨基酸2671-3045。
62．权利要求60的分离的核酸分子，其中所述核苷酸序列与SEQID NO:1的核苷酸51534-52657基本相似。
63．权利要求60的分离的核酸分子，其中所述核苷酸序列包含与SEQ ID NO:1的核苷酸51534-52657中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分。
64．权利要求60的分离的核酸分子，其中所述核苷酸序列是SEQID NO:1的核苷酸51534-52657。
65．权利要求29的分离的核酸分子，其中所述epothilone合酶结构域是硫酯酶结构域，其包含与SEQ ID NO:7的氨基酸2165-2439基本相似的氨基酸序列。
66．权利要求65的分离的核酸分子，其中所述硫酯酶结构域包含SEQ ID NO:7的氨基酸2165-2439。
67．权利要求65的分离的核酸分子，其中所述核苷酸序列与SEQID NO:1的核苷酸61427-62254基本相似。
68．权利要求65的分离的核酸分子，其中所述核苷酸序列包含与SEQ ID NO:1的核苷酸61427-62254中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分。
69．权利要求65的分离的核酸分子，其中所述核苷酸序列是SEQID NO:1的核苷酸61427-62254。
70．一种分离的核酸分子，其包含编码非核糖体肽合成酶的核苷酸序列，其中所述非核糖体肽合成酶包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:3,SEQ ID NO:3的氨基酸72-81,SEQ ID NO:3的氨基酸118-125,SEQ ID NO:3的氨基酸199-212,SEQ ID NO:3的氨基酸353-363,SEQ ID NO:3的氨基酸549-565,SEQ ID NO:3的氨基酸588-603,SEQ ID NO:3的氨基酸669-684,SEQ ID NO:3的氨基酸815-821,SEQ ID NO:3的氨基酸868-892,SEQ ID NO:3的氨基酸903-912,SEQ ID NO:3的氨基酸918-940,SEQ ID NO:3的氨基酸1268-1274,SEQ ID NO:3的氨基酸1285-1297,SEQ ID NO:3的氨基酸973-1256，和SEQ ID NO:3的氨基酸1344-1351。
71．权利要求70的分离的核酸分子，其中所述非核糖体肽合成酶包含选自下组的氨基酸序列SEQ ID NO:3,SEQ ID NO:3的氨基酸72-81,SEQ ID NO:3的氨基酸118-125,SEQ ID NO:3的氨基酸199-212,SEQ ID NO:3的氨基酸353-363,SEQ ID NO:3的氨基酸549-565,SEQ ID NO:3的氨基酸588-603,SEQ ID NO:3的氨基酸669-684,SEQ ID NO:3的氨基酸815-821,SEQ ID NO:3的氨基酸868-892,SEQ ID NO:3的氨基酸903-912,SEQ ID NO:3的氨基酸918-940,SEQ ID NO:3的氨基酸1268-1274,SEQ ID NO:3的氨基酸1285-1297,SEQ ID NO:3的氨基酸973-1256，和SEQ IDNO:3的氨基酸1344-1351。
72．权利要求70的分离的核酸分子，其中所述核苷酸序列与选自下组的核苷酸序列基本相似SEQ ID NO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQID NO:1的核苷酸14313-14334,SEQ ID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639，和SEQ IDNO:1的核苷酸15901-15924。
73．权利要求70的分离的核酸分子，其中所述核苷酸序列包含与选自下组的核苷酸序列中连续20个碱基对的部分序列相同的连续20个碱基对核苷酸部分SEQ ID NO:1的核苷酸11872-16104,SEQ IDNO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQ ID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:1的核苷酸14313-14334,SEQ ID NO:1的核苷酸14473-14547,SEQID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQ ID NO:1的核苷酸14788-15639，和SEQ ID NO:1的核苷酸15901-15924。
74．权利要求70的分离的核酸分子，其中所述核苷酸序列选自下组SEQ ID NO:1的核苷酸11872-16104,SEQ ID NO:1的核苷酸12085-12114,SEQ ID NO:1的核苷酸12223-12246,SEQ ID NO:1的核苷酸12466-12507,SEQ ID NO:1的核苷酸12928-12960,SEQID NO:1的核苷酸13516-13566,SEQ ID NO:1的核苷酸13633-13680,SEQ ID NO:1的核苷酸13876-13923,SEQ ID NO:1的核苷酸14313-14334,SEQ ID NO:1的核苷酸14473-14547,SEQ ID NO:1的核苷酸14578-14607,SEQ ID NO:1的核苷酸14623-14692,SEQ ID NO:1的核苷酸15673-15693,SEQ ID NO:1的核苷酸15724-15762,SEQID NO:1的核苷酸14788-15639，和SEQ ID NO:1的核苷酸15901-15924。
75．在重组宿主中异源表达epothilone的方法，包括a)将权利要求4的嵌合基因导入宿主；和b)在适合宿主生物合成epothilone的条件下培养宿主。
76．生产epothilone的方法，包括c)用权利要求75的方法在重组宿主中表达epothilone；和d)从重组宿主中提取epothilone。
77．一种分离的多肽，其包含由epothilone合酶结构域组成的氨基酸序列。
78．权利要求77的分离的多肽,其中所述epothilone合酶结构域是β-酮脂酰合酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸11-437,SEQ ID NO:4的氨基酸7-432,SEQ ID NO:5的氨基酸39-457,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸3024-3449,SEQ ID NO:5的氨基酸5103-5525,SEQ ID NO:6的氨基酸35-454,SEQ ID NO:6的氨基酸1522-1946，和SEQ ID NO:7的氨基酸32-450。
79．权利要求78的分离的多肽，其中所述β-酮脂酰合酶结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸11-437,SEQ IDNO:4的氨基酸7-432,SEQ ID NO:5的氨基酸39-457,SEQ ID NO:5的氨基酸1524-1950,SEQ ID NO:5的氨基酸3024-3449,SEQ IDNO:5的氨基酸5103-5525,SEQ ID NO:6的氨基酸35-454,SEQ IDNO:6的氨基酸1522-1946，和SEQ ID NO:7的氨基酸32-450。
80．权利要求77的分离的多肽，其中所述epothilone合酶结构域是酰基转移酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸543-864,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸3555-3876,SEQ ID NO:5的氨基酸5631-5951,SEQ ID NO:6的氨基酸561-881,SEQ ID NO:6的氨基酸2053-2373，和SEQ ID NO:7的氨基酸556-877。
81．权利要求80的分离的多肽，其中所述酰基转移酶结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸543-864,SEQ ID NO:4的氨基酸539-859,SEQ ID NO:5的氨基酸563-884,SEQ ID NO:5的氨基酸2056-2377,SEQ ID NO:5的氨基酸3555-3876,SEQ IDNO:5的氨基酸5631-5951,SEQ ID NO:6的氨基酸561-881,SEQ IDNO:6的氨基酸2053-2373，和SEQ ID NO:7的氨基酸556-877。
82．权利要求77的分离的多肽，其中所述epothilone合酶结构域是烯酰还原酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸6542-6837，和SEQ ID NO:7的氨基酸1478-1790。
83．权利要求82的分离的多肽，其中所述烯酰还原酶结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸974-1273,SEQ ID NO:5的氨基酸4433-4719,SEQ ID NO:5的氨基酸6542-6837，和SEQID NO:7的氨基酸1478-1790。
84．权利要求77的分离的多肽，其中所述epothilone合酶结构域是酰基载体蛋白结构域，所述多肽包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:2的氨基酸1314-1385,SEQ ID NO:4的氨基酸1722-1792,SEQ ID NO:5的氨基酸1434-1506,SEQ IDNO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸5010-5082,SEQID NO:5的氨基酸7140-7211,SEQ ID NO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸3673-3745，和SEQ ID NO:7的氨基酸2093-2164。
85．权利要求84的分离的多肽，其中所述酰基载体蛋白结构域包含选自下组的氨基酸序列SEQ ID NO:2的氨基酸1314-1385,SEQ IDNO:4的氨基酸1722-1792,SEQ ID NO:5的氨基酸1434-1506,SEQID NO:5的氨基酸2932-3005,SEQ ID NO:5的氨基酸5010-5082,SEQ ID NO:5的氨基酸7140-7211,SEQ ID NO:6的氨基酸1430-1503,SEQ ID NO:6的氨基酸3673-3745，和SEQ ID NO:7的氨基酸2093-2164。
86．权利要求77的分离的多肽，其中所述epothilone合酶结构域是脱水酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸5964-6132,SEQ ID NO:6的氨基酸2383-2551，和SEQ ID NO:7的氨基酸887-1051。
87．权利要求86的分离的多肽，其中所述脱水酶结构域包含选自下组的氨基酸序列SEQ ID NO:4的氨基酸869-1037,SEQ ID NO:5的氨基酸3886-4048,SEQ ID NO:5的氨基酸5964-6132,SEQ IDNO:6的氨基酸2383-2551，和SEQ ID NO:7的氨基酸887-1051。
88．权利要求77的分离的多肽，其中所述epothilone合酶结构域是β-酮还原酶结构域，其包含与选自下组的氨基酸序列基本相似的氨基酸序列SEQ ID NO:4的氨基酸1439-1684,SEQ ID NO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸2645-2895,SEQ ID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸6857-7101,SEQ IDNO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸3392-3636,和SEQ ID NO:7的氨基酸1810-2055。
89．权利要求88的分离的多肽，其中所述β-酮还原酶结构域包含选自下组的氨基酸序列SEQ ID NO:4的氨基酸1439-1684,SEQ IDNO:5的氨基酸1147-1399,SEQ ID NO:5的氨基酸2645-2895,SEQID NO:5的氨基酸4729-4974,SEQ ID NO:5的氨基酸6857-7101,SEQ ID NO:6的氨基酸1143-1393,SEQ ID NO:6的氨基酸3392-3636，和SEQ ID NO:7的氨基酸1810-2055。
90．权利要求77的分离的多肽，其中所述epothilone合酶结构域是甲基转移酶结构域，其包含与SEQ ID NO:6的氨基酸2671-3045基本相似的氨基酸序列。
91．权利要求90的分离的多肽，其中所述甲基转移酶结构域包含SEQ ID NO:6的氨基酸2671-3045。
92．权利要求77的分离的多肽，其中所述epothilone合酶结构域是硫酯酶结构域，其包含与SEQ ID NO:7的氨基酸2165-2439基本相似的氨基酸序列。
93．权利要求77的分离的多肽，其中所述硫酯酶结构域包含SEQ IDNO:7的氨基酸2165-2439。
全文摘要
由纤维堆囊菌分离得到编码epothilone生物合成必需的多肽的核酸分子。还公开了用经本发明的基因转化的重组宿主生产epothilone的方法。用这种方法生产的epothilone,足够用于它们的纯化和在药用制剂(诸如治疗癌症)中的使用。
文档编号C12R1/01GK1305530SQ99807421
公开日2001年7月25日 申请日期1999年6月16日 优先权日1998年6月18日
发明者T·斯彻普, J·M·利根, I·莫尔纳, R·泽克尔, J·戈拉彻, D·西尔 申请人:诺瓦提斯公司